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 transcr