Phage

J Bacteriol, 1997 May, 179(10), 3239 - 43
Comparative studies of the phage T2 and T4 DNA (N6-adenine)methyltransferases: amino acid changes that affect catalytic activity; Kossykh VG et al.; The bacteriophage T2 and T4 dam genes code for a DNA (N6-adenine)methyltransferase (MTase) . Nonglucosylated, hydroxymethylcytosine-containing T2gt- virion DNA has a higher level of methylation than T4gt- virion DNA does . To investigate the basis for this difference, we compared the intracellular enzyme levels following phage infection as well as the in vitro intrinsic methylation capabilities of purified T2 and T4 Dam MTases . Results from Western blotting (immunoblotting) showed that the same amounts of MTase protein were produced after infection with T2 and T4 . Kinetic analyses with purified homogeneous enzymes showed that the two MTases had similar Km values for the methyl donor, S-adenosyl-L-methionine, and for substrate DNA . In contrast, they had different k(cat) values (twofold higher for T2 Dam MTase) . We suggest that this difference can account for the ability of T2 Dam to methylate viral DNA in vivo to a higher level than does T4 Dam . Since the T2 and T4 MTases differ at only three amino acid residues (at positions 20 {T4, Ser; T2, Pro}, 26 {T4, Asn; T2, Asp}, and 188 {T4, Asp; T2, Glu}), we have produced hybrid proteins to determine which residue(s) is responsible for increased catalytic activity . The results of these analyses showed that the residues at positions 20 and 26 are responsible for the different k(cat) values of the two MTases for both canonical and noncanonical sites . Moreover, a single substitution of either residue 20 or 26 was sufficient to increase the k(cat) of T4 Dam.

J Bacteriol, 1997 May, 179(9), 3073 - 5
The integration host factor-DNA complex upstream of the early promoter of bacteriophage Mu is functionally symmetric; van Ulsen P et al.; Inversion of the ihf site in the promoter region of the early promoter of bacteriophage Mu did not influence the integration host factor (IHF)-mediated functions . IHF bound to this inverted site could counteract H-NS-mediated repression, directly activate transcription, and support lytic growth of bacteriophage Mu . This implies that the IHF heterodimer and its asymmetrical binding site form a functionally symmetrical complex.

J Bacteriol, 1997 May, 179(9), 2817 - 22
Cnr protein, the negative regulator of bacteriophage P4 replication, stimulates specific DNA binding of its initiator protein alpha; Ziegelin G et al.; Bacteriophage P4 DNA replication depends upon the phage-encoded alpha protein, which has DNA helicase and DNA primase activity and can specifically bind to the replication origin (ori) and to the cis replicating region (crr) . The P4 Cnr protein functions as a negative regulator of P4 replication, and P4 does not replicate in cells that overexpress cnr . We searched for P4 mutants that suppressed this phenotype (Cnr resistant {alpha cr}) . Eight independent mutants that grew in the presence of high levels of Cnr were obtained . None of these can establish the plasmid state . Each of these mutations lies in the DNA binding domain of gp alpha that occupies the C terminus of the protein . Five different sequence changes were found: T675M, G732V (three times), G732W (twice), L733V, and L737V . A TrxA-Cnr fusion protein does not bind DNA by itself but stimulates the ori and crr binding abilities of alpha protein in vitro . The alpha cr mutant proteins were still able to bind specifically to ori or crr, but specific DNA binding was less stimulated by the TrxA-Cnr protein . We present evidence that Cnr protein interacts with the gp alpha domain that binds specifically to DNA and that gp(alpha)cr mutations impair this interaction . We hypothesize that gp alpha-Cnr interaction is essential for the control of P4 DNA replication.

J Virol, 1997 May, 71(5), 3864 - 71
Sequential action of six virus-encoded DNA-packaging RNAs during phage phi29 genomic DNA translocation; Chen C et al.; A 120-base pRNA encoded by bacteriophage b29 has a novel and essential role in genomic DNA packaging . Six DNA-packaging RNAs (pRNAs) were bound to the sixfold symmetrical portal vertex of procapsids during the DNA translocation process and left the procapsid after the DNA-packaging reaction was completed, suggesting that the pRNA participated in the translocation of genomic DNA into procapsids . To further investigate the mechanism of DNA packaging, it is crucial to determine whether these six pRNA molecules work as an integrated entity or each pRNA acts as a functional individual . If pRNAs work individually, then do they work in sequence with communication or in random order without interaction? Results from compensation and complementation analysis did not support the integrated model . Computation of the probability of combination between wild-type and mutant pRNAs and experimental data of competitive inhibition excluded the random model while favoring the proposal that the six pRNAs functioned sequentially . Sequential action of the pRNA also explains why the pRNA is so sensitive to mutation, since the effect of a pRNA mutation will be amplified by 6 orders of magnitude after six consecutive steps, resulting in the observed complete loss of DNA-packaging activity caused by small alterations . When any one of the six pRNAs was replaced with an inactive one, complete blockage of DNA packaging resulted, strongly supporting the speculation that individual pRNAs, presumably together with other components such as the packaging ATPase gp16, take turns mediating successive steps of packaging . Although the data provided here could not exclude the integrated model completely, there is no evidence so far to argue against the model of sequential action.

Gene, 1997 Apr 29, 190(1), 11 - 5
An artificial regulatory circuit for stable expression of DNA-binding proteins in a T7 expression system; Paul BD et al.; We had earlier overproduced the transcription activator protein C of bacteriophage Mu in a phage-T7 expression system . Although we achieved a high level of overproduction, the expression was not consistent . This could be due to the leaky expression of T7 RNA polymerase in the uninduced state . Introduction of pLysS, a plasmid encoding T7 lysozyme, a natural inhibitor of T7 RNA polymerase, resulted in consistent, but extremely low production of the C protein . To overcome this problem, we have devised an artificial regulatory circuit to obtain stabilised, consistent overproduction of C protein . The C-binding site was cloned downstream from the transcription start point of T7 lys . Upon induction, the C protein produced binds to its site with a very high affinity, possibly acting as a transcriptional roadblock for lys . This would overcome the inhibitory effect of T7 lysozyme on T7 RNA polymerase.

Mol Gen Genet, 1997 Apr 28, 254(4), 358 - 64
Role of the coat protein-RNA interaction in the life cycle of bacteriophage MS2; Peabody DS; The coat protein of the RNA bacteriophage MS2 interacts with viral RNA to translationally repress replicase synthesis . This protein-RNA interaction is also thought to play a role in genome encapsidation . In this study the strength of the interaction was perturbed by constructing a recombinant genome containing a super-repressing coat mutation . Because replicase synthesis is prematurely repressed, the mutant produces plaques about five orders of magnitude less efficiently than wild-type . The few plaques obtained are second-site revertants of the original coat mutation and fall into two categories . Those of the first type contain nucleotide substitutions within the translational operator that reduce or destroy its ability to bind coat protein, showing that this interaction is not necessary for genome encapsidation . Revertants of the second type are double mutants in which one substitution converts the coat initiator AUG to AUA and the other substitutes an A for the G normally present two nucleotides upstream of the coat start codon . The mutation of the coat protein gene AUG to AUA, by itself, reduces coat protein synthesis to a few percent of the wild-type level . The second substitution destabilizes the coat initiator stem-loop and restores coat protein synthesis to within a few fold of wild-type levels.

J Biol Chem, 1997 Apr 25, 272(17), 11550 - 6
Specific function of DNA ligase I in simian virus 40 DNA replication by human cell-free extracts is mediated by the amino-terminal non-catalytic domain; Mackenney VJ et al.; The joining of Okazaki fragments during lagging strand DNA replication in mammalian cells is believed to be due to DNA ligase I . This enzyme is composed of a 78-kDa carboxyl-terminal catalytic domain and a 24-kDa amino-terminal region that is not required for ligation activity in vitro . Extracts of the human cell line 46BR.1G1, in which DNA ligase I is mutationally altered, supported aberrant in vitro SV40 DNA replication; the joining of Okazaki fragments was defective, and unligated intermediates were unstable . Human DNA ligase I, but not DNA ligase III or bacteriophage T4 DNA ligase, complemented both defects in 46BR.1G1 extracts . The catalytic domain of DNA ligase I was 10-fold less effective in complementation experiments than the full-length protein, indicating that the amino-terminal region of the enzyme is required for efficient lagging strand DNA replication . Moreover, in vitro SV40 DNA replication in normal human cell extracts was inhibited by an excess of either full-length DNA ligase I or the amino-terminal region of the protein, but not by the catalytic domain . This inhibition may be mediated by the interaction of the amino-terminal region of DNA ligase I with other replication proteins.

J Mol Biol, 1997 Apr 18, 267(5), 1089 - 103
Recognition of bacteriophage Qbeta plus strand RNA as a template by Qbeta replicase: role of RNA interactions mediated by ribosomal proteins S1 and host factor; Miranda G et al.; RNA-protein interactions between bacteriophage Qbeta plus strand RNA and the components of the Qbeta replicase system were studied by deletion analysis . Internal, 5'-terminal and 3'-terminal deletions were assayed for template activity with replicase in vitro . Of the two internal binding sites previously described for replicase, we found that the S-site (map position 1247 to 1346) could be deleted without any significant effect on template activity, whereas deletion of the M-site (map position 2545 to 2867) resulted in a strong inactivation and a high salt sensitivity of the residual activity . Binding complexes of the deletion mutant RNAs with the different proteins involved in Qbeta RNA replication were analysed by electron microscopy . The formation of looped complex structures, previously reported and explained as simultaneous interactions with replicase at the S and the M-site, was abolished by deleting the S-site but, surprisingly, not by deleting the M-site . The same types of complexes observed with replicase were also formed with purified protein S1 (the alpha subunit of replicase), suggesting that these internal interactions with Qbeta RNA are mediated by the S1 protein . The Qbeta host factor, a protein required for the template activity of the Qbeta plus strand, was reported earlier to form similar complexes by binding to the S and M-sites (or adjacent sites) and in addition to the 3'-end, resulting in double-looped structures . The patterns of looped complexes observed with the deletion mutant RNAs suggest that the binding of host factor might not involve the S and M-sites themselves but adjacent downstream sites . An additional internal host factor interaction near map position 2300 was detected with several mutant RNAs . Qbeta RNA molecules with 3'-truncations formed 3'-terminal loops with similar efficiency as wild-type RNA, indicating that recognition of the 3'-end by host factor is not dependent on a specific 3'-terminal base sequence.

Mol Gen Genet, 1997 Apr 16, 254(3), 304 - 11
Impaired lysogenisation of the Escherichia coli rpoA341 mutant by bacteriophage lambda is due to the inability of CII to act as a transcriptional activator; Obuchowski M et al.; The C-terminus of the alpha subunit of Escherichia coli RNA polymerase is known to function in transcriptional activation at certain promoters . This region was previously shown to be necessary for full activation of the pE promoter by the phage lambda CII protein in vitro . In this work we investigated the inability of phage lambda to follow the lysogenic pathway in cells carrying the point mutation rpoA341 (a change of lysine 271 to glutamic acid) . We found that neither overexpression of the cII gene nor stabilisation of the CII protein by the can1 mutation or by cIII gene overexpression was able to suppress the block in lysogenisation . In contrast, the lambda cin1 phage, which carries a CII-independent promoter for the expression of the cI gene, was able to efficiently lysogenise the rpoA341 mutant strain . Furthermore, the rpoA341 mutation prevented the activation of pE-lacZ and pI-lacZ transcriptional fusions by CII . Therefore we conclude that transcriptional activation by the cII gene product is abolished by the rpoA341 mutation, most probably due to impaired interaction between the CII activator and mutant RNA polymerase . The inability of RNA polymerase to respond to CII results in the impairment of lysogenisation of the rpoA341 mutant by phage lambda.

EMBO J, 1997 Apr 15, 16(8), 1992 - 2003
The activation domain of the MotA transcription factor from bacteriophage T4; Finnin MS et al.; Bacteriophage T4 encodes a transcription factor, MotA, that binds to the -30 region of middle-mode promoters and activates transcription by host RNA polymerase . We have solved the structure of the MotA activation domain to 2.2 A by X-ray crystallography, and have also determined its secondary structure by NMR . An area on the surface of the protein has a distinctive patch that is populated with acidic and hydrophobic residues . Mutations within this patch cause a defective T4 growth phenotype, arguing that the patch is important for MotA function . One of the mutant MotA activation domains was purified and analyzed by NMR, and the spectra clearly show that the domain is properly folded . The mutant full-length protein appears to bind DNA normally but is deficient in transcriptional activation . We conclude that the acidic/hydrophobic surface patch is specifically involved in transcriptional activation, which is reminiscent of eukaryotic acidic activation domains.

Proc Natl Acad Sci U S A, 1997 Apr 15, 94(8), 4074 - 9
Stoichiometric packaging of the three genomic segments of double-stranded RNA bacteriophage phi6; Qiao X et al.; A model that explains the stoichiometric packaging of the chromosomes of phi6, a bacteriophage with a genome of three unique double-stranded RNA segments, is proposed and supported . Ordered switches in packaging specificity and RNA synthesis are determined by the amount of RNA within the procapsid . The plus strand of segment S binds to one of several sites on the outside of the empty procapsid . The RNA enters and the procapsid expands so that the S sites are lost and M sites appear . Packaging of segment M results in the loss of the M sites and the appearance of the L sites . Packaging of L readies the particle for minus-strand synthesis . If any of the segments is less than normal size, packaging of that class of segments continues until the normal content of RNA for that segment is packaged and the binding sites then change.

Proc Natl Acad Sci U S A, 1997 Apr 15, 94(8), 4068 - 73
A permeabilized cell system that assembles filamentous bacteriophage; Feng JN et al.; A permeabilized cell system has been developed that is capable of assembling filamentous phage only upon addition of exogenous thioredoxin . The in vitro system exhibits the same component requirements seen in vivo: functional thioredoxin, an intact packaging signal in the substrate DNA, and the assembly protein, pIV . This crude in vitro system is insensitive to inhibitors of protein or DNA synthesis, demonstrating that particle assembly uses components that had accumulated before cell permeabilization . The temporal separation of the synthetic period, during which phage proteins and DNA accumulate, from the assembly period enabled us to examine the energy requirement for assembly . We show here that ATP hydrolysis is required for filamentous phage assembly and that the proton motive force is also important.

Proc Natl Acad Sci U S A, 1997 Apr 15, 94(8), 3679 - 84
The T/t common exon of simian virus 40, JC, and BK polyomavirus T antigens can functionally replace the J-domain of the Escherichia coli DnaJ molecular chaperone; Kelley WL et al.; The N-terminal 70 residue "J-domain" of the Escherichia coli DnaJ molecular chaperone is the defining and highly conserved feature of a large protein family . Based upon limited, yet significant, amino acid sequence homology to the J-domain, the DNA encoding the T/t common exon of the simian virus 40 (SV40), JC, or BK polyoma virus T antigen oncoproteins was used to construct J-domain replacement chimeras of the E . coli DnaJ chaperone . The virally encoded J-domains successfully substituted for the bacterial counterpart in vivo as shown by (i) complementation for viability at low and high temperature of a hypersensitive bacterial reporter strain, and (ii) the restoration of bacteriophage lambda plaque forming ability in the same strain . The amino acid change, H42Q, in the SV40 T/t and the JC virus T/t exon, which is positionally equivalent to the canonical dnaJ259 H33Q mutation within the E . coli J-domain, entirely abolished complementing activity . These results strongly suggest that the heretofore functionally undefined viral T/t common exon represents a bona fide J-domain that preserves critical features of the characteristic domain fold essential for J-domain interaction with the ATPase domain of the Hsp70 family . This finding has implications for the regulation of DNA tumor virus T antigens by molecular chaperones.

Nucleic Acids Res, 1997 Apr 15, 25(8), 1649 - 57
Direct genetic selection of two classes of R17/MS2 coat proteins with altered capsid assembly properties and expanded RNA-binding activities; Wang S et al.; RNA challenge phages are derivatives of bacteriophage P22 that enable direct genetic selection for a specific RNA-protein interaction . The bacteriophage P22 R17 encodes a wild-type R17 operator site and undergoes lysogenic development following infection of susceptible bacterial strains that express the R17/MS2 coat protein . A P22 R17 derivative with an OcRNA site (P22 R17 {A(-10)U}) develops lytically following infection of these strains . RNA challenge phages can be used to isolate second-site coat protein suppressors that recognize an OcRNA sequence by selecting for lysogens with a P22 R17 {Oc} phage derivative . The bacteriophage derivative P22 R17 {A(-10)U} was used in one such scheme to isolate two classes of genes that encode R17 coat proteins with altered capsid assembly properties and expanded RNA-binding characteristics . These mutations map outside the RNA-binding surface and include amino acid substitutions that interfere with interactions between coat protein dimers in the formation of the stable phage capsid . One class of mutants encodes substitutions at the highly conserved first and second positions of the mature coat protein . N-terminal sequence analysis of these mutants reveals that coat proteins with substitutions only at position 1 are defective in post-translational processing of the initiator methionine . All selected proteins possess expanded RNA-binding properties since they direct efficient lysogen formation for P22 R17 and P22 R17 {A(-10)U}; however, bacterial strains that express the protein mutants remain sensitive to lytic infection by other P22 R17 {Oc} bacteriophages . The described selection strategy provides a novel genetic approach to dissecting protein structure within RNA-binding proteins.

Virology, 1997 Apr 14, 230(2), 292 - 9
The two P2 Ogr-like domains of the delta protein from bacteriophage P4 are required for activity; Julien B et al.; The satellite P4 phage Delta protein positively regulates the late genes of its helper bacteriophage P2, as well as its own late genes . Delta is a member of a class of activators associated with P2-or P4-like phages and is the largest member of this family . It resembles a covalently joined head-to-tail dimer of the other members of this family of activators . We have analyzed the requirement for both standard domains of Delta through the isolation of amber mutants and the insertion of amber linkers . We show that both domains of Delta are required for DNA binding in vivo and for transcriptional activity . Proper spacing between the two domains is important for activity at two of the four P2 promoters . Expression of both domains from different plasmids causes activation of late gene transcription in vivo of all six late promoters of P2 and P4 . A monomric Delta from another satellite phage, phi R73, can function efficiently as a covalent dimer but when this Delta is made dimeric with the second half of P4 delta, it activates less efficiently.

Biochim Biophys Acta, 1997 Apr 12, 1360(2), 169 - 76
The molecular basis for cross-reaction of an anti-dystrophin antibody with alpha-actinin; James M et al.; The epitope recognised by the anti-dystrophin monoclonal antibodies MANDYS141 and MANDYS142 has been characterised using a phage display peptide library and a bacteriophage lambda cDNA library . Using a phage display library of random 15-mer peptides, the epitope recognised by the two antibodies was identified as EEXF . A lambda gt11 clone obtained by screening a human muscle cDNA library was shown to contain part of the out-of-frame human mitochondrial succinyl CoA synthetase (alpha-subunit) cDNA sequence which contains the sequence EEPL, suggesting a minimum requirement of EEXF/L for antibody binding . The sequence EEDF is located in the helical rod region of dystrophin and the N-terminal domain of alpha-actinin; this may explain why native dystrophin is not detected, since the alpha-helical, coiled-coil folding of the rod region of dystrophin may obscure the epitope in the native protein . The antibody cross-reaction between dystrophin and alpha-actinin is likely to be fortuitous and not due to any structural homology that exists between these two members of the spectrin superfamily.

Biochemistry, 1997 Apr 8, 36(14), 4223 - 32
Kinetic mechanism of transcription initiation by bacteriophage T7 RNA polymerase; Jia Y et al.; The kinetic mechanism of transcription initiation by bacteriophage T7 RNA polymerase was investigated using transient state kinetic methods . Transcription by bacteriophage T7 RNA polymerase occurs in three stages consisting of initiation, promoter clearance, and elongation . Abortive products, up to 6-8-mer, were synthesized during the initiation phase; the transition from initiation to elongation occurred between the synthesis of 6-8-mer and 11-12-mer, and the processive elongation phase began after the synthesis of 12-mer RNA . Our results show that the synthesis of elongation product from the phi 10 promoter is limited both by the efficiency of initiation and by the frequency at which the polymerase escapes the promoter . Studies with heparin trap suggest that the polymerase maintains contact with the promoter region during multiple turnovers of abortive RNA synthesis; thus, the polymerase does not completely dissociate from the promoter after each event of abortive RNA synthesis . The pre-steady-state kinetics of RNA synthesis indicate that initiation occurs at a rate constant (3.5 s(-1)) that is about 30 times faster than the steady-state rate constant of RNA synthesis (0.1 s(-1)) . The steady-state rate constant of RNA synthesis is limited largely by the cycling of the RNA polymerase, whereas initiation is limited by the formation of pppGpG, the first RNA product . We show that the synthesis of pppGpG is not limited by steps associated with GTP binding, DNA binding, or the melting of the promoter DNA . Instead, the kinetic results indicate that initiation at the phi10 promoter is limited either by the first phosphodiester bond formation step or more likely by a conformational change prior to pppGpG formation . Such a conformational change could play a role in proper alignment of the initiating and elongating NTPs for efficient phosphodiester bond formation and in maintaining the fidelity of RNA synthesis.

Mol Biotechnol, 1997 Apr, 7(2), 153 - 63
The use of RNA probes for the analysis of gene expression; Belin D; The monomeric bacteriophage RNA polymerases allow the synthesis of virtually any RNA molecule in unlimited quantity . In this protocol, we describe the preparation of plasmid and PCR-derived templates . A basic transcription protocol is provided with several optional modifications . The use of RNA probes in Northern blot hybridization and in RNase protection assays is described . The relative advantages and pitfalls of these two methods to quantitatively detect mRNA targets are discussed.

Int J Biol Macromol, 1997 Apr, 20(2), 115 - 21
Secondary structure of T4 gene 33 protein . Fourier transform infrared and circular dichroic spectroscopic studies; Shao W et al.; The secondary structure of bacteriophage T4 gene 33 protein (gp33) has been quantitatively examined by using Fourier transform infrared (FT-IR) and circular dichroism (CD) spectroscopy . Resolution enhancement techniques, including Fourier deconvolution and derivative spectroscopy were used to quantitate the spectral information from the amide I bands . The relative areas of these component bands indicate 21% alpha-helix, 25% beta-sheet, 34% turn, 12% random coil and 8% other undefined structures in gp33 . An analysis of the CD spectrum of gp33 at the same pH and temperature revealed 19% alpha-helix, 25% beta-sheet, 13% turn and 43% random coil structures . The possible reasons for the discrepancies in estimates of the contributions to the secondary structure from turns and random coils are discussed.

Mol Microbiol, 1997 Apr, 24(2), 341 - 53
Mutations of the coat protein gene of bacteriophage lambda that overcome the necessity for the Fl gene; the EFi domain; Murialdo H et al.; The functions of most of the 10 genes involved in phage lambda capsid morphogenesis are well understood . The function of the FI gene is one of the exceptions . Mutants in FI fail to mature and package DNA . The gene product (gpFI) seems to act as a catalyst for the formation of an intermediate in capsid assembly called complex II, which contains a procapsid (an empty capsid precursor), terminase (the enzyme that cleaves the DNA precursor and packages it into the procapsid) and DNA . The mechanism for this stimulation remains unknown . It has also been reported that gpFI appeared to stimulate terminase-mediated cos cleavage, in the absence of procapsids, by increasing enzyme turnover . In comparison with other head-gene mutants, FI mutants are leaky, producing approx . 0.1 phage per infected cell . Some second-site revertants of FI- phages, called 'fin', that bypass the necessity for gpFI, have been isolated and found to harbour a mutation in the genes that code for the two subunits of terminase . In the course of mapping additional fin mutants, it was discovered that some mapped outside the terminase genes . To localize the mutations, restriction fragments of fin mutant DNAs were subcloned into plasmids and their ability to contribute to fin function was determined by marker-rescue analysis . The location of the fin mutation was further delineated by deletion analysis of a plasmid that was positive for fin . This showed that some fin mutations mapped to a region comprising genes E, D and a portion of C . The sequencing of this entire region in several fin isolates showed that the fin mutations are clustered in a small region of gene E corresponding to a portion of 26 amino acid residues of the coat protein (gpE) . We have called this region of the protein the EFI domain . All the mutations result in an increase in positive charge relative to the wild-type protein . These results suggest that DNA maturation and packaging are in part controlled by an interaction between gpFI and capsid gpE.

EMBO J, 1997 Apr 1, 16(7), 1795 - 805
The oligomeric structure of nucleoid protein H-NS is necessary for recognition of intrinsically curved DNA and for DNA bending; Spurio R et al.; Escherichia coli hns, encoding the abundant nucleoid protein H-NS, was subjected to site-directed mutagenesis either to delete Pro115 or to replace it with alanine . Unlike the wild-type protein, hyperproduction of the mutant proteins did not inhibit macromolecular syntheses, was not toxic to cells and caused a less drastic compaction of the nucleoid . Gel shift and ligase-mediated circularization tests demonstrated that the mutant proteins retained almost normal affinity for non-curved DNA, but lost the wild-type capacity to recognize preferentially curved DNA and to actively bend non-curved DNA, a property of wild-type H-NS demonstrated here for the first time . DNase I foot-printing and in vitro transcription experiments showed that the mutant proteins also failed to recognize the intrinsically bent site of the hns promoter required for H-NS transcription autorepression and to inhibit transcription from the same promoter . The failure of the Pro115 mutant proteins to recognize curved DNA and to bend DNA despite their near normal affinity for non-curved DNA can be attributed to a defect in protein-protein interaction resulting in a reduced capacity to form oligomers observed in vitro and by a new in vivo test based on functional replacement by H-NS of the oligomerization domain (C-domain) of bacteriophage lambda cI repressor.

J Virol Methods, 1997 Apr, 65(1), 45 - 54
Development of an improved product enhanced reverse transcriptase assay; Chang A et al.; A PCR based reverse transcriptase (RT) assay was developed that has 10(4)-fold higher sensitivity than conventional nucleotide incorporation assays and allows discrimination between false positive results generated by cellular polymerases and positives resulting from authentic RT activity . Recently, several reverse transcriptase (RT) assays have been developed where a reverse transcriptase reaction is performed on an RNA template/DNA primer combination . A specific region of the cDNA product is then amplified by the polymerase chain reaction to increase the sensitivity of cDNA detection . These reverse transcriptase assays up to 10(6)-fold more sensitive at detecting retroviruses than conventional methods . The drawback to these assays with increased sensitivity is the increased incidence of false positive results generated by cellular polymerases that can reverse transcribe . The MS2 bacteriophage RNA template and primers from one of the recently developed assays were used as the basis to develop the assay . A simple high resolution agarose gel was used as the endpoint for the assay without compromising sensitivity . In addition, the pH of the RT reaction was lowered to pH 5.5, the RT incubation was 1 h, and protease inhibitors were added to the RT reaction components . These modifications yield an assay that can discriminate between authentic RT activity and contaminating cellular polymerases.

Methods, 1997 Apr, 11(4), 371 - 81
Synthesis and purification of single-stranded RNA for use in experiments with PKR and in cell-free translation systems; Pe'ery T et al.; The biosynthesis of RNA in vitro using bacteriophage RNA polymerases has opened up many avenues of research . Large amounts of specific RNA species can be readily produced but small amounts of contaminants that are simultaneously generated can interfere with biological assays, PKR, a ribosome-associated and double-stranded (ds) RNA-dependent protein kinase, is an important regulator of the initiation of protein synthesis . It can be activated by very low concentrations of dsRNA and inhibited by small structured RNAs or high concentrations of dsRNA . The best-studied inhibitor of PKR activation is adenovirus VA RNA1 . Its gene was cloned into a plasmid under the control of the T7 RNA polymerase promoter, and the optimization of VA RNA transcription is described . A dsRNA by-product of the transcription reaction activates PKR in kinase autophosphorylation assays, and hence a purification protocol that allows the separation and removal of dsRNA contaminants was developed . A scheme to analyze the RNA product with specific nucleases is discussed . In a reticulocyte cell-free translation system the activation of PKR by dsRNA contaminating a synthetic mRNA preparation is likely to lead to shut-off of translation . An assay to directly visualize and measure the level of PKR phosphorylation in the lysate is detailed.

Arthritis Rheum, 1997 Apr, 40(4), 615 - 23
T cell receptor alpha-chain and beta-chain junctional region homology in clonal CD3+, CD8+ T lymphocyte expansions in Felty's syndrome; Bowman SJ et al.; OBJECTIVE: Up to 42% of patients with Felty's syndrome (FS) have peripheral blood expansions of CD3+,CD8+ large granular lymphocytes (LGLs) . The aim of this study was to determine whether the T cell receptor (TCR) alpha- and beta-chain sequences of these expansions from different patients have features in common that would support the hypothesis of an antigen-driven process . METHODS: Extraction of RNA from peripheral blood lymphocytes followed by synthesis of complementary DNA, inverse polymerase chain reaction (PCR) with TCR-specific primers, bacteriophage transformation, and sequencing of PCR products . RESULTS: Structural analysis of TCR beta-chain usage in such patients demonstrated a junctional region motif comprising the amino acids -LG- or -RG- in 7 of 14 clonal sequences and the motif -GXG- in 8 of 14 . A biased alpha-chain junctional region usage of a hydrophobic and/or basic amino acid at position 2 was seen in 5 of 8 expanded sequences . These features differed significantly from control sequences . CONCLUSION: Given current models of TCR-peptide-major histocompatibility complex interaction, these observations are consistent with an antigen-driven, rather than a superantigen-driven, process in at least a subgroup of patients with FS.

J Bacteriol, 1997 Apr, 179(8), 2479 - 85
Mutations in Nu1, the gene encoding the small subunit of bacteriophage lambda terminase, suppress the postcleavage DNA packaging defect of cosB mutations; Cai ZH et al.; The linear double-stranded DNA molecules in lambda virions are generated by nicking of concatemeric intracellular DNA by terminase, the lambda DNA packaging enzyme . Staggered nicks are introduced at cosN to generate the cohesive ends of virion DNA . After nicking, the cohesive ends are separated by terminase; terminase bound to the left end of the DNA to be packaged then binds the empty protein shell, i.e., the prohead, and translocation of DNA into the prohead occurs . cosB, a site adjacent to cosN, is a terminase binding site . cosB facilitates the rate and fidelity of the cosN cleavage reaction by serving as an anchoring point for gpNu1, the small subunit of terminase . cosB is also crucial for the formation of a stable terminase-DNA complex, called complex I, formed after cosN cleavage . The role of complex I is to bind the prohead . Mutations in cosB affect both cosB functions, causing mild defects in cosN cleavage and severe packaging defects . The lethal cosB R3- R2- R1- mutation contains a transition mutation in each of the three gpNu1 binding sites of cosB . Pseudorevertants of lambda cosB R3- R2- R1- DNA contain suppressor mutations affecting gpNu1 . Results of experiments that show that two such suppressors, Nu1ms1 and Nu1ms3, do not suppress the mild cosN cleavage defect caused by the cosB R3- R2- R1- mutation but strongly suppress the DNA packaging defect are presented . It is proposed that the suppressing terminases, unlike the wild-type enzyme, are able to assemble a stable complex I with cosB R3- R2- R1- DNA . Observations on the adenosine triphosphatase activities and protease susceptibilities of gpNu1 of the Nu1ms1 and Nu1ms3 terminases indicate that the conformation of gpNu1 is altered in the suppressing terminases.

Hum Mol Genet, 1997 Apr, 6(4), 615 - 25
Identification of the gene encoding the human mitochondrial RNA polymerase (h-mtRPOL) by cyberscreening of the Expressed Sequence Tags database; Tiranti V et al.; A gene cloning strategy based on the screening of the Expressed Sequence Tags database (dbEST) using sequences of mitochondrial housekeeping proteins of yeast was employed to identify the cDNA encoding the precursor of the human mitochondrial RNA polymerase (h-mtRPOL) . The 3831 bp h-mtRPOL cDNA is located on chromosome 19p13.3 and encodes a protein of 1230 amino acid residues . The protein sequence shows significant homologies with sequences corresponding to mitochondrial RNA polymerases from lower eukaryotes, and to RNA polymerases from several bacteriophages . The mitochondrial RNA polymerase carries out the central activity of mitochondrial gene expression and, by providing the RNA primers for replication-initiation, is also implicated in the maintenance and propagation of the mitochondrial genome . Genes involved in the control of mtDNA replication and gene expression are attractive candidates for human disorders due to abnormalities of nucleo-mitochondrial intergenomic signalling . The availability of the h-mtRPOL cDNA will allow us to test its role in mitochondrial pathology . In addition, we propose the 'cyberscreening' of dbEST, based on yeast/human cross-species comparison, as a powerful, simple, rapid and inexpensive method, that may accelerate several-fold the molecular dissection of the human mitochondrial proteome.

Appl Environ Microbiol, 1997 Apr, 63(4), 1551 - 6
Chlorella virus PBCV-1 encodes a homolog of the bacteriophage T4 UV damage repair gene denV; Furuta M et al.; The bacteriophage T4 denV gene encodes a well-characterized DNA repair enzyme involved in pyrimidine photodimer excision . We have discovered the first homologs of the denV gene in chlorella viruses, which are common in fresh water . This gene functions in vivo and also when cloned in Escherichia coli . Photodamaged virus DNA can also be photoreactivated by the host chlorella . Since the chlorella viruses are continually exposed to solar radiation in their native environments, two separate DNA repair systems, one that functions in the dark and one that functions in the light, significantly enhance their survival.

Proc Natl Acad Sci U S A, 1997 Apr 1, 94(7), 2987 - 92
Gene 4 helicase of bacteriophage T7 mediates strand transfer through pyrimidine dimers, mismatches, and nonhomologous regions; Kong D et al.; In bacteriophage T7 the gene 2.5 single-stranded DNA-binding protein and the gene 4 helicase together promote the annealing of homologous regions of two DNA partners to form a joint molecule and subsequent strand transfer . In this reaction T7 gene 2.5 protein is essential for joint molecule formation, but is not required for T7 gene 4 protein-mediated strand transfer . T7 gene 4 helicase alone is able to mediate strand transfer, provided that a joint molecule is available . The present paper shows that, in addition, strand transfer proceeds at a normal rate even when both DNA partners contain ultraviolet-induced pyrimidine dimers (0.6 dimer per 100 nt) . An insert of a relatively long (842-nt) segment of nonhomologous DNA in the single-stranded DNA partner has no effect on strand transfer, whereas its presence in the double-stranded partner prevents strand transfer . A short insert (37 nt) can be tolerated in either partner . Thus, DNA helicase is able to participate in recombinational DNA repair through its role in strand exchange, providing a pathway distinct from nucleotide excision repair.

Proc Natl Acad Sci U S A, 1997 Apr 1, 94(7), 2921 - 6
In vitro evolution of terminal protein-containing genomes; Esteban JA et al.; A new self-sustained terminal protein-primed DNA amplification system has been used to describe in vitro evolutionary changes affecting maintenance of the genome size of bacteriophage phi29 . These changes involve generation and efficient amplification of short palindromic molecules containing an inverted duplication of one of the original DNA ends . A template-switching mechanism is proposed to account for the appearance of these molecules . After their formation, they would replicate by means of hairpin intermediates . Relevant kinetic information about this DNA replication system has been obtained from the competition between the input full-length phi29 DNA and its derived truncated versions . The physiological relevance of these molecules and the mechanisms to control their formation are discussed.

Biochemistry, 1997 Apr 1, 36(13), 4015 - 26
Backbone dynamics of the major coat protein of bacteriophage M13 in detergent micelles by 15N nuclear magnetic resonance relaxation measurements using the model-free approach and reduced spectral density mapping; Papavoine CH et al.; The backbone dynamics of the major coat protein (gVIIIp) of the filamentous bacteriophage M13, solubilized in detergent micelles, have been studied using 15N nuclear magnetic resonance spectroscopy at three frequencies . Motional parameters and overall and internal correlation times were derived with the model-free approach . It was also checked whether these parameters had to be modified due to anisotropic motion of the protein/micelle complex . Reduced spectral density mapping was used to calculate the spectral densities at J(O), J(omegaN), and {J(omegaH)} . The spectral densities were interpreted by mapping a linear or scaled linear combination of two Lorentzians onto a J(O)-J(omega) plot . The major coat protein of bacteriophage M13 consists of two alpha-helices, one of which is hydrophobic and located within the micelle, while the other is amphipathic and located on the surface of the micelle . Our results indicate that the motion of the hydrophobic helix is restricted such that it corresponds to the overall tumbling of the protein/micelle complex . The interpretation of the relaxation data of the amphipathic helix by means of the model-free approach and the reduced spectral density mapping indicate that in addition to the overall motion all residues in this helix are subject to motion on the fast nanosecond and picosecond time scales . The motions of the vectors in the low nanosecond range are characterized by similar values of the spectral densities and correlation times and represent the motion of the amphipathic helix on and away from the surface of the micelle . The relaxation data of the residues in the hinge region connecting the helices show that there is an abrupt change from highly restricted to less restricted motion . Both the C-terminal and N-terminal residues are very mobile.

Virology, 1997 Mar 31, 230(1), 72 - 81
A novel mechanism of virus-virus interactions: bacteriophage P2 Tin protein inhibits phage T4 DNA synthesis by poisoning the T4 single-stranded DNA binding protein, gp32; Mosig G et al.; P2 prophages have been known to inhibit DNA replication and growth of T-even phages . We show here that this inhibition is due to poisoning of the T-even single-stranded DNA binding protein gp32 by the product of the nonessential P2 tin gene . Synthesis of Tin protein from a gene cloned in a multicopy plasmid is necessary and sufficient to completely prevent de novo DNA replication and growth of wild-type T2 or T4 phage . We isolated more than 20 independent mutants that render T-even phages resistant to poisoning by the P2 Tin protein . In all of these mutants, which we call asp, Asp codon 163 of gene 32 is changed to a Gly or Asn codon . The mutant alleles are recessive; i.e., when wild-type and asp mutants coinfect the same host cells, most DNA replication is poisoned by P2 Tin protein . To explain our results, we propose that the P2 Tin protein interacts with T-even gp32 at position 163 and distorts the helical filament of gene 32 protein on single-stranded DNA . Thereby Tin protein inhibits either assembly or function, or both, of the T4 replisome . The inhibition of late gene expression by P2 Tin protein may be an indirect consequence of inhibition of DNA replication.

J Mol Biol, 1997 Mar 28, 267(2), 403 - 17
An aromatic stacking interaction between subunits helps mediate DNA sequence specificity: operator site discrimination by phage lambda cI repressor; Huang YT et al.; Sequence specific DNA binding by regulatory proteins provides the basis for regulation of initiation of transcription . A great deal of progress has been made toward understanding sequence specific recognition by individual protein subunits . An additional level of control that needs to be understood is that due to coupling between the subunits of oligomeric regulatory proteins . An example is the bacteriophage lambda cI repressor, a dimeric protein that regulates the lysogenic to lytic genetic switch of the phage . Two levels of specificity are critical to this regulation . First, like all transcriptional regulators, dimers distinguish operator from nonspecific DNA . Direct readout of the DNA sequence by the recognition helix is considered the well understood mechanism for this . However, differential affinity for O(R)1, O(R)2 and O(R)3 is equally critical to the switch because it mediates opposing regulation of divergent promoters . Site specificity at this second level is less well understood . Conformational adaptation by both the repressor and the different operators appears to be important . To evaluate how subunit-subunit interactions are involved in this process, we investigated the effects on both dimer stability and operator binding of amino acid substitutions at the contacts between the symmetrically related helices-5 in the dimer interface . Substitutions for Tyr88 alter dimer stability and greatly perturb differential operator affinity, but generally do not affect operator versus non-operator specificity . The pattern of these effects suggests that the geometry of the face-to-face aromatic stacking interaction between symmetrically related Tyr88 in each subunit, a group in the dimer interface but far removed from the DNA binding interface, plays a critical role in operator discrimination . Conformational changes in the tertiary structure of the subunits appears to be involved . By contrast, the significant effect of I84S substitution is to greatly decrease affinity for all three operators . Presumably, the altered packing of the dimer interface causes a quarternary structural change that moves the two helix-turn-helix motifs out of register with successive DNA major grooves.

J Mol Biol, 1997 Mar 28, 267(2), 237 - 49
The genome of the pseudo T-even bacteriophages, a diverse group that resembles T4; Monod C et al.; Polymerase chain reaction analysis of a large collection of bacteriophages with T-even morphology revealed four phages that are distantly related to all the others . The genomes of these pseudo T-even phages hybridized under stringent conditions to only a limited portion of the T4 genome that encodes virus head, head-to-tail joining and contractile tail genes . Except for this region, no extensive hybridization was detected between most pairs of the different pseudo T-even genomes . Sequencing of this conserved region of the pseudo T-even phage RB49 revealed substantial nucleotide sequence divergence from T4 (approximately 30% to 40%), and random genomic sequencing of this phage indicated that more than a third of its sequences had no detectable homology to T4 . Among those sequences related to the T-even genes were virion structural components including the constituents of the phage base plate . Only a few sequences had homology to T4 early functions; these included ribonucleotide diphosphatase reductase, DNA ligase and the large subunit of DNA topoisomerase . The genomes of the pseudo T-even phage were digested by restriction enzymes that are unable to digest the T-even DNAs which contain glucosylated hydroxymethyl-cytosine residues . This suggests that only limited nucleotide modifications must be present in the pseudo T-even genomes . Conservation of much of the morphogenetic region of these diverse phage genomes may reflect particularly strong sequence constraints on these gene products . However, other explanations are considered, including the possibility that the various morphogenetic segments were acquired by the pseudo T-even genomes by modular evolution . These results support the notion that phage evolution may proceed within a network of both closely and distantly related genomes.

J Biol Chem, 1997 Mar 28, 272(13), 8380 - 7
Role of the bacteriophage T7 and T4 single-stranded DNA-binding proteins in the formation of joint molecules and DNA helicase-catalyzed polar branch migration; Kong D et al.; Bacteriophage T7 gene 2.5 single-stranded DNA-binding protein and gene 4 DNA helicase together promote pairing of two homologous DNA molecules and subsequent polar branch migration (Kong, D., and Richardson, C . C . (1996) EMBO J . 15, 2010-2019) . In this report, we show that gene 2.5 protein is not required for the initiation or propagation of strand transfer once a joint molecule has been formed between the two DNA partners, a reaction that is mediated by the gene 2.5 protein alone . A mutant gene 2.5 protein, gene 2.5-Delta21C protein, lacking 21 amino acid residues at its C terminus, cannot physically interact with gene 4 protein . Although it does bind to single-stranded DNA and promote the formation of joint molecule via homologous base pairing, subsequent strand transfer by gene 4 helicase is inhibited by the presence of the gene 2.5-Delta21C protein . Bacteriophage T4 gene 32 protein likewise inhibits T7 gene 4 protein-mediated strand transfer, whereas Escherichia coli single-stranded DNA-binding protein does not . The 63-kDa gene 4 protein of phage T7 is also a DNA primase in that it catalyzes the synthesis of oligonucleotides at specific sequences during translocation on single-stranded DNA . We find that neither the rate nor extent of strand transfer is significantly affected by concurrent primer synthesis . The bacteriophage T4 gene 41 helicase has been shown to catalyze polar branch migration after the T4 gene 59 helicase assembly protein loads the helicase onto joint molecules formed by the T4 UvsX and gene 32 proteins (Salinas, F., and Kodadek, T . (1995) Cell 82, 111-119) . We find that gene 32 protein alone forms joint molecules between partially single-stranded homologous DNA partners and that subsequent branch migration requires this single-stranded DNA-binding protein in addition to the gene 41 helicase and the gene 59 helicase assembly protein . Similar to the strand transfer reaction, strand displacement DNA synthesis catalyzed by T4 DNA polymerase also requires the presence of gene 32 protein in addition to the gene 41 and 59 proteins.

J Mol Biol, 1997 Mar 21, 267(1), 150 - 62
Inhibition of Holliday structure resolving endonuclease VII of bacteriophage T4 by recombination enzymes UvsX and UvsY; Birkenkamp-Demtroder K et al.; Proteins UvsX, UvsY and Endonuclease VII (Endo VII) of bacteriophage T4 are required for DNA recombination, replication and repair . Endo VII is the product of gene 49 (gp49) and essential for resolution of branches from newly made DNA, prior to packaging into preformed heads . The ability of Endo VII to resolve Holliday structures in vitro suggested an in vivo function for the resolution of recombination intermediates, generated by UvsX and UvsY during the early infection cycle . Here we report results which contrast with this hypothesis . It is shown that the potent endonucleolytic activity of Endo VII with branched DNAs is inhibited in strand transfer reactions by the strand transferase UvsX, and more strongly by the accessory protein UvsY in vitro . The inhibitory effect of UvsX or UvsY is also seen in reactions with Endo VII using two synthetic cruciform DNAs and a C/C-mismatch containing substrate . Low concentrations of UvsY protein (12 ng or 0,76 pmol) were sufficient to reduce the cleavage efficiency of 30 units of Endo VII (about 16 fmol) to 50% . The inhibition is due to a direct protein-protein interaction between Endo VII, UvsX and UvsY as suggested by electrophoretic mobility shift assays (EMSAs) . These results were confirmed through affinity chromatography, where UvsX and UvsY bound to Endo VII, immobilized on a NHS-activated Sepharose matrix . This is the first identification of phage-encoded proteins which modulate the potent endonucleolytic activity of gp49 in vitro.

J Mol Biol, 1997 Mar 21, 267(1), 75 - 87
In vivo packaging of bacteriophage lambda monomeric chromosomes; Thomason LC et al.; There is an apparent paradox between the reported requirements for lambda DNA packaging in vivo and in vitro . In vivo, DNA concatemers are required for packaging . On the other hand, in vitro, packaging extracts can encapsidate either linear or circular monomeric lambda DNA . Perhaps cellular nucleases restrict the in vivo ability of monomers to package by degrading a free double chain end present as an intermediate in the packaging reaction . Consistent with this hypothesis, enhanced packaging of monomers was found in an ExoV- host . No additional enhancement was noted in a host also mutant for sbcB and sbcC . We isolated a mutant phage for which in vivo packaging of monomeric lambda chromosomes is increased about 10(3)-fold . The responsible mutation (plm1 for packages lambda monomers) was mapped to cro, sequenced, and found to cause a change from Ala29 to Ser in the alpha3 helix of Cro's DNA binding domain . Density transfer experiments showed that packaging of both plm1 and wild-type lambda was aided by allowing some DNA synthesis . However, the packaged chromosomes had not themselves undergone a full round of replication and therefore were not part of a canonical concatemer made by replication . Other tests showed that packaged phage had not been part of concatemers made by recombination or by annealing at cos . Our results with wild-type lambda also favor models in which two cos sites are needed for packaging, but these sites need not be in cis . In lambda plm1, replication intermediates may serve as substrates for encapsidation.

Proc Natl Acad Sci U S A, 1997 Mar 18, 94(6), 2174 - 9
Supercoil-induced extrusion of a regulatory DNA hairpin; Dai X et al.; Bacteriophage N4 virion RNA polymerase (N4 vRNAP) promoters contain inverted repeats, which form a 5- to 7-base-pair stem, 3-base loop hairpin that is required for vRNAP recognition . We show that, contrary to certain theoretical predictions, hairpin extrusion can occur at physiological superhelical densities in a Mg2+-dependent manner . Specific sequences on the template strand are required for hairpin extrusion . These sequences define stable DNA hairpins that are relatively unreactive to single strand-specific probes . In addition, a specific stable hairpin-inducing sequence can regulate transcription in vivo . Thus, a DNA structure, in its natural environment, is involved in transcriptional regulation.

Biochemistry, 1997 Mar 18, 36(11), 3404 - 16
Incorporation of trifluoromethionine into a phage lysozyme: implications and a new marker for use in protein 19F NMR; Duewel H et al.; Much interest is currently focused on understanding the detailed contribution that particular amino acid residues make in protein structure and function . Although the use of site-directed mutagenesis has greatly contributed to this goal, the approach is limited to the standard repertoire of twenty amino acids . Fluorinated amino acids have been utilized successfully to probe protein structure and dynamics as well as point to the importance of specific residues to biological function . In our continuing investigations on the importance of the amino acid methionine in biological systems, the successful incorporation of L-S-(trifluoromethyl)homocysteine (L-trifluoromethionine; L-TFM) into bacteriophage lambda lysozyme (LaL), an enzyme containing three methionine residues, is reported . The L isomer of TFM was synthesized in an overall yield of 33% from N-acetyl-D,L-homocysteine thiolactone and trifluoromethyl iodide . An expression plasmid giving strong overproduction of LaL was prepared and transformed into an Escherichia coli strain auxotrophic for methionine permitting the expression of LaL in the presence of L-TFM . The analogue would not support growth of the auxotroph and was found to be inhibitory to cell growth . However, cells that were initially grown in a Met-rich media followed by protein induction under careful control of the respective concentrations of L-Met and L-TFM in the media, were able to overexpress TFM-labeled LaL (TFM-LaL) at both high (70%) and low (31%) levels of TFM incorporation . TFM-LaL at both levels of incorporation exhibited analogous activity to the wild type enzyme and were inhibited by chitooligosaccharides indicating that incorporation of the analogue did not hinder enzyme function . Interestingly, the 19F solution NMR spectra of the TFM-labeled enzymes consisted of four sharp resonances spanning a chemical shift range of 0.9 ppm, with three of the resonances showing very modest shielding changes on binding of chitopentaose . The 19F NMR analysis of TFM-LaL at both high and low levels of incorporation suggested that one of the methionine positions gives rise to two separate resonances . The intensities of these two resonances were influenced by the extent of incorporation which was interpreted as an indication that subtle conformational changes in protein structure are induced by incorporated TFM . The similarities and differences between Met and TFM were analyzed using ab initio molecular orbital calculations . The methodology presented offers promise as a new approach to the study of protein-ligand interactions as well as for future investigations into the functional importance of methionine in proteins.

Nucleic Acids Res, 1997 Mar 15, 25(6), 1130 - 5
An NMR and mutational study of the pseudoknot within the gene 32 mRNA of bacteriophage T2: insights into a family of structurally related RNA pseudoknots; Du Z et al.; NMR methods were used to investigate a series of mutants of the pseudoknot within the gene 32 messenger RNA of bacteriophage T2, for the purpose of investigating the range of sequences, stem and loop lengths that can form a similar pseudoknot structure . This information is of particular relevance since the T2 pseudoknot has been considered a representative of a large family of RNA pseudoknots related by a common structural motif, previously referred to as 'common pseudoknot motif 1' or CPK1 . In the work presented here, a mutated sequence with the potential to form a pseudoknot with a 6 bp stem2 was shown to adopt a pseudoknot structure similar to that of the wild-type sequence . This result is significant in that it demonstrates that pseudoknots with 6 bp in stem2 and a single nucleotide in loop1 are indeed feasible . Mutated sequences with the potential to form pseudoknots with either 5 or 8 bp in stem2 yielded NMR spectra that could not confirm the formation of a pseudoknot structure . Replacing the adenosine nucleotide in loop1 of the wild-type pseudoknot with any one of G, C or U did not significantly alter the pseudoknot structure . Taken together, the results of this study provide support for the existence of a family of similarly structured pseudoknots with two coaxially stacked stems, either 6 or 7 bp in stem2, and a single nucleotide in loop1 . This family includes many of the pseudoknots predicted to occur downstream of the frameshift or readthrough sites in a significant number of viral RNAs.

J Mol Biol, 1997 Mar 14, 266(5), 927 - 38
Single-stranded DNA binding properties of the uvsY recombination protein of bacteriophage T4; Sweezy MA et al.; The uvsY protein is an essential component of the bacteriophage T4 general recombination machinery . The properties of this 16 kDa protein include selective binding to ssDNA, as well as specific protein-protein interactions with other T4 recombination proteins including uvsX (general recombinase) and gp32 (ssDNA-binding protein) . uvsY promotes the assembly of uvsX-ssDNA filaments, the active species in uvsX-catalyzed DNA rearrangements, apparently by helping uvsX displace gp32 from the ssDNA . To better understand the role of uvsY in the T4 recombination system, here we characterize the thermodynamic and molecular properties of the interaction of the uvsY protein with a model single-stranded polynucleotide, epsilonDNA, which is a fluorescent, etheno-modified form of random-sequence ssDNA . We have found that the binding of uvsY protein enhances the fluorescence of the epsilonDNA lattice and that the maximal amount of fluorescence enhancement observed is dependent on salt concentration . In addition, we have used the epsilonDNA fluorescence enhancement assay to establish thermodynamic parameters of binding and to define some of the molecular details of uvsY-epsilonDNA interactions . We show that uvsY binds to epsilonDNA in a non-cooperative manner, with a binding site size of four nucleotide residues per monomer of uvsY, and that this binding is salt-sensitive and involves the displacement of anions from the uvsY protein . We further show that uvsY protein binds preferentially to epsilonDNA over unmodified ssDNA . The significance of these results is discussed in light of current models of uvsY action in the T4 recombination system.

J Mol Biol, 1997 Mar 14, 266(5), 915 - 26
RNA-DNA hybrid formation at a bacteriophage T4 replication origin; Carles-Kinch K et al.; The bacteriophage T4 replication origins ori(uvsY) and ori(34) each contain two distinct components: a T4 middle-mode promoter that is strictly required for replication and a downstream region of about 50 bp that is required for maximal levels of replication . Here, we present evidence that structure of the downstream region is important for replication initiation . Based on sensitivity to a single-stranded DNA-specific nuclease in vitro the downstream region behaves as a DNA unwinding element . The propensity to unwind is probably important for origin activity in vivo, because replication activity is maintained when the native downstream region is replaced with a heterologous DNA unwinding element from pBR322 in either orientation . We analyzed the origin DNA for possible unwinding in vivo by using potassium permanganate, a chemical that reacts with unpaired pyrimidine bases . The non-template strand, but not the template strand, became hypersensitive to permanganate after T4 infection regardless of whether replication could occur . Strand-specific permanganate hypersensitivity was also observed in artificial origins containing the pBR322 DNA unwinding element in either orientation . Hypersensitivity was only detected when the origin contained a promoter that would be active during T4 infection . Furthermore, the origin transcript itself appears to be necessary for hypersensitivity since insertion of a transcriptional terminator abolishes hypersensitivity downstream of the termination site . Our results strongly suggest that the downstream region functions as a DNA unwinding element during replication initiation, leading to the formation of a persistent RNA-DNA hybrid at the origin.

J Mol Biol, 1997 Mar 14, 266(5), 901 - 14
The bacteriophage phi29 packaging proteins supercoil the DNA ends; Grimes S et al.; Bacteriophage phi29 DNA with covalently bound terminal protein (DNA-gp3) and its left and right-end restriction fragments (L and R-DNA-gp3) sedimented faster in sucrose density gradients than their proteinase K-treated counterparts, and the faster sedimentation was both gp3 and Mg2+-dependent . Addition of gp16, the phi29 DNA packaging ATPase, further increased the sedimentation rates of both intact DNA-gp3 and L and R-DNA-gp3 fragments . Thus, DNAs with gp3 were more compact than gp3-free DNA, and gp16 further condensed the DNA-gp3 forms . {35S}gp16 cosedimented with the fast-sedimenting DNA-gp3 fragments, and the putative L-DNA-gp3-gp16 complexes were packaged preferentially into proheads in the defined in vitro system . Lariats of DNA-gp3 and L and R-DNA-gp3 observed by electron microscopy rationalized the sedimentation results, and lariats with multiple loops or coils increased tenfold in a preparation of L-DNA-gp3-gp16 complexes . The rapid sedimentation and the structure of the DNA-gp3-gp16 complexes were consistent with supercoiling of lariat loops, and treatment with topoisomerase I shifted fast-sedimenting complexes toward the uncoiled lariat position in sucrose density gradients . DNA-gp3 has a maturation pathway in which the packaging proteins gp3 and gp16 supercoil the DNA ends, probably as a prerequisite for efficient interaction with the prohead.

J Mol Biol, 1997 Mar 14, 266(5), 891 - 900
Protein P7 of phage phi6 RNA polymerase complex, acquiring of RNA packaging activity by in vitro assembly of the purified protein onto deficient particles; Juuti JT et al.; The RNA polymerase complex of double-stranded RNA bacteriophage phi6 is composed of four proteins, P1, P2, P4 and P7 . These four proteins are capable of performing all the functions required for the replication of the double-stranded RNAs of the phi6 genome . The polymerase complex containing the three genomic dsRNA segments is the core particle of the phi6 virion . In this study purified protein P7 was found to form highly asymmetric dimers . Using polyclonal anti-P7 antibody, P7 was shown to be accessible on the surface of the nucleocapsid . Treatment of nucleocapsids with polyclonal anti-P7 antibody released coat protein P8 with ensuing activation of the plus strand RNA synthesis from the resulting core particles . Purified P7 could be assembled onto particles lacking P7 and particles lacking both P2 (RNA polymerase) and P7 . In both cases RNA packaging activity was acquired . Assembly of P7 onto deficient particles took place also in the absence of host proteins . Protein P7 is known to be necessary for stable packaging of the three genomic phi6 plus strand RNAs into preformed polymerase complex particles . Additionally, protein P7 seems to be involved in the regulation of plus strand synthesis (i.e . transcription) as a fidelity factor . Particles lacking protein P7 produce anomalous size transcripts . Analysis of the polymerase complex stability revealed that proteins P2, P4 and P7 are independently associated with the major structural protein P1 . The number of P7 molecules in one virion was estimated to be 60 and a location at the 5-fold symmetry position is proposed.

Biochemistry, 1997 Mar 11, 36(10), 2908 - 18
Initiation, elongation, and processivity of carboxyl-terminal mutants of T7 RNA polymerase; Gardner LP et al.; Bacteriophage T7 RNA polymerase is a single-subunit enzyme which has a C-terminal amino acid sequence of Phe-Ala-Phe-Ala883 (FAFA883) . Closely related hydrophobic sequences are present at the C termini of seven other single-subunit RNA polymerases, including the mitochondrial RNA polymerase . Mutations at any of the four C-terminal residues depress initiation rates of T7 RNA polymerase from 50 to 95%, accompanied by large increases in the K(m) values for the initiating nucleotide, GTP, as well as the K(m)'s for promoter DNA . The dramatic drops in initiation rates shown by the mutant enzymes remain after correcting for any alteration in saturation of the enzyme by the initiating nucleotide or the promoter DNA resulting from the changes in K(m) . In contrast, the high processivity of the enzyme is not altered by mutations in the last four residues . However, the propensity for the enzyme to add an untemplated nucleotide at the 3'-ends of transcripts is abolished by the A880AFA883 mutation . The C-terminal FAFA sequence or foot appears to interact both with the initiating NTP and with the most downstream nucleotides of the promoter, possibly through hydrophobic interactions with the minor groove, in the region where free radical footprinting of the polymerase-promoter DNA complex suggests that the enzyme binds across the minor groove.

Biochemistry, 1997 Mar 11, 36(10), 2744 - 52
Assembly of a nucleoprotein complex required for DNA packaging by bacteriophage lambda; Yang Q et al.; A critical step in the assembly of bacteriophage lambda is the excision of a single genome from a concatemeric DNA precursor and insertion of genomic DNA into an empty viral capsid . DNA packaging is mediated by the lambda proteins gpNu1 and gpA, which form an enzyme complex known as terminase . Initiation of the packaging process requires assembly of the terminase subunits onto cos, the lambda DNA packaging sequence, and nicking of the duplex, thus forming the 12-base-pair "sticky" ends of the mature genome . We have utilized gel-retardation techniques to examine the interaction of gpNu1, gpA, and terminase holoenzyme with DNA . Our data demonstrate that gpNu1 interacts specifically with cos-containing DNA, forming three gel-retarded complexes . Similarly, the larger gpA subunit binds to DNA, forming two complexes; however, this subunit forms similar complexes with DNA substrates of random sequence . All of the nucleoprotein complexes examined are disrupted by elevated concentrations of NaCl and we suggest that altered DNA binding is responsible for the extreme salt sensitivity of the endonuclease activity of the enzyme {Tomka, M . A., & Catalano, C . E . (1993) J . Biol . Chem . 268, 3056-3065} . DNA binding by each subunit is strongly affected by the presence of the other, with 10- and 3-fold increases in the affinity of gpNu1 and gpA, respectively, for DNA . Moreover, our data suggest that the terminase subunits interact in solution prior to DNA binding . Finally, we provide evidence that complex I, the first stable intermediate in the packaging pathway, is composed of the mature left genome end bound to the terminase subunits and demonstrate that dissociation of the complex is quite slow (t1/2 > 8 h) . The significance of these data with respect to terminase-mediated genome packaging is discussed.

Biochemistry, 1997 Mar 11, 36(10), 2733 - 43
Mechanism of bacteriophage T4 DNA holoenzyme assembly: the 44/62 protein acts as a molecular motor; Berdis AJ et al.; The role of ATP hydrolysis by the 44/62 protein in formation of the stable holoenzyme DNA replication complex has been further elucidated by specifically examining the role that the 44/62 protein plays in loading the 45 protein onto the DNA substrate . A stable phospho-45 protein or phosphorylated holoenzyme complex was not detected or isolated, suggesting that the 44/62 protein may not act as a protein kinase . Product and dead-end inhibition data are consistent with an ordered kinetic mechanism with respect to product release in which phosphate is released from the 44/62 protein prior to ADP . Positional isotope effect studies support this mechanism and failed to demonstrate that ATP hydrolysis by the 44/62 protein is reversible . Steady-state ATPase assays using aluminum tetrafluoride as an inhibitor are also consistent with release of ADP being partially rate-limiting . Aluminum tetrafluoride acts to trap ADP on the enzyme after turnover, forming a stable transition state analog that dissociates slowly from the enzyme . Processive DNA synthesis does not occur using the accessory proteins in the presence of pre- or post-hydrolysis analogs of ATP nor in the presence of ADP-AlF4, indicating that turnover of the 44/62 protein is absolutely required for formation of the holoenzyme complex . Collectively, data obtained regarding ATP hydrolysis by the 44/62 protein are described in terms of the clamp loading protein functioning as a molecular motor, similar to other systems including myosin and kinesin.

J Mol Biol, 1997 Mar 7, 266(4), 649 - 55
NMR structure of the principal neutralizing determinant of HIV-1 displayed in filamentous bacteriophage coat protein; Jelinek R et al.; An NMR approach for structure determination of short peptides displayed on the surface of filamentous bacteriophage virions is demonstrated using the hexapeptide GPGRAF that constitutes the principal neutralizing determinant of HIV-1 . This peptide was inserted near the N terminus of the major coat protein of bacteriophage fd . NMR studies of the recombinant protein solubilized in detergent micelles showed that the inserted peptide adopts a double bend S-shaped conformation that is similar to the antibody-bound structure determined by X-ray crystallography . This indicates that a peptide displayed on the bacteriophage coat protein has an enhanced propensity to adopt a conformation similar to that found in the native protein from which it is derived . This approach may be generally applicable to the structure determination of peptide epitopes and other small peptides.

J Biol Chem, 1997 Mar 7, 272(10), 6599 - 606
Amino acid changes in a unique sequence of bacteriophage T7 DNA polymerase alter the processivity of nucleotide polymerization; Yang XM et al.; T7 gene 5 DNA polymerase forms a complex with Escherichia coli thioredoxin (its processivity factor), and a 76-amino acid sequence (residues 258-334), unique to gene 5 protein, has been implicated in this interaction . We have examined the effect of amino acid substitution(s) in this region on T7 phage growth and on the interaction of the polymerase with thioredoxin . Among the mutations in gene 5, we found that a substitution of either Glu or Ala for Lys-302 yielded a protein that could not complement T7 phage lacking gene 5 (T7Delta5) to grow on E . coli having reduced thioredoxin levels . One triple mutant (K300E,K302E,K304E) could not support the growth of T7Delta5 even in wild type cells . This altered polymerase is stimulated 4-fold less by thioredoxin than is the wild type enzyme and the polymerase-thioredoxin complex has reduced processivity . The exonuclease activity of the altered polymerase is not stimulated to the same extent as that of the wild type enzyme by thioredoxin . The observed dissociation constant of the gene 5 protein K(300,302,304)E-thioredoxin complex is 7-fold higher than that of the wild type complex . The altered polymerase also has a lower binding affinity for double-stranded DNA.

Biochemistry, 1997 Mar 4, 36(9), 2478 - 84
Inhibition of bacteriophage T7 RNA polymerase in vitro transcription by DNA-binding pyrrolo{2,1-c}{1,4}benzodiazepines; Puvvada MS et al.; The interactions of several pyrrolo{2, 1-c}{1,4}benzodiazepine (PBD) antitumor antibiotics with linearized plasmid pGEM-2-N-ras DNA have been analyzed by quantitative in vitro transcription (QIVT) and in vitro transcription footprinting (IVTF) methods . A concentration-dependent inhibitory effect of the PBDs on transcription is observed using both techniques . The rank order for overall inhibition of transcription by the QIVT method is found to be: sibiromycin > tomaymycin > anthramycin > DC-81 > neothramycin, whereas the IVTF experiments show a different ranking: sibiromycin > anthramycin > neothramycin > tomaymycin . In addition, stimulation of transcription was observed at low PBD concentrations in both the QIVT and IVTF experiments . These results demonstrate unequivocally that the formation of PBD-DNA adducts at AGA-5' base sequences on the transcribed strand results in transcription blockage for all PBDs examined . Furthermore, the sequence of flanking base pairs appears to influence the degree of blocking, with the sequences ACAGAAA-5', AAAGATG-5', AGAGATA-5', and CAAGAAC-5' providing the most pronounced blocks for all PBDs studied in this system . Neothramycin and tomaymycin cause additional blocks at some GGA-5' and TGA-5' sequences . Parallel MPE-Fe(II) footprinting studies have revealed PBD binding sites on both the transcribing and nontranscribing strands, although all transcription blocks determined from the IVTF assays are due to drug bound on the transcribing DNA template strand.

J Photochem Photobiol B, 1997 Mar, 38(1), 48 - 53
Toxicity and repair of psoralen adducts in bacteriophage T7; Mamet-Bratley MD et al.; Psoralens react photochemically with DNA to form interstrand crosslinks as well as two types of monoadduct (furan-side and pyrone-side adducts) . To investigate the relative roles of these adducts in toxicity, we have studied the interaction of 4,5',8-trimethylpsoralen (TMP) and 8-methoxypsoralen (8-MOP) with bacteriophage T7 . These two derivatives differ in the fraction of pyrone-side monoadducts formed, TMP producing very small amounts of this type of adduct . The results show similar phage survival for the two psoralen analogs at equivalent numbers of crosslinks per DNA molecule . However, the survival fraction of treated phage is significantly lower than the fraction of noncrosslinked DNA molecules . Phage survival decreases after secondary irradiation which is used to transform monoadducts into crosslinks, but this decrease is not due solely to crosslinks; at doses beyond that required to transform all crosslinkable monoadducts into crosslinks, phage survival continues to decrease, pointing to the production of other genotoxic lesions during secondary irradiation . These results indicate that, although crosslinks can kill phage T7, as shown by the secondary irradiation results, they are not sufficient in number to explain the psoralen toxicity after primary irradiation . Therefore monoadducts, both furan-side and pyrone-side types, must in large part be responsible for phage inactivation.

Sex Transm Dis, 1997 Mar, 24(3), 161 - 4
An in vitro evaluation of condoms as barriers to a small virus; Lytle CD et al.; BACKGROUND: Because of the possible presence of small holes, the effectiveness of condoms as barriers to virus transmission is controversial . GOALS: To determine the proportion of condoms that allow virus penetration and the amounts of virus that penetrate . STUDY DESIGN: A sensitive, static test was used to evaluate different condom types as barriers to a small virus, including brand with or without lubrication and ones of different materials . The test included some physiologic-based parameters and some parameters that exaggerated expected actual use conditions . RESULTS: Under test conditions, 2.6% (12 of 470) of the latex condoms allowed some virus penetration; the median level of penetration was 7 x 10(-4) ml . Lubricated condoms performed similarly to nonlubricated ones . Polyurethane condoms yielded results higher than but not statistically different from those for latex condoms . CONCLUSIONS: Few condoms allowed any virus penetration . The median amount of penetration for latex condoms when extrapolated to expected actual use conditions was 1 x 10(-5) ml (volume of semen) . Thus, even for the few condoms that do allow virus penetration, the typical level of exposure to semen would be several orders of magnitude lower than for no condom at allPIP: Nine brands and 470 samples of latex condoms and two brands and 76 samples of polyurethane condoms bought from retail distributors were tested in vitro for their ability to block the penetration of virus . A sensitive, static test apparatus was designed for and used in the evaluation . The test included some physiological-based parameters as well as some which exaggerated the expected actual use conditions . Both lubricated and nonlubricated condoms were tested . Before testing, however, most of the lubrication was removed from the lubricated condoms through rinsing with Dulbecco's phosphate-buffered saline and blotting with sterile paper towels . The 0X174 bacteriophage of 27 nm particle diameter, 32 nm including its bulky spikes, was used as the proxy challenge virus . Under test conditions, 12 of the latex condoms (2.6%) allowed some virus penetration of median quantity 0.0007 ml . Just two of the latex condoms were responsible for 99.8% of the total penetration among latex condoms overall . The performance of lubricated condoms was similar to that of nonlubricated ones . Four of the polyurethane condoms allowed penetration, but only one condom was responsible for 98.6% of total penetration . The difference in performance between latex and polyurethane condoms is not statistically significant . The median amount of penetration for latex condoms when extrapolated to expected actual use conditions was 0.00001 ml of semen . Therefore, even for the few condoms which allow virus penetration, the typical level of exposure to semen is several orders of magnitude lower than the amount of exposure expected when not using a condom .

Bioorg Med Chem, 1997 Mar, 5(3), 581 - 90
Expanding the 43C9 class of catalytic antibodies using a chain-shuffling approach; Miller GP et al.; We employed a chain-shuffling technique to determine if the light chain of the catalytic antibody, 43C9, provides the best partner for the 43C9 heavy chain . Previously, we reported construction and screening of a 43C9 HC CROSS library, where the 43C9 heavy-chain gene was crossed with a library of light-chain genes in a lambda bacteriophage system . The library contained a high frequency of reconstituted antibodies recognizing the transition-state analogue . Here, we report the isolation and characterization of four of these clones . Recovered light-chain proteins share 92-96% sequence identity to the 43C9 light-chain protein . Somatic mutations of these light chains occur randomly at positions distant from the active site . Residues required for binding and catalysis were conserved . Mutations affected the topology of the binding site . Nevertheless, catalysis was not affected . Isolation of these light chains suggests the best partner for the 43C9 heavy chain is the original light chain . These clones attempt to broaden a class of 43C9-like antibodies, where the catalytic residues, His91 and Arg96, have been reproducibly selected . Similar catalytic properties between the 43C9-like antibodies suggests binding has been optimized, thus further maturation of the light chain would not lead to a better catalyst . To improve catalysis, other approaches must be considered.

Proteins, 1997 Mar, 27(3), 405 - 9
Shuffling of structural elements in filamentous bacteriophages; Kishchenko G et al.; All class II filamentous bacteriophage coat proteins contain a conserved, 12-amino acid sequence highly homologous to the loop portion of the EF-hand Ca(2+)-binding motif . The Pf3 coat protein contains two regions of homology to this sequence . The 12-amino acid sequence corresponds to a region of the Pf1 coat protein whose structure is controversial . In some models of the virus structure, this region is alpha-helical . In others, it forms a loop that folds back on itself . The similarity of this region to the loop in the helix-loop-helix Ca(2+)-binding motif suggests that it takes on a loop structure in the virion . Each filamentous phage lacks at least one residue normally involved in Ca(2+)-coordination, consistent with the relatively weak Ca(2+)-binding properties of the filamentous phages . Consideration of the structure of the coat protein in the membrane and in the virus particle indicates that the protein may be more effective in binding cations in its membrane-bound form than in the virus particle . This suggests that release of cations from this loop may be an obligate step during assembly of the proteins into the virus particle.

Microbiology, 1997 Mar, 143 ( Pt 3), 749 - 53
Bacterial capsules: no barrier against Bdellovibrio; Koval SF et al.; Bdellovibrio bacteriovorus 109J attached to both capsulated and non-capsulated Escherichia coli K29 cells . Electron microscopy revealed penetration of the thick polysaccharide capsule without any major disintegration of the neighbouring capsular matrix . The capsule remained intact during bdelloplast formation and lysis was unaffected by capsulation of the prey cell . This study shows that, in contrast to its effect on bacteriophage penetration and its protective activities against immune defence mechanisms, the capsule of E . coli does not serve as a barrier against invasion by B . bacteriovorus.

J Bacteriol, 1997 Mar, 179(6), 1852 - 6
Modulation of EcoKI restriction in vivo: role of the lambda Gam protein and plasmid metabolism; Salaj-Smic E et al.; Two novel types of alleviation of DNA restriction by the EcoKI restriction endonuclease are described . The first type depends on the presence of the gam gene product (Gam protein) of bacteriophage lambda . The efficiency of plating of unmodified phage lambda is greatly increased when the restricting Escherichia coli K-12 host carries a gam+ plasmid . The effect is particularly striking in wild-type strains and, to a lesser extent, in the presence of sbcC and recA mutations . In all cases, Gam-dependent alleviation of restriction requires active recBCD genes of the host and recombination (red) genes of the infecting phage . The enhanced capacity of Gam-expressing cells to repair DNA strand breaks might account for this phenomenon . The second type is caused by the presence of a plasmid in a restricting host lacking RecBCD enzyme . Commonly used plasmids such as the cloning vector pACYC184 can produce such an effect in strains carrying recB single mutations or in recBC sbcBC strains . Plasmid-mediated restriction alleviation in recBC sbcBC strains is independent of the host RecF, RecJ, and RecA proteins and phage recombination functions . The presence of plasmids can also relieve restriction in recD strains . This effect depends, however, on the RecA function in the host . The molecular mechanism of the plasmid-mediated restriction alleviation remains unclear.

J Bacteriol, 1997 Mar, 179(6), 1846 - 51
Isolation and characterization of a molecular chaperone, gp57A, of bacteriophage T4; Matsui T et al.; A molecular chaperone of bacteriophage T4, gp57A, which facilitates the formation of the long and short tail fibers, was isolated and characterized by peptide analysis, sedimentation equilibrium, and circular dichroism (CD) . Sequence analysis confirmed the predicted sequence of 79 amino acids from the nucleotide sequence of the gene with the N-terminal methionine removed . The result led to the conclusion that the apparent smaller molecular weight of 6,000 from Tricine-sodium dodecyl sulfate-polyacrylamide gel electrophoresis than the expected molecular weight of 8,710 was due to its abnormal electrophoretic behavior instead of cleavage or processing of the gene product . Estimation of the secondary structure from far-UV CD indicated a 94% alpha-helix content, which was in accord with the prediction from the primary structure . A sedimentation equilibrium study, on the other hand, revealed that gp57A assumes a tetrameric subunit structure.

RNA, 1997 Mar, 3(3), 315 - 23
Use of circular permutation to assess six bulges and four loops of DNA-packaging pRNA of bacteriophage phi29; Zhang C et al.; A 120-base phage phi29 encoded RNA (pRNA) has a novel role in DNA packaging . This pRNA possesses five single-base bulges, one three-base bulge, one bifurcation bulge, one bulge loop, and two stem loops . Circularly permuted pRNAs (cpRNA) were constructed to examine the function of these bulges and loops as well as their adjacent sequences . Each of the five single-base bulges was nonessential . The bifurcation bulge could be deleted and replaced with a new opening to provide flexibility for maintaining an overall correct folding in three-way junction . All of these nonessential bulges or their adjacent bases could be used as new termini for cpRNAs . The three-base (C18C19A20) bulge was dispensable for procapsid binding, but was indispensable for DNA packaging . The secondary structure around this CCA bulge and the phylogenetically conserved bases within or around it were investigated . Bases A14C15U16 were confirmed, by compensatory modification, to pair with U103G102A101 . A99 was needed only to allow the proper folding of CCA bulge in the appropriate sequence order and distance constraints . Beyond these, the seemingly phylogenetic conservation of other bases has little role in pRNA activity . Each of the three stem loops was essential for procapsid binding, DNA packaging, and phage assembly . Disruption of the middle of any one of the loops resulted in dramatic reductions in procapsid binding, subsequent DNA packaging, and phage assembly activities . However, disruption of the loops at sequences that were close to double-stranded regions of the RNA did not interfere with pRNA activity significantly . Our results suggest that double-stranded helical regions near these loops were most likely not involved in interactions with components of the DNA-packaging machinery . Instead, these regions appear to be merely present to serve as a scaffolding to display the single-stranded loops that are important for pRNA tertiary structure or for interaction with the procapsid or other packaging components.

Arch Biochem Biophys, 1997 Mar 1, 339(1), 79 - 84
The functional display of interleukin-2 on filamentous phage; Buchli PJ et al.; We report the novel display of interleukin-2 (IL-2) and an IL-2 analog, D126, on the surface of filamentous bacteriophage using a phagemid vector system . A synthetic human IL-2 gene and its D126 analog were fused to the carboxyl-terminal domain of the gene III minor phage coat protein . Expression of IL-2 and D126 was verified by their reactivity with an IL-2-specific antibody . Biological response of IL-2 phage on murine CTLL-2 cells was comparable to that of recombinant soluble IL-2, while the D126 phage displayed a reduced biological response similar to that previously measured by soluble D126 protein . Biosensor surface plasmon resonance was employed to verify binding of the IL-2 and D126 phage to the IL-2 alpha beta cc receptor complex . A 41-fold enrichment of IL-2 phage over R408 helper phage was demonstrated in biopanning affinity selection studies employing biotinylated alpha beta cc receptor complex . These biopanning studies are the first reports of affinity selection of IL-2 phage and demonstrate a novel use for the alpha beta cc receptor complex . Together, these studies confirm that the structural integrity of IL-2 and D126 is maintained when they are displayed as a gIIIp fusion protein on phage particles and provide the foundation for further selection studies employing IL-2 analog phage libraries.

J Immunol, 1997 Mar 1, 158(5), 2174 - 82
Both resting and activated B lymphocytes expressing engineered peptide-Ig molecules serve as highly efficient tolerogenic vehicles in immunocompetent adult recipients; Zambidis ET et al.; To test the potential for genetically transferring foreign sequences into autologous cells for specific modulation of immunity, we have generated transgenic mice that express an engineered peptide-IgG construct in the peripheral B cell compartment . B cells from these mice express and can be stimulated to secrete a murine IgG1 chain grafted with residues 12-26 from bacteriophage A cI repressor protein in-frame at the heavy chain N terminus . As expected, 12-26-IgG transgenic mice are profoundly tolerant to the peptide at both the T and B cell levels . Importantly, the injection of transgenic whole spleen, purified B cells, or even bone marrow cells into normal, immunocompetent adults results in profound peptide-specific T cell tolerance, as well as partial B cell tolerance . Injection of LPS-activated peptide-Ig-expressing B cells was uniquely effective at diminishing an ongoing humoral immune response typical of both Th1 and Th2 help . Since fixed transgenic B cells were tolerogenic, this suggests that secretion of the fusion protein is not required for tolerogenicity . These results show that an engineered self Ig, as well as B lymphocytes expressing epitopes from such a fusion protein, can regulate both cellular and humoral immune responses . Moreover, these studies provide the basis for expressing foreign epitopes on engineered IgG for the induction of gene-transferred tolerogenesis in autoimmune states.

J Virol, 1997 Mar, 71(3), 2157 - 62
In vitro selection of packaging sites in a double-stranded RNA virus; Yao W et al.; The Saccharomyces cerevisiae double-stranded RNA virus ScVL1 recognizes a small sequence in the viral plus strand for both packaging and replication . Viral particles will bind to this viral binding sequence (VBS) with high affinity in vitro . An in vitro selection procedure has been used to optimize binding, and the sequences isolated have been analyzed for packaging and replication in vivo . The selected sequence consists of a stem with a bulged A residue topped by a loop of several bases . Four residues of the 18 bases are absolutely conserved for tight binding . These all fall in regions that appear to be single stranded . Eight more residues have preferred identities, and six of these are in the stem . The VBS is similar to the R17 bacteriophage coat protein binding site . Packaging and replication require tight binding to viral particles.

J Biol Chem, 1997 Feb 28, 272(9), 5943 - 51
Template recognition and ribonucleotide specificity of the DNA primase of bacteriophage T7; Kusakabe T et al.; The 63-kDa gene 4 DNA primase of phage T7 catalyzes the synthesis of oligoribonucleotides on single-stranded DNA templates . At the sequence, 5'-GTC-3', the primase synthesizes the dinucleotide pppAC; the cytidine residue of the recognition sequence is cryptic . Only tetraribonucleotides function as primers, but the specificity for the third and fourth position is not as stringent with a preference of CMP > AMP >> UMP > GMP . The predominant recognition sites on M13 DNA are 5'-(G/T)GGTC-3' and 5'-GTGTC-3' . Synthesis is usually limited to tetranucleotides, but T7 primase can synthesize longer oligoribonucleotides on templates containing long stretches of guanosine residues 5' to the recognition sequence . The specificity beyond the first two positions of the primer increases as the length of the template on the 3'-side of 5'-GTC-3' increases . On an oligonucleotide having 20 3'-flanking cytidine residues GMP is incorporated at the third position; incorporation is reduced 4-fold when the flanking sequence reaches 65 residues, and little is incorporated on M13 templates . The presence of the 56-kDa gene 4 helicase decreases the incorporation of GMP on long templates . We propose that pausing is required for the incorporation of less preferred nucleotides and that pausing is decreased by the ability of the primase to translocate 5' to 3' on templates having long 3'-flanking sequences.

J Biol Chem, 1997 Feb 28, 272(9), 5695 - 702
The Cre recombinase cleaves the lox site in trans; Shaikh AC et al.; The Cre protein is a conservative site-specific recombinase that is encoded by bacteriophage P1 . Its function in vivo is to resolve dimeric lysogenic P1 plasmids that arise by general recombination . In this way Cre facilitates effective partition of the P1 prophage . Cre is a member of the integrase family of conservative site-specific recombinases . Cleavage of the DNA by the integrases involves covalent attachment of a conserved nucleophilic tyrosine to the 3'-phosphoryl end at the site of the break . We have used in vitro complementation tests to show that the Cre protein, like the Flp protein of the 2-microm plasmid of Saccharomyces cerevisiae, cleaves its target lox site in trans . Moreover, the data are compatible with two modes of cleavage; one requires the reconstitution of a pseudo full-site from half-sites and the other requires the assembly of a higher order complex that resembles a synaptic complex.

Cancer Lett, 1997 Feb 26, 113(1-2), 71 - 6
In vitro mutagenicity of the plasmacytomagenic agent pristane (2,6,10,14-tetramethylpentadecane); Felix K et al.; Pristane is known to induce a distinct type of B-cell-derived malignant lymphoma, plasmacytoma, after administration into the peritoneal cavity of genetically susceptible BALB/cAnPt mice . Since the mechanism of pristane-induced plasmacytoma development is poorly understood, we chose to examine the possibility that pristane is mutagenic in rodent cells and decided to use bacteriophage lambda-derived lacI/lacZ genes as target/reporter to quantitate mutagenesis . Here we show that in vitro exposure to micromolar amounts of pristane, delivered as an inclusion complex with beta-cyclodextrin, resulted in 1.7-fold and 6.2-fold increases of mutant frequencies over controls in a cell line of rat fibroblasts and primary mouse B lymphocytes, respectively . We conclude that pristane can be mutagenic to mammalian cells, yet are currently unable to explain the mechanism of mutagenicity . It is suggested that B-cell mutagenesis contributes to the plasmacytomagenic activity of pristane in vivo.

Biochem Biophys Res Commun, 1997 Feb 24, 231(3), 600 - 5
The gene 32 single-stranded DNA-binding protein is not bound stably to the phage T4 presynaptic filament; Jiang H et al.; A central reaction in homologous recombination is synapsis, which involves invasion of duplex DNA by a homologous single strand . A key intermediate in this process is the presynaptic filament, a protein-DNA complex composed of a "strand transferase" polymerized along the invading single strand . In this report, the organization and mechanism of assembly of the bacteriophage T4 presynaptic filament are explored . Three T4 proteins, encoded by the uvsX, uvsY and 32 genes, are involved in this process . It is demonstrated that a well-defined series of events involving multiple protein-DNA and protein-protein interactions is required to mediate a transition from an initial gene 32-DNA complex to a mature presynaptic filament in which the UvsX and UvsY proteins are in contact with the DNA and each other, while most or all of the gene 32 protein is removed from the complex.

J Mol Biol, 1997 Feb 21, 266(2), 217 - 22
Structure and subunit composition of the RuvAB-Holliday junction complex; Yu X et al.; The E . coli RuvA and RuvB proteins, which are involved in the late stages of recombination and the recombinational repair of damaged DNA, bind to Holliday junctions and promote branch migration . We have used electron microscopy and image analysis to examine RuvA and RuvB bound to model Holliday structures . The two hexameric rings of RuvB are oriented in a bipolar manner, so that the large end of each faces the junction . The results suggest a model for branch migration in which DNA is pumped out of the small end of each ring as ATP is hydrolyzed . The same structural polarity has been established for the bacteriophage T7 gp4 replicative helicase . Mass and image analysis of the RuvAB-junction complex suggests that two tetramers of RuvA form a symmetrical sandwich about the plane of the junction.

Gene, 1997 Feb 20, 186(1), 67 - 72
Hypermutagenic in vitro transcription employing biased NTP pools and manganese cations; Pezo V et al.; In vitro DNA-dependent RNA transcription using bacteriophage T3 RNA polymerase may be rendered hypermutagenic by employing biased nucleoside triphosphate (NTP) concentrations and manganese cations . Using the E . coli R67 plasmid-encoded dihydrofolate reductase (DHFR) gene as target substitution rates approaching 4 x 10(-2) per base per reaction could be achieved, on a par with hypermutagenic reverse transcription . In all cases the majority of substitutions was that expected from the NTP pool bias . The addition of manganese ions increased the frequency of mutations, particularly the proportion of transversions . Functional DHFR hypermutants with up to 8% amino acid substitutions were readily obtained from a single reaction which, given the unique mutation matrix allows exploration of sequence space complementary to that accessed by other hypermutagenic protocols.

Proc Natl Acad Sci U S A, 1997 Feb 18, 94(4), 1154 - 9
Cryptic single-stranded-DNA binding activities of the phage lambda P and Escherichia coli DnaC replication initiation proteins facilitate the transfer of E . coli DnaB helicase onto DNA; Learn BA et al.; The bacteriophage lambda P and Escherichia coli DnaC proteins are known to recruit the bacterial DnaB replicative helicase to initiator complexes assembled at the phage and bacterial origins, respectively . These specialized nucleoprotein assemblies facilitate the transfer of one or more molecules of DnaB helicase onto the chromosome; the transferred DnaB, in turn, promotes establishment of a processive replication fork apparatus . To learn more about the mechanism of the DnaB transfer reaction, we investigated the interaction of replication initiation proteins with single-stranded DNA (ssDNA) . These studies indicate that both P and DnaC contain a cryptic ssDNA-binding activity that is mobilized when each forms a complex with the DnaB helicase . Concomitantly, the capacity of DnaB to bind to ssDNA, as judged by UV-crosslinking analysis, is suppressed upon formation of a P x DnaB or a DnaB x DnaC complex . This novel switch in ssDNA-binding activity evoked by complex formation suggests that interactions of P or DnaC with ssDNA may precede the transfer of DnaB onto DNA during initiation of DNA replication . Further, we find that the lambda O replication initiator enhances interaction of the P x DnaB complex with ssDNA . Partial disassembly of a ssDNA:O x P x DnaB complex by the DnaK/DnaJ/GrpE molecular chaperone system results in the transfer in cis of DnaB to the ssDNA template . On the basis of these findings, we present a general model for the transfer of DnaB onto ssDNA or onto chromosomal origins by replication initiation proteins.

Structure, 1997 Feb 15, 5(2), 265 - 75
A conserved infection pathway for filamentous bacteriophages is suggested by the structure of the membrane penetration domain of the minor coat protein g3p from phage fd; Holliger P et al.; BACKGROUND: . Gene 3 protein (g3p), a minor coat protein from bacteriophage fd mediates infection of Escherichia coli bearing an F-pilus . Its N-terminal domain (g3p-D1) is essential for infection and mediates penetration of the phage into the host cytoplasm presumbly through interaction with the Tol complex in the E . coli membranes . Structural knowledge of g3p-D1 is both important for a molecular understanding of phage infection and of biotechnological relevance, as g3p-D1 represents the primary fusion partner in phage display technology . RESULTS: . The solution structure of g3p-D1 was determined by NMR spectroscopy . The principal structural element of g3p-D1 is formed by a six-stranded beta barrel topologically identical to a permutated SH3 domain but capped by an additional N-terminal alpha helix . The presence of structurally similar domains in the related E . coli phages, lke and 12-2, as well as in the cholera toxin transducing phage ctxφ is indicated . The structure of g3p-D1 resembles those of the recently described PTB and PDZ domains involved in eukaryotic signal transduction . CONCLUSIONS: . The predicted presence of similar structures in membrane penetration domains from widely diverging filamentous phages suggests they share a conserved infection pathway . The widespread hydrogen-bond network within the beta barrel and N-terminal alpha helix in combination with two disulphide bridges renders g3p-D1 a highly stable domain, which may be important for keeping phage infective in harsh extracellular environments.

Nucleic Acids Res, 1997 Feb 15, 25(4), 915 - 6
Simultaneous display of different peptides on the surface of filamentous bacteriophage; Malik P et al.; We have developed a new system for producing hybrid virions of filamentous bacteriophage fd simultaneously displaying two different peptides by infecting cells harbouring a plasmid containing a modified gene VIII with an engineered bacteriophage carrying a second and different copy of a modified gene VIII . The simultaneous display of different peptides has many potential applications in exploring the immune response and studying protein-protein interaction.

Nucleic Acids Res, 1997 Feb 15, 25(4), 727 - 34
Domain structure of vaccinia DNA ligase; Sekiguchi J et al.; The 552 amino acid vaccinia virus DNA ligase consists of three structural domains defined by partial proteolysis: (i) an amino-terminal 175 amino acid segment that is susceptible to digestion with chymotrypsin and trypsin; (ii) a protease-resistant central domain that contains the active site of nucleotidyl transfer (Lys-231); (iii) a protease-resistant carboxyl domain . The two protease-resistant domains are separated by a protease-sensitive interdomain bridge from positions 296 to 307 . Adenylyltransferase and DNA ligation activities are preserved when the N-terminal 200 amino acids are deleted . However, the truncated form of vaccinia ligase has a reduced catalytic rate in strand joining and a lower affinity for DNA than does the full-sized enzyme . The 350 amino acid catalytic core of the vaccinia ligase is similar in size and protease-sensitivity to the full-length bacteriophage T7 DNA ligase.

Mutat Res, 1997 Feb 14, 388(2-3), 197 - 212
Toward an understanding of the use of transgenic mice for the detection of gene mutations in germ cells; Douglas GR et al.; Recently-developed transgenic models have provided unprecedented access to rodent somatic and germ line tissues for the study of gene mutation in vivo . While mutations in germ cells are considered an important aspect of any regulatory assessment of the risks posed by chemicals, currently-available conventional tests, which involve the study of thousands of offspring make it impractical to test large numbers of chemicals, for the induction of inherited gene mutations . When effects in germ cells per se, rather than offspring are acceptable targets, transgenic mouse assays may provide a practical alternative . As part of an international collaborative study to begin to determine the reliability, efficacy, and role of such assays, lacZ transgenic mice (Muta Mouse) were treated with single i.p . doses of ethylnitrosourea (ENU), methyl methanesulfonate (MMS), and isopropyl methanesulfonate (iPMS), and mutant frequencies determined using phenyl-beta-D-galactoside (p-gal) positive selection . For studies using germ cells, the selection of sampling times and target cells is crucial . Spermatagonial stem cells and cells in post-spermatagonial stem cell stages are the critical target cell populations of regulatory importance . Cell populations within these categories were studied by sampling germ cells isolated from seminiferous tubules and spermatozoa from the epididymis at 91 days and 25 days after treatment . The data show that ENU and iPMS induced mutations in post-spermatagonial stem cells and spermatagonial stem cells . However, MMS did not induce mutations in either cell type, or at either sampling time, at doses approaching lethality . This result is possibly because MMS induces preferentially large lesions and chromosomal aberrations (as opposed to point mutations), which are not readily detectable with bacteriophage-based shuttle vectors . Since MMS-induced specific locus and dominant lethal mutations are induced only after the mid-spermatid stage, it is also possible that the timing used missed this effect . While the ENU and iPMS data in this study demonstrate the suitability of the lacZ male transgenic mice for the study of gene mutations in post-spermatagonial stem cells and spermatagonial stem cells by sampling cells isolated from seminiferous tubules at selected times after treatment, the MMS results do not answer fully whether transgenic mouse mutation assays can detect mutations resulting from lesions induced after the mid-spermatid stage when most cellular processing is retarded . Nevertheless, it appears clear from presently available information, that the bacteriophage-based lacZ transgenic model is suitable for the detection of gene mutations in spermatogonial stem cells, spermatocytes, and early spermatids.

J Mol Biol, 1997 Feb 14, 266(1), 1 - 7
Analysis of phage MS2 coat protein mutants expressed from a reconstituted phagemid reveals that proline 78 is essential for viral infectivity; Hill HR et al.; A full-length cDNA copy of the RNA genome of bacteriophage MS2 was assembled by the in-frame ligation of the central portion of the genome into a plasmid containing the 5' and 3' ends . Upon transformation of the ligation reaction into Escherichia coli, infectious phage particles were released into the medium . The plaquing ability of the phage produced from the cDNA construct was assessed against various bacterial strains confirming that the bacteriophage produced were male-specific . Sensitivity to RNase in agar overlay was used to confirm that the phage contained RNA . In addition, the phage were unable to infect piliated cells overexpressing MS2 coat protein, a resistance conferred by the binding of recombinant coat protein to the infecting strand of RNA at the replicase initiation region, thus preventing translation of the replicase gene . The phage capsids were visualised after negative staining by transmission electron microscopy, and appeared as spherical particles of approximately 25 nm diameter . The capsid proteins were examined by Western blotting, confirming the presence of a single protein of approximately 14 kDa, which bound anti-MS2 coat protein antibodies . The genomic RNA from single plaques was analysed by reverse transcription-PCR and the presence of the MS2 coat protein gene confirmed by DNA sequencing . The production of replicative MS2 phage from cDNA fragments was used to assess the viability of MS2 coat protein mutants, which had previously been shown to assemble into T = 3 capsid-like particles when expressed in vivo from a bacterial vector . The E76D mutation did not appear to affect phage viability, whilst replacement of the completely conserved P78 residue with asparagine abolished the production of infectious particles, suggesting that P78 may be involved in interactions with the phage maturation protein.

J Biomol Struct Dyn, 1997 Feb, 14(4), 407 - 19
Recognition of promoter DNA by subdomain 4.2 of Escherichia coli sigma 70: a knowledge based model of -35 hexamer interaction with 4.2 helix-turn-helix motif; Reddy BV et al.; In Escherichia coli, subdomains 2.4 and 4.2 of the primary transcription factor sigma 70 are the most highly conserved regions and are responsible for the recognition of -10 and -35 promoter elements respectively . Mutational studies provide evidence to this end and indicate that the side chains of subdomain 4.2 make specific contacts with the nucleotides at -35 . Subdomain 4.2 is highly conserved among group-1 sigma factors and is strongly homologous to the classical helix-turn-helix (HTH) motif shared by bacteriophage lembda cl, Cro, the CAP protein and other homeodomain proteins, suggesting that sigma factor also belongs to the HTH class of proteins . In this study, a single point mutation of the conserved hydrophobic residue valine at position 576, in the 4.2 subdomain results in a mutant that is transcriptionally inefficient although conformationally similar to wild-type sigma . The mutant sigma, like wild-type, migrates as a 87 kDa protein on SDS gels and has 50% helicity . However, transcription at "extended -10 promoter' by RNA polymerase containing mutant sigma 70-V576G, synthesized appreciable amount of RNA product, when compared with that generated by sigma 70-W434G, a mutation in -10 DNA binding domain . A model of HTH motif for the conserved 20 residue region of 4.2 domain of E . coli sigma 70 as well as its mutant sigma 70-V576G and sigma 70-V576T were constructed based on five other homologous HTH motifs from DNA-protein complexes for which X-ray or NMR structure is available . A B-DNA structure was designed for -35 region using sequence dependent base pair parameters . The modeled HTH structure was docked into the major groove formed by the -35 hexamer DNA using the DNA-recognition rules and amino acid-nucleotide base contact information of homologous DNA-protein complexes . Analysis of the residue contact information of the model was tested and found to have good agreement with the experimental reports.

Biochemistry (Mosc), 1997 Feb, 62(2), 217 - 20
Development of immunoenzymatic reactions of peptides using recombinant hybrid proteins; Mandrika IK et al.; A recombinant hybrid protein comprising the bacteriophage fr coat protein, a short linker, and atrial natriuretic factor (ANF) as well as native and recombinant phage coat proteins, ANF, and variants of the hybrid recombinant protein were used in the development of various immunological reactions including immunization, preparation of affinity columns, purification of antibodies, synthesis of conjugates, and immunoenzyme assay of ANF and the recombinant protein . The hybrid protein is effective in competitive assay of ANF and other constructs that include the phage protein.

Bioorg Khim, 1997 Feb, 23(2), 83 - 90
{Nucleotide sequence of cDNA and organization of the gene for alpha-subunit of photoreceptor phosphodiesterase of cyclic GMP in human retinal cones}; Feshchenko EA et al.; Five clones were isolated from a human retina cDNA library whose cDNA inserts allowed reconstruction of the total sequence of the human clone cGMP phosphodiesterase alpha'-subunit cDNA comprising 3455 bp . The protein's deduced sequence contains 858 amino acids residues with molecular mass 99,169 Da . A substantial homology was revealed between the amino acid sequence of the human cones cGMP-phosphodiesterase alpha'-subunit and the corresponding sequences of alpha, beta, and alpha' subunits of visual cGMP-phosphodiesterase of bovine, murine, chicken and human retinas . Four recombinant bacteriophages were isolated from a genomic library whose inserts made it possible to reconstruct a 32-kb fragment of the human cones cGMP-phosphodiesterase alpha'-subunit gene . 5'-Flanking region of the gene and first 14 exons, encoding an N-terminal segment of the protein, along with the adjacent intron segments were sequenced.

Oral Microbiol Immunol, 1997 Feb, 12(1), 40 - 6
Occurrence of temperate bacteriophages in different Actinobacillus actinomycetemcomitans serotypes isolated from periodontally healthy individuals; Willi K et al.; The occurrence of temperate bacteriophages was studied in 34 isolates of Actinobacillus actinomycetemcomitans derived from 27 periodontally healthy Finnish individuals both by lysis/plaque assays and by DNA hybridizations . In addition the serotype, the ribotype and the arbitrarily primed polymerase chain reaction (AP-PCR) profile were determined for each A . actinomycetemcomitans strain . Fourteen isolates showed hybridization patterns very similar to that of a known lysogen when probed with the genome of the previously characterized temperate phage Aa phi 23 . Only 6 of these 14 strains had produced lysis or single plaques on suitable indicator strains . Phage Aa phi 247 derived from one of these lysogens was indistinguishable from Aa phi 23 by electron microscopy, and the genomes showed highly related DNA hybridization patterns . The remaining 20 isolates exhibited hybridization patterns very different from that of Aa phi 23 DNA . Seven of these strains also gave lysis or single plaques, suggesting that 21 of the 34 strains were lysogenic . These data indicate that the prophages per se do not represent a virulence factor exclusively associated with periodontal disease . Presence of an Aa phi 23-related prophage correlated with serotype a and AP-PCR type 1 of the bacterial host . This may indicate that Aa phi 23 and related phages have a limited host range.

J Struct Biol, 1997 Feb, 118(1), 73 - 82
Multiple conformational states of the bacteriophage T4 capsid surface lattice induced when expansion occurs without prior cleavage; Kocsis E et al.; The maturation pathway of bacteriophage T4 capsid provides a model system for the study of largescale conformational changes, in that the precursor capsid progresses through four long-lived and widely differing states . The surface lattice first assembled (uncleaved/unexpanded state: hexagonal lattice constant, a = 11.8 nm) undergoes proteolytic cleavage (cleaved/unexpanded state), then expands (cleaved/ expanded state: a = 14.0 nm), and then binds accessory proteins . The most profound change, expansion, normally follows cleavage of the major capsid protein gp23 to gp23* (the 65-residue N-terminal "delta-domain" is removed), but can be induced in vitro in the absence of cleavage by treatment with 0.25 M guanidine-HCl (uncleaved/expanded state) . We have studied this alternative pathway by negative staining electron microscopy of polyheads (tubular capsid variants) . We find that uncleaved/expanded polyheads encompass four discrete states, called G1-G4, distinguished by their lattice constants of 12.6 nm (G1), 13.4 nm (G2), and 14.0 nm (G3, G4) and by the structures of their hexameric capsomers . Viewed in projection, the G4 capsomer differs from the cleaved/ expanded capsomer only in the presence of additional mass at one site per protomer . This mass correlates with the presence of the delta-domain, which translocates from the inner to the outer surface when the uncleaved lattice expands . Based on proximity of resemblance among these capsomers, we suggest that G1 to G4 represent a sequence of transitional states whose endpoint is G4 . G1, G2, and G3 may correspond to intermediates that are too short-lived to be observed when the cleaved lattice expands, but are trapped by the retention of delta-domains at the interfaces between subunits in the uncleaved lattice.

Gan To Kagaku Ryoho, 1997 Feb, 24(4), 460 - 5
{Modification of gene targeting method for functional analysis of the target gene in vivo}; Shibata H et al.; Gene targeting in ES cells is a powerful tool to generate mice bearing predesigned mutations in the germ line . However, these mice carrying such constitutional mutations are often lethal and, therefore, we cannot study other functional aspects of the gene at later stages or in particular tissues . To inactivate the target gene in particular tissues or at particular stages of development, conditional gene inactivation based on the Cre-loxP recombination system of bacteriophage P1 is considered one of the applicable techniques . To express the Cre gene in a cell-type or developmental stage specific manner, either transgenic or adenoviral technology is most considerable technique in vivo.

Mol Microbiol, 1997 Feb, 23(3), 603 - 16
Characterization of Natronobacterium magadii phage phi Ch1, a unique archaeal phage containing DNA and RNA; Witte A et al.; A novel archaeal bacteriophage, phi Ch1, was isolated from a haloalkalophilic archaeon Natronobacterium magadii upon spontaneous lysis . The phage-cured strain N . magadii(L13) was used to demonstrate infectivity of phage phi Ch1 . The turbid-plaque morphology and the fact that N . magadii cells isolated from plaques were able to produce phage indicated that phi Ch1 is a temperate phage . The phage morphology resembles other members of Myoviridae-infecting Halobacterium species . In solution below 2M NaCl, the phage lost its morphological stability and infectivity . One- and two-dimensional SDS-PAGE of phage particles revealed at least four major and five minor proteins with molecular masses ranging from 15 to 80 kDa and acidic isoelectric points . Southern blot analysis of chromosomal DNA of a lysogenic N . magadii strain showed that phi Ch1 exists as a chromosomally integrated prophage . The phage particles contain both double-stranded, linear DNA (approx . 55 kbp) as well as several RNA species (80-700 nucleotides) . Hybridization of labelled RNA fragments to total DNA from N . magadii and phi Ch1 showed that the virion-associated RNA is host encoded . Part of the phage DNA population is modified and restriction analysis revealed evidence for adenine methylation . Phage phi Ch1 is the first virus described for the genus natronobacterium, and the first phage containing DNA and RNA in mature phage particles.

Microbiology, 1997 Feb, 143 ( Pt 2), 553 - 62
Analysis of sequences flanking the vap regions of Dichelobacter nodosus: evidence for multiple integration events, a killer system, and a new genetic element; Bloomfield GA et al.; Dichelobacter nodosus is the causative agent of ovine footrot . The vap regions of the D . nodosus genome may have arisen by the integration of a genetic element and may have a role in virulence . The virulent D . nodosus strain A198 has multiple copies of the vap regions . In the present study, sequences to the left and right of vap regions 1, 2 and 3 of strain A198 were analysed by Southern blotting and DNa sequencing . The results suggest that vap regions 1 and 2 rose by independent integration events into different tRNA genes . The discovery of a second integrase gene (intB), a gene with similarity to bacteriophage repressor proteins (regA), and a gene similar to an ORF from a conjugative transposon (gepA), suggests that a second genetic element, either a bacteriophage or a conjugative transposon, is integrated next to vap region 3 in the D . nodosus genome . The arrangement of intB and the vap regions in three other virulent strains and one benign strain was determined using using Southern blotting and PCR . One strain, H1215, contained vapE' and not vapE, and thus resembles vap region 3, suggesting that vap region 3 also may have arisen by an independent integration event . In all strains, a copy of intB was found next to the vap regions . The vap regions contain two genes, vapA and toxA, with similarity to the hig genes of the killer plasmid Rts1 . Evidence is presented that vapA and toxA have a similar function in D . nodosus.

RNA, 1997 Feb, 3(2), 157 - 74
Antisense RNA control of gene expression in bacteriophage P22 . II . Kinetic mechanism and cation specificity of the pairing reaction; Schaefer KL et al.; The bacteriophage P22 sar RNA-ant mRNA pairing reaction was characterized kinetically . The pairing reaction proceeds by a three-step pathway . First, reversible base pairs form between complementary hairpin loops in sar RNA and ant RNA (Kd = 270 nM) . Next, stable duplex formation initiates between single-stranded nucleotides in sar RNA and ant RNA; the ant RNA nucleotides are at the bottom of a hairpin stem that is partially accessible . Concomitant unwinding of one sar RNA hairpin and the complementary ant RNA hairpin then occurs, to form a partially paired intermediate (k2 = 12 min(-1) . Finally, a complete duplex forms after unwinding of the other sar RNA hairpin and the complementary ant RNA hairpin (k3 = 7 min(-1) . Experiments with sar RNA sequence and length variants demonstrate that the precise structures of both sar RNA hairpins affect the kinetic parameters . The pairing reaction is Mg2+-dependent, and shows high specificity for the required cation . Maximal pairing rates are achieved when more than one Mg2+ ion is bound . The cation-dependence and specificity indicate a requirement for Mg2+-dependent tertiary structure.

RNA, 1997 Feb, 3(2), 141 - 56
Antisense RNA control of gene expression in bacteriophage P22 . I . Structures of sar RNA and its target, ant mRNA; Schaefer KL et al.; Sar RNA is an antisense RNA that is partly responsible for the negative regulation of antirepressor synthesis during development of bacteriophage P22 (Liao SM et al., 1987, Genes & Dev 1:197-203; Wu Th, Liao SM, McClure WR, Susskind MM, Genes & Dev 1:204-212) . The structures of sar RNA and its target, ant mRNA, were probed using limited RNase digestion as a function of Mg2+ concentration . Sar RNA forms two hairpins that are present at all Mg2+ concentrations (Mg2+-independent hairpins) . One of the hairpins contains three tandem U x U base pairs . Ant RNA forms three Mg2+-independent hairpins and one Mg2+-dependent hairpin . In addition, many nucleotides in sar RNA and ant RNA appear to be involved in tertiary interactions . The effects of RNA structure on the pairing reaction are considered in the accompanying paper (Schaefer KL, McClure WR, 1997, RNA 3:157-174).

J Virol Methods, 1997 Feb, 64(1), 57 - 64
Rescue of measles virus using a replication-deficient vaccinia-T7 vector; Schneider H et al.; A system which allows the reconstitution of measles virus (MV) from cloned cDNA is described . The severely host cell restricted vaccinia vector MVA-T7 expressing bacteriophage T7 RNA polymerase was used to generate full-length antigenomic MV RNA and simultaneously the mRNAs encoding the viral N, P and L proteins in order to produce replicationally and transcriptionally active nucleocapsids . The functionality of the N, P and L proteins was demonstrated first by their ability to rescue MV specific subgenomic RNAs . Assembly and budding of reconstituted MV was shown by syncytia formation and subsequently by virus isolation . The inability of MVA-T7 to produce progeny virus in most mammalian cells circumvents the necessity to separate the reconstituted MV from the MVA-T7 helper virus . Since all components are expressed transiently, this system is especially suitable for studying the functions of N, P and L . Furthermore, it is useful for investigating later steps in the MV life cycle.

J Cell Biochem, 1997 Feb, 64(2), 258 - 72
Purification and substrate specificity of polydeoxyribonucleotide kinases isolated from calf thymus and rat liver; Karimi-Busheri F et al.; Damage to DNA can result in strand breaks with 5'-hydroxyl and 3'-phosphate termini . Before DNA polymerases and ligases can rejoin the broken strands, such termini have to be restored to 5'-phosphate and 3'-hydroxyl groups . Polydeoxynucleotide kinase is an enzyme that may fulfil this function . We have purified the kinases from calf thymus and rat liver to near homogeneity . Based on SDS-polyacrylamide gel electrophoresis and activity gels, the enzymes from both sources are approximately 60-kDa polypeptides . Both enzymes have an acidic pH optimum (5.5-6.0) for kinase activity, and similar pl values (8.5-8.6), and a specificity for DNA . The calf thymus kinase possesses a 3'-phosphatase activity, as has previously been shown for the rat liver enzyme . The minimum size of oligonucleotide that can be labelled is 7-8 nucleotides in length, but the optimal size appears to be > 18 nucleotides . Comparison of phosphorylation of oligo(dA)24 and oligo(dT)24 with oligonucleotides containing a varied nucleotide sequence indicated that the homopolymers are poorer substrates . Unlike the bacteriophage T4 polynucleotide kinase, the mammalian kinases exhibit no preference for 5'-overhanging termini when acting at DNA termini produced by restriction enzymes . With double-stranded oligonucleotide complexes designed to mode single-strand gaps and nicks, the mammalian kinases preferentially phosphorylate the 5'-terminus associated with the gap or nick, in keeping with the idea that the kinases are involved in the repair of DNA single-strand breaks.

J Bacteriol, 1997 Feb, 179(4), 1253 - 61
Dimerization specificity of P22 and 434 repressors is determined by multiple polypeptide segments; Donner AL et al.; The repressor protein of bacteriophage P22 binds to DNA as a homodimer . This dimerization is absolutely required for DNA binding . Dimerization is mediated by interactions between amino acids in the carboxyl (C)-terminal domain . We have constructed a plasmid, p22CT-1, which directs the overproduction of just the C-terminal domain of the P22 repressor (P22CT-1) . Addition of P22CT-1 to DNA-bound P22 repressor causes the dissociation of the complex . Cross-linking experiments show that P22CT-1 forms specific heterodimers with the intact P22 repressor protein, indicating that inhibition of P22 repressor DNA binding by P22CT-1 is mediated by the formation of DNA binding-inactive P22 repressor:P22CT-1 heterodimers . We have taken advantage of the highly conserved amino acid sequences within the C-terminal domains of the P22 and 434 repressors and have created chimeric proteins to help identify amino acid regions required for dimerization specificity . Our results indicate that the dimerization specificity region of these proteins is concentrated in three segments of amino acid sequence that are spread across the C-terminal domain of each of the two phage repressors . We also show that the set of amino acids that forms the cooperativity interface of the P22 repressor may be distinct from those that form its dimer interface . Furthermore, cooperativity studies of the wild-type and chimeric proteins suggest that the location of cooperativity interface in the 434 repressor may also be distinct from that of its dimerization interface . Interestingly, changes in the dimer interface decreases the ability of the 434 repressor to discriminate between its wild-type binding sites, O(R)1, O(R)2, and O(R)3 . Since 434 repressor discrimination between these sites depends in large part on the ability of this protein to recognize sequence-specific differences in DNA structure and flexibility, this result indicates that the C-terminal domain is intimately involved in the recognition of sequence-dependent differences in DNA structure and flexibility.

J Bacteriol, 1997 Feb, 179(4), 1059 - 67
Mutational analysis of protein binding sites involved in formation of the bacteriophage lambda attL complex; MacWilliams M et al.; Bacteriophage lambda site-specific recombination requires the formation of higher-order protein-DNA complexes to accomplish synapsis of the partner attachment (att) sites as well as for the regulation of the integration and excision reactions . The att sites are composed of a core region, the actual site of strand exchange, and flanking arm regions . The attL site consists of two core sites (C and C'), an integration host factor (IHF) binding site (H'), and three contiguous Int binding arm sites (P'1, P'2, and P'3) . In this study, we employed bacteriophage P22 challenge phages to determine which protein binding sites participate in attL complex formation in vivo . The C', H', and P'1 sites were critical, because mutations in these sites severely disrupted formation of the attL complex . Mutations in the C and P'2 sites were less severe, and alteration of the P'3 site had no effect on complex formation . These results support a model in which IHF, bound to the H' site, bends the attL DNA so that the Int molecule bound to P'1 also interacts with the C' core site . This bridged complex, along with a second Int molecule bound to P'2, helps to stabilize the interaction of a third Int with the C core site . The results also indicate that nonspecific DNA binding is a significant component of the Int-core interactions and that the cooperativity of Int binding can overcome the effects of mutations in the individual arm sites and core sites.

Mol Reprod Dev, 1997 Feb, 46(2), 109 - 13
Efficient removal of loxP-flanked DNA sequences in a gene-targeted locus by transient expression of Cre recombinase in fertilized eggs; Sunaga S et al.; The bacteriophage P1 Cre/loxP site-specific recombination system is a useful tool for engineering chromosomal changes in animal cells . Transient expression of the Cre recombinase gene directly introduced into fertilized eggs by pronuclear injection has been reported to provide an efficient method of transgene modulation in fertilized eggs . In the present study, we examined the efficacy of this method to remove loxP-flanked DNA sequences in a gene-targeted locus in fertilized eggs . We replaced a part of the T-cell receptor gamma (TCR V gamma) locus with homologous sequences containing a loxP-flanked neogene in mouse embryonic stem (ES) cells by gene-targeting technique . The resulting ES cell clones containing the mutant allele (V gamma LNL) were used to generate chimeric mice by blastocyst injection . Eight male chimeras were bred with superovulated wild-type female mice . One hundred and seventy-six fertilized eggs were collected, and subjected to pronuclear injection of the Cre expression plasmid, pCAGGS-Cre, of a covalently closed circular form . Three out of 11 pups inherited the targeted V gamma locus . The inherited targeted allele of these 3 mice was shown to have undergone Cre-mediated recombination, resulting in a deletion of the loxP-flanked sequences (V gamma delta) as shown by Southern blot analysis of DNA from tail biopsies . All 3 founder mutant mice were capable of transmitting the V gamma delta locus to their offspring . The other 8 pups carried only wild-type alleles . There were no pups carrying the unrecombined V gamma LNL locus . Thus, the frequency of Cre-mediated recombination was 100% (3/3) with this method . In contrast, when closed circular pCAGGS-Cre plasmid was introduced into ES cells by electroporation, the recombination frequency of the V gamma LNL locus was 9.6% . These results indicated that our system based on transient expression of the Cre recombinase gene directly introduced into fertilized eggs by pronuclear injection provides a fast and efficient method for generating mutant mice with desired deletions or translocations in target genes.

Biophys J, 1997 Feb, 72(2 Pt 1), 958 - 63
The conformation of DNA packaged in bacteriophage G; Sun M et al.; When packaged in a bacteriophage capsid, double-stranded DNA occupies a cavity whose volume is roughly twice the volume of the DNA double helix . The data thus far have not revealed whether the compactness of packaged bacteriophage DNA is achieved by folding of the DNA, undirectional winding of the DNA, or a combination of both folding and winding . To assist in discriminating among these possibilities, the present study uses electron microscopy, together with ultraviolet light-induced DNA-DNA cross-linking, to obtain the following information about the conformation of DNA packaged in the comparatively large bacteriophage, G: 1) At the periphery of some negatively stained particles of bacteriophage G, electron microscopy reveals standards of DNA that are both parallel to each other and parallel to the polyhedral bacteriophage G capsid . However, these strands are not visible toward the center of the zone of packaged DNA . 2) Within some positively stained particles, electron microscopy reveals DNA-associated stain in relatively high concentration at corners of the polyhedral bacteriophage G capsid . 3) When cross-linked DNA is expelled from its capsid during preparation for electron microscopy, some DNA molecules consist primarily of a compacted central region, surrounded by DNA strands that appear to be unravelling at multiple positions uniformly distributed around the compacted DNA region . The above results are explained by a previously presented model in which DNA is compacted by folding to form 12 icosahedrally arranged pear-shaped rings.

Nucleic Acids Res, 1997 Feb 1, 25(3), 648 - 53
Metal ion interaction with cosubstrate in self-splicing of group I introns; Sjogren AS et al.; The catalytic mechanism for self-splicing of the group I intron in the pre-mRNA from the nrdB gene in bacteriophage T4 has been investigated using 2'-amino- 2'-deoxyguanosine or guanosine as cosubstrates in the presence of Mg2+, Mn2+and Zn2+ . The results show that a divalent metal ion interacts with the cosubstrate and thereby influences the efficiency of catalysis in the first step of splicing . This suggests the existence of a metal ion that catalyses the nucleophilic attack of the cosubstrate . Of particular significance is that the transesterification reactions of the first step of splicing with 2'-amino-2'-deoxyguanosine as cosubstrate are more efficient in mixtures containing either Mn2+or Zn2+together with Mg2+than with only magnesium ions present . The experiments in metal ion mixtures show that two (or more) metal ions are crucial for the self-splicing of group I introns and suggest the possibility that more than one of these have a direct catalytic role . A working model for a two-metal-ion mechanism in the transesterification steps is suggested.

J Mol Biol, 1997 Jan 31, 265(4), 372 - 84
Rapid evolution of translational control mechanisms in RNA genomes; Klovins J et al.; We have introduced 13 base substitutions into the coat protein gene of RNA bacteriophage MS2 . The mutations, which are clustered ahead of the overlapping lysis cistron, do not change the amino acid sequence of the coat protein, but they disrupt a local hairpin, which is needed to control translation of the lysis gene . The mutations decreased the phage titer by four orders of magnitude but, upon passaging, the virus accumulated suppressor mutations that raised the fitness to almost wild-type level . Analysis of the pseudorevertants showed that the disruption of the local hairpin, controlling expression of the lysis gene, had apparently been so complete that its restoration by chance mutations could not be achieved . Instead, alternative foldings initiated by the starting mutations were further stabilized and optimized . Strikingly, in the pseudorevertants analyzed, translational control of the lysis gene had been restored . This feat was accomplished by, on average, four suppressor mutations that generally occurred at codon wobble positions . We also introduced 11 mutations in a hairpin more upstream in the coat protein gene and not implicated in lysis control . Here the titer dropped by three logs, but pseudorevertants with a fitness close to wild-type were soon generated . These pseudorevertants again were the result of the optimization of alternative foldings induced by the mutations . The transition of the secondary structure from wild-type to pseudorevertant could be visualized by structure probing . Our study shows that the folding of the RNA is an important phenotypic property of RNA viruses . However, its distortion can easily be overcome by optimizing alternative base-pairings . These new structures are not qualitatively equivalent to the original one, since they do not successfully compete with the wild-type.

Gene, 1997 Jan 31, 185(1), 5 - 9
A novel bacterial vector system for monitoring protein-protein interactions in the cAMP-dependent protein kinase complex; Cairns MT et al.; A bacterial expression vector is described for investigation of protein-protein interactions . Important features of the vector include partition of the cI repressor of bacteriophage lambda into two functional domains separated by a multicloning site, and low level auto-regulated expression of human genes as C-terminal fusions to the DNA-binding domain of cI . Two different reporter systems have been employed; expression of either a suppressor tRNA or the alkaline phosphatase gene is dependent in both cases on the extent of repression of the major leftward promoter of lambda (lambdaP(L)) . The cAMP-dependent protein kinase (PKA) has been used as a model protein complex because both homodimer and heterodimer interactions are known to occur and because cAMP acts as a modulator of these interactions . It has been shown that the product of the repressor gene with newly incorporated expressed polylinker restriction sites still functions as a repressor . Substitution of the dimerisation domain of the cI repressor with the regulatory subunit of PKA does not diminish the ability of a cI fusion protein to repress expression of the reporter gene from lambdaP(L), indicating that the regulatory subunit of PKA dimerises the fusion protein in the Escherichia coli cytoplasm . Substitution instead with the catalytic subunit of PKA destroys the repression ability of cI, which is partially restored by separate expression of the regulatory subunit within the same cell . Complete restoration is achieved using a host E . coli strain which has lost its ability to synthesise cAMP and again this can be reversed by the addition of exogenous cAMP to these cells . Human PKA has been reconstituted in the E . coli cytoplasm, where all subunit interactions appear functional and respond as expected to the allosteric modulator cAMP.

J Biol Chem, 1997 Jan 31, 272(5), 2861 - 5
Regulation of T4 phage aerobic ribonucleotide reductase . Simultaneous assay of the four activities; Hendricks SP et al.; We have devised an assay procedure that permits simultaneous monitoring of the four activities of ribonucleotide reductase . Using this assay, we have compared the reduction of all four substrates by the T4 bacteriophage aerobic ribonucleotide reductase within different allosteric environments . Specifically, we compared the relative turnover rates by the enzyme when activated with "in vivo" concentrations of the known allosteric effectors versus activation by ATP alone . Consistent with the known allosteric properties of this enzyme, our results show that ATP does act as a general activator, although the rate of purine nucleotide reduction was approximately 5% of the rate for the pyrimidine nucleotides . However, addition of the allosteric effectors at their estimated physiological concentrations dramatically changed the relative rates of substrate reduction, creating a more "balanced" pool of products . Addition of the substrates at their respective in vivo concentrations further pushed rates of product formation toward a ratio similar to the base composition of the T4 genome . The similarity of the product profile produced under in vivo conditions to the genomic composition of T4 phage is discussed.

J Mol Biol, 1997 Jan 24, 265(3), 261 - 5
The activation defect of a lambda cI positive control mutant; Whipple FW et al.; "Positive control" mutants of the cI protein of bacteriophage lambda (lambda cI) bind DNA but, unlike the wild-type protein, fail to activate transcription . According to the original interpretation of Ptashne and co-workers, these mutants bear amino acid substitutions that disrupt a stimulatory interaction between lambda cI bound at operator site O(R)2 and RNA polymerase bound at promoter P(RM), an idea supported by kinetic analysis in one case . Genetic analysis has suggested that one residue in particular, glutamate 34 (E34), is critical for the stimulatory effect of wild-type lambda cI . More recently, however, Kolkhof and Muller-Hill have challenged this view, suggesting that mutant E34K fails to activate because it binds at unusually low concentrations to O(R)3, a site that mediates repression of P(RM) . To test this hypothesis, we have examined the behaviour of the lambda cI-E34K mutant both in vitro and in vivo by assaying transcription from P(RM) and monitoring operator site occupancy over a range of protein concentrations . Our results are inconsistent with the interpretation of Kolkhof and Muller-Hill, and demonstrate that under conditions where lambda operator O(R)2 is fully occupied and operator O(R)3 is vacant, wild-type lambda cI activates transcription from promoter P(RM) whereas the mutant does not.

Proc Natl Acad Sci U S A, 1997 Jan 21, 94(2), 479 - 84
The thioredoxin binding domain of bacteriophage T7 DNA polymerase confers processivity on Escherichia coli DNA polymerase I; Bedford E et al.; Bacteriophage T7 DNA polymerase shares extensive sequence homology with Escherichia coli DNA polymerase I . However, in vivo, E . coli DNA polymerase I is involved primarily in the repair of DNA whereas T7 DNA polymerase is responsible for the replication of the viral genome . In accord with these roles, T7 DNA polymerase is highly processive while E . coli DNA polymerase I has low processivity . The high processivity of T7 DNA polymerase is achieved through tight binding to its processivity factor, E . coli thioredoxin . We have identified a unique 76-residue domain in T7 DNA polymerase responsible for this interaction . Insertion of this domain into the homologous site in E . coli DNA polymerase I results in a dramatic increase in the processivity of the chimeric DNA polymerase, a phenomenon that is dependent upon its binding to thioredoxin.

Virology, 1997 Jan 20, 227(2), 409 - 19
PCR-directed formation of viral hybrids in vitro; Khan SA et al.; When constructing viruses that have desired hybrid phenotypes, anticipated difficulties include the nonviability of many, possibly most, of the hybrid genomes that can be constructed by incorporation of DNA fragments . Therefore, many different hybrid genomes may have to be constructed in order to find one that is viable . To perform this combinatorial work in a single experiment, we have used bacteriophage T7-infected cell extracts to transfer DNA in vitro . In an extract, we have incubated T7 DNA, together with DNA obtained by polymerase chain reaction (PCR) amplification of the gene (gene 17) for the tail fiber of the T7-related bacteriophage, T3 . After in vitro packaging of DNA in the extract, hybrid progeny bacteriophage were detected by probing with a T3-specific oligonucleotide; hybrids are found at a frequency of 0.1% . By determination of the nucleotide sequence of the entire gene 17 of 14 independently isolated hybrids, both right and left ends of the PCR fragment are found to be truncated in all hybrids . For all 14 hybrids, the right end is in the same location; the left end is found at 3 different locations . The nonrandom location of the ends is explained by selection among different inserts for viability; that is, most of the hybrid genomes are nonviable . Some hybrids acquire from T3 the desirable phenotype of nonadherence to agarose gels during agarose gel electrophoresis.

Virology, 1997 Jan 20, 227(2), 400 - 8
Isolation of a gp20-complex and its role in in vitro assembly of both prohead and core of bacteriophage T4; Kato H et al.; The product of gene 20 (gp20) in bacteriophage T4 forms the proximal vertex of the head . It has been suggested that gp20-rich fractions derived from the proheads can initiate core assembly in vitro and that gp20 plays an important role in prohead and core assembly . However, the gp20 isolated from proheads or overproducing cells harboring gene 20 did not work in vitro, owing to a strong tendency to aggregate . Native gp20 was purified from T4-infected Escherichia coli hdB3-1 cells, since it was shown that gp20 might not associate with membrane in this infection . Isolated gp20 is associated with two other proteins in a gp20-complex, composed of a connector and a neck joined to six fibers . The gp20-complex is also produced in T4 infection of E . coli groEL . Moreover, the complex can efficiently induce prohead and core assembly and is very stable.

J Biol Chem, 1997 Jan 17, 272(3), 1646 - 53
DNA-based loss of specificity mutations . Effects of DNA sequence on the contacted and non-contacted base preferences of bacteriophage P22 repressor; Hilchey SP et al.; Although the two central bases of the P22 operator are not contacted by the P22 repressor, changes in these bases alter the affinity of operator for repressor . Previous studies (Wu, L., and Koudelka, G . B . (1993) J . Biol . Chem . 268, 18975-18981) show that the structure of the P22 repressor-operator complex varies with central base sequence . Here we show that central base sequence composition affects the strength of two, and likely all, specific amino acid-base pair contacts between synthetic P22 operators and P22 repressor . However, altering a specific protein-DNA contact via a loss-of-contact mutation in repressor results in a loss of specificity at only one contacted position . Thus, only changing the sequence of non-contacted bases affects repressor's global base specificity . The observed effects of ionic concentration on the affinities of various operators for repressor and the DNase I patterns of protein complexes with these binding sites indicate certain central base sequences facilitate optimal juxtaposition of repressor with its contacted bases, while others prevent it . The existence of different structural forms of the repressor-operator complexes explains how the relative energetic importance of specific amino acid-base pair edge contacts is modulated.

J Biol Chem, 1997 Jan 17, 272(3), 1448 - 51
Modeling ligand-gated receptor activity . FhuA-mediated ferrichrome efflux from lipid vesicles triggered by phage T5; Letellier L et al.; An in vitro assay of iron-ferrichrome translocation across the FhuA protein of outer membranes from Escherichia coli has been devised . Upon reconstitution into large lipid vesicles, bacteriophage T5 binds to this polyvalent receptor, triggering a conformational change that resulted in channel opening . This facilitates the translocation of an iron(III)-siderophore, without the complexities involved in the in vivo process . Efflux of 55Fe(III)-ferrichrome across FhuA channels was determined quantitatively by monitoring the release of trapped radioactivity . The assay is rapid, reliable, and specific, because other bacteriophages, such as Phi80, fail to trigger channel opening of the FhuA receptor.

Nature, 1997 Jan 16, 385(6613), 269 - 72
Extension of chromatin accessibility by nuclear matrix attachment regions; Jenuwein T et al.; Transcription of the variable region of the rearranged immunoglobulin mu gene is dependent on an enhancer sequence situated within one of the introns of the gene . Experiments with transgenic mice have shown that activation of the promoter controlling this transcription also requires the matrix-attachment regions (MARs) that flank the intronic enhancer . As this mu gene enhancer can establish local areas of accessible chromatin, we investigated whether the MARs can extend accessibility to more distal positions . We eliminated interactions between enhancer- and promoter-bound factors by linking mu enhancer/MAR fragments to the binding sites for bacteriophage RNA polymerases that were either close to or one kilobase distal to the enhancer . The mu enhancer alone mediated chromatin accessibility at the proximal site but required a flanking MAR to confer accessibility upon the distal promoter . This long-range accessibility correlates with extended demethylation of the gene construct but not with whether it is being actively transcribed . MARs thus collaborate with the mu enhancer to generate an extended domain of accessible chromatin.

J Immunol, 1997 Jan 15, 158(2), 842 - 50
Bispecific monoclonal antibody complexes bound to primate erythrocyte complement receptor 1 facilitate virus clearance in a monkey model; Taylor RP et al.; We investigated the feasibility of using bispecific mAb complexes to redirect and improve the efficiency of the primate E complement receptor 1-based clearance reaction to remove a virus from the circulation . As an initial approach, we used bacteriophage phiX174 as an immunologic model for mammalian viruses . Bispecific complexes were prepared by chemically cross-linking a mAb specific for complement receptor 1 with a mAb specific for the bacteriophage phiX174 . In a monkey model these complexes facilitate rapid and quantitative binding of the target bacteriophage to E in vitro and in vivo . Moreover, after in vivo binding to E, the complexes containing mAb and prototype virus are rapidly cleared from the circulation of rhesus and cynomolgus monkeys without loss of E . Our findings suggest that bispecific mAb complexes, in concert with primate E complement receptor 1, may have therapeutic utility in the treatment of diseases associated with blood-borne pathogens.

J Biol Chem, 1997 Jan 10, 272(2), 1291 - 6
A model for the double-stranded RNA (dsRNA)-dependent dimerization and activation of the dsRNA-activated protein kinase PKR; Wu S et al.; Binding of double-stranded RNA (dsRNA) to PKR induces autophosphorylation and activation . However, the requirement for dsRNA in promoting dimerization and the requirement for dimerization in PKR activation are controversial . We have studied the dsRNA binding and dimerization requirements for the activation of PKR in vivo . Co-expression and immunoprecipitation experiments detected an interaction between the K296P mutant and a bacteriophage T7-epitope-tagged K64E mutant of dsRNA binding domain . In contrast, the K64E/K296P double mutant did not form a detectable dimer with the wild-type dsRNA binding domain . These results support that dimerization of intact PKR with the isolated dsRNA binding domain requires dsRNA binding activity . Expression of the isolated PKR kinase domain (residues 228-551) reduced translation of the reporter mRNA even in the presence of PKR inhibitors . Furthermore, the isolated kinase domain (residues 228-551) undergoes autophosphorylation and sequentially transphosphorylates both mutant K296P PKR and wild-type eIF-2alpha in vitro . In contrast, the isolated kinase domain (residues 264-551) lacking the third basic region was not active . These observations lead us to propose that the dsRNA binding domains on intact PKR inhibit kinase activity and that dsRNA binding to intact PKR induces a conformational change to expose dimerization sites within the dsRNA binding domain thereby promoting dimerization and facilitating trans-phosphorylation and activation.

Virology, 1997 Jan 6, 227(1), 207 - 10
Assembly of membrane-containing bacteriophage PRD1 is dependent on GroEL and GroES; Hanninen AL et al.; Assembly of the broad-host-range bacteriophage PRD1 involves translocation of the virus-specific membrane to the inside of the icosahedral protein shell formed of trimeric coat proteins . The formation of PRD1 particles is, in addition to the virus-encoded assembly factors P10 and P17, dependent on GroEL/GroES chaperonins . The chaperonins assist in the folding of the capsid proteins P3 and P5 and in the assembly of viral membrane proteins.

Virology, 1997 Jan 6, 227(1), 131 - 41
In vivo and in vitro evidence for an anti-Rho activity induced by the phage P4 polarity suppressor protein Psu; Linderoth NA et al.; The polarity suppression (Psu) protein of bacteriophage P4 causes suppression of transcriptional polarity in Escherichia coli by overcoming Rho termination factor activity . Two new psu mutants defective in polarity suppression are described . The psu5 mutation deletes codons 95-98 from about the middle of the gene, and the mutant protein is inactive . The psu6 mutation changes Phe169 to Val and encodes a temperature-sensitive protein . Constitutive overexpression of psu+ from a plasmid prevents colony formation, but overexpression of mutant genes (psu5, psu6) does not, suggesting that Psu disturbs essential host function(s) . Rho protein synthesis is enhanced several-fold in cells containing wild-type Psu, due to readthrough at the rho attenuator, while the physical stability of Rho is maintained . As a consequence, Psu-producing cells accumulate significantly more Rho than normal cells, reminiscent of termination-defective rho mutants . The polarity suppression activity induced by Psu is demonstrated in vitro by the efficient readthrough of Rho-dependent terminators lambda tR1 and TIS2 during coupled transcription-translation . Purified Rho protein restores termination at TIS2 when added to Psu-containing reactions but NusG does not . The data support the hypothesis that Psu has or elicits an anti-Rho function.

Virology, 1997 Jan 6, 227(1), 103 - 10
An in vitro system for the investigation of heterologous RNA recombination; Qiao X et al.; Bacteriophage Phi6 has a genome of three segments of double-stranded RNA enclosed in a polyhedral procapsid . Purified procapsids are capable of the specific packaging of viral plus strands and the synthesis of their complementary minus strands . The genomic segments of Phi6 are capable of heterologous recombination . We have prepared an in vitro system containing purified procapsids that is capable of packaging plus strands of the genomic segments and synthesizing minus strands on these templates . The system generates heterologous recombination products when stimulated by having one of the plus strands incapable of serving as a template for minus strand synthesis . Recombinants were produced upon transfection of spheroplasts with the in vitro packaged and replicated RNA . Sites of recombination were not found to be localized in particular regions of either the donor or the recipient strands.

Biochim Biophys Acta, 1997 Jan 3, 1350(1), 89 - 97
Functional and sequence analysis of splicing defective nrdB mutants of bacteriophage T4 reveal new bases and a new sub-domain required for group I intron self-splicing; Lal SK et al.; The nrdB gene of bacteriophage T4 codes for the small subunit of ribonucleotide reductase and contains a 598 nuclelotide group 1 self splicing intron . In order to study the functional domains for self-splicing of this intron, 23 nrdB splicing defective intron mutants were analyzed for both sequence and functional changes . These mutants cluster towards the ends in regions of conserved structural elements of the intron . These 23 mutants have single base changes at 14 different sites . Interestingly two of these sites that seemed to map within the intron are actually located on the flanking exon sequences on both sides of the intron . A high frequency (4/12) of the mutation sites are in bases not thought to be base-paired in the standard model of group I intron structure . The mutation sites in pairing regions P3, P7, P8, P9 and between P6{3'} and P7{5'} are identical to changes found in the well studied td (encoding dTMP synthase) intron . However, five new mutation sites (S61, SL1, S29, SL11, SL196 and SL126) are unique to the nrdB intron and disrupt self-splicing . A mutation (S61) in the P7.1 pairing region is especially significant because no mutations have been found in this pairing, thus defining a new sub-domain essential for RNA splicing . Like the td intron, the mutation site in P9 of the nrdB intron is a hot spot for mutations, but unlike td, the nrdB intron does not show a mutational hot spot in the P6{5'} region . Our molecular dissection of the nrdB intron also supports the P9.0 and P10 pairings that have been postulated to help form a complex tertiary structure required to give the RNA sequence its catalytic activity: particularly 3' splice site selection, cleavage and exon ligation.

J Biol Chem, 1997 Jan 3, 272(1), 534 - 8
Bacterial protease Lon is a site-specific DNA-binding protein; Fu GK et al.; The product of the Escherichia coli lon gene is the ATP-dependent Lon protease . Lon contributes to the regulation of several important cellular functions, including radiation resistance, cell division, filamentation, capsular polysaccharide production, lysogeny of certain bacteriophages, and proteolytic degradation of certain regulatory and abnormal proteins . Lon homologues are also found in several widely divergent bacteria, as well as in the mitochondria of yeast and humans . E . coli Lon has long been known to bind to DNA, but this interaction has not been further characterized and has generally been assumed to be nonspecific . We now demonstrate that E . coli Lon can bind to a TG-rich DNA promoter element in a sequence-specific manner . This finding is based on the results of experiments employing SouthWestern blotting, protein purification, "shift-shift" electrophoretic mobility shift assays, electrophoretic mobility shift assays using in vitro transcribed and translated Lon, and DNase footprinting . Site-specific DNA binding is likely to be an additional important biochemical characteristic of the multifaceted Lon protease.

Annu Rev Biophys Biomol Struct, 1997, 26, 401 - 24
Bacteriophage display and discovery of peptide leads for drug development; Lowman HB; Phage display makes large-peptide diversity libraries readily attainable for identifying novel peptide ligands for receptors and other protein or non-protein targets . This technology kindles enthusiasm for the idea that large and protein-protein interaction surfaces (epitopes) can be distilled down to small pharmacophores . These may be accessible to organic scaffolding, yielding new orally active drugs that might otherwise have taken greater time and effort to be discovered through chemical-library screening . This review, though not comprehensive with respect to the explosive volume of phage display work over the last few years, focuses on recent developments in phage-displayed peptide technology.

Virus Genes, 1997, 14(2), 137 - 46
Identification of a strong promoter of bacteriophage MB78 that lacks consensus sequence around minus 35 region and interacts with phage specific factor; Zargar MA et al.; A strong promoter of bacteriophage MB78 does not have minus 35 consensus sequence although it has a TGn motif immediately upstream of minus 10 sequence as well as the AT rich UP element . It is efficiently recognised by the sigma 70 RNA polymerase, however, a phage-specific factor competes with sigma 70 RNA polymerase for binding to this region, the binding of the factor being stronger than that of the polymerase . Contrary to the reports in the literature the polymerase appears not to bind to the UP element whereas the phage-specific factor does . The latter seems to be involved in the regulation of the promoter activity.

Arch Virol, 1997, 142(6), 1139 - 54
Mutations in the SIV env and the M13 lacZa gene generated in vitro by reverse transcriptases and DNA polymerases; Stuke AW et al.; To investigate the accuracy of retroviral in vitro DNA replication we have examined with two fidelity assays the reverse transcriptases (RTs) from SIVagm, HIV-1, MoMLV as well for comparison the Klenow fragment from E . coli and DNA polymerase a from calf-thymus . These forward mutation assays measured the loss of bacteriophage M13 lacZa gene function by mutations . In the EnvlacZa assay frameshift mutations occurring during polymerisation of a 176 b long simian immunodeficiency virus (SIV) envelope (env) sequence were phenotypically detected by blue/white-plaque screening . To measure in addition substitutions, a 116 b long M13 lacZa gene DNA template was used as the mutational target (LacZa assay) . With the SIVagm env gene DNA template, we observed similar levels of frameshift fidelity for all three RTs . Nevertheless, the SIVagm RT was slightly more accurate than the other RTs and nearly all frameshifts were observed at two homopolymeric runs of its homologous template . Measuring also substitution errors at the lacZa template the mutation frequency of the SIVagm RT increased 2.5 fold and that of the HIV-1 RT was enhanced by a factor of 3.

Plasmid, 1997, 37(2), 141 - 53
Transfection of Actinomyces spp . by genomic DNA of bacteriophages from human dental plaque; Yeung MK et al.; Bacteriophages that produced turbid or clear zones of lysis in strains of Actinomyces were isolated from 22 of 124 samples of fresh human dental plaque . All human and nonhuman strains of Actinomyces viscosus or Actinomyces naeslundii tested in this study were sensitive to infection by one or more of these phages . In contrast, none of the Actinomyces odontolyticus, Actinomyces israelii, or Actinomyces bovis strains tested were susceptible . Results of restriction endonuclease analyses indicated that the genomes of these phages consisted of double-stranded DNA molecules ranging in size between 16 and 60 kbp . Sequence homology under hybridization conditions of high stringency was observed among a few of the isolated phages . A lysogenized isolate of A . viscosus MG-1 was obtained following infection with a temperate phage, designated phi 225 . Results of Southern blot analyses indicated that phi 225 replicated as a plasmid in the lysogenized strain . Genomic DNA from several lytic phages was used to establish conditions for transfection by electroporation of strains of Actinomyces spp . Efficiencies of DNA transfer ranged from 10(2) to 10(5) plaque-forming units per microgram of DNA were obtained under optimal transfection conditions . The results of these studies demonstrate that transfer of genetic information in Actinomyces spp . can be achieved by transfection.

Immunogenetics, 1997, 45(6), 365 - 70
CpG-rich sequences close to the site of duplication within the human IGH constant region; Sadhu A et al.; Human immunoglobulin heavy chains are encoded by a highly complex locus, IGH, which encompasses many transcriptional units, including nine alternative constant region genes . Much of the constant region gene cluster was duplicated during hominoid evolution . In rodents, IGH is known to be regulated by multiple elements, including several enhancers 3' of the alpha chain-encoding A constant region gene, CA, the last transcribed region . Sequences downstream of the two human CA genes, possibly containing homologous enhancer elements, have not yet been reported . By long-range mapping of genomic DNA, a cluster of unmethylated rare restriction sites, representing a potential CpG island, was previously reported downstream of each CA gene, close to the 3' end of the duplicated region . Such potential CpG islands are candidates for additional IGH regulatory elements . We isolated bacteriophage clones containing these clusters of methylation-sensitive restriction sites, which lie within short CpG-rich stretches . Some of these sites showed tissue-specific methylation . 3' of the unmethylated CpG-rich sequences, clones derived from the 5' (telomeric) copy of the duplicated region, contained, in order, endogenous retroviral sequences, a processed ELK1 pseudogene, and the pseudogene IGHGP . Comparison with the presumed 3' (centromeric) copy of the duplicated region showed that similarity was lost exactly at the end of the retroviral long terminal repeat sequences . These results imply that an endogenous retroviral integration was present immediately 3' of IGH in the common hominoid ancestor and suggest models for the duplication of the region.

Photochem Photobiol, 1997 Jan, 65(1), 161 - 5
The effects of methylene blue and oxygen concentration on the photoinactivation of Q beta bacteriophage; Lee D et al.; Concentration effects of methylene blue (MB) and oxygen on the photoinactivation rate of Q beta bacteriophage were examined . The effect of initial virus concentration was verified on the similar f2 phage . The inactivation rate, kappa, is an increasing function of MB and O2 concentration and shows saturation with respect to MB concentration . Thus the results suggest that MB must adsorb to Q beta sites and oxygen must be present for photoinactivation to occur . The inactivation rate is independent of the initial number of phage particles present before inactivation, indicating that inactivation does not depend upon interaction among viral particles or on surface effects . The results indicate that at least two different viral phenotypes exist within the wild-type Q beta and f2 populations: one susceptible and the other resistant.

Nat Biotechnol, 1997 Jan, 15(1), 74 - 8
Efficient epitope mapping by bacteriophage lambda surface display; Kuwabara I et al.; A bacteriophage lambda surface expression system, lambda foo, was used for epitope mapping of human galectin-3 . We constructed random epitope and peptide libraries and compared their efficiencies in the mapping . The galectin-3 cDNA was randomly digested by DNase 1 to make random epitope libraries . The libraries were screened by affinity selection using a microtiter plate coated with monoclonal antibodies . Direct DNA sequencing of the selected clones defined two distinct epitope sites consisting of nine and 11 amino-acid residues . Affinity selection of random peptide libraries recovered a number of sequences that were similar to each other but distinct from the galectin-3 sequence . These results demonstrate that a single affinity selection of epitope libraries with antibodies is able to define an epitope determinant as small as nine residues long and is more efficient in epitope mapping than random peptide libraries.

Microbiology, 1997 Jan, 143 ( Pt 1), 179 - 85
Bacteriophage T4 development depends on the physiology of its host Escherichia coli; Hadas H et al.; Several parameters of phage T4 adsorption to and growth in Escherichia coli B/r were determined . All changed monotonously with the bacterial growth rate (mu), which was modified by nutritional conditions . Adsorption rate was faster at higher mu values, positively correlated to cell size, and increased by pretreatment with low penicillin (Pn) concentrations; it was directly proportional to total cellular surface area, indicating a constant density of T4 receptors on cell envelopes irrespective of growth conditions . Parameters of phage development and cell lysis were mu-dependent . The rate of phage release and burst size increased, while the eclipse and latent periods decreased with increasing mu . Differentiation between the contribution of several physiological parameters to the development of T4 was performed by manipulating the host cells . A competitive inhibitor of glucose uptake, methyl alpha-D-glucoside, was exploited to reduce the growth rate in the same effective carbon source . Synchronous cells were obtained by the "baby-machine' and large cells were obtained by pretreatment with low Pn concentrations . Lysis was delayed by superinfection, and DNA content and concentration were modified by growing a thy mutant in limiting thymine concentrations . The results indicate that burst size is not limited by cell size or DNA composition, nor directly by the rate of metabolism, but rather by the rates of synthesis and assembly of phage components and by lysis time . The rates of synthesis and assembly of phage components seem to depend on the content of the protein-synthesizing system and lysis time seems to depend on cellular dimensions.

Genes Chromosomes Cancer, 1997 Jan, 18(1), 30 - 41
Alu-polymerase chain reaction genomic fingerprinting technique identifies multiple genetic loci associated with pancreatic tumourigenesis; McKie AB et al.; DNA fingerprinting can be used to detect genetic rearrangements in cancer that may be associated with activation of oncogenes and inactivation of tumour suppressor genes . We have developed a fingerprinting strategy based on polymerase chain reaction (PCR) amplification of genomic DNA with primers specific for the Alu repeat sequences, which are highly abundant in the human genome . This has been applied to DNA from pancreatic cancer and paired normal samples to isolate and identify fragments of genomic DNA rearranged in the malignant cells . These fragments have been sequenced and used as probes to isolate hybridising clones from gridded bacteriophage P1, phage artificial chromosome, and cosmid libraries for fluorescent in situ hybridisation mapping and the identification of expressed sequences . Further characterisation has identified a putative novel gene (ART1) that is up-regulated specifically in pancreatic cancer as well as another sequence with similarity to genes involved in differentiation (POU domains) . In conclusion, we suggest that Alu-PCR fingerprinting may be a useful technique for the identification of genes involved in tumourigenesis.

RNA, 1997 Jan, 3(1), 57 - 67
Analysis of bacteriophage N protein and peptide binding to boxB RNA using polyacrylamide gel coelectrophoresis (PACE); Cilley CD et al.; The antitermination protein N from bacteriophage lambda (Nlambda) interacts with the nut site in its own mRNA, as well as host factors, to facilitate formation of a termination-resistant transcription complex . The conserved, amino-terminal arginine-rich domain of Nlambda protein is known to interact with a small RNA hairpin (boxB) derived from the nut site RNA . We have examined the binding of Nlambda protein, peptides derived from the amino terminus of Nlambda, and the related phage P22 N protein to lambda boxB RNAs . To facilitate the study of complexes that are not amenable to gel retardation assays, a new polyacrylamide affinity coelectrophoresis technique (PACE) was developed . Using the PACE assay, we have demonstrated that a 19-amino acid peptide from the amino terminus of Nlambda protein binds lambda boxB RNA with a Kd,app of 5.2 nM . PACE was also used to study the binding affinity of a number of Nlambda peptide and lambda boxB RNA mutants . The PACE technique is complementary to the traditional gel retardation assay for direct measurement of binding interactions, and will be useful for any procedure that requires a pool of RNAs to be resolved based on their relative affinities for proteins or peptides.

J Bacteriol, 1997 Jan, 179(2), 358 - 63
The HflB protease of Escherichia coli degrades its inhibitor lambda cIII; Herman C et al.; The cIII protein of bacteriophage lambda is known to protect two regulatory proteins from degradation by the essential Escherichia coli protease HflB (also known as FtsH), viz., the lambda cII protein and the host heat shock sigma factor sigma32 . lambda cIII, itself an unstable protein, is partially stabilized when the HflB concentration is decreased, and its half-life is decreased when HflB is overproduced, strongly suggesting that it is degraded by HflB in vivo . The in vivo degradation of lambda cIII (unlike that of sigma32) does not require the molecular chaperone DnaK . Furthermore, the half-life of lambda cIII is not affected by depletion of the endogenous ATP pool, suggesting that lambda cIII degradation is ATP independent (unlike that of lambda cII and sigma32) . The lambda cIII protein, which is predicted to contain a 22-amino-acid amphipathic helix, is associated with the membrane, and nonlethal overproduction of lambda cIII makes cells hypersensitive to the detergent sodium dodecyl sulfate . This could reflect a direct lambda cIII-membrane interaction or an indirect association via the membrane-bound HflB protein, which is known to be involved in the assembly of certain periplasmic and outer membrane proteins.

J Bacteriol, 1997 Jan, 179(2), 323 - 9
Purification and characterization of VSH-1, a generalized transducing bacteriophage of Serpulina hyodysenteriae; Humphrey SB et al.; Serpulina hyodysenteriae B204 cells treated with mitomycin (20 microg of mitomycin/ml of culture broth) lysed and released bacteriophages . Bacteriophage particles, precipitated by using polyethylene glycol and purified by CsC1 density gradient ultracentrifugation, had a buoyant density of 1.375 g/cm3 and consisted of a head (45-nm diameter) and an ultrastructurally simple (noncontractile) tail (64 by 9 nm) composed of at least 13 proteins with molecular masses ranging between 13 and 101 kDa . The purified bacteriophage has been designated VSH-1 (VSH for virus of S . hyodysenteriae) . VSH-1 was incapable of lytic growth on any of five intestinal spirochete strains, representing three Serpulina species . VSH-1 nucleic acid was determined to be approximately 7.5 kb in size and to be linear, double-stranded DNA based on differential staining with acridine orange, DNase I sensitivity, electrophoretic mobility, and contour length as measured by electron microscopy . Phage DNA digested by the restriction enzymes SspI, AseI, EcoRV, and AflII gave electrophoretic banding patterns nearly identical to those of digested chromosomal DNA from S . hyodysenteriae . Additionally, VSH-1 DNA fragments hybridized with probes complementary to S . hyodysenteriae chromosomal genes nox and flaA1 . When purified bacteriophages induced from cultures of S . hyodysenteriae A203 (deltaflaA1 593-762::cat) were added to growing cells of strain A216 (deltanox 438-760::kan), transductants (Cmr Kmr) were obtained at a frequency of 1.5 x l0(-6) per phage particle (enumerated by electron microscopy) . These findings indicate that induced VSH-1 virions package DNA of S . hyodysenteriae and are capable of transferring host genes between cells of that spirochete . To our knowledge, this is the first report of genetic transduction of a spirochete.

J Virol, 1997 Jan, 71(1), 495 - 500
Magnesium-induced conformational change of packaging RNA for procapsid recognition and binding during phage phi29 DNA encapsidation; Chen C et al.; Bacteriophage phi29 is typical of double-stranded DNA viruses in that its genome is packaged into a preformed procapsid during maturation . An intriguing feature of phi29 assembly is that a virus-encoded RNA (pRNA) is required for the packaging of its genomic DNA . Psoralen cross-linking, primer extension, and T1 RNase partial digestion revealed that pRNA had at least two conformations; one was able to bind procapsids, and the other was not . In the presence of Mg2+, one stretch of pRNA, consisting of bases 31 to 35, was confirmed to be proximal to base 69, as revealed by its efficient cross-linking by psoralen . Two cross-linking sites in the helical region were identified . Mg2+ induced a conformational change of pRNA that exposes the portal protein binding site by promoting the refolding of two strands of the procapsid binding region, resulting in the formation of pRNA-procapsid complexes . The procapsid binding region in this binding-competent conformation could not be cross-linked with psoralen . When the two strands of the procapsid binding region were fastened by cross-linking, pRNA could neither bind procapsids nor package phi29 DNA . A pRNA conformational change was also discernible by comparison of migration rates in native EDTA and Mg2+ polyacrylamide gel electrophoresis and was revealed by T1 RNase probing . The Mg2+ concentration required for the detection of a change in pRNA cross-linking patterns was 1 mM, which was the same as that required for pRNA-procapsid complex formation and DNA packaging and was also close to that in normal host cells.

J Virol, 1997 Jan, 71(1), 487 - 94
Approaches to determine stoichiometry of viral assembly components; Trottier M et al.; Due to the rapidity of biological reactions, it is difficult to isolate intermediates or to determine the stoichiometry of participants in intermediate reactions . Instead of determining the absolute amount of each component, this study involved the use of relative parameters, such as dilution factors, percentages probabilities, and slopes of titration curves, that can be more accurately quantified to determine the stoichiometry of components involved in bacteriophage phi29 assembly . This work takes advantage of the sensitive in vitro phage phi29 assembly system, in which 10(8) infectious virions per ml without background can be assembled from eight purified components . It provides a convenient assay for quantification of the stoichiometry of packaging components, including the viral procapsid, genomic DNA, DNA-packaging pRNA, and other structural proteins and enzymes . The presence of a procapsid binding domain and another essential functional domain within the pRNA makes it an ideal component for constructing lethal mutants for competitive procapsid binding . Two methods were used for stoichiometry determination . Method 1 was to determine the combination probability of mutant and wild-type pRNAs bound to procapsids . The probability of procapsids that possess a certain amount of mutant and a certain amount of wild-type pRNA, both with an equal binding affinity, was predicted with the binomial equation {EQUATION IN TEXT} where Z is the total number of pRNAs per procapsid, M is the number of mutant pRNAs bound to one procapsid, and (ZM) is equal to {FORMULA IN TEXT} . With various ratios of mutant to wild-type pRNA in in vitro viral assembly, the percent mutant pRNA versus the yield of virions was plotted and compared to a series of predicted curves to find a best fit . It was determined that five or six copies of pRNA were required for one DNA-packaging event, while only one mutant pRNA per procapsid was sufficient to block packaging . Method 2 involved the comparison of slopes of curves of dilution factors versus the yield of virions . Components with known stoichiometries served as standard controls . The larger the stoichiometry of the component, the more dramatic the influence of the dilution factor on the reaction . A slope of 1 indicates that one copy of the component is involved in the assembly of one virion . A slope larger than 1 would indicate multiple-copy involvement . By this method, the stoichiometry of gp11 in phi29 particles was determined to be approximately 12 . These approaches are useful for the determination of the stoichiometry of functional units involved in viral assembly, be they single molecules or oligomers . However, these approaches are not suitable for the determination of exact copy numbers of individual molecules involved if the functional unit is composed of multiple subunits prior to assembly.

J Bacteriol, 1997 Jan, 179(1), 63 - 71
Characterization of a region of the IncHI2 plasmid R478 which protects Escherichia coli from toxic effects specified by components of the tellurite, phage, and colicin resistance cluster; Whelan KF et al.; The IncHI2 plasmid R478 specifies resistance to potassium tellurite (Te(r)), to some bacteriophages (Phi), and to pore-forming colicins (PacB) . The genes encoding the three phenotypes are linked, and an 8.4-kb fragment of R478 DNA encoding them cannot be subcloned unless cocloned with a second section of the plasmid . Subclone pKFW4A contains a 5.9-kb BamHI-EcoRI fragment which caused some toxicity when present in Escherichia coli cells . Bacterial cells containing freshly transformed pKFW4A, examined by light microscopy and electron microscopy, had a filamentous morphology consistent with a block in septation . Insertion of transposon Tn1000 into terZ, -A, -B, and -C genes of pKFW4A resulted in the loss of the filamentation phenotype . Deletion of several regions of the clone confirmed that these latter components are involved in the filamentation phenotype . The region specifying protection from toxicity caused by the larger 8.4-kb fragment (encompassing this cluster and the entire 5.9-kb section of pKFW4A) was sequenced and analyzed by T7 polymerase expression and Tn1000 mutagenesis . Three open reading frames, terW, terY, and terX, were identified in a 2.6-kb region . Two polypeptides with approximate molecular masses of 18 and 28 kDa were expressed in CSRDE3 cells and were consistent with TerW (17.1 kDa; 155 amino acids {aa}) and TerY (26.9 kDa; 248 aa), whereas a protein of 213 aa deduced from terX was not observed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis . The terX gene product shows strong identity with the previously identified TerE, TerD, and TerZ polypeptides, and there is a conserved motif of 13 residues, GDN(R/L)TG(E/A)GDGDDE, within this group of polypeptides . Complementation analysis indicated that terW, located approximately 6.0 kb upstream of terZ, brings about protection of cells from toxic effects of components of the Te(r), Phi, and PacB cluster.

Biochemistry, 1996 Dec 24, 35(51), 16652 - 64
Gene V protein dimerization and cooperativity of binding of poly(dA); Terwilliger TC; Gene V protein of bacteriophage f1 is a dimeric protein that binds cooperatively to single-stranded nucleic acids . In order to determine whether a monomer-dimer equilibrium has an appreciable effect upon the thermodynamics of gene V protein binding to nucleic acids, the dissociation constant for the protein dimer was investigated using size-exclusion chromatography . At concentrations ranging from 5 x 10(-10) to 1.2 x 10(-5) M, the Stokes radius of the protein was that expected of the dimer of the gene V protein . The Stokes radius of the protein was also independent of salt concentration from 0.2 to 1.0 M NaCl in a buffer containing 10 mM Tris-HCl, pH 7.4, and 1 mM EDTA . The binding of the dimeric gene V protein to poly(dA) was studied using a simplified lattice model for protein-protein interactions adapted for use with a dimeric protein that binds simultaneously to two strands of nucleic acid . Interpretation of the salt dependence, C = {d log(Kint omega)}/{d log(NaCl)}, of binding of such a dimeric protein to nucleic acid using the theory of Record et al . (Record, M . T., et al . (1976) J . Mol . Biol . 107, 145-158) indicates that C is a function of the numbers of cations and anions released from protein and nucleic acid upon binding of the dimer, not of the monomer . Cooperativity of gene V protein binding to poly(dA) was studied with titration experiments that are sensitive to the degree of cooperativity of binding . The cooperativity factor omega, defined as the ratio of the binding constant for a site adjacent to a previously bound dimer to that for an isolated site, was found to be relatively insensitive to salt, with a value in the range of 2000-7000 for binding to poly(dA) at 3 degrees C and at 23 degrees C . This high cooperativity factor supports the suggestion that protein-protein contacts play a major role in the formation of the superhelical gene V protein-single-stranded nucleic acid complex.

Biochemistry, 1996 Dec 24, 35(51), 16621 - 9
Functional consequences and exonuclease kinetic parameters of point mutations in bacteriophage T4 DNA polymerase; Abdus Sattar AK et al.; Three groups of T4 DNA polymerase mutants were prepared and characterized . In the first group, Ala and Asn were substituted for four acidic residues in the exonuclease domain that were chosen on the basis of their sequence alignment with the Klenow fragment from Escherichia coli DNA polymerase I . Two divalent metal ions required for catalyzing the 3'-5' exonuclease reaction are ligated by carboxyl groups from these conserved Asp and Glu residues . The Ala and Asn replacements have a profound effect on the exonuclease activity of T4 DNA polymerase and also have a significant, but less pronounced influence on its polymerase activity which is located in a domain distal to the exonuclease region . The kcat values for the exonuclease reaction were reduced by 3-4 orders of magnitude by these replacements, but the values of Km(app) did not differ greatly from the wild-type enzyme . The second group consists of replacements of other residues, that are conserved in the exonuclease domain of eukaryotic DNA polymerases, but do not contribute to divalent metal ion coordination . Many of these alterations resulted in decreased exonuclease and/or polymerase activity . Mutants in the third group have substitutions of conserved residues in the polymerase domain which diminished polymerase and altered exonuclease activities . Our results, combined with structural data on crystals of protein N388, a truncated form of T4 DNA polymerase (Wang et al., 1996), show that: (i) the reduction in the relative specific exonuclease activities of mutants in the first group was significantly less than that of mutants in the Klenow fragment, despite the nearly identical geometric arrangement of the metal liganding groups in two proteins; (ii) altered residues, that affect exonuclease and/or polymerase activities in mutants of the second group, cluster within a small area of the exonuclease domain, suggesting that this area may be directly or indirectly involved in polymerase activity; (iii) mutations in the third group, which affect polymerase and exonuclease activities, may participate in DNA and dNTP binding . Our results point to the functional interdependence of the polymerase and exonuclease domains in T4 DNA polymerase, a property not observed with the Klenow fragment.

Proc Natl Acad Sci U S A, 1996 Dec 24, 93(26), 15400 - 4
Rescue of a segmented negative-strand RNA virus entirely from cloned complementary DNAs; Bridgen A et al.; We provide the first report, to our knowledge, of a helper-independent system for rescuing a segmented, negative-strand RNA genome virus entirely from cloned cDNAs . Plasmids were constructed containing full-length cDNA copies of the three Bunyamwera bunyavirus RNA genome segments flanked by bacteriophage T7 promoter and hepatitis delta virus ribozyme sequences . When cells expressing both bacteriophage T7 RNA polymerase and recombinant Bunyamwera bunyavirus proteins were transfected with these plasmids, full-length antigenome RNAs were transcribed intracellularly, and these in turn were replicated and packaged into infectious bunyavirus particles . The resulting progeny virus contained specific genetic tags characteristic of the parental cDNA clones . Reassortant viruses containing two genome segments of Bunyamwera bunyavirus and one segment of Maguari bunyavirus were also produced following transfection of appropriate plasmids . This accomplishment will allow the full application of recombinant DNA technology to manipulate the bunyavirus genome.

J Biol Chem, 1996 Dec 20, 271(51), 33148 - 55
T4 endonuclease VII . Importance of a histidine-aspartate cluster within the zinc-binding domain; Giraud-Panis MJ et al.; The DNA junction-resolving enzyme endonuclease VII of bacteriophage T4 contains a zinc-binding region toward the N-terminal end of the primary sequence . In the center of this 39-amino acid section (between residues 38 and 44) lies the sequence HLDHDHE, termed the His-acid cluster . Closely related sequences are found in three other proteins that have similar zinc-binding motifs . We have analyzed the function of these residues by a site-directed mutagenesis approach, modifying single amino acids and studying the properties of the resulting N-terminal protein A fusions . No sequence changes within the His-acid cluster led to a change in zinc content of the protein, indicating that these residues are not involved in the coordination of zinc . We found that the N-terminal aspartate residue (Asp-40) and the two histidine residues (His-41 and His-43) within the cluster are essential for junction-cleavage activity of the proteins . However, all sequence variations within this region generate proteins that retain their ability to bind to four-way DNA junctions (with minor changes in binding affinity in some cases) and to distort their global structure in the same manner as active enzymes . We conclude that the process of cleavage can be uncoupled from those of binding to and distortion of the junction . It is probable that some amino acid side chains of the His-acid cluster participate in the phosphodiester cleavage mechanism of endonuclease VII . The essential aspartate residue might be required for coordination of catalytic metal ions.

FEBS Lett, 1996 Dec 16, 399(3), 237 - 40
Tight-binding inhibitory sequences against pp60(c-src) identified using a random 15-amino-acid peptide library; Nishi T et al.; A bacteriophage peptide library containing a random 15-amino-acid insert was screened for identification of peptide sequence(s) that bind pp60(c-src) . Sequencing the random insert from more than 100 virions indicated that more than 60% of the phage virions that bound to this enzyme contained a GXXG sequence motif in which X was frequently a hydrophobic residue . The GXXG sequence was often repeated as GXXGXXG . Two nonameric peptides were synthesized to determine whether or not the peptide inhibits pp60(c-src) tyrosine kinase activity and the importance of the glycine residues within this sequence . The peptide containing glycine had a Ki of 24 microM, whereas replacing the glycines with proline increased the Ki value to 3.1 mM.

Eur J Biochem, 1996 Dec 15, 242(3), 720 - 6
In vitro alpha-complementation of beta-galactosidase on a bacteriophage surface; Dunn IS; Surface display of large multimeric non-secreted proteins is advantageous on the bacteriophage lambda compared with the widely used filamentous phage systems . A model system, the alpha-complementation of beta-galactosidase, was used for both further general characterization of protein-protein interactions on the lambda tail tube surface and for specifically probing the structure of the phage-displayed beta-galactosidase tetramer . In this complementation system, dimeric enzymatically inactive N-terminal deletion mutants of beta-galactosidase (alpha-acceptors) interact with peptides whose sequences span the region of the deletion (alpha-peptides) with the subsequent formation of tetramers and restoration of activity . The lambda phage could tolerate incorporation into their tail tubes of a limited number of copies of V protein (gpV) subunits C-terminally modified with an active alpha-peptide . Purified alpha-peptide phage showed specific in vitro alpha-complementation with an alpha-acceptor extract; the features of this reaction suggested that each complemented monomer can directly associate with an alpha-peptide displayed within the same tail tube structure . In contrast to the alpha-peptide, attempts to surface display an alpha-acceptor protein in a similar manner were unsuccessful . The implications of this work for surface-display cDNA libraries are discussed.

Genes Dev, 1996 Dec 15, 10(24), 3170 - 82
SurA, a periplasmic protein with peptidyl-prolyl isomerase activity, participates in the assembly of outer membrane porins; Rouviere PE et al.; Little is known about either the process of periplasmic protein folding or how information concerning the folding state in this compartment is communicated . We present evidence that SurA, a periplasmic protein with peptidyl-prolyl isomerase activity, is involved in the maturation and assembly of LamB . LamB is a trimeric outer membrane porin for maltodextrins as well as the bacteriophage lambda receptor in Escherichia coli . We demonstrate that SurA is involved in the conversion of unfolded monomers into a newly identified intermediate in LamB assembly, which behaves as a folded monomer . The absence of SurA blocks the assembly pathway and leads to accumulation of species prior to the folded monomer . These species also accumulate when the stress sigma factor sigmaE is induced by LamB overexpression . We suggest that accumulation of species prior to the generation of folded monomer is a stress signal sensed by sigmaE.

Mol Gen Genet, 1996 Dec 13, 253(3), 362 - 9
A second site-specific recombination event in the lambdoid bacteriophage HK022; Kolot M et al.; An in vitro site-specific recombination reaction of the lambdoid phage HK022 has revealed two supercoiled products that proved to be Holliday intermediates . One of them is the Holliday intermediate which has resulted from an attP x attB reaction . The other is an intermediate which has resulted from a recombination reaction between attP and the attL site of the product from the first reaction . The preferential attL x attP over attR x attP reaction was confirmed in vitro and in vivo by challenging attP sites with attL and attR sites . The biased attP x attL over attP x attR reaction in phage HK022 is discussed.

Biochem Biophys Res Commun, 1996 Dec 13, 229(2), 590 - 5
Ultraviolet B radiation-induced DNA lesions in mouse epidermis: an assessment using a novel 32P-postlabelling technique; Chatterjee ML et al.; Ultraviolet B (UVB) component of the sunlight is the major cause of nonmelanoma skin cancer (NMSC) in humans . UVB is absorbed directly by cellular DNA and produces lesions that may cause mutation(s) in target gene(s) ultimately leading to cancer . Early detection of these lesions, therefore, may help to identify individuals at a high risk to develop NMSC, and devise approaches for the prevention of this common malignancy . Employing mouse skin as a model, we applied a 32P postlabelling method to detect UVB-induced DNA lesions in the epidermis in nanomole quantities . Autoradiography maps showed that epidermal DNA from UVB exposed mice at 24 h contain up to five DNA lesions; the quantitation of these lesions showed that their formation increased in a UVB dose-dependent manner . Treatment of DNA samples with the bacteriophage DNA repair enzyme T4 endonuclease V confirmed that four of these lesions are pyrimidine dimers . While, some of these lesions were repaired 18 h after UVB irradiation, 30% of them persisted even 48 h post-irradiation . Application of a sunscreen containing ethylhexyl-p-methoxycinnamate or chemopreventive agent green tea polyphenols or silymarin to the skin of the mice prior to UVB exposure was found to prevent the formation of pyrimidine dimers.

J Biol Chem, 1996 Dec 13, 271(50), 32343 - 8
Transcription activation by the bacteriophage Mu Mor protein requires the C-terminal regions of both alpha and sigma70 subunits of Escherichia coli RNA polymerase; Artsimovitch I et al.; Middle transcription of bacteriophage Mu requires Escherichia coli RNA polymerase and a Mu-encoded protein, Mor . Consistent with these requirements, the middle promoter, Pm, has a -10 hexamer but lacks a recognizable -35 hexamer . Interactions between Mor and RNA polymerase were studied using in vitro transcription, DNase I footprinting, and the yeast interaction trap system . We observed reduced promoter activity in vitro using reconstituted RNA polymerases with C-terminal deletions in alpha or sigma70 . As predicted if alpha were binding to Pm, we detected a polymerase-dependent footprint in the -60 region . Reconstituted RNA polymerases containing Ala substitutions in the alpha C-terminal domain were used to assay Mor-dependent transcription from Pm in vitro . The D258A substitution and alpha deletion gave large reductions in activation, whereas the L262A, R265A, and N268A substitutions caused smaller reductions . The interaction trap assay revealed weak interactions between Mor and both alpha and sigma70; consistent with a key role of alpha-D258, the D258A substitution abolished interaction, whereas the R265A substitution did not . We propose that: (i) alpha-D258 is a Mor "contact site"; and (ii) residues Leu-262, Arg-265, and Asn-268 indirectly affect Mor-polymerase interaction by stabilizing the ternary complex via alpha-DNA contact.

J Biol Chem, 1996 Dec 13, 271(50), 31839 - 45
The RNA-binding site of bacteriophage Qbeta coat protein; Lim F et al.; The coat proteins of the RNA bacteriophages Qbeta and MS2 are specific RNA binding proteins . Although they possess common tertiary structures, they bind different RNA stem loops and thus provide useful models of specific protein-RNA recognition . Although the RNA-binding site of MS2 coat protein has been extensively characterized previously, little is known about Qbeta . Here we describe the isolation of mutants that define the RNA-binding site of Qbeta coat protein, showing that, as with MS2, it resides on the surface of a large beta-sheet . Mutations are also described that convert Qbeta coat protein to the RNA binding specificity of MS2 . The results of these and other studies indicate that, although they bind different RNAs, the binding sites of the two coat proteins are sufficiently similar that each is easily converted by mutation to the RNA binding specificity of the other.

Gene, 1996 Dec 12, 183(1-2), 15 - 21
Total modification of the bacteriophage lambda tail tube major subunit protein with foreign peptides; Dunn IS; The bacteriophage lambda has been shown previously to tolerate a high multiplicity of peptide additions to the C-terminus of the major tail tube subunit protein (gpV, the product of the V gene) . However, it was not clear whether all gpV copies within a functional virion could tolerate such modification . Complementation tests with phage bearing either V gene amber mutations or a precisely deleted V gene were used to test the extent of possible tail tube peptide display . Expression of plasmid-encoded gpV fused C-terminally with certain foreign peptides allowed rescue of such V gene-defective phage to essentially wild-type levels . After extensive purification such phage were shown by sensitive Western blotting to contain only the modified form of gpV . Peptide-modified gpV could also form indefinite tail tube polymeric structures (polytubes).

J Mol Biol, 1996 Dec 6, 264(3), 440 - 52
The kinetic mechanism of formation of the bacteriophage T4 DNA polymerase sliding clamp; Young MC et al.; DNA replication in bacteriophage T4 requires the assembly of a structure called the "sliding clamp" near the 3' end of the DNA strand that is to be extended . This structure is a trimer ring of the T4 gene 45 product (gp45) and serves to regulate the processivity of the DNA polymerase within the T4 DNA replication system . The placement of this ring is performed by an ATPase complex of the products of T4 genes 44 and 62 (gp44/62) that consists of four gp44 subunits and one gp62 subunit . In an effort to understand the role of ATP hydrolysis in processes occurring during the formation of the phage T4 DNA sliding clamp, we have performed direct substrate and product binding experiments and steady-state and presteady kinetic experiments on the gp44/62-gp45 system . Substrate (ATP) and product (ADP) binding studies show that the gp44/62 complex binds 4(+/-1) ATP molecules with a Kd of 34(+/-12) microM, and 3.7(+/-0.3) ADP molecules with a Kd of 14(+/-7) microM . The binding of the other reaction product (inorganic orthophosphate) could not be detected . Presteady-state kinetic analysis of ATP hydrolysis during the sliding-clamp-loading process indicates a biphasic progress curve, consisting of an initial rapid "burst" phase with an amplitude of four ATP molecules per gp44/62 complex and a rate of 15 s(-1), followed by a second slower phase corresponding to the steady-state rate of ATP hydrolysis by this complex . The rate of the burst phase is kinetically consistent with the previously observed rate of T4 DNA polymerase holoenzyme formation . The burst amplitude depends solely on the concentration of gp44/62 ATP binding sites present . These results suggest that the formation of a single T4 sliding clamp requires the hydrolysis of four ATP molecules by one gp44/62 complex in a process requiring 0.5 to 1 second . A model describing the clamp-loading process is discussed in the context of these results.

Indian J Exp Biol, 1996 Dec, 34(12), 1274 - 5
Indirect enzyme-linked immunosorbent assay (ELISA) for monitoring antibodies in rainbow trout Oncorhynchus mykiss; Saifi AI et al.; An indirect enzyme-linked immunosorbent assay (ELISA) has been described to detect and measure the antibodies in O . mykiss against a lambda bacteriophage antigen (strain c1187 Sam7) . Antibodies were first detected six days after the first intraperitoneal injection and by day 21 all fish were positive . Maximum mean titres were detected on day 30 (3 days after the second injection) . The mean titre declined by day 33 which was 6 days after second injection and remained constant from day 39 to day 45 . The results suggest that immune response in a rainbow trout occurred faster and the fish were able to mount a very good response to viruses.

C R Acad Sci III, 1996 Dec, 319(12), 1079 - 85
A bacteriophage T3 promoter can be linked to a lethal gene without detectable toxicity for eukaryotic cells . Interest for inducible transgenes; Amouyal M; The bacteriophage T3 promoter can be selectively transcribed by the corresponding RNA polymerase in eukaryotic cells . A toxic gene can in principle be linked to this promoter in a "dormant" and innocuous transgene in a transgenic animal . In this scheme, the activating strain expresses the RNA polymerase . When expression of the gene is needed in the progeny, the 2 lines are crossed . However, when a single molecule is sufficient to kill the cell--as with the diphtheria toxin--transcriptional "leakage" from the promoter may not be tolerated by the cell, even when extremely weak . Therefore, prior to more elaborate studies, diphtheria toxin, as a prototype of a gene toxic to the organism, has been linked to the bacteriophage T3 promoter in a T3-E-DTA construct . The T3-E-DTA plasmid has been transiently transfected into human embryonic kidney derived cells together with a lacZ plasmid . By co-transfection, the T3-E-DTA cells can be readily identified as lacZ positive, and their fate followed by the production of beta-galactosidase at the single cell or overall population level . In spite of the extreme toxicity of the toxin, the cells tolerate the presence of the T3-E-DTA construct, and are only killed--with a high efficiency--when the T3 RNA polymerase is present . Transactivation is usually restricted to the auxiliary factors of transcription . With this study, the promoter and the polymerase are revealed as potential and efficient inducible and activating elements of a very simple binary system.

Int J Biol Macromol, 1996 Dec, 19(4), 279 - 85
On the importance of van der Waals interaction in the groove binding of DNA with ligands: restrained molecular dynamics study; Chang DK et al.; Comparison of interaction energy between an oligonucleotide and a DNA-binding ligand in the minor and major groove modes was made by use of restrained molecular dynamics . Distortion in DNA was found for the major groove mode whereas less significant changes for both ligand and DNA were detected for the minor groove binding after molecular dynamics simulation . The conformation of the ligand obtained from the major groove modes resembles that computed with the ligand soaked in water . The van der Waals contact energy was found to be as significant as electrostatic energy and more important for difference in binding energy between these two binding modes . The importance of van der Waals force in groove binding was supported by computations on the complex formed by the repressor peptide fragment from the bacteriophage 434 and its operator oligonucleotide.

Protein Eng, 1996 Dec, 9(12), 1211 - 7
Study of antibody-antigen interaction through site-directed mutagenesis of the VH region of a hybrid phage-antibody fragment; Li Y et al.; An important aspect of the study of antibody structure-function relationships involves analysis of natural or synthetic mutations of antigen-combining sites . The anti-hen egg lysozyme monoclonal antibody HyHEL-10 has been a focus for antibody structure-function studies . We have displayed on bacteriophage of a hybrid single chain Fv, containing the light chain variable region of HyHEL-10 and the heavy chain variable region of a structurally related but functionally distinct antibody, AS32 . By using a combination of site-directed mutagenesis, complementary determining region grafting and molecular modeling, we have identified a number of contact and non-contact residues that are important in the affinity of HyHEL-10 for lysozyme . In particular, the heavy chain variable region framework residue at position 94 was shown to be an important determinant of high-affinity binding . The phage display approach eliminates the need for purification of antibodies and, when used in combination with polymerase chain reaction for variable region sequence mutagenesis, facilitates the rapid generation and characterization of mutant antibodies.

Cell Mol Biol (Noisy-le-grand), 1996 Dec, 42(8), 1219 - 27
Low sample volume causes differentiation in human rhabdomyosarcoma cell line RD subjected to electroporation; Aranega A et al.; Gene transfection has been accomplished with a variety of techniques such as DEAE dextran, calcium phosphate coprecipitation, protoplast fusion, liposomes, microinjection and recombinant bacteriophages . However, transfection by electroporation, consisting of the reversible permeabilization of cell membranes after exposure to a pulsed electric field, has been shown to be the most rapid, simple and efficient method for the stable incorporation of genes in different cell lines . We studied rhabdomyosarcoma cells subjected to electroporation in two different vol . {400 microliters (group 1) and 150 microliters (group 2} of 140 mM NaCl/15 mM Hepes buffer, pH 7.2) and evaluated the effects of electroporation volume on growth and differentiation . Low sample volumes induced a terminal process of morphological and ultrastructural myogenic differentiation in rhabdomyosarcoma cells, which concluded with cell death . Our results suggest that in electroporation low sample vol . of rhabdomyosarcoma cells induced morphological and phenotypic differentiation, with increased expression of desmin, alpha-actinin and tropomyosin.

Protein Sci, 1996 Dec, 5(12), 2485 - 93
Crystal structure of the coat protein from the GA bacteriophage: model of the unassembled dimer; Ni CZ et al.; There are four groups of RNA bacteriophages with distinct antigenic and physicochemical properties due to differences in surface residues of the viral coat proteins . Coat proteins also play a role as translational repressor during the viral life cycle, binding an RNA hairpin within the genome . In this study, the first crystal structure of the coat protein from a Group II phage GA is reported and compared to the Group I MS2 coat protein . The structure of the GA dimer was determined at 2.8 A resolution (R-factor = 0.20) . The overall folding pattern of the coat protein is similar to the Group I MS2 coat protein in the intact virus (Golmohammadi R, Valegard K, Fridborg K, Liljas L . 1993, J Mol Biol 234:620-639) or as an unassembled dimer (Ni Cz, Syed R, Kodandapani R . Wickersham J, Peabody DS, Ely KR, 1995, Structure 3:255-263) . The structures differ in the FG loops and in the first turn of the alpha A helix . GA and MS2 coat proteins differ in sequence at 49 of 129 amino acid residues . Sequence differences that contribute to distinct immunological and physical properties of the proteins are found at the surface of the intact virus in the AB and FG loops . There are six differences in potential RNA contact residues within the RNA-binding site located in an antiparallel beta-sheet across the dimer interface . Three differences involve residues in the center of this concave site: Lys/Arg 83, Ser/Asn 87, and Asp/Glu 89 . Residue 87 was shown by molecular genetics to define RNA-binding specificity by GA or MS2 coat protein (Lim F . Spingola M, Peabody DS, 1994, J Biol Chem 269:9006-9010) . This sequence difference reflects recognition of the nucleotide at position -5 in the unpaired loop of the translational operators bound by these coat proteins . In GA, the nucleotide at this position is a purine whereas in MS2, it is a pyrimidine.

J Virol, 1996 Dec, 70(12), 8660 - 8
Replication and packaging of coronavirus infectious bronchitis virus defective RNAs lacking a long open reading frame; Penzes Z et al.; The construction of a full-length clone of the avian coronavirus infectious bronchitis virus (IBV) defective RNA (D-RNA), CD-91 (9,080 nucleotides {Z . Penzes et al., Virology 203:286-293}), downstream of the bacteriophage T7 promoter is described . Electroporation of in vitro T7-transcribed CD-91 RNA into IBV helper virus-infected primary chick kidney cells resulted in the production of CD-91 RNA as a replicating D-RNA in subsequent passages . Three CD-91 deletion mutants were constructed--CD-44, CD-58, and CD-61--in which 4,639, 3,236, and 2,953 nucleotides, respectively, were removed from CD-91, resulting in the truncation of the CD-91 long open reading frame (ORF) from 6,465 to 1,311, 1,263, or 2,997 nucleotides in CD-44, CD-58, or CD-61, respectively . Electroporation of in vitro T7-transcribed RNA from the three constructs into IBV helper virus-infected cells resulted in the replication and packaging of CD-58 and CD-61 but not CD-44 RNA . The ORF of CD-61 was further truncated by the insertion of stop codons into the CD-61 sequence by PCR mutagenesis, resulting in constructs CD-61T11 (ORF: nucleotides 996 to 1,058, encoding 20 amino acids), CD-61T22 (ORF: nucleotides 996 to 2,294, encoding 432 amino acids), and CD-61T24 (ORF: nucleotides 996 to 2,450, encoding 484 amino acids), all of which were replicated and packaged to the same levels as observed for either CD-61 or CD-91 . Analysis of the D-RNAs showed that the CD-91- or CD-61-specific long ORFs had not been restored . Our data indicate that IBV D-RNAs based on the natural D-RNA, CD-91, do not require a long ORF for efficient replication . In addition, a 1.4-kb sequence, corresponding to IBV sequence at the 5' end of the 1b gene, may be involved in the packaging of IBV D-RNAs or form part of a cis-acting replication element.

J Virol, 1996 Dec, 70(12), 8492 - 501
Immunogenicity of an aphthovirus chimera of the glycoprotein of vesicular stomatitis virus; Grigera PR et al.; An oligodeoxynucleotide coding for amino acids 139 through 149 of antigenic site A (ASA) of the VP1 capsid protein of the foot-and-mouth disease virus C3 serotype (FMDV C3) was inserted into three different in-frame sites of the vesicular stomatitis virus New Jersey serotype (VSV-NJ) glycoprotein (G) gene cDNA present in plasmid pKG97 under control of the bacteriophage T7 polymerase promoter . Transfection of these plasmids into CV1 cells coinfected with the T7 polymerase-expressing vaccinia virus recombinant vTF1-6,2 resulted in expression of chimeric proteins efficiently reactive with both anti-FMDV and anti-VSV G antibodies . However, in vitro translation of transcripts of these VSV-G/FMDV-ASA chimeric plasmids resulted in proteins that were recognized by anti-G serum but not by anti-FMDV serum, indicating a requirement for in vivo conformation to expose the ASA antigenic determinant . Insertion of DNA coding for a dimer of the ASA unidecapeptide between the VSV-NJ G gene region coding for amino acids 160 and 161 gave rise to a chimeric ASA-dimer protein designated GF2d, which reacted twice as strongly with anti-FMDV antibody as did chimeric proteins in which the ASA monomer was inserted in the same position or two other G-gene positions . For even greater expression of chimeric VSV-G/FMDV-ASA proteins, plasmid pGF2d and a deletion mutant p(delta)GF2d (G protein deleted of 324 C-terminal amino acids) were inserted into baculovirus vectors expressing chimeric proteins GF2d-bac and deltaGF2d-bac produced in Sf9 insect cells . Mice vaccinated with three booster injections of 30 microg each of partially purified GF2d-bac protein responded by enzyme-linked immunosorbent assay with FMDV antibody titers of 1,000 units, and those injected with equivalent amounts of deltaGF2d-bac protein showed serum titers of up to 10,000 units . Particularly impressive were FMDV neutralizing antibody titers in serum of mice vaccinated with deltaGF2d-bac protein, which approached those in the sera of mice vaccinated with three 1-microg doses of native FMDV virions . Despite excellent reactivity with native FMDV, the anti-deltaGF2d-bac antibody present in vaccinated mouse serum showed no capacity to bind to sodium dodecyl sulfate (SDS)-denatured FMDV virions and only minimal reactivity with VP1 protein by Western blotting (immunoblotting) after SDS-polyacrylamide gel electrophoresis . It was also shown in a competitive binding assay that a synthetic ASA unidecapeptide, up to concentrations of 200 microg/ml, was quite limited in its ability to inhibit binding of anti-deltaGF2-bac antibody to native FMDV virions . These results suggest that the chimeric VSV-G/FMDV-ASA proteins mimic the capacity of FMDV to raise and react with neutralizing antibodies to a restricted number of ASA conformations present on the surface of native FMDV particles.

J Pediatr, 1996 Dec, 129(6), 898 - 903
Humoral immunity in steroid-dependent children with asthma and hypogammaglobulinemia; Lack G et al.; OBJECTIVE: To determine primary and secondary antibody responses in children with hypogammaglobulinemia attributed to corticosteroid use . RESULTS: In seven patients with steroid-dependent asthma and significant hypogammaglobulinemia (IgG concentration, 275 to 443 mg/dl), antibody responses to protein and polysaccharide antigens were shown to be normal, as were primary and secondary responses to a neoantigen, bacteriophage phi X174 . CONCLUSIONS: Patients with asthma, and with hypogammaglobulinemia resulting from steroid therapy, have normal humoral immunity, and immunoglobulin replacement therapy is not indicated.

Clin Chem, 1996 Dec, 42(12), 1961 - 9
Epitope mapping of prostate-specific antigen with monoclonal antibodies; Jette DC et al.; Prostate-specific antigen (PSA) is a widely used marker for screening and monitoring prostate cancer . We identified and characterized the epitopes of two anti-PSA monoclonal antibodies (mAbs) designated B80 and B87 . The epitopes were initially mapped as nonoverlapping by developing a sandwich immunoassay to measure PSA with the two anti-PSA mAbs . The two antibodies do not cross-react with homologous pancreatic kallikrein, but recognize epitopes unique to PSA . B80 and B87 can recognize both free and complexed PSA and hence measure total PSA . Epitope scanning and bacteriophage peptide library affinity selection procedures were used to identify and locate an epitope on PSA . A possible epitope for B80 was identified as being located on or near PSA amino acid residues 50-58 (-GRH-SLFHP-) . The epitope for B87 was likely on an exposed nonlinear conformational determinant, unique to PSA, and not masked by the binding of B80 or alpha 1-antichymotrypsin.

Biophys J, 1996 Dec, 71(6), 3454 - 66
Theory, design, and characterization of a microdialysis flow cell for Raman spectroscopy; Tuma R et al.; The theory, design, and application of a dialysis flow cell for Raman spectroscopy are described . The flow cell permits rapid collection of Raman spectra concurrent with the efflux of small solute molecules or ions into a solution of macromolecules and is well suited to acquisition of data during hydrogen-isotope exchange reactions of biological molecules . Kinetic parameters of the device are described by a diffusion model, which accounts satisfactorily for the observed rates of efflux of deuterium oxide (K2H = 0.30 min-1), calcium ions (KCa = 0.10 min-1) and EGTA (KEGTA = 0.07 min-1) . Application to the kinetics of glutamate protonation in a peptide copolymer {poly(Glu, Lys, Tyr)} shows that pH-titration rates as high as 3.3 pH units/min can be monitored . It is also shown that one can extract first-order hydrogen-isotope exchange rate constants from measured second-order exchanges by taking into account the rate of entry of 2H2O effluent into the bulk H2O solution . Deuterium exchanges of the single-stranded polyribonucleotides poly(rA) and poly(rU) and of the double-stranded RNA genome from bacteriophage phi 6 have been investigated . The measured nucleotide base exchange rates are comparable with those determined previously by other methods . The results indicate that base exchanges as fast as approximately 2 min-1 can be determined reliably with the present design . Application of the Raman flow cell to hydrogen-isotope exchange of the basic pancreatic trypsin inhibitor confirms consistency with results obtained previously on this protein by tritiation and NMR techniques.

J Bacteriol, 1996 Dec, 178(23), 6991 - 3
Escherichia coli rpoC397 encodes a temperature-sensitive C-terminal frameshift in the beta' subunit of RNA polymerase that blocks growth of bacteriophage P2; Christie GE et al.; Escherichia coli 397c is temperature sensitive for growth at 43.5 degrees C and unable to plate bacteriophage P2 at 33 degrees C . The mutation conferring these phenotypes was mapped to the rpoC gene . RNA synthesis is temperature sensitive in the mutant strain, and the beta' subunit of RNA polymerase isolated from this strain exhibits increased electrophoretic mobility . DNA sequence analysis revealed that the mutation is a deletion of 16 bp, resulting in a frameshift that leads to truncation of the beta' subunit at the carboxy terminus.

J Bacteriol, 1996 Dec, 178(23), 6945 - 51
Upstream interactions at the lambda pRM promoter are sequence nonspecific and activate the promoter to a lesser extent than an introduced UP element of an rRNA promoter; Tang Y et al.; The rightward regulatory region of bacteriophage lambda contains two promoters, pRM and pR, which direct the synthesis of nonoverlapping divergent transcripts from start sites 82 bp apart . Each of the two promoters has an upstream (A+T)-rich region (ATR) within the sequence from -40 to -60 where in the rrnB P1 promoter a stretch of 20 (A+T) bp greatly stimulates promoter function . Here we present an investigation of the possible functional significance of pRM's ATR . We determined the effects on RNA polymerase-pRM promoter interaction both of (G+C) substitutions in the ATR and of amino acid substitutions in the alpha subunit, known to affect the upstream interaction . We find small (two- to threefold) effects of selected mutations in the alpha subunit on open complex formation at pRM . However, the (presumably upstream) interactions underlying these effects are sequence nonspecific, as they are not affected by (G+C) substitutions in the ATR . Substitution of the 20-bp UP element of the rrnB P1 promoter between positions -40 and -60 at pRM stimulates open complex formation to a considerably greater extent (5- to 10-fold) . Results from kinetic studies indicate that on this construct the UP element mainly accelerates a step subsequent to the binding of RNA polymerase, although it may also facilitate the binding event itself . Less extensive studies likewise provide evidence for a two- to threefold activation of pR by upstream interactions . The possible involvement of the alpha subunit in the previously characterized (e.g., B . C . Mita, Y . Tang, and P . L . deHaseth, J . Biol . Chem . 270:30428-30433, 1995) interference of pR-bound RNA polymerase with open complex formation at pRM is discussed.

J Bacteriol, 1996 Dec, 178(23), 6921 - 9
Transcription-independent DNA translocation of bacteriophage T7 DNA into Escherichia coli; Garcia LR et al.; Penetration of wild-type T7 DNA into the host cell occurs in two steps . The phage particle ejects a few hundred base pairs of the left end of the genome into the host . Translocation of the remainder of the DNA is then coupled to transcription . In a normal infection, transcription-coupled translocation of wild-type T7 DNA is initiated at the major A1, A2, and A3 promoters for Escherichia coli RNA polymerase . At 37 degrees C, various deletion mutants lacking these three promoters grow at the same efficiency as wild-type T7 because the minor B promoter is efficiently transferred from the phage head into the cell . As the temperature of the phage infection decreases, the latent periods of (A1, A2, A3)- phages increase relative to that of wild-type T7; nevertheless, (A1, A2, A3)- phages have normal plating efficiencies at reduced temperatures . Lengthening of the latent period at low temperatures is due to a delay in transferring the complete (A1, A2, A3)- genome into the host cell . The (A1, A2, A3)- phages eject the leading end of their genome into the host, but at low temperature, insufficient DNA is transferred into the cell to allow RNA polymerase immediate access the B promoter . However, by an inefficient transcription-independent process, the B promoter eventually translocates into the cell . Mutant derivatives of (A1, A2, A3)- phages that have growth profiles at low temperatures similar to that of wild-type T7 have been isolated . The mutations allow both (A1, A2, A3)- and (A1, A2, A3)+ phages to translocate their entire genomes into the cell by a transcription-independent mechanism . The mutations are located in gene 16, a gene that encodes a component of the internal virion core . We postulate that gp16 is directly involved with the process of DNA translocation from the virion into the cell.

J Bacteriol, 1996 Dec, 178(23), 6873 - 81
Cloning and characterization of the gene encoding 1-cyclohexenylcarbonyl coenzyme A reductase from Streptomyces collinus; Wang P et al.; We report the cloning of the gene encoding the 1-cyclohexenylcarbonyl coenzyme A reductase (ChcA) of Streptomyces collinus, an enzyme putatively involved in the final reduction step in the formation of the cyclohexyl moiety of ansatrienin from shikimic acid . The cloned gene, with a proposed designation of chcA, encodes an 843-bp open reading frame which predicts a primary translation product of 280 amino acids and a calculated molecular mass of 29.7 kDa . Highly significant sequence similiarity extending along almost the entire length of the protein was observed with members of the short-chain alcohol dehydrogenase superfamily . The S . collinus chcA gene was overexpressed in Escherichia coli by using a bacteriophage T7 transient expression system, and a protein with a specific ChcA activity was detected . The E . coli-produced ChcA protein was purified and shown to have similar steady-state kinetics and electrophoretic mobility on sodium dodecyl sulfate-polyacrylamide gels as the enoyl-coenzyme A reductase protein prepared from S . collinus . The enzyme demonstrated the ability to catalyze, in vitro, three of the reductive steps involved in the formation of cyclohexanecarboxylic acid . An S . collinus chcA mutant, constructed by deletion of a genomic region comprising the 5' end of chcA, lost the ChcA activity and the ability to synthesize either cyclohexanecarboxylic acid or ansatrienin . These results suggest that chcA encodes the ChcA that is involved in catalyzing multiple reductive steps in the pathway that provides the cyclohexanecarboxylic acid from shikimic acid.

J Bacteriol, 1996 Dec, 178(23), 6772 - 7
Either bacteriophage T4 RNase H or Escherichia coli DNA polymerase I is essential for phage replication; Hobbs LJ et al.; Bacteriophage T4 rnh encodes an RNase H that removes ribopentamer primers from nascent DNA chains during synthesis by the T4 multienzyme replication system in vitro (H . C . Hollingsworth and N . G . Nossal, J . Biol . Chem . 266:1888-1897, 1991) . This paper demonstrates that either T4 RNase HI or Escherichia coli DNA polymerase I (Pol I) is essential for phage replication . Wild-type T4 phage production was not diminished by the polA12 mutation, which disrupts coordination between the polymerase and the 5'-to-3' nuclease activities of E . coli DNA Pol I, or by an interruption in the gene for E . coli RNase HI . Deleting the C-terminal amino acids 118 to 305 from T4 RNase H reduced phage production to 47% of that of wild-type T4 on a wild-type E . coli host, 10% on an isogenic host defective in RNase H, and less than 0.1% on a polA12 host . The T4 rnh(delta118-305) mutant synthesized DNA at about half the rate of wild-type T4 in the polA12 host . More than 50% of pulse-labelled mutant DNA was in short chains characteristic of Okazaki fragments . Phage production was restored in the nonpermissive host by providing the T4 rnh gene on a plasmid . Thus, T4 RNase H was sufficient to sustain the high rate of T4 DNA synthesis, but E . coli RNase HI and the 5'-to-3' exonuclease of Pol I could substitute to some extent for the T4 enzyme . However, replication was less accurate in the absence of the T4 RNase H, as judged by the increased frequency of acriflavine-resistant mutations after infection of a wild-type host with the T4 rnh (delta118-305) mutant.

J Mol Biol, 1996 Nov 29, 264(2), 233 - 42
Role of operator subsites in Arc repression; Smith TL et al.; By binding to adjacent subsites in its 21 base-pair operator, Arc represses transcription from two divergent promoters, Pant and Pmnt, in the immunity I operon of bacteriophage P22 . Arc dimers bind to each subsite with nanomolar affinities and interact through protein-protein interactions to stabilize binding further . Here, we show that an Arc dimer bound to a single subsite reduces the rate of RNA polymerase open-complex formation and represses transcription from Pant and Pmnt promoter variants to varying degrees . Occupancy of the subsite proximal to the Pant-35 region results in significantly greater repression than occupancy of the- 10 proximal subsite . For repression of Pmnt, Arc bound at the- 10 proximal subsite is more effective than Arc bound at the- 35 proximal subsite . Because of the divergent orientations of the two promoters, the-35 proximal site in Pant is the same as the- 10 proximal site in Pmnt . Thus, in both cases, the same operator subsite is primarily responsible for repression of transcription initiation.

J Mol Biol, 1996 Nov 29, 264(2), 213 - 9
Incorrect base insertion and prematurely terminated transcripts during T7 RNA polymerase transcription elongation past benzo{a}pyrenediol epoxide-modified DNA; Choi DJ et al.; DNA replication and transcription are affected adversely by the presence of bulky adducts that are generated by the covalent binding of a variety of metabolically activated environmental pollutants to cellular DNA . When these lesions are not cleared by cellular repair enzymes prior to replication, mutations and ultimately tumor initiation can occur . Transcription and DNA repair appear to be intimately connected, since certain adducts are more efficiently removed from the transcribed strands of active loci than from non-transcribed strands and other quiescent domains in the genome . The mechanism by which RNA polymerases deal with bulky adducts during DNA transcription is therefore of great interest . The availability of site-specifically modified and stereochemically defined oligodeoxyribonucleotides derived from the covalent reaction of 7r, 8t-dihydroxy-9, 10t-epoxy- 7,8,9,10-tetrahydrobenzo{a}pyrene (anti-BPDE) with guanine residues prompted us to study the efficiencies of transcription past these lesions using bacteriophage T7 RNA polymerase . We show here that T7 RNA polymerase can bypass such lesions in a DNA template, providing that a cytosine residue is incorporated opposite anti-BPDE-modified guanine . However, when an incorrect base (most frequently a purine) is inserted opposite the modified site, the RNA polymerase stalls, and the complex dissociates, resulting in a truncated transcript . The ability of the T7 RNA polymerase to discriminate between a correct and an incorrect inserted base and, accordingly, to continue or terminate transcription, might constitute an important mechanism that ensures the fidelity of transcription past a modified base present on the transcribed strand of the DNA template.

J Biol Chem, 1996 Nov 29, 271(48), 30451 - 8
Equilibrium and stopped-flow kinetic studies of interaction between T7 RNA polymerase and its promoters measured by protein and 2-aminopurine fluorescence changes; Jia Y et al.; The mechanism of bacteriophage T7 RNA polymerase binding to its promoter DNA was investigated using stopped-flow and equilibrium methods . To measure the kinetics of protein-DNA interactions in real time, changes in tryptophan fluorescence in the polymerase and 2-aminopurine (2-AP) fluorescence in the promoter DNA upon binary complex formation were used as probes . The protein fluorescence changes measured conformational changes in the polymerase whereas the fluorescence changes of 2-AP base, substituted in place of dA in the initiation region (-4 to +4), measured structural changes in the promoter DNA, such as DNA melting . The kinetic studies, carried out in the absence of the initiating nucleotide, are consistent with a two-step DNA binding mechanism, {formula: see text} where the RNA polymerase forms an initial weak EDa complex rapidly with an equilibrium association constant K1 . The EDa complex then undergoes a conformational change to EDb, wherein RNA polymerase is specifically and tightly bound to the promoter DNA . Both the polymerase and the promoter DNA may undergo structural changes during this isomerization step . The isomerization of EDa to EDb is a fast step relative to the rate of transcription initiation and its rate does not limit transcription initiation . To understand how T7 RNA polymerase modulates its transcriptional efficiency at various promoters at the level of DNA binding, comparative studies with two natural T7 promoters, Phi10 and Phi3.8, were conducted . The results indicate that kinetics, the bimolecular rate constant of DNA binding, kon (K1k2), and the dissociation rate constant, koff (k-2), and thermodynamics, the equilibrium constants of the two steps (K1 and k2/k-2) both play a role in modulating the transcriptional efficiency at the level of DNA binding . Thus, the 2-fold lower kon, the 4-fold higher koff, and the 2-5-fold weaker equilibrium interactions together make Phi3.8 a weaker promoter relative to Phi10.

Gene, 1996 Nov 28, 181(1-2), 127 - 33
Point mutations in a transcription terminator, lambda tI, that affect both transcription termination and RNA stability; Cisneros B et al.; The terminator tI is located approx . 280 nucleotides beyond the int gene of bacteriophage lambda . Besides its role as a transcription terminator, tI may confer stability to the int message by protecting it from 3' exonucleolytic degradation . In order to study the role of the tI sequence in transcription termination and RNA stability, three different point mutations tI1, tI2, and tI3 were isolated and characterized . All the tI mutations map in the G + C-rich region of dyad symmetry in the terminator and decrease the transcriptional termination of tI in vivo from 99% for the wild type terminator to 81-93% as determined by galactokinase activity and in vitro from 80% for the wild type terminator to 8-12% using the E . coli RNA polymerase . Additionally, the tI mutations cause upstream transcript instability in vivo . This instability defect caused by tI mutations is compensated by the host mutant deficient in polynucleotide phosphorylase resulting in increased steady state levels of these mutant transcripts . The results show that the intact hairpin of tI is essential for efficient transcription termination and for maintaining mRNA stability by blocking the 3' to 5' exonucleolytic activity of polynucleotide phosphorylase.

J Biol Chem, 1996 Nov 22, 271(47), 29599 - 604
Identification and characterization of the N-ethylmaleimide-sensitive site in lambda-integrase; Tirumalai RS et al.; Integrase (Int) of bacteriophage lambda is a heterobivalent DNA-binding protein and a type I topoisomerase . Upon modification with N-ethylmaleimide (NEM), a sulfhydryl-directed reagent, Int loses its capacity to bind "arm-type" DNA sequences and, consequently, to carry out recombination; however, its ability to bind "core-type" sequences and its topoisomerase activity are unaffected . In this report, the NEM-sensitive site was identified by modifying Int with {14C}NEM . Following cleavage by formic acid, which cleaves Asp-Pro bonds, and fractionation on a Fractogel HW-50 (F) sizing column, the fragment containing the primary site of {14C}NEM incorporation was subjected to amino acid sequencing . The results indicate that the primary site of {14C}NEM incorporation is in the peptide-spanning amino acid residues 1-28, which contains a cysteine at position 25 . To confirm that Cys-25 is the target of NEM reactivity, site-directed mutagenesis was used to change this cysteine to alanine or serine . The mutant protein is not chemically modified by NEM and shows no loss of activity after NEM treatment . The fact that C25A and C25S both retain full recombination activity indicates that the SH group of Cys-25 does not provide any critical contacts, either with arm-type DNA or with other parts of the Int protein to form the arm-type recognition pocket . The loss of arm-type DNA binding and the concomitant loss of recombination function as a result of NEM modification must be due to the presence of the maleimide moiety and not due to loss of a critical cysteine contact.

Gene, 1996 Nov 21, 180(1-2), 225 - 7
An Escherichia coli system for assay of F1p site-specific recombination on substrate plasmids; Snaith MR et al.; We have developed an Escherichia coli system for testing the behaviour of plasmids carrying target sites for the F1p site-specific recombinase . The E . coli strain BL-FLP is described, which carries a chromosomally integrated bacteriophage T7 RNA polymerase gene expressed from a lac promoter, and harbours the plasmid pMS40.pMS40 has the features: (i) it carries the FLP recombinase gene under the control of a bacteriophage T7 promoter, (ii) it confers kanamycin resistance, and (iii) it uses an R6K origin of replication; these two latter features make it compatible with most conventional cloning vectors . Substrate plasmids carrying F1p-recognition targets (FRT) are transformed into BL-FLP, and the consequences of F1p-mediated recombination can be analysed after subsequent extraction of plasmid DNA . We show that this system is capable of base-perfect F1p-mediated recombination on plasmid substrates . We also present a corrected sequence of the commonly used F1p substrate plasmid, pNEO beta GAL (O'Gorman et al . (1991) Science 251, 1351-1355).

FEBS Lett, 1996 Nov 18, 397(2-3), 169 - 72
Converging antigenic structure of a recombinant viral peptide displayed on different frameworks of carrier proteins; Carbonell X et al.; A peptide reproducing the G-H loop amino acid sequence of foot-and-mouth disease virus VP1 protein was fused to the solvent-exposed C-terminus of the bacteriophage P22 tailspike protein {Carbonell and Villaverde (1996) Gene, in press}, a homotrimeric polypeptide with a strong beta-helical structure . This fusion does not interfere with the biological activities of the phage tail . The antigenic profile of the complex antigenic site A within the G-H loop has been determined by competitive ELISA with a panel of monoclonal antibodies directed against different overlapping B-cell epitopes . The antigenic data have been compared with those obtained with a set of 12 chimeric beta-galactosidases displaying the G-H loop on different exposed regions . A high coincidence has been evidenced between the antigenicity of the viral peptide fused to the phage protein and that of some peptides inserted in an exposed loop of the activating interface of beta-galactosidase . This indicates that completely different structural frameworks of carrier proteins can provide similar constraints that allow the recombinant peptide to successfully mimic the antigenicity, and probably conformational features, of the natural peptide on the virion surface.

FEBS Lett, 1996 Nov 18, 397(2-3), 143 - 8
Formation of bacteriophage MS2 infectious units in a cell-free translation system; Katanaev VL et al.; We show that a simple cell-free translation system from Escherichia coli, programmed with phage MS2 RNA, is able to infect F+ E . coli cells . The plaques appearing on the E . coli host strain are morphologically indistinguishable from those derived from normal phage MS2 infection . This effect is strictly translation-dependent, since an incomplete translation system or the system inhibited by antibiotics leads to no infection . The cell-free based infection is maximal under conditions favouring the highest synthesis of maturation protein (one of the four phage-encoded proteins) . The infection is abolished when RNase A or trypsin treatment is included before addition of cells . Similarly, due to RNA and maturation protein degradation, the continued incubation of the translation mixture under protein synthesis conditions significantly decreases infectivity . These findings suggest the formation of 'minimal infectious units', simple complexes of MS2 RNA and maturation protein . Here we describe the first example of bacteriophage infectious unit formation directly performed in a cell-free translation system . A possible application of this phenomenon might be the construction of newly designed RNA vector delivery systems and, moreover, could be an approach for molecular evolution studies.

Arch Biochem Biophys, 1996 Nov 15, 335(2), 321 - 32
Structure of the human gene encoding the protein repair L-isoaspartyl (D-aspartyl) O-methyltransferase; DeVry CG et al.; The protein L-isoaspartyl/D-aspartyl O-methyltransferase (EC 2.1.1.77) catalyzes the first step in the repair of proteins damaged in the aging process by isomerization or racemization reactions at aspartyl and asparaginyl residues . A single gene has been localized to human chromosome 6 and multiple transcripts arising through alternative splicing have been identified . Restriction enzyme mapping, subcloning, and DNA sequence analysis of three overlapping clones from a human genomic library in bacteriophage P1 indicate that the gene spans approximately 60 kb and is composed of 8 exons interrupted by 7 introns . Analysis of intron/exon splice junctions reveals that all of the donor and acceptor splice sites are in agreement with the mammalian consensus splicing sequence . Determination of transcription initiation sites by primer extension analysis of poly(A)+ mRNA from human brain identifies multiple start sites, with a major site 159 nucleotides upstream from the ATG start codon . Sequence analysis of the 5'-untranslated region demonstrates several potential cis-acting DNA elements including SP1, ETF, AP1, AP2, ARE, XRE, CREB, MED-1, and half-palindromic ERE motifs . The promoter of this methyltransferase gene lacks an identifiable TATA box but is characterized by a CpG island which begins approximately 723 nucleotides upstream of the major transcriptional start site and extends through exon 1 and into the first intron . These features are characteristic of housekeeping genes and are consistent with the wide tissue distribution observed for this methyltransferase activity.

J Biol Chem, 1996 Nov 15, 271(46), 29170 - 81
Identification of specific carboxyl groups on uracil-DNA glycosylase inhibitor protein that are required for activity; Sanderson RJ et al.; The bacteriophage PBS2 uracil-DNA glycosylase inhibitor (Ugi) protein inactivates uracil-DNA glycosylase (Ung) by forming an exceptionally stable protein-protein complex in which Ugi mimics electronegative and structural features of duplex DNA (Beger, R . D., Balasubramanian, S., Bennett, S . E., Mosbaugh, D . W., and Bolton, P . H . (1995) J . Biol . Chem . 270, 16840-16847; Mol, C . D., Arvai, A . S., Sanderson, R . J., Slupphaug, G., Kavli, B., Krokan, H . E., Mosbaugh, D . W., and Tainer, J . A . (1995) Cell 82, 701-708) . The role of specific carboxylic amino acid residues in forming the Ung.Ugi complex was investigated using selective chemical modification techniques . Ugi treated with carbodiimide and glycine ethyl ester produced five discrete protein species (forms I-V) that were purified and characterized . Analysis by mass spectrometry revealed that Ugi form I escaped protein modification, and forms II-V showed increasing incremental amounts of acyl-glycine ethyl ester adduction . Ugi forms II-V retained their ability to form a Ung.Ugi complex but exhibited a reduced ability to inactivate Escherichia coli Ung, directly reflecting the extent of modification . Competition experiments using modified forms II-V with unmodified Ugi as a competitor protein revealed that unmodified Ugi preferentially formed complex . Furthermore, unmodified Ugi and poly(U) were capable of displacing forms II-V from a preformed Ung.Ugi complex but were unable to displace Ugi form I . The primary sites of acyl-glycine ethyl ester adduction were located in the alpha2-helix of Ugi at Glu-28 and Glu-31 . We infer that these two negatively charged amino acids play an important role in mediating a conformational change in Ugi that precipitates the essentially irreversible Ung/Ugi interaction.

J Biol Chem, 1996 Nov 15, 271(46), 28903 - 11
Using 2-aminopurine fluorescence and mutational analysis to demonstrate an active role of bacteriophage T4 DNA polymerase in strand separation required for 3' --> 5'-exonuclease activity; Marquez LA et al.; The fluorescence of 2-aminopurine deoxynucleotide positioned in a 3'-terminal mismatch was used to evaluate the pre-steady state kinetics of the 3' --> 5' exonuclease activity of bacteriophage T4 DNA polymerase on defined DNA substrates . DNA substrates with one, two, or three preformed terminal mispairs simulated increasing degrees of strand separation at a primer terminus . The effects of base pair stability and local DNA sequence on excision rates were investigated by using DNA substrates that were either relatively G + C- or A + T-rich . The importance of strand separation as a prerequisite to the hydrolysis of a terminal nucleotide was demonstrated by using a unique mutant DNA polymerase that could degrade single-stranded but not double-stranded DNA, unless two or more 3'-terminal nucleotides were unpaired . Our results led us to conclude that the reduced exonuclease activity of this mutant DNA polymerase on duplex DNA substrates is due to a defect in melting the primer terminus in preparation for the excision reaction . The mutated amino acid (serine substitution for glycine at codon 255) resides in a critical loop structure determined from a crystallographic study of an amino-terminal fragment of T4 DNA polymerase . These results suggest an active role for amino acid residues in the exonuclease domain of the T4 DNA polymerase in the strand separation step.

Proc Natl Acad Sci U S A, 1996 Nov 12, 93(23), 12822 - 7
The carboxyl terminus of the bacteriophage T4 DNA polymerase is required for holoenzyme complex formation; Berdis AJ et al.; To further elucidate the mechanism and dynamics of bacteriophage T4 holoenzyme formation, a mutant polymerase in which the last six carboxyl-terminal amino acids are deleted, was constructed, overexpressed, and purified to homogeneity . The mutant polymerase, designated delta C6 exo-, is identical to wild-type exo- polymerase with respect to kcat, kpol, and dissociation constants for nucleotide and DNA substrate . However, unlike wild-type exo- polymerase, the delta C6 exo- polymerase is unable to interact with the 45 protein to form the stable holoenzyme . A synthetic polypeptide corresponding to the carboxyl terminus of the wild-type exo- polymerase was tested as an in vitro inhibitor of bacteriophage T4 DNA replication . Surprisingly, the peptide does not directly inhibit holoenzyme complex formation by disrupting the interaction of the polymerase with the 45 protein . On the contrary, the peptide appears to disrupt the interaction of the 44/62 protein with the 45 protein, suggesting that the 44/62 protein and the polymerase use the same site on the 45 protein for functional interactions . Data presented are discussed in terms of a model correlating the functionality of the carboxyl terminus of the polymerase for productive interactions with the 45 protein as well as in terms of the 45 protein concomitantly interacting with the 44/62 protein and polymerase.

Biochemistry, 1996 Nov 12, 35(45), 14395 - 404
The N-terminal B-domain of T4 gene 32 protein modulates the lifetime of cooperatively bound Gp32-ss nucleic acid complexes; Villemain JL et al.; The N-terminal basic or B-domain (residues 1-21) of bacteriophage T4 gene 32 protein (gp32) provides a major determinant for highly cooperative binding by gp32 to single-stranded (ss) nucleic acids at equilibrium . In order to gain mechanistic insight into N-terminal domain function, the kinetics of dissociation of wild-type and previously characterized B-domain substitution mutant gp32s (R4K, R4Q, and K3A) from the model ribohomopolymer, poly(A), have been investigated under solution conditions identical to those used for equilibrium studies {Villemain, J . L., & Giedroc, D . P . (1993) Biochemistry 32, 11235-11246; Villemain, J . L., & Giedroc, D . P . (1996) J . Biol . Chem . 271, 27623-27629} . The dissociation of cooperatively bound gp32-poly(A) complexes was induced by sodium chloride concentration jumps and monitored by an increase in tryptophan fluorescence upon dissociation of the protein from poly(A) using stopped-flow techniques . The apparent dissociation rate constant, kd(app), for all mutant proteins studied was found to depend strongly on the initial fractional saturation of poly(A) just as was found previously for wild-type gp32 . This permitted application of Lohman's model for the irreversible dissociation of cooperatively bound gp32-nucleic acid complexes {Lohman, T . M . (1983) Biopolymers 22, 1697-1713} from which the molecular rate constant, ke, the rate of dissociation of a protein monomer from teh end of a gp32-ss nucleic acid complex or protein cluster, could be determined . From the {NaCl}-dependence of kd(app), ke determined at 0.45 M NaCl, pH 8.1, 20 degrees C, was found to be 62 +/- 23, 78 +/- 8, 328 +/- 36, and 384 +/- 34 s-1 for wild-type, R4K, K3A, and R4Q gp32s, respectively . With the exception of R4K gp32, we find a striking correlation between the relative magnitudes of ke and Kapp, suggesting that the molecular defect in the equilibrium binding properties of the N-terminal domain mutants resides in the increased rate at which gp32 monomers dissociate from singly contiguous binding sites at the ends of clusters . The bimolecular association rate constant measured for wild-type gp32 and a weakly binding B-domain mutant, R4T gp32, to poly(dT) was found to be nearly identical, further evidence that the primary defect is in the dissociation reaction . We conclude that the N-terminal domain strongly modulates the lifetime of cooperatively bound gp32-polynucleotide complexes . The mechanistic and functional implications of these findings are discussed.

Biochemistry, 1996 Nov 12, 35(45), 14216 - 24
Purification and structural and functional characterization of FhuA, a transporter of the Escherichia coli outer membrane; Boulanger P et al.; The Escherichia coli outer membrane ferrichrome transporter FhuA was purified chromatographically in a neutral detergent (octyl glucoside or dodecyl maltoside) . The amount of dodecyl maltoside bound to the protein (1.2 +/- 0.15 g/g of FhuA) and the Stokes radius of the FhuA-dodecyl maltoside complex (Rs = 4.2 nm) were determined using size exclusion chromatography . Sedimentation equilibrium and velocity experiments indicated that the FhuA preparation was monodisperse and that the protein was monomeric . The value found for the frictional coefficient of the protein-detergent complex (1.18) suggested a globular shape for the complex . Sedimentation experiments gave values for the molecular mass of the FhuA-dodecyl maltoside complex (180 kDa) and for the Stokes radius in complete agreement with those calculated from size exclusion chromatography . The circular dichroism spectrum indicated a 51% beta-sheet content . Functionality of the purified protein was assessed from fluorescence measurements using the DNA probe YO-PRO-1 . Interaction of nM concentrations of FhuA with bacteriophage T5 resulted in the release of 90 +/- 8% of the phage DNA . The limiting step in DNA ejection was binding of the phage to its receptor . Release of DNA took place in a few seconds . Ferrichrome (0.8 microM) competed with the phage for binding to FhuA and prevented DNA ejection.

J Mol Biol, 1996 Nov 8, 263(4), 539 - 50
Novel mutants in the 5' upstream region of the portal protein gene 20 overcome a gp40-dependent prohead assembly block in bacteriophage T4; Yap NL et al.; The exact mechanism by which the double-stranded (ds) DNA bacteriophages incorporate the portal protein at a unique vertex of the icosahedral capsid is unknown . In phage T4, there is evidence that this vertex, constituted by 12 subunits of gp20, acts as an initiator for the assembly of the major capsid protein and the scaffolding proteins into a prolate icosahedron of precise dimensions . Assembly of the T4 initiator vertex occurs on the membrane and is facilitated by the non-structural protein gp40 . gp40 apparently acts as a catalyst for the gp20 assembly and a direct interaction between gp20 and gp40 has been proposed based on the genetic evidence that second site suppressors of g40 mutants map in g20 . But, surprisingly, we found that these 40bypass mutants arose not by alterations in the g20 structural gene, but by alterations in the upstream non-coding region . At least six independent bypass mutants were isolated with all except one having mutations in the non-coding region . The only exception that had a mutation in the coding region was a silent mutation, since it did not alter the amino acid sequence of gp20 . The bypass mutants produced a three- to fivefold overexpression of gp20 . That the gp20 overexpression is directly responsible for 40bypass was shown by a number of approaches . The overexpression was apparently due to a secondary structural change in the g20 transcript resulting in an enhanced translational initiation of g20 message . The data suggest that the regulation of portal protein gene expression is an important regulator of prohead assembly in bacteriophage T4.

J Biol Chem, 1996 Nov 8, 271(45), 28045 - 51
Protein-protein and protein-DNA interactions at the bacteriophage T4 DNA replication fork . Characterization of a fluorescently labeled DNA polymerase sliding clamp; Sexton DJ et al.; The T4 DNA polymerase holoenzyme is composed of the polymerase enzyme complexed to the sliding clamp (the 45 protein), which is loaded onto DNA by an ATP-dependent clamp loader (the 44/62 complex) . This paper describes a new method to directly investigate the mechanism of holoenzyme assembly using a fluorescently labeled cysteine mutant of the 45 protein . This protein possessed unaltered function yet produced substantial changes in probe fluorescence intensity upon interacting with other components of the holoenzyme . These fluorescence changes provide insight into the role of ATP hydrolysis in holoenzyme assembly . Using either ATP or the non-hydrolyzable ATP analog, adenosine 5'-O-(3-thiophosphate), events in holoenzyme assembly were assigned as either dependent or independent of ATP hydrolysis . A holoenzyme assembly mechanism is proposed in which the 44/62 complex mediates the association of the 45 protein with DNA in an ATP-dependent manner not requiring ATP hydrolysis . Upon ATP hydrolysis, the 44/62 complex triggers a conformational change in the 45 protein that may be attributed to the clamp loading onto DNA.

Nature, 1996 Nov 7, 384(6604), 87 - 92
A nucleotide-flipping mechanism from the structure of human uracil-DNA glycosylase bound to DNA; Slupphaug G et al.; Any uracil bases in DNA, a result of either misincorporation or deamination of cytosine, are removed by uracil-DNA glycosylase (UDG), one of the most efficient and specific of the base-excision DNA-repair enzymes . Crystal structures of human and viral UDGs complexed with free uracil have indicated that the enzyme binds an extrahelical uracil . Such binding of undamaged extrahelical bases has been seen in the structures of two bacterial methyltransferases and bacteriophage T4 endonuclease V . Here we characterize the DNA binding and kinetics of several engineered human UDG mutants and present the crystal structure of one of these, which to our knowledge represents the first structure of any eukaryotic DNA repair enzyme in complex with its damaged, target DNA . Electrostatic orientation along the UDG active site, insertion of an amino acid (residue 272) into the DNA through the minor groove, and compression of the DNA backbone flanking the uracil all result in the flipping-out of the damaged base from the DNA major groove, allowing specific recognition of its phosphate, deoxyribose and uracil moieties . Our structure thus provides a view of a productive complex specific for cleavage of uracil from DNA and also reveals the basis for the enzyme-assisted nucleotide flipping by this critical DNA-repair enzyme.

Biochemistry, 1996 Nov 5, 35(44), 13878 - 84
Equilibrium stability and sub-millisecond refolding of a designed single-chain Arc repressor; Robinson CR et al.; Arc-L1-Arc is a single-chain variant of bacteriophage P22 Arc repressor in which a 15 residue linker joins the C-terminus of one subunit to the N-terminus of an otherwise identical subunit . Spectroscopic probes indicate that the native and denatured state of the single-chain protein are similar to those of the unlinked Arc dimer . In equilibrium experiments, Arc-L1-Arc denatures in a reaction without populated intermediate states as judged by the fits of the denaturation isotherms to a two-state model and by the coincidence of denaturation curves monitored by fluorescence and circular dichroism . Comparison of the equilibrium stabilities of Arc-L1-Arc and unlinked Arc gives an effective concentration of subunits in the denatured single-chain variant of 2.7 (+/- 0.7) mM . The kinetic refolding and unfolding reactions of Arc-L1-Arc also appear to proceed without populated intermediates . The rate constant for Arc-L1-Arc unfolding is about 2-fold faster than that of unlinked Arc, indicating that the linker mediates no significant contacts in the native structure that need to be broken to allow unfolding . As expected, the major effect of the linker occurs during the refolding reaction, where the effective subunit concentration calculated from the bimolecular and unimolecular refolding rate constants is 4.5 (+/- 1.8) mM . The transition states for the unfolding and refolding reactions of Arc-L1-Arc and wild-type Arc have similar solvent exposures as measured by the urea dependencies of the equilibrium and rate constants . In the absence of urea, the single-chain protein refolds very rapidly (kf approximately 10(4) s-1) in a reaction that is essentially complete in the sub-millisecond time regime.

Somat Cell Mol Genet, 1996 Nov, 22(6), 477 - 88
Production and characterization of human 293 cell lines expressing the site-specific recombinase Cre; Chen L et al.; We have constructed 293 cell lines expressing the site-specific Cre recombinase from bacteriophage P1, that acts on a 34 bp target sequence called loxP . Stably transformed cells were obtained by transfection with a plasmid containing Cre and a selectable marker under the control of viral promoters . The resulting 293Cre cell lines could be used to induce expression from adenovirus vectors containing reporter genes under the control of a Cre responsive "molecular switch." High efficiency recombination was observed for Ad viral DNA containing loxP sites . The Cre expressing cell lines described here are likely to be useful for several purposes: For expression of toxic gene products from Cre inducible viral vectors, to induce recombination between loxP sites in transfected plasmids, and to induce deletions or rearrangements of genes defined by loxP sites in viral genomes.

Plant Mol Biol, 1996 Nov, 32(4), 641 - 52
Cloning and developmental/stress-regulated expression of a gene encoding a tomato arabinogalactan protein; Li SX et al.; Arabinogalactan proteins (AGPs) represent a major class of plant hydroxyproline-rich glycoproteins (HRGPs) and are components of cell walls and plasma membranes . AGPs are thought to play roles in cell differentiation, development, and cell-cell interactions . Using a synthetic DNA oligonucleotide based upon an amino acid sequence motif common to AGPs from Lolium, rose, and carrot (i.e., Hyp-Ala-Hyp-Ala-Hyp), we have isolated and sequenced the first AGP gene from a partial Sau3A tomato genomic library packaged in bacteriophage charon 35 . The deduced 215 amino acid protein contains 20% Ala, 22% Pro, 10% Gly, and 11% Ser and consists of two Pro-Ala-Pro-Ala-Pro pentapeptide repeats and 16 Ala-Pro dipeptide repeats, consistent with known AGP amino acid compositions and sequences . Comparison of the genomic sequence to a reverse transcribed PCR product and tomato cDNA confirmed the AGP gene is expressed and contains one large intervening sequence . RNA blot hybridization analysis in tomato indicates this AGP gene is strongly expressed in stem and flower, moderately expressed in root and green fruit, and weakly expressed in leaves and red fruit as a 980 nucleotide transcript . Five-day-old seedlings also express this transcript; however, this expression is not regulated by light . More significantly, a gradient of AGP gene expression is observed in tomato stems, ranging from high levels of expression in young internodes to low levels of expression in old internodes . Wounding serves to down-regulate expression in young and old internodes . Heat shock also affects AGP gene expression in stems by transiently down-regulating mRNA levels.

Curr Biol, 1996 Nov 1, 6(11), 1389 - 91
Cholera: nice bacteria and bad viruses; Levin BR et al.; The genes coding for cholera toxin are borne on, and can be infectiously transmitted by, a filamentous bacteriophage, raising intriguing questions about the mechanisms and evolution of bacterial pathogenesis, and the taxonomy, epidemiology and control of cholera and other bacterial diseases.

Nucleic Acids Res, 1996 Nov 1, 24(21), 4319 - 26
Bacteriophage T4 regA protein binds RNA as a monomer, overcoming dimer interactions; Phillips CA et al.; The stoichiometry of the complex formed between the T4 translational repressor protein regA and the 16 nt gene 44 recognition element (gene 44RE) RNA has been determined . Under quantitative binding conditions, the association of wild-type regA protein with gene 44RE RNA exhibits saturation at a 1:1 ratio of protein to RNA . It is known that regA protein exists as a dimer in protein crystals . Thus, the stoichiometry may be indicative of a regA dimer bound to two RNAs or a regA monomer bound to one RNA . Gel filtration through Sephadex G-75 revealed that wild-type and R91L regA proteins (14.6 kDa) elute at a mass of 29 kDa, consistent with the mass of a dimer . However, wild-type regA preincubated with gene 44RE (1:1) resulted in a complex that eluted at approximately 20 kDa, consistent with a regA monomer-RNA complex . Covalent crosslinking of surface lysines with glutaraldehyde confirmed that wild-type and R91L proteins exist as dimers and higher oligomers in solution . However, the addition of RNA to wild-type regA protein prior to crosslinking inhibited the formation of crosslinked dimers . Thus, the regA protein-protein interactions observed in solution are disrupted or blocked in the presence of gene 44RE RNA . Together, these studies demonstrate that regA protein binds RNA as a monomer, although free protein exists predominantly as a dimer.

Nucleic Acids Res, 1996 Nov 1, 24(21), 4105 - 10
DNA damage by peroxynitrite characterized with DNA repair enzymes; Epe B et al.; The DNA damage induced by peroxynitrite in isolated bacteriophage PM2 DNA was characterized by means of several repair enzymes with defined substrate specificities . Similar results were obtained with peroxynitrite itself and with 3-morpholinosydnonimine (SIN-1), a compound generating the precursors of peroxynitrite, nitric oxide and superoxide . A high number of base modifications sensitive to Fpg protein which, according to HPLC analysis, were mostly 8-hydroxyguanine residues, and half as many single-strand breaks were observed, while the numbers of oxidized pyrimidines (sensitive to endonuclease III) and of sites of base loss (sensitive to exonuclease III or T4 endonuclease V) were relatively low . This DNA damage profile caused by peroxynitrite is significantly different from that obtained with hydroxyl radicals or with singlet molecular oxygen . The effects of various radical scavengers and other additives (t-butanol, selenomethionine, selenocystine, desferrioxamine) were the same for single-strand breaks and Fpg-sensitive modifications and indicate that a single reactive intermediate but not peroxynitrite itself is responsible for the damage.

J Bacteriol, 1996 Nov, 178(22), 6419 - 26
Short-range and long-range context effects on coliphage T4 endonuclease II-dependent restriction; Carlson K et al.; Synthetic sites inserted into a plasmid were used to analyze the sequence requirements for in vivo DNA cleavage dependent on bacteriophage T4 endonuclease II . A 16-bp variable sequence surrounding the cleavage site was sufficient for cleavage, although context both within and around this sequence influenced cleavage efficiency . The most efficiently cleaved sites matched the sequence CGRCCGCNTTGGCNGC, in which the strongly conserved bases to the left were essential for cleavage . The less-conserved bases in the center and in the right half determined cleavage efficiency in a manner not directly correlated with the apparent base preference at each position; a sequence carrying, in each of the 16 positions, the base most preferred in natural sites in pBR322 was cleaved infrequently . This, along with the effects of substitutions at one or two of the less-conserved positions, suggests that several combinations of bases can fulfill the requirements for recognition of the right part of this sequence . The replacements that improve cleavage frequency are predicted to influence helical twist and roll, suggesting that recognition of sequence-dependent DNA structure and recognition of specific bases are both important . Upon introduction of a synthetic site, cleavage at natural sites within 800 to 1,500 bp from the synthetic site was significantly reduced . This suggests that the enzyme may engage more DNA than its cleavage site and cleaves the best site within this region . Cleavage frequency at sites which do not conform closely to the consensus is, therefore, highly context dependent . Models and possible biological implications of these findings are discussed.

Curr Genet, 1996 Nov, 30(5), 389 - 95
A new point mutation in the nuclear gene of yeast mitochondrial RNA polymerase, RPO41, identifies a functionally important amino-acid residue in a protein region conserved among mitochondrial core enzymes; Lisowsky T et al.; The core enzyme of mitochondrial RNA polymerase in yeast is homologous to those of bacteriophages T3, T7 and SP6 . In previous studies the identification of the first conditional yeast mutant for this enzyme helped to identify the corresponding specificity factor and to elucidate their interaction inside mitochondria . In the present study we report the identification of a second nuclear mutation located in the gene for mitochondrial RNA polymerase . A comparison of the two temperature-sensitive mutants demonstrates that the new mutant has a phenotype distinct from the first one and characterizes a new important domain of the enzyme . Two different suppressor genes which both rescue the first mutant do not abolish the defect of the second one and, in addition, an extremely high instability of mitochondrial genomes is observed in the new mutant . The enzymatic defect is caused by a single nucleotide exchange which results in the replacement of the serine938 residue by phenylalanine . This amino acid is located in the middle part of the protein in an as yet poorly characterized region that is not highly conserved between mitochondrial core enzymes and bacteriophage-type RNA polymerases . However, the affected amino acid and the respective protein domain are specific for mitochondrial RNA polymerase core enzymes and may help to define enzymatic functions specific for the mitochondrial transcription apparatus.

J Mol Biol, 1996 Nov 1, 263(3), 447 - 62
The herpes simplex virus procapsid: structure, conformational changes upon maturation, and roles of the triplex proteins VP19c and VP23 in assembly; Trus BL et al.; The proteins coded by the five major capsid genes of herpes simplex virus 1, VP5 (gene UL19), VP19c (UL38), VP23 (UL18), pre-VP22a (UL26.5), and pre-VP21 (UL26), assemble into fragile roundish "procapsids", which mature into robust polyhedral capsids in a transition similar to that undergone by bacteriophage proheads . Here we describe the HSV-1 procapsid structure to a resolution of approximately 2.7 nm from three-dimensional reconstructions of cryo-electron micrographs . Comparison with the mature capsid provides insight into the large-scale conformational changes that take place upon maturation . In the procapsid, the elongated protomers (VP5 subunits) make little contact with each other except around the bases of the hexons and pentons, whereas they are tightly clustered into capsomers in the mature state; the axial channels, which are constricted or blocked in the mature capsid, are fully open; and unlike the well observed 6-fold symmetry of mature hexons, procapsid hexons are distorted into oval and triangular shapes . These deformations reveal a VP5 domain in the inner part of the protrusion wall which participates in inter-protomer bonding in the procapsid and is close to the site where the channel closes upon maturation . Remarkably, there are no direct contacts between neighboring capsomers; instead, interactions between them are mediated by the "triplexes" at the sites of local 3-fold symmetry . This observation discloses the mechanism whereby the triplex proteins, VP19c and VP23, play their essential roles in capsid morphogenesis . In the mature capsid, density extends continuously between neighboring capsomers in the inner "floor" layer . In contrast, there are large gaps in the corresponding region of the procapsid, implying that formation of the floor involves extensive remodeling . Inside the procapsid shell is the hollow spherical scaffold, whose radial density profile indicates that the major scaffold protein, pre-VP22a, is a long molecule (> 24 nm) composed of three domains . Since no evidence of icosahedral symmetry is detected in the scaffold, we infer that (unless higher resolution is required) the scaffold may not be an icosahedral shell but may instead be a protein micelle with a preferred radius of curvature.

Virology, 1996 Nov 1, 225(1), 82 - 96
Scaffolding mutants identifying domains required for P22 procapsid assembly and maturation; Greene B et al.; Assembly of the icosahedral shells of the dsDNA bacteriophages, herpesviruses, and adenoviruses requires proteins not found in the mature virion, termed scaffolding proteins . The bacteriophage P22 precursor procapsid contains approximately 300 scaffolding molecules within a shell composed of 420 coat protein subunits . Though nonsense mutants are common, few mutants affecting the functions of the scaffolding protein have been recovered . We report here the isolation and characterization of new missense mutants unable to form infectious virions under restrictive conditions . These mutant scaffolding subunits were competent for protein folding and capsid assembly under restrictive conditions . Two mutants were defective in assembly into the procapsid of the portal complex, which serves as the channel through which DNA is packaged . These mutations may identify a region of the scaffolding protein required for interaction with the portal subunits . Two mutants in a different region of the sequence were impaired in scaffolding release from the procapsid both in vivo and in vitro . These mutations may identify a new domain required for scaffolding release . Scaffolding release appeared to be required for capsid expansion; in turn, scaffolding release seemed to depend upon the presence of a portal . This may help to order the pathway of events in phage maturation.

J Biol Chem, 1996 Nov 1, 271(44), 27823 - 8
Bent DNA in the human adenovirus type 2 E1A enhancer is an architectural element for transcription stimulation; Ohyama T; The upstream half of the human adenovirus type 2 enhancer adopts a curved DNA structure . Most of the enhancer elements are within the curvature, suggesting that this unusual structure is linked to enhancer function . To verify this experimentally, I constructed in vitro transcription assay systems which could distinguish any effects generated by conformational changes in a DNA template . The curved DNA conformation in the enhancer clearly affected the extent of the stimulation of the E1A gene transcription: assays using the wild-type DNA template showed that the moderately curved enhancer was superior to the highly curved enhancer in transcriptional stimulation . In additional experiments, the enhancer region was substituted with a curved DNA derived from the bacteriophage lambda origin of replication . Assays using this mutant revealed that this curved segment could also act as an enhancer when it had the proper conformation . Consequently, DNA conformation may play a general role in transcriptional stimulation.

J Bacteriol, 1996 Nov, 178(21), 6133 - 9
An N-terminal mutation in the bacteriophage T4 motA gene yields a protein that binds DNA but is defective for activation of transcription; Gerber JS et al.; The bacteriophage T4 MotA protein is a transcriptional activator of T4-modified host RNA polymerase and is required for activation of the middle class of T4 promoters . MotA alone binds to the -30 region of T4 middle promoters, a region that contains the MotA box consensus sequence {(t/a)(t/a)TGCTT(t/c)A} . We report the isolation and characterization of a protein designated Mot21, in which the first 8 codons of the wild-type motA sequence have been replaced with 11 different codons . In gel retardation assays, Mot21 and MotA bind DNA containing the T4 middle promoter P(uvsX) similarly, and the proteins yield similar footprints on P(uvsX) . However, Mot21 is severely defective in the activation of transcription . On native protein gels, a new protein species is seen after incubation of the sigma70 subunit of RNA polymerase and wild-type MotA protein, suggesting a direct protein-protein contact between MotA and sigma70 . Mot21 fails to form this complex, suggesting that this interaction is necessary for transcriptional activation and that the Mot21 defect arises because Mot21 cannot form this contact like the wild-type activator.

J Biol Chem, 1996 Oct 25, 271(43), 26825 - 34
Biochemical analysis of mutant T7 primase/helicase proteins defective in DNA binding, nucleotide hydrolysis, and the coupling of hydrolysis with DNA unwinding; Washington MT et al.; We characterized nine helicase-deficient mutants of bacteriophage T7 helicase-primase protein (4A') prepared by random mutagenesis as reported in the accompanying paper (Rosenberg, A . H., Griffin, K., Washington, M . T., Patel, S . S., and Studier, F . W . (1996) J . Biol . Chem . 271, 26819-26824) . Mutants were selected from each of the helicase-conserved motifs for detailed analysis to understand better their function . In agreement with the in vivo results, the mutants were defective in helicase activity but were active in primase function . dTTP hydrolysis, DNA binding, and hexamer formation were examined . Three classes of defective mutants were observed . Group A mutants (E348K, D424N, and S496F), defective in dTTP hydrolysis, lie in motifs 1a, 2, and 4 and are possibly involved in NTP binding/hydrolysis . Group B mutants (R487C and G488D), defective in DNA binding, lie in motif 4 and are responsible directly or indirectly for DNA binding . Group C mutants (G116D, A257T, S345F, and G451E) were not defective in any of the activities except the helicase function . These mutants, scattered throughout the protein, appear defective in coupling dTTPase activity to helicase function . Secondary structural predictions of 4A' and DnaB helicases resemble the known structures of RecA and F1-ATPase enzymes . Alignment shows a striking correlation in the positions of the amino acids that interact with NTP and DNA.

J Biol Chem, 1996 Oct 25, 271(43), 26819 - 24
Selection, identification, and genetic analysis of random mutants in the cloned primase/helicase gene of bacteriophage T7; Rosenberg AH et al.; T7 gene 4 specifies two overlapping proteins 4A, a 566-amino acid primase/helicase, and 4B, a 503-amino acid helicase whose initiation codon is the 64th codon of the 4A protein . The 4A' gene, which has a leucine codon replacing the 4B initiation codon, specifies a single 566-amino acid protein that can provide the primase and helicase functions required for normal T7 growth . We selected N-methyl-N'-nitro-N-nitrosoguanidine mutants in the cloned 4A' gene that no longer support the growth of a phage that completely lacks gene 4 . Genetic mapping of the 76 mutations found them to be distributed throughout the protein, including both the N-terminal and C-terminal halves of the molecule thought to represent primase and helicase domains, respectively . Complementation tests with partially and completely defective phage showed that all but five of the mutants lacked helicase function but retained primase function . The other five, which lacked both functions, all made short proteins, including one missing only 60 amino acids . No mutations lacked only primase function, and none mapped within the first 105 amino acids, which includes the 63-amino acid region unique to 4A that contains elements required to recognize primase sites . Forty-six mutations were sequenced and included 27 missense mutations affecting 25 amino acids . Many mutations in the N-terminal half of the protein affected its solubility in cell extracts . Mutations in the C-terminal half clustered in or near five helicase consensus sequences . Biochemical analysis of nine of the mutant proteins is described in the accompanying paper (Washington, M . T., Rosenberg, A . H., Griffin, K., Studier, F . W., and Patel, S . S . (1996) J . Biol . Chem . 271, 26825-26834).

Mutat Res, 1996 Oct 25, 357(1-2), 57 - 66
A novel lacI transgenic mutation-detection system and its application to establish baseline mutation frequencies in the scid mouse; Andrew SE et al.; To assess DNA mutations in vivo, we have established a new transgenic mouse line, BC-1, carrying a lacI target gene for mutation detection within a bacteriophage shuttle-vector . The lacI gene was positioned within sequences derived from a rearranged murine immunoglobulin gene locus, a feature that distinguishes the BC-1 transgene from other shuttle vector systems . As mutations in lacI transgenes likely reflect mutations occurring throughout the genome, these systems have been successfully used to investigate spontaneous and induced mutations in a variety of tissues . An important additional application of the transgenic systems is the characterization of lacI mutations occurring in murine strains having specific DNA repair defects . For this study, scid (severe combined immunodeficiency) mice were selected as animals with this mutation have a defect in double-strand DNA break repair . To determine what impact the scid mutation might have on spontaneous mutation frequencies within DNA recovered from various tissues, these mice were crossed with the BC-1 line . Interestingly, mutation frequencies within BC-1/scid mouse DNA were not significantly different from those of BC-1 control mice . Furthermore, spontaneous lacI mutations obtained from BC-1 and from BC-1/scid liver DNA were similar in spectrum . As spontaneous BC-1 liver mutations were similar to those reported previously for other lacI systems, such as the Big Blue transgenic line, this suggested that the nature of the DNA sequences flanking the reporter gene did not modify lacI mutation rate or character.

Gene, 1996 Oct 24, 177(1-2), 179 - 89
Expression of the bacteriophage T4 DNA terminase genes 16 and 17 yields multiple proteins; Franklin JL et al.; The products of the bacteriophage T4 terminase genes 16 and 17 are known to mediate cutting and packaging of concatemeric vegetative DNA . We show here that the larger of these genes, 17, yields multiple protein species . The complex expression of the T4 terminase genes includes overlapping transcripts, probably initiated from multiple promoters, RNA processing at certain preferred sites and translation initiation from multiple ribosome binding sites (RBS) . Translation initiation from these RBS may be modulated by inverted repeat (IR) sequences whose folding can be predicted to differ in different RNA species . In T4 infected bacteria, genes 16 and 17 are probably co-transcribed from several near-consensus late promoters upstream from gene 16, and processed at multiple sites . Additional 5' ends of late transcripts are located downstream from a near-consensus late promoter inside gene 17 and further downstream, unrelated to any known promoter consensus sequence . The gene 17 transcripts that are initiated or cleaved internally contain RBS for shorter open reading frames (ORFs) in the same frame as full-length gene product (gp) 17 of 70 kDa . The truncated proteins, a 59-kDa gp17' and a 45-kDa gp17", are synthesized from cloned gene 17 segments in which the first gene 17 RBS is deleted . Expression of gene 17 is different in BL21(DE3) or W3110{pACT7} host bacteria . The gp17' and gp17" proteins are predicted to contain one or more of the ATPase motifs that are common among large subunits of other phage terminases . They lack a predicted single stranded (ss) DNA binding motif that is unique the large terminase proteins in T4 gp17, and that has been implicated in recognizing ssDNA regions in replicating and recombining T4DNA destined to be packaged . We hypothesize that a truncated gene 17' is an evolutionary precursor of the full-size T4 gene 17 . Its function may have been maintained to allow processive packaging from double stranded (ds) DNA ends.

Gene, 1996 Oct 17, 176(1-2), 131 - 7
Deletion of the myristylation signal allows high-level production of the hepatitis B virus large surface glycoprotein preS1 with vaccinia virus recombinants; Pfleiderer M et al.; We have investigated the requirements necessary for high-level production of the hepatitis B virus (HBV strain ayw) large surface glycoprotein preS1 with vaccinia virus (VV) recombinants . In earlier studies, only nanogram amounts of preS1 could be obtained from cells infected with an appropriate recombinant VV carrying the preS1 gene under the transcriptional control of a conventional VV promoter (p7.5) . Here, we report that the use of an improved promoter system, i.e., the bacteriophage T7 polymerase/VV hybrid expression system (T7/EMC system) in combination with a G-C conversion at position 5 of the preS1 open reading frame, deleting the myristylation motif of the polypeptide, results in an at least 12-fold increase in preS1 expression compared to the wild-type preS1 expressed with the strongest homologous VV promoter system known so far . Although the T7/EMC promoter system was most effective, improved expression of the modified preS1 (preS1dMyr) is independent from the promoter system used, from the insertion locus of the modified preS1 within the VV genome and also from the cell line used for expression studies.

Gene, 1996 Oct 17, 176(1-2), 49 - 53
Stable high-copy-number bacteriophage lambda promoter vectors for overproduction of proteins in Escherichia coli; Love CA et al.; The construction of new high-copy-number (hcn) lambda-promoter expression vectors is described . All these vectors (1) contain tandem lambda pR and pL promoters upstream of an extensive multiple cloning site (MCS) for insertion of genes, (2) direct expression of the lambda cIts857 gene, enabling their use in any Escherichia coli host strain for thermal induction of gene overexpression, and (3) bear the par locus of plasmid pSC101, ensuring their stable maintenance at hcn in the absence of continuous antibiotic selection . Six of the vectors also contain efficient ribosome-binding sites upstream of unique HpaI or NdeI sites in their MCS regions, and two contain sequences that encode N-terminal poly-His . The performance of these vectors was assessed by using them to overproduce the E . coli HMP flavohaemoprotein and the bacteriophage M13 gene II replicator protein.

Nucleic Acids Res, 1996 Oct 15, 24(20), 4042 - 9
Escherichia coli OxyR protein represses the unmethylated bacteriophage Mu mom operon without blocking binding of the transcriptional activator C; Sun W et al.; Transcription of the bacteriophage Mu mom operon requires transactivation by the phage-encoded C protein . DNase I footprinting showed that in the absence of C, Escherichia coli RNA polymerase E(sigma)70 (RNAP) binds to the mom promoter (Pmom) region at a site, P2 (from -64 to -11 with respect to the transcription start site), on the top (non-transcribed) strand . This is slightly upstream from, but overlapping P1 (-49 to +16), the functional binding site for rightward transcription . Host DNA-{N6-adenine} methyltransferase (Dam) methylation of three GATCs immediately upstream of the C binding site is required to prevent binding of the E.coli OxyR protein, which represses mom transcription in dam- strains . OxyR, known to induce DNA bending, is normally in a reduced conformation in vivo, but is converted to an oxidized state under standard in vitro conditions . Using DNase I footprinting, we provide evidence supporting the proposal that the oxidized and reduced forms of OxyR interact differently with their target DNA sequences in vitro . A mutant form, OxyR-C199S, was shown to be able to repress mom expression in vivo in a dam- host . In vitro DNase I footprinting showed that OxyR-C199S protected Pmom from -104 to -46 on the top strand and produced a protection pattern characteristic of reduced wild-type OxyR . Prebinding of OxyR-C199S completely blocked RNAP binding to P2 (in the absence of C), whereas it only slightly decreased binding of C to its target site (-55 to -28, as defined by DNase I footprinting) . In contrast, OxyR-C199S strongly inhibited C-activated recruitment of RNAP to P1 . These results indicate that OxyR repression is mediated subsequent to binding by C . Mutations have been isolated that relieve the dependence on C activation and have the same transcription start site as the C-activated wild-type promoter . One such mutant, tin7, has a single base change at -14, which changes a T6 run to T3GT2 . OxyR-C199S partially inhibited RNAP binding to the tin7 promoter in vitro, even though the OxyR and RNAP-P1 binding sites probably do not overlap, and in vivo expression of tin7 was reduced 5- to 10-fold in dam- cells . These results suggest that OxyR can repress tin7.

Proc Natl Acad Sci U S A, 1996 Oct 15, 93(21), 11622 - 7
Interplay of structure and disorder in cochaperonin mobile loops; Landry SJ et al.; Protein-protein interactions typically are characterized by highly specific interfaces that mediate binding with precisely tuned affinities . Binding of the Escherichia coli cochaperonin GroES to chaperonin GroEL is mediated, at least in part, by a mobile polypeptide loop in GroES that becomes immobilized in the GroEL/GroES/nucleotide complex . The bacteriophage T4 cochaperonin Gp31 possesses a similar highly flexible polypeptide loop in a region of the protein that shows low, but significant, amino acid similarity with GroES and other cochaperonins . When bound to GroEL, a synthetic peptide representing the mobile loop of either GroES or Gp31 adopts a characteristic bulged hairpin conformation as determined by transferred nuclear Overhauser effects in NMR spectra . Thermodynamic considerations suggest that flexible disorder in the cochaperonin mobile loops moderates their affinity for GroEL to facilitate cycles of chaperonin-mediated protein folding.

Virology, 1996 Oct 15, 224(2), 568 - 72
The N-terminal part of bacteriophage P2 capsid protein is essential for postassembly maturation of P2 and P4 capsids; Bjerve K et al.; During capsid assembly of bacteriophage P2 and its satellite phage P4, gpN undergoes proteolytic cleavage with the removal of the first 31 amino acids . The truncated protein gpN* is unable to support formation of viable phages in complementation tests . A c-myc antigenic epitope (EQKLISEEDL) exchanged for eleven amino acids in the amino terminal part of gpN results in both proteolytic processing of gpN::c-myc as well as assembly of P2 and P4 procapsid-like structures, but gpN::c-myc failed, like N*, to support the production of infectious P2 and P4 particles.

FEMS Microbiol Lett, 1996 Oct 15, 144(1), 21 - 7
Drastically decreased transcription from CII-activated promoters is responsible for impaired lysogenization of the Escherichia coli rpoA341 mutant by bacteriophage lambda; Szalewska-Palasz A et al.; It was demonstrated previously that a mutation, rpoA341, in the gene encoding the alpha subunit of Escherichia coli RNA polymerase prevents lysogenization by bacteriophage lambda . The rpoA341 allele is known to be responsible for impaired transcription of some positively regulated E . coli chromosomal operons . Here we demonstrate that the inhibition of lysogenization of the rpoA341 mutant is a result of drastically decreased transcription from positively regulated phage promoters . We were unable to detect any transcripts originating from the CII-activated pE, pI and paQ promoters (important for lysogenic development) in the phage-infected rpoA341 mutant, in contrast to an otherwise isogenic rpoA+ strain . The results are discussed in the light of other reports showing that activation of the pE promoter by CII protein in vitro is decreased only about fivefold when the native alpha subunit is replaced by truncated alpha polypeptides.

Gene, 1996 Oct 10, 175(1-2), 151 - 5
The bacteriophage P1 lytic replicon: directionality of replication and cis-acting elements; Cohen G et al.; We have identified the direction of replication of a bacteriophage P1 lytic replicon . This was accomplished by constructing lambda P1 lysogens that contain a functional P1 lytic replicon and analysing which of two nearby bacterial DNA markers flanking the lambda prophage were amplified when that replicon was activated . We demonstrate that both DNA markers are coordinately amplified, a result consistent with lytic replication proceeding in a bidirectional fashion . To analyze the role of various elements comprising the lytic replicon, we assessed the ability of a wild type replicon to complement a defective replicon that contains a transposon inserted between an essential lytic replication gene (repL) and the promoter (P53) at which transcription of that gene is initiated . We show that the wild type replicon cannot complement the mutant replicon . The simplest hypothesis to explain this result is that either P53 or repL protein functions primarily in cis for the replicon to operate.

Biochim Biophys Acta, 1996 Oct 9, 1288(2), F79 - 99
A new concept in (adenoviral) oncogenesis: integration of foreign DNA and its consequences; Doerfler W; A new concept for viral oncogenesis is presented which is based on experimental work on the chromosomal integration of adenovirus DNA into mammalian genomes . The mechanism of adenovirus DNA integration is akin to non-sequence-specific insertional recombination in which patch homologies between the recombination partners are frequently observed . This reaction has been imitated in a cell-free system by using nuclear extracts from hamster cells and partly purified fractions derived from them . As a consequence of foreign DNA insertion into the mammalian genome, the foreign DNA is extensively de novo methylated in specific patterns, presumably as part of a mammalian host cell defense mechanism against inserted foreign DNA which can be permanently silenced in this way . A further corollary of foreign (adenovirus or bacteriophage lambda) DNA integration is seen in extensive changes in cellular DNA methylation patterns at sites far remote from the locus of insertional recombination . Repetitive cellular, retrotransposon-like sequences are particularly, but not exclusively, prone to these increases in DNA methylation . It is conceivable that these changes in DNA methylation are a reflection of a profound overall reorganization process in the affected genomes . Could these alterations significantly contribute to the transformation events during viral or other types of oncogenesis? These sequelae of foreign DNA integration into established mammalian genomes will have to be critically considered when interpreting results obtained with transgenic, knock-out, and knock-in animals and when devising schemes for human somatic gene therapy . The interpretation of de novo methylation as a cellular defense mechanism has prompted investigations on the fate of food-ingested foreign DNA . The gastrointestinal (GI) tract provides a large surface for the entry of foreign DNA into any organism . As a tracer molecule, bacteriophage M13 DNA has been fed to mice . Fragments of this DNA can be found in small amounts (about 1% of the administered DNA) in all parts of the intestinal tract and in the feces . Furthermore, M13 DNA can be traced in the columnar epithelia of the intestine, in Peyer's plaque leukocytes, in peripheral white blood cells, in spleen, and liver . Authentic M13 DNA has been recloned from total spleen DNA . If integrated, this DNA might elicit some of the described consequences of foreign DNA insertion into the mammalian genome . Food-ingested DNA will likely infiltrate the organism more frequently than viral DNA.

Mol Divers, 1996 Oct, 2(1-2), 13 - 8
Identification of peptide sequences that bind the Thomsen-Friedenreich cancer-associated glycoantigen from bacteriophage peptide display libraries; Peletskaya EN et al.; The goal of this study was to determine if polypeptides that bind specifically to the carcinoma-associated Thomsen-Friedenreich (T) antigen could be isolated from a random peptide bacteriophage display library . T antigen is a carbohydrate antigen that is exposed and immunoreactive on the surfaces of most primary carcinomas and their metastases, while it is masked on normal cells . Tumor-specific surface carbohydrates are often used as markers of cell differentiation and play a role in cell aggregation, which is an important step in the metastatic process . Therefore, peptides that bind and mask T antigen may yield useful carbohydrate-specific probes and provide insight into carbohydrate-mediated tumor-cell aggregation . A 15-amino acid random peptide bacteriophage display library was screened for polypeptides that exhibited high specificity to two glycoproteins which display T antigen on their surfaces . The results suggest that synthetic peptides identified from the bacteriophage display library have high affinities (Kd approximately 1 microM) and specificities for proteins and human tumor cells which present T antigen . Thus, random bacteriophage peptide display libraries may be a rich source of sequences that bind to carbohydrate antigen structures.

Mol Biotechnol, 1996 Oct, 6(2), 155 - 62
Selection of proteins and peptides from libraries displayed on filamentous bacteriophage; McGregor D; This article attempts to review recent developments in the rapidly developing field of phage display libraries . The current state of peptide, antibody, and cDNA libraries, as well as current and future applications of phage display libraries are discussed . The main focus of the article is on the methods for selecting binding ligands against targets in a variety of different formats . These include solid phase and in-solution selection methods, and the strategies used to select for higher affinity, and binding ligands against impure and cellular target proteins.

Curr Opin Biotechnol, 1996 Oct, 7(5), 547 - 53
Phage display of proteins; Dunn IS; Phage display of proteins continues to be an important technology with a variety of applications . In the past year, advances have been made in coupling rational protein design with the power of the display selection process . In addition to the widely used filamentous phage, other bacteriophage surface expression systems have now been developed, some of which may be of particular use for the selection of surface-display cDNA clones.

Curr Biol, 1996 Oct 1, 6(10), 1307 - 16
Bypass of lethality with mosaic mice generated by Cre-loxP-mediated recombination; Betz UA et al.; BACKGROUND: The analysis of gene function based on the generation of mutant mice by homologous recombination in embryonic stem cells is limited if gene disruption results in embryonic lethality . Mosaic mice, which contain a certain proportion of mutant cells in all organs, allow lethality to be circumvented and the potential of mutant cells to contribute to different cell lineages to be analyzed . To generate mosaic animals, we used the bacteriophage P1-derived Cre-loxP recombination system, which allows gene alteration by Cre-mediated deletion of loxP-flanked gene segments . RESULTS: We generated nestin-cre transgenic mouse lines, which expressed the Cre recombinase under the control of the rat nestin promoter and its second intron enhancer . In crosses to animals carrying a loxP-flanked target gene, partial deletion of the loxP-flanked allele occurred before day 10.5 post coitum and was detectable in all adult organs examined, including germ-line cells . Using this approach, we generated mosaic mice containing cells deficient in the gamma-chain of the interleukin-2 receptor (IL-2R gamma); in these animals, the IL-2R gamma-deficient cells were underrepresented in the thymus and spleen . Because mice deficient in DNA polymerase beta die perinatally, we studied the effects of DNA polymerase beta deficiency in mosaic animals . We found that some of the mosaic polymerase beta-deficient animals were viable, but were often reduced in size and weight . The fraction of DNA polymerase beta-deficient cells in mosaic embryos decreased during embryonic development, presumably because wild-type cells had a competitive advantage . CONCLUSIONS: The nestin-cre transgenic mice can be used to generate mosaic animals in which target genes are mutated by Cre-mediated recombination of loxP-flanked target genes . By using mosaic animals, embryonic lethality can be bypassed and cell lineages for whose development a given target gene is critical can be identified . In the case of DNA polymerase beta, deficient cells are already selected against during embryonic development, demonstrating the general importance of this protein in multiple cell types.

Mol Microbiol, 1996 Oct, 22(2), 283 - 92
The Mu strong gyrase-binding site promotes efficient synapsis of the prophage termini; Pato ML et al.; A strong DNA gyrase-binding site (SGS) is located midway between the termini of the bacteriophage Mu genome and is required for efficient replicative transposition . We have proposed that the SGS promotes the efficient synapsis of the Mu prophage ends (an obligate early step in replicative transposition), and that it does so by helping to organize the prophage DNA into a supercoiled loop with the SGS at the apex of the loop and the prophage termini at the base . The positioning of the synapsing termini equidistant from the SGS is a key element in the proposed model . To test this proposal, we have constructed prophages with a second, internal right end and asked whether the natural, external right end or the internal right end is used for synapsis with the left end in the presence and absence of the SGS . In the presence of the central SGS, the natural, or outside, right end was used exclusively and very efficiently . In the absence of the central SGS, the internal right end was used preferentially and inefficiently: the efficiency of transposition decreased with increasing distance between the internal right end and the left end . Repositioning the SGS midway between the left end and an internal right end allowed highly efficient use of the internal right end . These results support a model in which gyrase can influence long-range DNA interactions to promote efficient synapsis of Mu prophage ends.

Am J Physiol, 1996 Oct, 271(4 Pt 2), H1565 - 75
Isolation, mapping, and regulated expression of the gene encoding mouse C-type natriuretic peptide; Huang H et al.; Genomic sequences encoding mouse C-type natriuretic peptide (CNP) were isolated from bacteriophage libraries and characterized by restriction enzyme and sequence analysis . The mouse CNP gene (Nppc) comprised at least two exons and one intron and included several cis-regulatory elements in the 5'-flanking sequence . The deduced amino acid sequence of mouse CNP-22 was identical to other mammalian CNPs . Analysis of allele distributions in interspecific back-cross and recombinant inbred strains assigned Nppc to chromosome 1 . CNP transcripts were detected by ribonuclease protection analysis in brain, ovary, and uterus, with lower levels in testes and epididymus . Uterine CNP transcripts and protein were low in sexually immature mice and adults at estrus and increased at proestrus, but similar variations in ovarian CNP expression were not statistically significant . Atrial natriuretic peptide and B-type natriuretic peptide transcripts were not detected in mouse ovary or uterus . Thus CNP gene expression is regulated by tissue-specific and inducible mechanisms in female reproductive organs . Correlations between CNP expression and uterine fluid content suggest that CNP may regulate uterine fluid balance in mice and other mammals.

Genetics, 1996 Oct, 144(2), 715 - 26
Transgene Coplacement and high efficiency site-specific recombination with the Cre/loxP system in Drosophila; Siegal ML et al.; Studies of gene function and regulation in transgenic Drosophila are often compromised by the possibility of genomic position effects on gene expression . We have developed a method called transgene coplacement, in which any two sequences can be positioned at exactly the same site and orientation in the genome . Transgene coplacement makes use of the bacteriophage P1 system of Cre/loxP site-specific recombination, which we have introduced into Drosophila . In the presence of a cre transgene driven by a dual hsp70-Mos1 promoter, a white reporter gene flanked by loxP sites is excised with virtually 100% efficiency both in somatic cells and in germ cells . A strong maternal effect, resulting from Cre recombinase present in the oocyte, is observed as white or mosaic eye color in F1 progeny . Excision in germ cells of the F1 yields a strong grand-maternal effect, observed as a highly skewed ratio of eye-color phenotypes in the F2 generation . The excision reactions of Cre/loxP and the related FLP/FRT system are used to create Drosophila lines in which transgenes are at exactly allelic sites in homologous chromosomes.

Microbiology, 1996 Oct, 142 ( Pt 10), 2803 - 13
Mutants of Streptomyces roseosporus that express enhanced recombination within partially homologous genes; Hosted TJ et al.; Streptomyces roseosporus mutants that express enhanced recombination between partially homologous (homeologous) sequences were isolated by selection for recombination between the bacteriophage phi C31 derivative KC570 containing the Streptomyces coelicolor glucose kinase (glk) gene and the S . roseosporus chromosome . The frequencies of homeologous recombination in the ehr mutants were determined by measuring the chromosomal insertion frequencies of plasmids containing S . coelicolor glnA or whiG genes . S . roseosporus ehr mutants showed 10(2)- to 10(4)-fold increases in homeologous recombination relative to Ehr+ strains, but no increase in homologous recombination . Southern hybridization analysis revealed single unique sites for the insertion of each of the plasmids, and the crossovers occurred in frame and in proper translational register, yielding functional chimeric glnA and whiG genes.

Int J Radiat Biol, 1996 Oct, 70(4), 459 - 65
Mutations induced by gamma-irradiation of M13 bacteriophages containing single-stranded DNA; Braun JE et al.; Oxygenated suspensions of M13 bacteriophages, containing single-stranded M13mp10 DNA, were gamma-irradiated followed by infection of E . coli cells . Mutants in the mutational target sequence, which consists of the lac promoter /operator region, the lacZ alpha gene, and a 144 bp inframe insert in the lacZ alpha gene, were selected and characterized . Except for three one-base deletions, all of the 51 mutations characterized were base substitutions . All base substitutions appeared to involve guanines and cytosines and none affect adenines and thymines . Since most of the known repair systems do not act on single-stranded DNA, the conclusion can be drawn that radiation induces under these conditions only mutagenic damages on guanine and cytosine . Although all possible G- and C-transversions and transitions were found, there is a strong preference for G-->C and G-->T transversions (21 and 25% of all base substitutions, respectively) and C-->T transitions (48% of all base substitutions) . These results indicate, that the G/C-->C/G and G/C-->T/A transversions, found after irradiation of double-stranded M13 DNA, are mainly due to radiation guanine products, whereas cytosine damage is mainly responsible for G/C-->A/T transitions.

J Bacteriol, 1996 Oct, 178(19), 5668 - 75
Bacteriophage PSP3 and phiR73 activator proteins: analysis of promoter specificities; Julien B et al.; Transcription from the late promoters of bacteriophage P2 and its satellite phage P4 is activated by a unique class of small, zinc-binding proteins . Using plasmid expression systems, we compared activators from two P2-like (helper) phages with those encoded by two satellite phages . The helper phage activators have more activity on the P4 phage sid promoter . In contrast, the satellite phage activators function better on the four late P2 promoters and on the P4 late leftward promoter . We purified one activator encoded by a P2-like phage and an activator from a satellite phage and determined their binding sites within the P2 and P4 late promoters . Differences in activity levels correlate with binding specificities; promoters that function best with the satellite phage activators have only one activator binding site centered at -55, while the P4 sid promoter, which has more activity with helper phage activators, has a second binding site centered at -18 . Surprisingly, DNase I footprinting revealed only very minor differences in promoter binding by the two activators reported here and the P4 activator reported previously . Thus, the differences in transcriptional activity are probably due to interactions between the activators and RNA polymerase, rather than interactions between the activators and DNA.

J Bacteriol, 1996 Oct, 178(19), 5568 - 72
Bacteriophage PSP3 and phi R73 activator proteins: analysis of promoter specificities; Clerch B et al.; The effect of plasmid pKM101 on the survival of Escherichia coli AB1157, growing in minimal medium, in the presence of a 4-quinolone DNA gyrase inhibitor was investigated . The presence of this plasmid decreased susceptibility to the quinolone ciprofloxacin, whereas mucAB genes present in a multicopy plasmid did not . The same effect of pKM101 was detected in a recA430 mutant, confirming that it was not really related to the SOS response . In contrast, when survival assays were performed under amino acid starvation conditions, pKM101 did not confer protection against ciprofloxacin . All of these results indicated that the synthesis of a product(s), different from MucAB, which was encoded by the plasmid pKM101 increased the rate of survival of the AB1157 strain in the presence of quinolone . To identify the gene(s) responsible for this phenotype, several plasmid derivatives carrying different portions of pKM101 were constructed . The 2.2-kb region containing korB, traL, korA, and traM genes was sufficient to decrease susceptibility to quinolone . This plasmidic fragment also made the AB1157 host strain grow more slowly (the Slo phenotype) . Moreover, the suppression of the Slo phenotype by addition of adenine to the cultures abolished the decreased susceptibility to quinolone . These results are evidence that the protection against quinolone conferred by this region of pKM101 in strain AB1157 is a direct consequence of the slow growth rate.

J Biol Chem, 1996 Sep 27, 271(39), 24262 - 9
Properties of a primer RNA-DNA hybrid at the mouse mitochondrial DNA leading-strand origin of replication; Lee DY et al.; Primers for vertebrate mitochondrial leading-strand DNA replication are products of transcription synthesized by mitochondrial RNA polymerase . The precursor primer RNA exists as a persistent RNA-DNA hybrid, known as an R-loop, formed during transcription through the replication origin (Xu, B., and Clayton, D . A . (1996) EMBO J . 15, 3135-3143) . In an effort to examine the precise structure of this primer RNA intermediate, we have used two methods to reconstitute model R-loops containing the mouse mitochondrial DNA origin sequence . First, we demonstrate that bacteriophage SP6 RNA polymerase can efficiently catalyze the formation of an R-loop at the mouse mtDNA origin sequence . Second, the R-loop can be assembled by annealing presynthesized RNA and supercoiled DNA template in the presence of formamide . R-loop formation by either method is dependent on specific template sequences . The reconstituted R-loop is exceptionally stable and exhibits an unexpected structure . Structural studies indicate that the RNA strand is organized within the RNA-DNA base-paired region, suggesting that the heteroduplex interaction occurs through a specific conformation . We propose that the organized structure of the R-loop is critical for primer RNA function in vivo with important implications for the RNA processing and DNA replication machinery.

J Biol Chem, 1996 Sep 27, 271(39), 24207 - 12
Structural and functional organization of the DNA polymerase of bacteriophage T7; Yang X et al.; The 80-kDa gene 5 protein encoded by bacteriophage T7 shares significant amino acid homology with the large fragment of Escherichia coli DNA polymerase I (Klenow fragment) . Like the Klenow fragment, T7 gene 5 protein has both DNA polymerase and 3' to 5' exonuclease activities . However, unlike the Klenow fragment, T7 gene 5 protein binds tightly to E . coli thioredoxin to form a complex that has a high processivity of nucleotide polymerization . In order to identify the domains of gene 5 protein responsible for polymerization, hydrolysis, and binding of thioredoxin, we have analyzed proteolytic fragments of gene 5 protein . Cleavage of the protein within one protease-sensitive region (residue 250-300) yields two molecular weight species of peptides of 32-37 and 43-51 kDa derived from the N-terminal and C-terminal region, respectively . DNA polymerase activity is found within the C-terminal fragments and exonuclease activity within the N-terminal fragments . Thioredoxin stimulates the DNA polymerase activity of the C-terminal fragments . All fragments bind to DNA . In addition to delineating the polymerase and exonuclease domains, the protease-sensitive region appears to interact with E . coli thioredoxin . Thioredoxin protects this region from proteolysis, and alteration of this region reduces the ability of thioredoxin to stimulate polymerase activity.

Gene, 1996 Sep 26, 174(1), 27 - 34
Structure of the rabbit kappa-casein encoding gene: expression of the cloned gene in the mammary gland of transgenic mice; Baranyi M et al.; The rabbit kappa-casein (kappa-Cas) encoding gene has been isolated as a series of overlapping DNA fragments cloned from a rabbit genomic library constructed in bacteriophage lambda EMBL3 . The clones harboured the 7.5-kb gene flanked by about 2.1 kb upstream and 9 kb downstream sequences . The cloned gene is the most frequently occurring of two kappa-Cas alleles identified in New Zealand rabbits . Comparison of the corresponding domains in rabbit and bovine kappa-Cas shows that both genes comprise 5 exons and that the exon/intron boundary positions are conserved whereas the introns have diverged considerably . The first three introns are shorter in the rabbit, the second intron showing the greatest difference between the two species: 1.35 kb instead of 5.8 kb in the bovine gene . Repetitive sequence motives reminiscent of the rabbit C type repeat and the complementary inverted C type repeat were identified in the fourth and first introns, respectively . Transgenic mice were produced by microinjecting into mouse oocytes an isolated genomic DNA fragment which contained the entire kappa-Cas coding region, together with 2.1-kb 5' and 4.0-kb 3' flanking region . Expression of transgene rabbit kappa-Cas mRNA could be detected in the mammary gland of lactating transgenic mice and the production of rabbit kappa-Cas was detected in milk using species-specific antibodies . The cloned gene is thus functional.

Biochemistry, 1996 Sep 24, 35(38), 12329 - 37
Spectroscopic and calorimetric characterizations of DNA duplexes containing 2-aminopurine; Law SM et al.; The base analog 2-aminopurine (AP) strongly promotes A.T to G.C and G.C to A.T transitions in bacteria and bacteriophage . During DNA replication, the primary mutagenic event involves formation of a heteroduplex with an AP.C site at a much higher frequency than formation of the corresponding heteroduplex with an A.C site . It is not known if AP-induced mutagenesis correlates with differences in the thermodynamic properties of an AP.C versus an A.C site, or whether interactions involving DNA polymerases are controlling . To address this specific question, and more generally to characterize AP-containing duplexes, we have used a combination of spectroscopic and calorimetric techniques to determine the thermodynamic properties of six 11-mer duplexes . The sequences of these duplexes are identical except for the identity of the variable central base pair which can be either A.T, A.C, AP.T, AP.C, AP.A, or AP.G, and which we use to designate each duplex . Analyses and interpretation of the optically and calorimetrically derived thermal and thermodynamic data on these six duplexes reveal the relative stabilizing influence of the central base pairs to be A.T > AP.T > AP.C > AP.A > AP.G > A.C, with the AP.C-containing duplex being significantly more stable than the A.C-containing duplex . In the aggregate, our results suggest that during incorporation, base pair discrimination by DNA polymerases is influenced, in part, by differences in the thermodynamic stabilities of the newly formed base pairs.

J Biol Chem, 1996 Sep 20, 271(38), 23506 - 11
Purification and characterization of the vaccinia virus deoxyuridine triphosphatase expressed in Escherichia coli; Roseman NA et al.; The deoxyuridine triphosphatase gene of vaccinia virus, encoded by the open reading frame F2L, was cloned into Escherichia coli and expressed under the control of a bacteriophage T7 promoter . After induction of T7 RNA polymerase by isopropyl beta-D-thiogalactopyranoside, a 16.5-kDa peptide accumulated to high levels . This 16.5-kDa protein was purified to homogeneity and characterized . Gel filtration of the purified protein revealed a trimeric native structure . Biochemical analysis revealed the enzyme to be a metalloenzyme; enzymatic activity is inhibited by EDTA . This inhibition was reversed by the addition of Mg2+, Mn2+, or Zn2+ . While the enzyme activity was highly specific for dUTP with an apparent Km of 0.94 microM, inhibition studies show that 8-azido-ATP acted as a competitive inhibitor of dUTP with a Ki of approximately 173 microM . Also, protection studies demonstrated that nucleotide competitors inhibit photoincorporation of the photoaffinity analogues {gamma-32P}5-azido-dUTP and {gamma-32P}8-azido-ATP . This suggests that while catalytic activity is limited to dUTP, other nucleotides can bind the active site.

Gene, 1996 Sep 16, 173(2), 179 - 81
LambdaZLG6: a phage lambda vector for high-efficiency cloning and surface expression of cDNA libraries on filamentous phage; Jespers LS et al.; We describe a vector, lambdaZLG6, combining the high efficiency of cDNA library cloning in bacteriophage lambda with filamentous phage display of cDNA-encoded products . The cDNAs are expressed as fusions to the 3' end of M13 gene VI . The lambdaZLG library is converted to a pZLG6-cDNA phagemid library by in vivo mass excision . Helper phage infection generates a library of phagemid particles displaying the cDNA-encoded products and containing the corresponding nucleotide sequences within.

EMBO J, 1996 Sep 16, 15(18), 5032 - 9
A direct interaction between a DNA-tracking protein and a promoter recognition protein: implications for searching DNA sequence; Tinker-Kulberg RL et al.; Bacteriophage T4 gene 45 protein, gp45, serves as the sliding clamp of viral DNA replication and as the activator of T4 late gene transcription . In the latter context, DNA tracking is an essential feature of the unique mechanism of action . T4 late promoters, which consist of a simple TATA box, TATAAATA, are recognized by the small sigma-family gene 55 protein, gp55, which binds to Escherichia coli RNA polymerase core . A direct and RNA polymerase-independent interaction of gp45 with gp55 has been demonstrated in two ways . (i) gp45 tracks along DNA; co-tracking of gp55 requires the previously documented DNA-loading process of gp45, and can be detected by photochemical crosslinking . (ii) The dynamics of DNA tracking by gp45 can be followed by footprinting; the catenated DNA-tracking state of gp45 is short-lived, but is stabilized by gp55 . The ability of this topologically linked DNA-tracking transcriptional activator to interact directly with a promoter recognition protein suggests the existence of multiple pathways of promoter location, which are discussed.

EMBO J, 1996 Sep 16, 15(18), 4806 - 16
Structure-function analysis of the Escherichia coli GrpE heat shock protein; Wu B et al.; We have isolated various missense mutations in the essential grpE gene of Escherichia coli based on the inability to propagate bacteriophage lambda . To better understand the biochemical mechanisms of GrpE action in various biological processes, six mutant proteins were overexpressed and purified . All of them, GrpE103, GrpE66, GrpE2/280, GrpE17, GrpE13a and GrpE25, have single amino acid substitutions located in highly conserved regions throughout the GrpE sequence . The biochemical defects of each mutant GrpE protein were identified by examining their abilities to: (i) support in vitro lambda DNA replication; (ii) stimulate the weak ATPase activity of DnaK; (iii) dimerize and oligomerize, as judged by glutaraldehyde crosslinking and HPLC size chromatography; (iv) interact with wild-type DnaK protein using either an ELISA assay, glutaraldehyde crosslinking or HPLC size chromatography . Our results suggest that GrpE can exist in a dimeric or oligomeric form, depending on its relative concentration, and that it dimerizes/oligomerizes through its N-terminal region, most likely through a computer predicted coiled-coil region . Analysis of several mutant GrpE proteins indicates that an oligomer of GrpE is the most active form that interacts stably with DnaK and that the interaction is vital for GrpE biological function . Our results also demonstrate that both the N-terminal and C-terminal regions are important for GrpE function in lambda DNA replication and its co-chaperone activity with DnaK.

EMBO J, 1996 Sep 16, 15(18), 4785 - 8
Intrinsic versus imposed curvature in cyclical oligomers: the portal protein of bacteriophage SPP1; van Heel M et al.; Large cyclical oligomers may be formed by (curvi-) linear polymerization of monomers until the n(th) monomer locks in with the first member of the chain . The subunits in incomplete structures exhibit a natural curvature with respect to each other which can be perturbed when the oligomer closes cyclically . Using cryo-electron microscopy and multivariate statistical image processing we report herein a direct structural observation of this effect . A sub-population (approximately 15%) of incomplete oligomers was found within a sample of SPP1 bacteriophage portal proteins embedded in vitreous ice . Whereas the curvature between adjacent subunits of the closed circular 13-fold symmetric oligomer is 27.7 degrees, in these incomplete oligomers the angle is only 25.8 degrees, a value which almost allows for a 14-subunit cyclical arrangement . A simple model for the association of large cyclical oligomers is suggested by our data.

Virology, 1996 Sep 15, 223(2), 303 - 17
Analysis of 76 kb of the chlorella virus PBCV-1 330-kb genome: map positions 182 to 258; Kutish GF et al.; Analysis of 76 kb of newly sequenced DNA, located between map positions 182 and 258 kb in the 330-kb chlorella virus PBCV-1 genome, revealed 175 open reading frames (ORFs) of 65 codons or longer . One hundred and five of these 175 ORFs were considered major ORFs . Twenty-one of the 105 major ORFs resembled proteins in databases including ribonucleotide reductase small subunit, RNase III, thioredoxin, glutaredoxin, protein disulfide isomerase, deoxynucleoside kinase, frog virus 3 ATPase, Acetobacter cellulose synthase, a bacteriophage encoded endonuclease, and two C-5 cytosine DNA methyltransferases . One of the ORFs was the PBCV-1 major capsid protein . The 105 major ORFs were evenly distributed along the genome . One set of ORFs was separated by 543 nucleotides whereas 75 of the ORFs were separated by fewer than 100 nucleotides . Nineteen of the 175 ORFs resembled other PBCV-1 ORFs, suggesting that they represent either gene duplications or gene families.

Genetika, 1996 Sep, 32(9), 1172 - 5
{Attenuation of bacteriophage phi80 repression}; Kozyrev DP et al.; Data are obtained suggesting the involvement of the cor gene of the {symbol: see text} 80 phage in regulation of phage operons . Mutations in the cor gene, which is located outside the region of phage {symbol: see text} 80 immunity, cause disturbances in the establishment and maintenance of the phage lysogenic state . The cor gene was shown to affect RecA-inactivation of the phage repressor induced by UV-irradiation . A model suggesting cor gene involvement in {symbol: see text} 80 operon regulation is discussed.

Bioorg Khim, 1996 Sep, 22(9), 664 - 70
{Phage mimotopes of monoclonal antibodies against plasma membrane Ca2+-ATPase}; Pestov NB et al.; Libraries of random phage-displayed pentadeca- and hexapeptides were screened with the use of four monoclonal antibodies against the human plasma membrane Ca2(+)-ATPase . Bacteriophages specifically binding the antibodies were selected, and the amino acid sequences of the expressed peptides (mimotopes) were determined . Mimotopes for three antibodies (8B8, 2D8, F9) did not correspond to the Ca2(+)-ATPase sequence . Pentadecapeptides for the 7C8 antibodies displayed similarity to the fragment Glu1097-Arg1113 of the Ca2(+)-ATPase calmodulin-binding site . However, these antibodies failed to bind recombinant fragment Leu1069-Leu1220; therefore, the structure of this epitope remains obscure . This work opens a series of studies of the plasma membrane Ca2(+)-ATPase structure by means of monoclonal antibodies and the phage display method.

Biologicals, 1996 Sep, 24(3), 243 - 53
A validatible porosimetric technique for verifying the integrity of virus-retentive membranes; Phillips MW et al.; The verification of membrane integrity for filtration processes specifically designed for the removal of adventitious virus from biotherapeutics is of the utmost importance to both biomanufacturers and regulatory agencies . Although conventional bubble-point and air-diffusion tests are widely accepted for integrity testing of bacteria-retentive membranes, these tests have severe limitations in their ability to assess the integrity of virus-retentive membranes . A novel membrane integrity test based upon liquid-liquid porosimetric principles (CorrTest) has been specifically designed to correlate and predict the virus retention capabilities of Viresolve virus removing membranes . To optimize test sensitivity for both Viresolve/70 and Viresolve/180 membrane types, two distinct porosimetric correlations at different transmembrane pressures have been developed . Based upon an 80% prediction interval, an integrity test performed at either test pressure can reliably predict the ability of Viresolve membranes to remove the bacteriophage phi X174 to within 0.4 log removal value (LRV) units . To maintain test sensitivity and provide greater flexibility for conducting the liquid-liquid intrusion integrity test, appropriate pressure- and temperature-correction equations have been established . The two immiscible fluids employed in the developed technology are easily flushed from the membrane structure and are generally regarded as acceptable, non-toxic reagents for pharmaceutical applications . Consequently, the CorrTest integrity test can reliably and non-destructively measure both pre- and post-use membrane integrity to verify virus removal performance with the Viresolve module.

Ultramicroscopy, 1996 Sep, 65(1-2), 81 - 93
Analysis of structural variability within two-dimensional biological crystals by a combination of patch averaging techniques and self organizing maps; Fernandez JJ et al.; We study in this work the use of self organizing maps to analyze the structural variability that can be found along two-dimensional crystals of biological macromolecules . Small areas of the crystals, termed "patches" by previous researchers, are used to obtain local average images that are then used as the input of a Self Organizing Map . This procedure allows for a fast and accurate image classification . Multivariate Statistical Analysis is then used on the resulting code vectors producing a very condensed data representation . This methodology is applied to previously studied crystals of bacteriophage phi 29 p10 connector, finding a crystalline heterogeneity probably associated to multilayers in some areas of the crystal.

Proteins, 1996 Sep, 26(1), 72 - 80
A comparative study of dynamic structures between phage 434 Cro and repressor proteins by normal mode analysis; Wako H et al.; Two DNA binding proteins, Cro and the amino-terminal domain of the repressor of bacteriophage 434 (434 Cro and 434 repressor) that regulate gene expression and contain a helix-turn-helix (HTH) motif responsible for their site-specific DNA recognition adopt very similar three-dimensional structures when compared to each other . To reveal structural differences between these two similar proteins, their dynamic structures, as examined by normal mode analysis, are compared in this paper . Two kinds of structural data, one for the monomer and the other for a complex with DNA, for each protein, are used in the analyses . From a comparison between the monomers it is found that the interactions of Ala-24 in 434 Cro or Val-24 in 434 repressor, both located in the HTH motif, with residues 44, 47, 48, and 51 located in the domain facing the motif, and the interactions between residues 17, 18, 28, and 32, located in the HTH motif, cause significant differences in the correlative motions of these residues . From the comparison between the monomer and the complex with DNA for each protein, it was found that the first helix in the HTH motif is distorted in the complex form . While the residues in the HTH motif in 434 Cro have relatively larger positive correlation coefficients of motions with other residues within the HTH motif, such correlations are not large in the HTH motif of 434 repressor . It is suggestive to their specificity because the 434 repressor is less specific than 434 Cro . Although a structural comparison of proteins has been performed mainly from a static or geometrical point of view, this study demonstrates that the comparison from a dynamic point of view, using the normal mode analysis, is useful and convenient to explore a difference that is difficult to find only from a geometrical point of view, especially for proteins very similar in structure.

Protein Sci, 1996 Sep, 5(9), 1833 - 43
Phage display of intact domains at high copy number: a system based on SOC, the small outer capsid protein of bacteriophage T4; Ren ZJ et al.; Peptides fused to the coat proteins of filamentous phages have found widespread applications in antigen display, the construction of antibody libraries, and biopanning . However, such systems are limited in terms of the size and number of the peptides that may be incorporated without compromising the fusion proteins' capacity to self-assemble . We describe here a system in which the molecules to be displayed are bound to pre-assembled polymers . The polymers are T4 capsids and polyheads (tubular capsid variants) and the display molecules are derivatives of the dispensable capsid protein SOC . In one implementation, SOC and its fusion derivatives are expressed at high levels in Escherichia coli, purified in high yield, and then bound in vitro to separately isolated polyheads . In the other, a positive selection vector forces integration of the modified soc gene into a soc-deleted T4 genome, leading to in vivo binding of the display protein to progeny virions . The system is demonstrated as applied to C-terminal fusions to SOC of (1) a tetrapeptide; (2) the 43-residue V3 loop domain of gp120, the human immunodeficiency virus type-1 (HIV-1) envelope glycoprotein; and (3) poliovirus VP1 capsid protein (312 residues) . SOC-V3 displaying phage were highly antigenic in mice and produced antibodies reactive with native gp120 . That the fusion protein binds correctly to the surface lattice was attested in averaged electron micrographs of polyheads . The SOC display system is capable of presenting up to approximately 10(3) copies per capsid and > 10(4) copies per polyhead of V3-sized domains . Phage displaying SOC-VP1 were isolated from a 1:10(6) mixture by two cycles of a simple biopanning procedure, indicating that proteins of at least 35 kDa may be accommodated.

Genetics, 1996 Sep, 144(1), 7 - 14
Destabilization of bacteriophage T4 mRNAs by a mutation of gene 61.5; Kai T et al.; We identified a novel gene of bacteriophage T4, gene 61.5, which appears to be involved in protein synthesis late in infection . Northern blot analysis revealed that a mutant of 61.5 accumulated truncated transcripts of representative late genes . Using a double mutant of genes 61.5 and 55, which prevents transcription of late genes, we demonstrate that even transcripts of middle genes, while full-length when initially expressed, are similarly truncated at later stages of infection . These results indicate that the abnormality in transcript length occurs late in infection, regardless of whether the transcript derives from a middle or a late gene . Primer-extension analysis revealed that the 5' ends of the late gene 23 transcripts that accumulated in gene 61.5 mutant-infected cells were located at internal discrete sites as well as at the expected transcription start site . Moreover, the decay rates of full-length transcripts from genes uvsY or 45 were more than twofold faster in the absence of a functional gene 61.5 . These results suggest that mutation of gene 61.5 activates endonucleolytic cleavage of middle and late transcripts, probably by RNase M.

Trends Genet, 1996 Sep, 12(9), 364 - 9
The coevolution of gene family trees; Fryxell KJ; Gene duplication mutants arise spontaneously at a high rate in bacteria, bacteriophages, insects and mammalian cells, and are generally viable . Thus, the rate-limiting step in the evolutionary process of gene duplication and divergence was probably not gene duplication per se . Rather, it is likely that only a small fraction of all duplicated genes were retained, and were able to diverge into new specificities . Furthermore, gene duplications and functionally related gene families often show similarities in divergence dates, functional specificities, and phylogenetic tree topologies . These correlations suggest that the family trees of functionally related gene families co-evolved because functionally complementary gene duplication and divergence events tended to be retained by natural selection.

J Leukoc Biol, 1996 Sep, 60(3), 397 - 404
Osteopontin inhibits nitric oxide production and cytotoxicity by activated RAW264.7 macrophages; Rollo EE et al.; Osteopontin (OPN), a secreted acidic phosphoglycoprotein found in many tissues and body fluids, is produced in increased amounts in response to certain infections and after malignant transformation . In this study we examined the action of OPN on macrophage cytotoxicity and nitric oxide (NO) synthesis . A human OPN cDNA was cloned into the bacteriophage T7-based vector, pET8C, and the encoded protein purified from an induced culture of Escherichia coli carrying the plasmid . Recombinant OPN inhibited NO production by macrophage-like RAW264.7 cells stimulated with lipopolysaccharide plus interferon-gamma . OPN also inhibited the cytolytic activity of the activated macrophages toward NO-sensitive P815 mastocytoma cells, an action that was blocked by the NO synthase inhibitor, NG-monomethyl-L-arginine . Inhibition of NO production correlated with an OPN-dependent decrease in the abundance of inducible NO synthase mRNA . The shape of the dose-response curve, with a maximal effect over a narrow range of OPN concentrations, suggested a complex interaction of OPN with cell surface receptors . Our data support the hypothesis that tumor-cell-derived OPN functions to protect the tumor cells from macrophage-mediated destruction.

Microbiology, 1996 Sep, 142 ( Pt 9), 2363 - 73
Gene transfer and transposition mutagenesis in Streptomyces roseosporus: mapping of insertions that influence daptomycin or pigment production; McHenney MA et al.; Streptomyces reseosporus, the producer of the cyclic lipopeptide antibiotic daptomycin, was shown to be a suitable host for molecular genetic manipulation . S . roseosporus does not appear to express significant restriction barriers based upon bacteriophage plaque formation studies . Plasmid DNA can be introduced into S . roseosporus by bacteriophage-FP43-mediated transduction and by conjugation from Escherichia coli . The streptomycete transposons Tn5096 and Tn5099, derived from IS493, transpose in S . roseosporus, and Tn5099-induced transposition mutants altered in the production of daptomycin, red pigment or black pigment were identified, and mapped to Dral and Asnl fragments . Three auxotrophic mutations (argB1, ade-1 and metB1) were identified among 100 individual Tn5096 insertions . Alignment and physical mapping of several Tn5099 insertions in Dral-E and Asnl-B fragments was facilitated by the presence of Dral and Asnl cleavage sites in Tn5099.

J Gen Virol, 1996 Sep, 77 ( Pt 9), 2109 - 119
Screening for membrane-permeabilizing mutants of the poliovirus protein 3AB; Lama J et al.; Synthesis of the poliovirus polypeptide 3AB in bacterial cells results in an increase in membrane permeability . The alterations observed resemble those elicited by bacteriophage lytic proteins, which are presumed to cause pore formation in biological membranes . This property has been exploited in the development of an in vivo screening system that allows morphological differentiation of Escherichia coli clones expressing either wild-type 3AB or variant 3AB proteins lacking the ability to permeabilize bacteria . Expression of the wild-type 3AB gene in the presence of a chromogenic beta-galactosidase substrate causes E . coli clones to stain dark blue . In contrast, bacterial mutants that synthesize 3AB proteins with alterations in the hydrophobic domain lack pore-forming activity and stain to a light blue colour, allowing differentiation from wild-type clones . This phenotypic property correlates with the rate of entry of the beta-galactosidase substrate into the bacteria . The method developed here was used to screen more than 8000 E . coli clones after random PCR mutagenesis of the poliovirus 3AB gene . Our results show the existence of three different domains involved in the permeabilizing activity of 3AB protein . Twenty individual amino acid substitutions were identified in clones that showed the mutant phenotype and such bacteria displayed different reduced levels of permeability towards ONPG, hygromycin B, lysozyme and uridine . The procedure reported here may be of general interest to understand structure-function relationships in other eukaryotic proteins known to form pores.

J Bacteriol, 1996 Sep, 178(18), 5513 - 21
Polynucleotide phosphorylase of Escherichia coli is required for the establishment of bacteriophage P4 immunity; Piazza F et al.; Bacteriophage P4's superinfection immunity mechanism is unique among those of other known bacteriophages in several respects: (i) the P4 immunity factor is not a protein but a short, stable RNA (CI RNA); (ii) in the prophage the expression of the replication operon is prevented by premature transcription termination rather than by repression of transcription initiation; (iii) transcription termination is controlled via RNA-RNA interactions between the CI RNA and two complementary target sequences on the nascent transcript; and (iv) the CI RNA is produced by processing of the same transcript it controls . It was thought that several host-encoded factors may participate in the molecular events required for P4 immunity expression, i.e., RNA processing, RNA-RNA interactions, and transcription termination . To identify such factors we searched for Escherichia coli mutations that affect P4 lysogenization . One such mutation, bfl-1, severely reduced P4's lysogenization frequency and delayed both the disappearance of the long transcripts that cover the entire replication operon and the appearance of the CI RNA . By physical mapping and genetic analysis we show that bfl-1 is allelic to pnp, which codes for polynucleotide phosphorylase, a 3'-to-5' exonucleolytic enzyme . A previously isolated pnp null mutant (pnp-7) exhibited a phenotype similar to that of bfl-1 . These results indicate that the polynucleotide phosphorylase of E . coli is involved with the maturation pathway of bacteriophage P4's RNA immunity factor.

Arch Biochem Biophys, 1996 Sep 1, 333(1), 207 - 13
Stability studies of nucleic acid-binding Fab isolated from combinatorial bacteriophage display libraries; Deutscher SL et al.; This study examined the global stability and activity properties of recombinant DNA-binding antibody fragments that were obtained from a bacteriophage combinatorial display library . The goal of this study was to determine whether the combinatorial approach of heavy and light chain assembly in E . coli and subsequent affinity selection preferentially selects for antibody fragments with unusual structural stabilities . Specifically, the binding properties and stability of recombinant antibody fragments with or without a C-terminal His tag to temperature, pH, and guanidine-HCI were examined . Both Fab exhibited almost identical Kd (120-130, 140-170, and 450-560 nM) and maximal fluorescence quenching (20-25%) values for binding to (dT)20, (dT)15, and (dT)10, respectively . Thermal denaturation data obtained by CD spectroscopy demonstrated that both Fab possessed structural properties comparable to well-folded proteins with defined tertiary structures which were stable below 70 degrees C (Tm 73 degrees C) . These results were confirmed by differential scanning calorimetry . Both Fab exhibited the same rate of irreversible thermal inactivation (0.061-0.069 min-1) at 75 degrees C and could be reversibly renatured from guanidine-HCI and pH extremes . Crystallization trials with one recombinant DNA-binding Fab yielded diffraction quality crystals also suggesting a well-defined tertiary structure.

Virology, 1996 Sep 1, 223(1), 57 - 67
A Rho-dependent transcription termination site regulated by bacteriophage P4 RNA immunity factor; Briani F et al.; The genes required for replication of the temperate bacteriophage P4, which are coded by the phage left operon, are expressed from a constitutive promoter (PLE) . In the lysogenic state, repression of the P4 replication genes is achieved by premature transcription termination . The leader region of the left operon encodes all the genetic determinants required for prophage immunity, namely: (i) the P4 immunity factor, a short, stable RNA (CI RNA) that is generated by processing of the leader transcript; (ii) two specific target sequences that exhibit complementarity with the CI RNA . RNA-RNA interactions between the CI RNA and the target sites on the mRNA leader region are essential for transcription termination . To understand how transcription termination is elicited by the P4 immunity mechanism, it is relevant to identify the transcription termination site . This, however, could not be directly inferred from the 3'-end of the transcription products because of the extensive and complex processing and degradation of the leader RNA . In this work, by making use of a tRNA gene as a reporter, we identify the termination site of the immunity transcripts (timm) . This is a Rho-dependent terminator located within the first translated gene of the left operon and is regulated by P4 immunity . Analysis of the P4 transcription pattern in Escherichia coli rho mutants suggests that termination at timm may also be important for the efficient processing of the CI RNA.

Nat Med, 1996 Sep, 2(9), 979 - 84
Clinical evidence of efficient tumor targeting based on single-chain Fv antibody selected from a combinatorial library; Begent RH et al.; We present a system for cancer targeting based on single-chain Fv (scFv) antibodies selected from combinatorial libraries, produced in bacteria and purified by using an engineered tag . Combinatorial libraries of scFv genes contain great diversity, and scFv antibodies with characteristics optimized for a particular task can be selected from them using filamentous bacteriophage . We illustrate the benefits of this system by imaging patients with carcinoembryonic antigen (CEA)-producing cancers using an iodine-123 labeled scFv anti-CEA selected for high affinity . All known tumor deposits were located, and advantages over current imaging technology are illustrated . ScFvs are produced in a cloned form and can be readily engineered to have localizing and therapeutic functions that will be applicable in cancer and other diseases.

Mol Biol Evol, 1996 Sep, 13(7), 1012 - 22
A molecular and evolutionary study of the beta-globin gene family of the Australian marsupial Sminthopsis crassicaudata; Cooper SJ et al.; Beta-globin gene families in eutherians (placental mammals) consist of a set of four or more developmentally regulated genes which are closely linked and, in general, arranged in the order 5'-embryonic/fetal genes-adult genes-3' . This cluster of genes is proposed to have arisen by tandem duplication of ancestral beta-globin genes, with the first duplication occurring 200 to 155 MYBP just prior to a period in mammalian evolution when eutherians and marsupials diverged from a common ancestor . In this paper we trace the evolutionary history of the beta-globin gene family back to the origins of these mammals by molecular characterization of the beta-globin gene family of the Australian marsupial Sminthopsis crassicaudata . Using Southern and restriction analysis of total genomic DNA and bacteriophage clones of beta-like globin genes, we provide evidence that just two functional beta-like globin genes exist in this marsupial, including one embryonic-expressed gene (S.c-epsilon) and one adult-expressed gene (S.c-beta), linked in the order 5'-epsilon-beta-3' . The entire DNA sequence of the adult beta-globin gene is reported and shown to be orthologous to the adult beta-globin genes of the North American marsupial Didelphis virginiana and eutherian mammals . These results, together with results from a phylogenetic analysis of mammalian beta-like globin genes, confirm the hypothesis that a two-gene cluster, containing an embryonic- and an adult-expressed beta-like globin gene, existed in the most recent common ancester of marsupials and eutherians . Northern analysis of total RNA isolated from embryos and neonatals indicates that a switch from embryonic to adult gene expression occurs at the time of birth, coinciding with the transfer of the marsupial from a uterus to a pouch environment.

J Mol Biol, 1996 Aug 30, 261(4), 524 - 35
Mutations affecting the high affinity ATPase center of gpA, the large subunit of bacteriophage lambda terminase, inactivate the endonuclease activity of terminase; Hwang Y et al.; Phage lambda terminase carries out the cos cleavage reaction that generates mature chromosomes from immature concatemeric DNA . The ATP-stimulated endonuclease activity of terminase is located in gpA, the large terminase subunit . There is a high affinity ATPase center in gpA, and a match to the conserved P-loop of known ATPases is found starting near residue 490 . Changing the conserved P-loop lysine at residue 497 of gpA affects the high affinity ATPase activity of terminase . In the present work, mutations causing the gpA changes K497A and K497D were found to be lethal, and phages carrying these mutations were defective in cos cleavage, in vivo . Purified K497A and K497D enzymes cleaved cos in vitro at rates reduced from the wild-type rate by factors of 1000 and 2000, respectively . The strong defects in cos cleavage are sufficient to explain the lethality of the K497A and K497D defects . In in vitro packaging studies using mature (cleaved) phage DNA, the K497A enzyme was indistinguishable from the wild-type enzyme, and the K497D enzyme showed a mild packaging defect under limiting terminase conditions . In a purified DNA packaging system, the wild-type and K497D enzymes showed similar packaging activities that were stimulated to half-maximal levels at about 3 microM ATP, indicating that the K497D change does not affect DNA translocation . In sum, the work indicates that the high affinity ATPase center of gpA is involved in stimulation of the endonuclease activity of terminase.

J Mol Biol, 1996 Aug 23, 261(3), 386 - 98
Efficient inhibition of transcription elongation in vitro by oligonucleotide phosphoramidates targeted to proviral HIV DNA; Giovannangeli C et al.; Triplex-forming oligophosphoramidates containing thymines and cytosines or 5-methyl cytosines (5' T4CT4C6T 3') bind strongly to a 16 basepair oligopurine.oligopyrimidine sequence of HIV proviral DNA even at neutral pH . These triple-helical complexes formed with oligonucleotide analogues with N3'-->P5' phosphoramidate linkages are remarkably stable compared to oligonucleotides with natural phosphodiester linkages . In transcription assays the (T,C)-containing phosphoramidate oligomers induce an efficient arrest of both bacteriophage and eukaryotic transcriptional machineries under conditions where the isosequential phosphodiesters have no inhibitory effect . In both cases the RNA polymerase (SP6, T7 or Pol II) is physically blocked by the non-covalent triplex and RNA synthesis is stopped at the triplex site . However the eukaryotic transcription machinery is blocked more efficiently (at submicromolar concentration) than the bacteriophage polymerases . The analysis of the 3'-ends of the truncated transcripts provides evidence for differences in the termination patterns induced by the triplex barrier for the bacteriophage and the eukaryotic systems . This in vitro comparative study provides the basis for the rational design of strong transcriptional inhibitors . The efficient in vitro inhibition obtained using the phosphoramidate oligomers in the eukaryotic transcription assay makes them good candidates for the development of sequence-specific antigene agents.

J Mol Biol, 1996 Aug 23, 261(3), 357 - 71
Ribosomal protein L9 interactions with 23 S rRNA: the use of a translational bypass assay to study the effect of amino acid substitutions; Adamski FM et al.; During translation of bacteriophage T4 gene 60 mRNA, ribosomes bypass 50 nucleotides with high efficiency . One of the mRNA signals for bypass is a stem-loop in the first part of the coding gap . When the length of this stem-loop is extended by 36 nucleotides, bypass is reduced to 0.35% of the wild-type level . Bypass is partially restored by a mutation in the C-terminal domain of Escherichia coli large ribosomal subunit protein L9 . Previous work has shown that L9 is an elongated protein with an alpha-helix that connects and orients the N and C-terminal domains that both contain a predicted RNA binding site . We have determined two binding sites of L9 on 23 S rRNA . A 778 nucleotide RNA fragment encompassing domain V (nucleotides 1999 to 2776) of the 23 S rRNA is retained on filters by L9 and contains both sites . The N and C-terminal domains of L9 were shown to interact with nucleotides just 5' to nucleotide 2231 and 2179 of the 23 S rRNA, respectively, using the toeprint assay . These L9 binding sites on 23 S rRNA suggest that L9 functions as a brace across helix 76 to position helices 77 and 78 relative to the peptidyl transferase center . In this study, bypass on a mutant gene 60 mRNA has been used as an assay to probe the importance of particular L9 amino acids for function . Amino acid substitutions in the C-terminal domain are shown to partially restore bypass . These mutant L9 proteins have reduced binding to a 23 S rRNA fragment (nucleotides 1999 to 2274) containing domain V, to which L9 binds . They partially retain both the N and C-terminal domain interactions . On the other hand, substitutions of amino acids in the N-terminal domain, which greatly reduce RNA binding, do not restore bypass . The latter mutants have completely lost the N-terminal domain interaction . Addition of an amino acid to the alpha-helix also restores gene 60 bypass . RNA binding by this mutant is similar to that observed for the C-terminal domain mutants that partially restore bypass.

J Biol Chem, 1996 Aug 23, 271(34), 20770 - 5
Bacteriophage T4 anaerobic ribonucleotide reductase contains a stable glycyl radical at position 580; Young P et al.; It has been recently recognized that the class III anaerobic ribonucleotide reductase requires the presence of a second activating gene product, NrdG . We have proposed that the role for NrdG involves the generation of an oxygen sensitive glycyl free radical within the NrdD enzyme . In this article we present the generation of such a glycyl free radical within the T4 NrdD subunit and its dependence upon the phage NrdG subunit . Initially, an overexpression system was created that allowed the joint production of T4 NrdD and T4 NrdG . With this system and in the presence of T4 NrdG, an oxygen-sensitive cleavage of NrdD was observed that mimicked the cleavage observed in phage infected Escherichia coli extracts . Under anaerobic conditions the presence of T4 NrdD with NrdG revealed a strong doublet EPR signal (g = 2.0039) . Isotope labeling of the NrdD with {2H}glycine and {13C}glycine, respectively, confirmed the presence of a stabilized glycine radical . The unpaired electron is strongly coupled to C-2 in glycine and the doublet splitting originates from one of the alpha-protons . The glycine residue at position 580 was determined to be the radical containing residue through site-directed mutagenesis studies involving a G580A NrdD mutant . The glycyl radical generation was specific for the T4 NrdG, and the host E . coli NrdG was found to be unable to activate the phage reductase . Finally, anaerobic purification revealed the holoenzyme complex to contain iron, whereas the NrdD polypeptide was found to lack the metal . Our results suggest a tetrameric structure for the T4 anaerobic ribonucleotide reductase containing one homodimer each of NrdD and NrdG, with a single glycyl radical present.

Proc Natl Acad Sci U S A, 1996 Aug 20, 93(17), 9073 - 8
Analysis of genetic instability during mammary tumor progression using a novel selection-based assay for in vivo mutations in a bacteriophage lambda transgene target; Jakubczak JL et al.; Genetic instability is thought to be responsible for the numerous genotypic changes that occur during neoplastic transformation and metastatic progression . To explore the role of genetic instability at the level of point mutations during mammary tumor development and malignant progression, we combined transgenic mouse models of mutagenesis detection and oncogenesis . Bitransgenic mice were generated that carried both a bacteriophage lambda transgene to assay mutagenesis and a polyomavirus middle T oncogene, mammary gland-targeted expression of which led to metastatic mammary adenocarcinomas . We developed a novel assay for the detection of mutations in the lambda transgene that selects for phage containing forward mutations only in the lambda cII gene, using an hfl- bacterial host . In addition to the relative ease of direct selection, the sensitivity of this assay for both spontaneous and chemically induced mutations was comparable to the widely used mutational target gene, lambda lacI, making the cII assay an attractive alternative for mutant phage recovery for any lambda-based mouse mutagenesis assay system . The frequencies of lambda cII- mutants were not significantly different in normal mammary epithelium, primary mammary adenocarcinomas, and pulmonary metastases . The cII mutational spectra in these tissues consisted mostly of G/C-->A/T transitions, a large fraction of which occurred at CpG dinucleotides . These data suggest that, in this middle T oncogene model of mammary tumor progression, a significant increase in mutagenesis is not required for tumor development or for metastatic progression.

Proc Natl Acad Sci U S A, 1996 Aug 20, 93(17), 8868 - 72
Dual regulation of open-complex formation and promoter clearance by Arc explains a novel repressor to activator switch; Smith TL et al.; In studies of variants of the P(ant) promoter of bacteriophage P22, the Arc protein was found not only to slow the rate at which RNA polymerase forms open complexes but also to accelerate the rate at which the enzyme clears the promoter . These dual activities permit Arc, bound at a single operator subsite, to act as an activator or as a repressor of different promoter variants . For example, Arc activates a P(ant) variant for which promoter clearance is rate limiting in the presence and absence of Arc but represses a closely related variant for which open-complex formation becomes rate limiting in the presence of Arc . The acceleration of promoter clearance by Arc requires occupancy of the operator subsite proximal to the -35 region and is diminished when Arc bears a mutation in Arg-23, a residue that makes a DNA-backbone contact in the operator complex.

Mutat Res, 1996 Aug 17, 355(1-2), 13 - 40
DNA adducts from chemotherapeutic agents; Lawley PD et al.; The guiding principle of early work was the hypothesis that the anti-cancer alkylating drugs acted through their ability to cross-link macromolecules essential for cell division . Not long afterwards, DNA was specified as the essential target, and support for the hypothesis came from evidence that the archetypal agent, mustard gas, could link guanine bases in DNA through their N-7 atoms . Quantitative correlations between alkylation of DNA and its inactivation as a template followed, with bacteriophage as a simple test object, showing that the mean lethal dose was close to a single cross-link in the genome . This conclusion applied to either mustard gas or the more recently introduced platinum drugs . Although both inter- and intra-strand cross-links were effective, it was thought that in cells the inter-strand cross-link would, by preventing the separation of the strands necessary for cell division, and by being more difficult to repair, constitute the more effectively lethal lesion . With repair-deficient bacteria, it also emerged that a single cross-link in the genome was lethal, but proficient bacteria could remove about 20 cross-links through excision repair . Mono-7-alkylguanines were not removed and were evidently inert . Thus, only a few percent of the total alkylation products were the most effective lesions . Parallel studies with cultured mammalian cells gave a rather different picture, in that the mean lethal doses of even hypersensitive cell lines were around 20 or more cross-links per genome, about the same as for resistant strains of bacteria . Most cells could withstand several hundreds of cross-links per genome, and although adducts were removed, there was incomplete removal of cross-links . Some, but not all, sensitive cell lines were deficient in excision repair . Methods were devised for measuring the extents of alkylation of DNA in cells of patients treated with chemotherapeutic drugs; these are mainly immunoassays, and were applied generally to peripheral blood leukocytes, although some tumours were studied . Extents of alkylation of leukocyte DNA were generally of the same order as, or rather less than the mean lethal doses of cultured cells of the 'normal' type, but in some reports for cisplatin-treated patients, very wide variability between individuals was found . A positive correlation between adduct levels, and particularly a very minor adduct recognised specifically by one antibody, and favourable therapeutic outcome was discerned, and suggested to have a pharmacogenetic basis . In several instances, extents of alkylation of tumours were significantly higher than the average for leukocytes; for ovarian and a testicular tumour for cisplatin, and for a plasma cell tumour for melphalan . Nevertheless, these favourable examples would not constitute more than three or four mean lethal doses in the tumour cells, assuming that they had the same sensitivity as 'normal' cell lines: the therapeutic effect would of course be much more favourable if the tumour cells resembled 'sensitive' cell lines . This lack of a favourable difference between extents of alkylation in DNA of patients and the mean lethal dose for normal cells was particularly obvious with the methylating drugs dacarbazine and procarbazine . These considerations stress the need for higher extents of alkylation to be achieved in target tumour DNA for successful chemotherapy . One approach is to give a higher overall dose, and to 'rescue' the bone marrow (known from the earliest report on mustard gas to be the most susceptible tissue) by autologous transplantation . The second, which has yet to reach the clinic, is to convert unreactive prodrugs through enzymic activation into alkylating agents specifically in tumours (see Bagshawe, 1994).

J Biol Chem, 1996 Aug 16, 271(33), 20198 - 207
The gene 59 protein of bacteriophage T4 . Characterization of protein-protein interactions with gene 32 protein, the T4 single-stranded DNA binding protein; Morrical SW et al.; The gene 59 protein (gp59) of bacteriophage T4 stimulates the activities of gene 41 protein (gp41), the T4 replicative DNA helicase, by promoting the assembly of gp41 onto single-stranded (ss)-DNA molecules that are covered with cooperatively bound gene 32 protein (gp32) . This helicase-ssDNA assembly process, which is important for the reconstitution of the primosome component of the T4 DNA replication fork, appears to require both gp59-gp41 and gp59-gp32 protein-protein interactions . In this study we characterize the physical and functional interactions of gp59 with gp32, the T4 ssDNA-binding protein . Experimental results presented herein indicate: 1) that gp59 binds specifically to both free and ssDNA-bound gp32 molecules; and 2) that in both cases binding involves contacts between gp59 and the acidic C-terminal domain of gp32 (the so-called "A-domain") . We further show that single-stranded DNA molecules coated with (gp32-A), a truncated form of gp32 lacking the A-domain, are refractory to gp59-dependent helicase assembly . The data indicate that specific contacts between gp59 molecules and the A-domains of gp32 molecules are essential for gp59-dependent assembly of gp41 onto gp32-ssDNA complexes . Our results are consistent with a model in which gp59 binds to gp32 molecules within the gp32-ssDNA complex and therein forms a target site for helicase-ssDNA assembly.

J Biol Chem, 1996 Aug 16, 271(33), 20088 - 95
Replication of O6-methylguanine-containing DNA by repair and replicative DNA polymerases; Reha-Krantz LJ et al.; The biological consequences of O6-methylguanine (m6G) in DNA are well recognized . When template m6G is encountered by DNA polymerases, replication is hindered and trans-lesion replication results in the preferential incorporation of dTMP opposite template m6G . Thus, unrepaired m6G in DNA is both cytotoxic and mutagenic . Yet, cell lines tolerant to m6G in DNA have been isolated, which indicates that some cellular DNA polymerases may replicate m6G-containing DNA with reasonable efficiency . Previous reports suggested that mammalian pol beta could not replicate m6G-containing DNA, but we find that pol beta can catalyze trans-lesion replication; however, the lesion must reside in the optimal context for pol beta activity, single- or short nucleotide gapped substrates . Primed single-stranded DNA templates, with or without template m6G, were poor substrates for pol beta as reported in earlier studies . In contrast, trans-lesion replication by bacteriophage T4 DNA polymerase was observed for primed single-stranded DNA templates . Replication of m6G-containing DNA by T4 DNA polymerase required the gp45 accessory protein that clamps the polymerase to the DNA template . The rate-limiting step in replicating m6G-containing DNAs by both DNA polymerases tested was incorporation of dTMP across from the lesion.

J Biol Chem, 1996 Aug 16, 271(33), 19999 - 20008
Amino acid residues critical for the interaction between bacteriophage T7 DNA polymerase and Escherichia coli thioredoxin; Himawan JS et al.; Upon infection of Escherichia coli, bacteriophage T7 annexes a host protein, thioredoxin, to serve as a processivity factor for its DNA polymerase, T7 gene 5 protein . In a previous communication (Himawan, J., and Richardson, C . C . (1992) Proc . Natl . Acad . Sci . U . S . A . 89, 9774-9778), we reported that an E . coli strain encoding a Gly-74 to Asp-74 (G74D) thioredoxin mutation could not support wild-type T7 growth and that in vivo, six mutations in T7 gene 5 could individually suppress this G74D thioredoxin defect . In the present study, we report the purification and biochemical characterization of the G74D thioredoxin mutant and two suppressor gene 5 proteins, a Glu-319 to Lys-319 (E319K) mutant of gene 5 protein and an Ala-45 to Thr-45 (A45T) mutant . The suppressor E319K mutation, positioned within the DNA polymerization domain of gene 5 protein, appears to suppress the parental thioredoxin mutation by compensating for the binding defect that was caused by the G74D alteration . We suggest that the Glu-319 residue of T7 gene 5 protein and the Gly-74 residue of E . coli thioredoxin define a contact point or site of interaction between the two proteins . In contrast, the A45T mutation in gene 5 protein, located within the 3' to 5' exonuclease domain, does not suppress the G74D thioredoxin mutation by simple restoration of binding affinity . Based upon our understanding of the mechanisms of suppression, we propose a model for the T7 gene 5 protein-E . coli thioredoxin interaction.

EMBO J, 1996 Aug 15, 15(16), 4414 - 22
Dynamics of DNA-tracking by two sliding-clamp proteins; Fu TJ et al.; Bacteriophage T4 gene 45 protein (gp45) and Escherichia coli beta are DNA-tracking sliding-clamp proteins that increase processivity by tethering their conjugate DNA polymerases to DNA . gp45 also activates T4 late transcription . DNA loading of gp45 and beta requires ATP or dATP hydrolysis; efficient loading at primer-template junctions is assisted by single-stranded DNA-binding proteins . The kinetics of gp45 loading and tracking have been examined by DNase I footprinting of linear DNA with one blunt end, one primer-template junction, and binding sites for proteins that block gp45 tracking . DNA loading of gp45 can also be interrupted by adding the non-hydrolyzable ATP analog ATP-gamma-S . At saturation, DNA is very closely packed with gp45 or beta . When gp45 loading is interrupted, or when a segment of the track is blocked off, the gp45 footprint dissipates within seconds, but the DNA-tracking state of beta is much more stable . The stability of the tracking state of gp45 is, however, increased by the macromolecular crowding agent polyethylene glycol . We suggest that labile gp45 catenation directly generates the coupling of late transcription to DNA replication during bacteriophage T4 multiplication.

Nucleic Acids Res, 1996 Aug 15, 24(16), 3173 - 80
Preparation of oligoribonucleotides containing 4-thiouridine using Fpmp chemistry . Photo-crosslinking to RNA binding proteins using 350 nm irradiation; McGregor A et al.; The preparation of a 4-thiouridine phosphoramidite suitable for RNA synthesis and its subsequent incorporation into oligoribonucleotides is described . The thiol group is protected with a 2-cyanoethyl group and the 2'-OH with a 1-(2-fluorophenyl)-4-methoxypiperidin-4-yl function . Thiouridine-containing oligoribonucleotides were used as 350 nm UV crosslinking probes for the photoaffinity labelling of RNA binding proteins . Specific crosslinking was demonstrated between the Rev protein of HIV-1 (as a glutathione S-transferase fusion protein) and its RNA target, the Rev-responsive element . It was not possible to generate crosslinks between the RNA bacteriophage MS2 coat protein and the initiator stem-loop of the replicase gene, to which it binds . These results are consistent with the structural data available on both systems.

Biochem J, 1996 Aug 15, 318 ( Pt 1), 75 - 84
Characterization of the rat glutathione S-transferase Yc2 subunit gene, GSTA5: identification of a putative antioxidant-responsive element in the 5'-flanking region of rat GSTA5 that may mediate chemoprotection against aflatoxin B1; Pulford DJ et al.; We have isolated and characterized genomic DNA encoding the rat glutathione S-transferase Yc2 subunit . This protein is now referred to as rGSTA5 and is noteworthy because of its high activity towards aflatoxin B1-8,9-epoxide, its marked inducibility by chemoprotectors, its sex-specific regulation, and its over-expression in hepatoma and preneoplastic nodules . The rGSTA5 gene, which was isolated on two overlapping bacteriophage lambda clones, is approx . 12 kb in length and, unlike other class Alpha genes described to date, it comprises six exons . The transcription start site has been identified 228 bp upstream from the ATG translational initiation codon, and is situated 51 bp downstream from a consensus TATA-box . Deletion analysis, using luciferase reporter constructs, has shown that the region between -177 bp and +65 bp from the transcriptional start site contains a functional promoter . Computer-assisted analysis of the upstream sequence has indicated the presence of an antioxidant-responsive element (ARE), and several elements thought to be required for tissue-specific expression of the enzyme . In addition, several putative oestrogen-responsive half sites were observed in both upstream and intronic sequences.

Biochemistry, 1996 Aug 13, 35(32), 10539 - 48
Specific inhibition of in vitro transcription elongation by triplex-forming oligonucleotide-intercalator conjugates targeted to HIV proviral DNA; Giovannangeli C et al.; A 16-base pair oligo(purine)-oligo(pyrimidine) sequence present in the coding region of two HIV 1 proviral genes (pol and nef) was chosen as a target for triplex-forming oligonucleotides in in vitro transcription assays . Inhibition of transcription elongation was observed with triplex-forming oligonucleotide-acridine conjugates (Acr-15-TCG:5'-Acr-T4CT4G6-3' and Acr-9-TC:5'-Acr-T4CT4-3' where C is 5-methylcytosine) under conditions where the unsubstituted oligomers did not exhibit any inhibitory effect . Both SP6 bacteriophage RNA polymerase and eukaryotic RNA polymerase II were physically blocked by such a triplex barrier . The polymerase arrest is caused by the triple-helical complex involving the hydrogen-bonded oligonucleotide stabilized by the intercalated moiety and not solely by the acridine molecule specifically intercalated at the duplex-triplex junction . The stability of the triple-helical complex formed by the 15-mer containing thymines, cytosine, and guanines (15-TCG) and involving the formation of six contiguous C.GxG base triplets was strongly enhanced in the presence of a benzopyridoindole derivative (BePI), which intercalates in triplex structures . This improvement of the binding affinity led to an increased inhibition of transcription elongation . The present results demonstrate the necessity to use triplex-forming oligonucleotides with high binding affinity and a long residence time on their double-stranded target to efficiently inhibit transcription elongation . These data provide a rational basis for the optimization and the development of triplex-forming oligonucleotides as transcriptional blockers, even when they are targeted to the transcribed portion of a gene, downstream of the transcription initiation site.

Biochemistry, 1996 Aug 13, 35(32), 10472 - 83
Biophysical characterization of wild-type and mutant bacteriophage IKe major coat protein in the virion and in detergent micelles; Williams KA et al.; Interactions between the filamentous bacteriophage major coat protein and its environment differ markedly between the membrane-bound assembly intermediate which spans the lipid bilayer and the phage coat protein which makes up the capsid of the virion . Nonetheless, both reflect successful strategies to sequester the hydrophobic regions of the coat protein away from the aqueous milieu . To characterize the roles of individual residues in the conformation, stability, and oligomerization of the coat protein in both the virion and in detergent micelles, wild-type IKe and M13 coat proteins, together with a library of over 40 IKe coat protein mutants, were studied using circular dichroism (CD), fluorescence, and solution nuclear magnetic resonance (NMR) spectroscopies . The largely helical conformations of coat protein in IKe wild-type and mutant virions were found to be very similar by CD, demonstrating that the overall organization of the phage can accommodate a diverse range of amino acid substitutions in the major coat protein . Intrinsic Trp fluorescence showed that the polarity of the Trp 29 environment in the virion was modulated by residues within one helical turn of this locus . Characterization of IKe phage growth and plaquing properties highlighted the importance of Pro 30 in maintaining viability . As well, the Pro 30 mutants were the only substitutions which rendered the detergent-solubilized coat protein less thermostable and additionally altered the polarity of the Trp 29 environment . The Pro 30 Gly mutant exhibited numerous 1H and 15N chemical shift changes between residues Ile 25 and Ala 38 in the 2D 1H-15N HSQC spectrum in myristoyllysophosphatidylglycerol (MPG) micelles, demonstrating that the effect of the substitution is propagated beyond adjacent residues . The overall results highlight the stabilizing effect of Pro in the first turn of a transmembrane helix and the importance of hydrophobicity in modulating the oligomerization and stability of coat protein both in the phage and in detergent micelles.

Biochemistry, 1996 Aug 13, 35(32), 10383 - 91
Accessibility and environment probing using cysteine residues introduced along the putative transmembrane domain of the major coat protein of bacteriophage M13; Spruijt RB et al.; The major coat protein of the filamentous bacteriophage M13 is located in the inner membrane of host cell Escherichia coli prior to assembly into virions . To identify the transmembrane domain of the coat protein, we have introduced unique cysteine residues along the putative transmembrane domain at position 25, 31, 33, 36, 38, 46, 47, 49, or 50 . The mutant major coat protein was solubilized by membrane-mimicking detergents or reconstituted into mixed bilayers of phospholipids . Information about the environmental polarity was deduced from the wavelength of maximum emission, using N-{{(iodoacetyl)-amino)ethyl}-1-sulfonaphthylamine (IAEDANS) attached to the SH groups of the cysteines as a fluorescent probe . Additional information was obtained by determining the accessibility of AEDANS for the fluorescence quencher molecules acrylamide and 5-doxylstearic acid, and the reactivity of the cysteine's sulfhydryl group toward 5,5'-dithiobis(2-nitrobenzoic acid) (DTNB) . Our data suggest transmembrane boundaries close to residue 25 and 46, with residue 25 inside the hydrophobic part of the membrane in very close proximity to the membrane-water interface and residue 46 located at the membrane-water interface . Domains of the mutant coat protein which are packed or coated by cholate molecules and various other detergents {except for sodium dodecyl sulfate (SDS)} are at least similarly packed by phospholipid molecules in bilayers . SDS is a good solubilizing detergent but badly mimics the typical nature of a membrane structure . The overall results are interpreted with respect to the established conformation of the coat protein and its membrane anchoring mechanism.

J Biol Chem, 1996 Aug 9, 271(32), 19625 - 31
The ATP-activated hexameric helicase of bacteriophage T4 (gp41) forms a stable primosome with a single subunit of T4-coded primase (gp61); Dong F et al.; We have examined the formation of the primosome subassembly of the bacteriophage T4-coded DNA replication (elongation) complex from its helicase, primase, and DNA components . Previously, we had shown that the T4 helicase (gp41) exists in solution in a stable monomer left and right arrow dimer equilibrium at physiological protein (and salt) concentrations and forms a hexamer upon activation by ATP (or GTP) binding (Dong, F., Gogol, E . P., and von Hippel, P . H.(1995) J . Biol . Chem . 270, 7462-7473) . Here we report that the T4 primase (gp61) is a monomer in solution under the same conditions, and that the ATP-activated helicase binds to a single gp61 primase molecule on appropriate DNA templates to reconstitute a stable primosome . We show that: (i) the gp41 helicase alone does not form a stable complex with DNA templates, although this helicase by itself can carry out moderately processive ATP-driven translocation along single-stranded DNA (Young, M . C., Schultz, D . E., Ring, D., and von Hippel, P . H.(1994) J . Mol . Biol . 235, 1447-1458); (ii) the primase alone does form a stable complex with DNA; (iii) the helicase can bind to the primase-DNA complex in the presence of ATP or GTP to form a stable ternary complex; (iv) this complex consists of six helicase subunits and one primase subunit; and (v) the reconstituted primosome is stable for at least 10 to 20 min after NTP cleavage and dissociation of the hydrolysis products . These results strongly suggest that the functional T4 DNA replication primosome consists of an integrated 6:1 helicase-primase complex bound to DNA, and that the ATP-activated helicase hexamer remains intact throughout the processive DNA replication process.

J Biol Chem, 1996 Aug 9, 271(32), 19578 - 84
Recombinant decorin glycoforms . Purification and structure; Ramamurthy P et al.; The vaccinia virus/T7 bacteriophage expression system was used to express human decorin in HT-1080 cells by co-infection with vTF7-3, encoding T7 RNA polymerase, and vDCN1, encoding the decorin core protein fused to a polyhistidine-insulin signal sequence fusion-protein cassette . Overexpression using the vaccinia virus/T7 phage system resulted in secretion of approximately 30 mg of decorin/10(9) cells per 24 h which enabled purification and separation of multiple glycoforms under native conditions . Cells were cultured in the presence of {35S}methionine or a mixture of {3H}glucosamine and {35S}sulfate, and recombinant glycoprotein purified by metal affinity chromatography which resolved the secreted decorin into two classes, a proteoglycan form and a core protein form . About 25% of the recombinant protein was secreted into the culture medium as core protein devoid of glycosaminoglycan chains . The decorin core protein was resolved into two forms (approximately 49 and approximately 53 kDa) that differed in the extent of N-linked oligosaccharide substitution (2 and 3 N-linked oligosaccharides, respectively) . Deglycosylation of the recombinant proteoglycans and core proteins resulted in a single band migrating with an apparent molecular mass approximately 43 kDa when analyzed by SDS-polyacrylamide gel electrophoresis . Far-UV circular dichroism spectra of native decorin proteoglycan showed a minima at 218 nm, consistent with a secondary structure that is predominantly beta-sheet . Circular dichroism spectra of bovine decorin extracted from articular cartilage and recombinant decorin similarly treated revealed a minima of 205 nm indicating a loss of secondary structure . The affinity of decorin proteoglycan and core protein for collagen-like molecules was demonstrated, with the complement component C1q exhibiting the most striking affinity for decorin, although adherence to collagen types I and V was also observed . The extensive secondary structure maintained in the purified recombinant protein is likely to be important for the biological function of decorin.

J Biol Chem, 1996 Aug 9, 271(32), 19571 - 7
Eukaryotic expression of recombinant biglycan . Post-translational processing and the importance of secondary structure for biological activity; Hocking AM et al.; Biglycan is a small chondroitin sulfate proteoglycan found in many tissues and is structurally related to decorin, fibromodulin, and lumican . The biological function of biglycan is poorly understood, although several studies have indicated interaction with other extracellular matrix components . We have initiated studies of structural and functional domains of biglycan by transient eukaryotic expression using the vaccinia virus/T7 bacteriophage expression system . A recombinant vaccinia virus, vBGN4 encoding the mature biglycan core protein as a polyhistidine fusion protein under control of the T7 phage promoter was expressed in HT-1080 cells and UMR106 cells . The structure of the recombinant biglycan secreted by these cells was defined by analyzing molecules labeled in the presence of {35S}sulfate, {3H}glucosamine, and {35S}methionine . Glycoforms of biglycan were separated by imidazole gradient elution, under non-denaturing conditions, and comprised: a large proteoglycan form substituted with two chondroitin sulfate chains of molecular mass approximately 34 kDa (HT-1080 cells) or approximately 40 kDa (UMR106 cells); a small proteoglycan form substituted with two chondroitin sulfate chains with a median molecular mass approximately 28 kDa; and a core protein form secreted devoid of glycosaminoglycan chains . All the glycoforms were substituted with two N-linked oligosaccharides, and the disaccharide composition of the two glycosaminoglycan populations were identical . Approximately 70% of the recombinant biglycan secreted by HT-1080 cells was substituted with chondroitin sulfate chains, whereas about 50% of the biglycan expressed by UMR106 cells was substituted with chondroitin sulfate chains . Infection with vBGN4 in both HT-1080 and UMR106 cells resulted in the production of approximately 10 mg of biglycan/10(9) cells per 24 h . The native recombinant biglycan was shown to bind to collagen type V and the complement protein, C1q . However, when the secondary structure of recombinant biglycan was disrupted by exposure to 4 M guanidine hydrochloride, the affinity for collagen type V was dramatically reduced . These data demonstrate the importance of secondary structure to the function of this small proteoglycan.

J Biol Chem, 1996 Aug 9, 271(32), 19563 - 70
The role of the zinc motif in sequence recognition by DNA primases; Kusakabe T et al.; The DNA primase of bacteriophage T7 has a zinc-binding motif that is essential for the recognition of the sequence 3'-CTG-5' . The T7 primase also catalyzes helicase activity, a reaction coupled to nucleotide hydrolysis . We have replaced the zinc motif of the T7 primase with those found in the gene 61 primase of phage T4 and the DnaG primase of Escherichia coli . The T4 and E . coli primases recognize the sequences 3'-T(C/T)G-5' and 3'-GTC-5', respectively . Both chimeric proteins can partially replace T7 primase in vivo . The two chimeric primases catalyze the synthesis of oligoribonucleotides albeit at a reduced rate and DNA dependent dTTPase activity is reduced by 3-10-fold . Both chimeric proteins recognize 3'-(A/G)CG-5' sites on single-stranded DNA, sites that differ from those recognized by the T7, T4, or E . coli primases, indicating that the zinc motif is only one determinant in site-specific recognition.

J Mol Biol, 1996 Aug 2, 260(5), 767 - 80
Stoichiometry and domainal organization of the long tail-fiber of bacteriophage T4: a hinged viral adhesin; Cerritelli ME et al.; The long-tail fibers (LTFs) form part of bacteriophage T4's apparatus for host cell recognition and infection, being responsible for its initial attachment to susceptible bacteria . The LTF has two parts, each approximately 70 to 75 nm long; gp34 (140 kDa) forms the proximal half-fiber, while the distal half-fiber is composed of gp37 (109 kDa), gp36(23 kDa) and gp35 (30 kDa) . LTFs have long been thought to be dimers of gp34, gp37 and gp36, with one copy of gp35 . We have used mass mapping by scanning transmission electron microscopy (STEM), quantitative SDS-PAGE, and computational sequence analysis to study the structures of purified LTFs and half-fibers of both kinds . These data establish that the LTF is, in fact, trimeric, with a stoichiometry of gp34: gp37: gp36: gp35 = 3:3:3:1 . Averaged images of stained and unstained molecules resolve the LTF into a linear stack of 17 domains . At the proximal end is a globular domain of approximately 145 kDa that becomes incorporated into the baseplate . It is followed by a rod-like shaft (33 x 4 mm; 151 kDa) which correlates with a cluster of seven quasi repeats, each 34 to 39 residues long . The proximal half-fiber terminates in three globular domains . The distal half-fiber consists of ten globular domains of variable size and spacing, preceding a needle-like end domain (15 x 2.5 nm; 31 kDa) . The LTF is rigid apart from hinges between the two most proximal domains, and between the proximal and distal half-fibers . The latter hinge occurs at a site of local non-equivalence (the "kneecap") at which density, correlated with the presence of gp35, bulges asymmetrically out on one side . Several observations indicate that gp34 participates in the sharing of conserved structural modules among coliphage tail-fiber genes to which gp37 was previously noted to subscribe . Two adjacent globular domains in the proximal half-fiber match a pair of domains in the distal half-fiber, and the rod domain in the proximal half-fiber resembles a similar domain in the T4 short tail-fiber (gp12) . Finally, possible structures are considered; combining our data with earlier observations, the most likely conformation for most of the LTF is a three-stranded beta-helix.

J Mol Biol, 1996 Aug 2, 260(5), 678 - 96
T4 endonuclease VII selects and alters the structure of the four-way DNA junction; binding of a resolution-defective mutant enzyme; Pohler JR et al.; Bacteriophage T4 endonuclease VII is a nuclease that is selective for four-way DNA junctions and related branched DNA species . Using site-directed mutagenesis we have isolated a mutant protein (E86A) that is inactive in the cleavage of DNA junctions while retaining full selectivity of binding . Using endonuclease VII E86A we have shown: (1) The protein binds as a dimer to DNA junctions, with rapid exchange of subunits in free solution . (2) Binding to junctions is highly selective for the structure of DNA junctions; the complex is not displaced by a 1000-fold excess of duplex competitor DNA . (3) On binding endonuclease VII E86A to junctions, the configuration of the helical arms is significantly altered to a structure that is independent of the presence or absence of metal ions . We suggest a model for the structure of the junction in the protein complex . (4) The protein can bind to the junction in two stereochemically equivalent ways, depending upon the sequence of the junction . T4 endonuclease VII is a junction-selective enzyme that both recognises and manipulates the structure of its substrate.

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