Phage

Nucleic Acids Res, 1995 Mar 25, 23(6), 1075 - 82
Deposition of histone H1 onto reconstituted nucleosome arrays inhibits both initiation and elongation of transcripts by T7 RNA polymerase; O'Neill TE et al.; The effect of histone H1 on transcription by bacteriophage T7 RNA polymerase was examined using reconstituted chromatin templates . A 3.8 kb linear DNA template consisting of a specific transcription promoter for T7 RNA polymerase placed upstream of 18 tandem repeats of a 207 bp nucleosome positioning sequence derived from the 5S rRNA gene of Lytechinus variegatus was used as a template for chromatin reconstitution . Regularly spaced arrays of nucleosome cores were assembled onto this DNA template from donor histone octamers by salt step dialysis . Histone H1 was incorporated onto free DNA or reconstituted chromatin templates and double label transcription assays were performed . The experiments indicated that histone H1 has a strong inhibitory effect on both transcription initiation and elongation . These effects are especially pronounced on chromatin templates, where both transcription initiation and elongation are virtually halted . The inhibition of transcription elongation appears to result from a dramatic increase in premature termination of transcripts . These experiments indicate that assembly of histone H1 into chromatin can result in structures which are completely repressed with respect to transcription.

J Biol Chem, 1995 Mar 24, 270(12), 6440 - 9
Rapid identification of highly active and selective substrates for stromelysin and matrilysin using bacteriophage peptide display libraries; Smith MM et al.; The discovery of useful peptide substrates for proteases that recognize many amino acids in their active sites is often a slow process due to the lack of initial substrate data and the expense of analyzing large numbers of peptide substrates . To overcome these obstacles, we have made use of bacteriophage peptide display libraries . We prepared a random hexamer library in the fd-derived vector fAFF-1 and included a "tether" sequence that could be recognized by monoclonal antibodies . We chose the matrix metalloproteinases stromelysin and matrilysin as the targets for our studies, as they are known to require at least 6 amino acids in a peptide substrate for cleavage . The phage library was treated in solution with protease and cleaved phage separated from uncleaved phage using a mixture of tether-binding monoclonal antibodies and Protein A-bearing cells followed by precipitation . Clones were screened by the use of a rapid screening assay that identified phage encoding peptide sequences susceptible to cleavage by the enzymes . The nucleotide sequence of the random hexamer region of 43 such clones was determined for stromelysin and 23 for matrilysin . Synthetic peptides were prepared whose sequences were based on some of the positive clones, as well as consensus sequences built from the positive clones . Many of the peptides have kcat/KM values as good or better than those of previously reported substrates, and in fact, we were able to produce stromelysin and matrilysin substrates that are both the most active and smallest reported to date . In addition, the phage data predicted selectivity in the P2 and P'1 positions of the two enzymes that were supported by the kinetic analysis of the peptides . This work demonstrates that the phage selection techniques enable the rapid identification of highly active and selective protease substrates without making any a priori assumptions about the specificity or the "physiological substrate" of the protease under study.

Gene, 1995 Mar 21, 155(1), 61 - 5
Evaluation of antibodies fused to minor coat protein III and major coat protein VIII of bacteriophage M13; Kretzschmar T et al.; A gene coding for an anti-(2-phenyl-5-oxazolone) single-chain Fv antibody (Ab) fragment (anti-phOx scFv) was cloned in-frame into phagemid vectors upstream from genes encoding (i) the wild-type (wt) minor coat protein (cp) III of the filamentous bacteriophage M13 of Escherichia coli, (ii) a truncated version of cpIII (amino acid (aa) positions 198-406), (iii) the wt major cpVIII, or (iv) a hybrid of interleukin-1 beta (IL-1 beta; aa 10-152) and wt cpVIII . Recombinant (re-) phage obtained by phagemid rescue were examined for the efficiency of displaying these various anti-phOx scFv::cp hybrids with commercially available anti-M13 enzyme-linked immunosorbent assays (ELISA), by immunoblotting with an anti-c-myc Ab, and by selection experiments . We found that the highest ELISA signals were obtained with the cpIII constructs and also that more immunoreactive material was detected by blotting than with Ab::cpVIII fusions . Consequently, more scFv::cpIII than scFv::cpVIII phage could be recovered in micropanning experiments with the antigen phOx as target.

Structure, 1995 Mar 15, 3(3), 255 - 63
Crystal structure of the MS2 coat protein dimer: implications for RNA binding and virus assembly; Ni CZ et al.; BACKGROUND: The coat protein in RNA bacteriophages binds and encapsidates viral RNA, and also acts as translational repressor of viral replicase by binding to an RNA hairpin in the RNA genome . Because of its dual function, the MS2 coat protein is an interesting candidate for structural studies of protein-RNA interactions and protein-protein interactions . In this study, unassembled MS2 coat protein dimers were selected to analyze repressor activity and virus assembly . RESULTS: The crystal structure of a mutant MS2 coat protein that is defective in viral assembly yet retains repressor activity has been determined at 2.0 A resolution . The unassembled dimer is stabilized by interdigitation of alpha-helices, and the formation of a 10-stranded antiparallel beta-sheet across the interface between monomers . The substitution of arginine for tryptophan at residue 82 results in the formation of two new inter-subunit hydrogen bonds that further stabilize the dimer . Residues that influence RNA recognition, identified by molecular genetics, were located across the beta-sheet . Two of these residues (Tyr85 and Asn87) are displaced in the unliganded dimer and are located in the same beta-strand as the Trp-->Arg mutation . CONCLUSIONS: When compared with the structure of the coat protein in the assembled virus, differences in orientation of residues 85 and 87 suggest conformational adjustment on binding RNA in the first step of viral assembly . The substitution at residue 82 may affect virus assembly by imposing conformational restriction on the loop that makes critical inter-subunit contacts in the capsid.

Cytometry, 1995 Mar 15, 22(1), 45 - 7
Assessment of aerosol containment on the ELITE flow cytometer; Ferbas J et al.; Biohazardous aerosols generated during cell sorting have been of increased concern recently because of interest in sorting specimens containing human immunodeficiency virus type 1 (HIV-1) . Current flow cytometers have features designed to contain such aerosols within the sorting chamber, but the efficacy of these features has not been established . Therefore, we tested aerosol containment by two ELITE flow cytometers (Coulter Cytometry, Inc., Hialeah, FL) during sorting of specimens containing high titers of bacteriophage . Agar plates confluent with susceptible Escherichia coli were used to detect infectious units released from the sorting chamber . Under recommended operating conditions very few infectious units were released from the sorting chambers . Release increased when the center stream was not optimally collected in a vacuum-exhausted tube or the chamber door was not completely closed . Failure of the negative pressure and high efficiency particle air (HEPA) filtration features had less of an effect . The data indicate that these standard safety features provide a rational expectation of safety for the flow cytometry operator.

Proc Natl Acad Sci U S A, 1995 Mar 14, 92(6), 2234 - 8
DNA nick processing by exonuclease and polymerase activities of bacteriophage T4 DNA polymerase accounts for acridine-induced mutation specificities in T4; Kaiser VL et al.; Acridine-induced frameshift mutagenesis in bacteriophage T4 has been shown to be dependent on T4 topoisomerase . In the absence of a functional T4 topoisomerase, in vivo acridine-induced mutagenesis is reduced to background levels . Further, the in vivo sites of acridine-induced deletions and duplications correlate precisely with in vitro sites of acridine-induced T4 topoisomerase cleavage . These correlations suggest that acridine-induced discontinuities introduced by topoisomerase could be processed into frameshift mutations . The induced mutations at these sites have a specific arrangement about the cleavage site . Deletions occur adjacent to the 3' end and duplications occur adjacent to the 5' end of the cleaved bond . It was proposed that at the nick, deletions could be produced by the 3'-->5' removal of bases by DNA polymerase-associated exonuclease and duplications could be produced by the 5'-->3' templated addition of bases . We have tested in vivo for T4 DNA polymerase involvement in nick processing, using T4 phage having DNA polymerases with altered ratios of exonuclease to polymerase activities . We predicted that the ratios of the deletion to duplication mutations induced by acridines in these polymerase mutant strains would reflect the altered exonuclease/polymerase ratios of the mutant T4 DNA polymerases . The results support this prediction, confirming that the two activities of the T4 DNA polymerase contribute to mutagenesis . The experiments show that the influence of T4 DNA polymerase in acridine-induced mutation specificities is due to its processing of acridine-induced 3'-hydroxyl ends to generate deletions and duplications by a mechanism that does not involve DNA slippage.

Proc Natl Acad Sci U S A, 1995 Mar 14, 92(6), 2184 - 8
Enhanced activity of the bacteriophage lambda PL promoter at low temperature; Giladi H et al.; The response of the early phage lambda PL promoter to temperature was investigated . Experiments with lacZ reporter gene fusions demonstrated that the activity of the phage lambda PL promoter is inversely dependent on temperature . The bacterial DNA-binding protein integration host factor (IHF) further enhances lambda PL promoter activity at low temperature, although no apparent changes in the cellular level of IHF protein were observed at the different temperatures . IHF protein binds DNA in vitro more avidly at low temperatures . In vitro transcription assays further revealed that the temperature response of PL is the result of an intrinsic property of the promoter as well as its activation by IHF.

J Biol Chem, 1995 Mar 10, 270(10), 5181 - 6
Homology dependence of UvsX protein-catalyzed joint molecule formation; Salinas F et al.; The bacteriophage UvsX protein is a "strand transferase" that promotes the pairing of homologous single and double-stranded DNAs . The efficiency of UvsX protein-mediated joint molecule formation between supercoiled duplex DNA and oligonucleotides is shown to have a sharp dependence on the degree of homology . The reaction proceeded efficiently with oligonucleotides containing 32 homologous positions but not with oligonucleotides containing only 24 homologous bases . This was shown to reflect an intrinsic homology requirement for the formation of stable joint molecules and was not caused by poor binding of the protein to short single-stranded DNAs . Even a single mismatch located in the middle of a region of 40 homologous nucleotides had a detectable effect on the efficiency of pairing . An in vitro recombinationally initiated DNA synthesis reaction that mimics the "secondary mode" of phage T4 DNA replication exhibited the same homology dependence.

J Biol Chem, 1995 Mar 10, 270(10), 5107 - 14
Mutagenesis of the COOH-terminal region of bacteriophage T4 regA protein; O'Malley SM et al.; The bacteriophage T4 regA protein is a translational repressor that regulates the synthesis of > 12 T4 proteins . Earlier studies demonstrated that photocross-linking of the 122-residue regA protein to (dT)16 occurs at two sites, with the major site occurring at Phe-106 . Amino acid substitutions were introduced at Phe-106 to evaluate its role in nucleic acid binding . Binding affinities of mutants F106C, F106V, and F106Y for nonspecific and specific RNA ligands indicated little difference between the Kapp of the mutants and wild type regA protein, for either poly(U) or for a specific gene 44 oligoribonucleotide . Thus, Phe-106 does not contribute measurably to the overall free energy of binding . Partial proteolysis of regA protein was carried out to further probe its domain structure . Chymotryptic cleavage produced a fragment of 11,095 Da that has reduced affinity for poly(U) and that contains the first 93 residues of regA protein . Interestingly, proteolysis of regA protein is reduced in the presence of the specific target, gene 44 RNA . Two deletion mutants, 1-->94 and 1-->109, have also been cloned and purified . The binding affinities of these deletion mutants indicated a 100-1000-fold reduction in their affinities for poly(U) . These studies indicate the last 13 amino acids in regA protein make a significant contribution to RNA binding.

Virology, 1995 Mar 10, 207(2), 400 - 8
In vitro transcription of the double-stranded RNA bacteriophage phi 6 is influenced by purine NTPs and calcium; Ojala PM et al.; The double-stranded RNA bacteriophage phi 6 contains a virion-associated RNA-dependent RNA polymerase complex . Removal of the virus envelope and the nucleocapsid surface protein, P8, reveals a nucleocapsid core particle (proteins P1, P2, P4, P7) which is the viral polymerase complex, capable of synthesizing RNA strands of positive polarity . The in vitro plus strand synthesis (transcription) reaction of the particle obtained from the mature virion was optimized and its activation and inactivation were investigated . Purine nucleoside triphosphates (NTPs), binding to a low-affinity binding site in the polymerase complex, activated plus strand synthesis . GTP was the preferred NTP, but dGTP, ddGTP, and the noncleavable analog GMP-PCP could also switch on transcription . This NTP-binding site is probably different from that of the unspecific viral NTPase found in protein P4 and also from that of the rNTP-specific RNA polymerase active site . Binding of purine NTPs was sufficient for the switch-on; hydrolysis of the NTP was not required . Besides nucleotides, divalent cations had an effect on phi 6 in vitro plus strand synthesis . Magnesium ions are required for the activity but calcium ions inhibit the reaction . Manganese ions are shown to dissipate the effect of magnesium and calcium ions, leading to uncontrolled, exceptionally high level plus strand synthesis.

Virology, 1995 Mar 10, 207(2), 392 - 9
Replication of transfected plasmid DNA by cells infected with African swine fever virus; Oliveira S et al.; Recombinant plasmids containing African swine fever virus (ASFV) DNA fragments covering all the virus genome were transfected into infected cells in order to detect viral origins of DNA replication . Plasmid replication was monitored by sensitivity to MboI, which cleaves only replicated, unmethylated DNA, and resistance to DpnI, which cleaves only the same methylated sequence . All the recombinants replicated to a similar extent, indicating that ASFV does not use a preferred origin for DNA replication . Circular plasmids without viral inserts were also replicated, but linearized plasmids or lambda bacteriophage DNA were not replicated . Replicated plasmid DNA began to accumulate with a time course similar to viral DNA, starting between 6 and 12 hr p.i . and increasing steadily for about 18 hr . This apparent dependence on viral functions was confirmed by the sensitivity of plasmid replication to phosphonoacetic acid and resistance to aphidicolin and by the reduction of replication in cells infected with a mutant defective in DNA replication . Replicated plasmid DNA present as unit length circles and as large dimension forms, probably head-to-tail concatemers . The results of two-dimensional electrophoresis (neutral/alkaline) favor a rolling-circle mechanism for plasmid DNA replication.

Virology, 1995 Mar 10, 207(2), 442 - 51
Circularly permuted viral pRNA active and specific in the packaging of bacteriophage phi 29 DNA; Zhang C et al.; A viral-encoded 120-base pRNA has been shown to have an essential role in the packaging of bacteriophage phi 29 DNA . The finding that both the 5'- and 3'-termini of the pRNA are proximate and crucial for biological function (C . Zhang, C . Lee, and P . Guo, 1994, Virology, 201, 77-85) prompted investigation of the activity of circularly permuted pRNAs (cpRNA) and of the expandability and essentiality of bases extending from the termini . A 117-base pRNA with a deletion of three bases downstream of the proximal terminus was active in DNA packaging . Concatemeric DNAs containing two tandem pRNA genes separated by a short or a long loop sequence were constructed . The cpRNAs from these DNA templates were transcribed in vitro and shown to be active in phi 29 DNA packaging, with activity comparable to the parental (noncircularly permuted) pRNA, indicating that neither of the loops tested affected the activity and folding of the cpRNA . As few as four bases were sufficient to serve as a loop for the terminal 180 degree turn, and a loop as long as 27 bases did not affect the cpRNA structure and function . Eight cpRNAs were constructed to assess the effect of openings within the wild-type pRNA structure . Opening of the bulge at residue 38 did not affect cpRNA activity, but opening the bulge at residue 55 greatly reduced it . Although the sequence of the 5',3'-terminal loop was not important for the folding and activity of the cpRNA, the activities of cpRNAs with openings at individual bulges or hairpins were different, indicating that each region plays a different role in pRNA folding and function . Our results indicate that it is possible to generate active circularly permuted pRNA by assigning internal sites of the pRNA as new 3'- and 5'-termini . The creation of new variable ends makes the labeling of internal bases of the pRNA molecule possible and will facilitate the analysis of pRNA secondary and tertiary structure.

Biochem Biophys Res Commun, 1995 Mar 8, 208(1), 339 - 44
Isolation and structural analysis of the 5' flanking region of the gene encoding the human glucagon receptor; Buggy J et al.; The gene encoding the human glucagon receptor, including several kb of upstream sequence, was isolated from a bacteriophage lambda FIX II library constructed from human placental DNA . We report here the novel sequence of the 5' flanking region of the gene, the identification of a previously unreported intron of 5 kb, and the identification of the transcription start point of the glucagon receptor-specific transcript, which estimates the length of the first exon to be 300 bp.

J Mol Biol, 1995 Mar 3, 246(4), 461 - 71
The bacteriophage N4-coded single-stranded DNA-binding protein (N4SSB) is the transcriptional activator of Escherichia coli RNA polymerase at N4 late promoters; Cho NY et al.; Transcription of the 72kb linear double-stranded DNA genome of coliphage N4 is carried out by the sequential activity of three different RNA polymerases . Early and middle viral transcripts are synthesized by two phage-coded RNA polymerases while late transcription is carried out by the Escherichia coli sigma 70-RNA polymerase . We have determined the sequences and sites of initiation of several N4 late transcripts; N4 late promoters share weak homology with the E . coli sigma 70 promoter consensus sequence . Indeed, N4 late promoters are weak templates for the host enzyme . We present evidence that the phage-coded, single-stranded DNA-binding protein (N4SSB), a protein that is required for phage DNA replication and recombination and does not bind with sequence specificity to DNA, is the activator of E . coli RNA polymerase at late N4 promoters . Models for the mechanism of action of N4SSB as a transcriptional activator are discussed.

J Biol Chem, 1995 Mar 3, 270(9), 4759 - 74
Gel kinetic analysis of DNA polymerase fidelity in the presence of proofreading using bacteriophage T4 DNA polymerase; Creighton S et al.; A gel fidelity assay, previously used in the analysis of DNA polymerases having no associated 3' to 5' exonuclease activity, has been generalized for use with polymerases that contain exonucleolytic proofreading . The main purpose of this study was the development of a general analysis, using a standard Markov model, to convert experimentally observed DNA primer gel bands arising from insertion and proofreading of right and wrong deoxyribonucleotides, into nucleotide incorporation velocities and, most importantly, fidelities . The model has been applied primarily to an analysis of polymerase kinetics and fidelity in the presence of a next correct rescue dNTP, but the model can be conveniently modified to investigate other experimental designs . In the presence of rescue dNTP, direct competition occurs between excision or extension of a mismatch . At concentrations of rescue dNTP sufficient to suppress the gel band intensity at the mismatch target site, nucleotide incorporation and misincorporation rates can be obtained from the ratios of gel band intensities 3' (downstream) and 5' (upstream) to the target site, measured as a function dNTP concentration for "wrong" and "right" dNTP substrates . The polymerase misincorporation efficiency, in the presence of proofreading, is given by the ratio of wrong to right incorporation efficiencies, Vmax/Km, obtained from the gel band ratios . The bacteriophage T4 polymerase with a highly active 3'-exonuclease activity was used to illustrate the assay . Nucleotide misincorporation efficiencies measured at several template sites were dCMP.A approximately equal to 10(-6), dGMP.A approximately equal to 10(-5), dTMP.T approximately equal to 2 x 10(-4), and dAMP.A < 10(-7) . Proofreading of the dGMP.A mispair was suppressed by about 3-fold in the presence of high concentrations of next correct "rescue" dNTP causing a concomitant reduction in the fidelity of dGMP.A to about 3 x 10(-5).

J Biol Chem, 1995 Mar 3, 270(9), 4563 - 9
Modulation of the ATPase activity of the molecular chaperone DnaK by peptides and the DnaJ and GrpE heat shock proteins; Jordan R et al.; Previous studies have demonstrated that the Escherichia coli DnaK, DnaJ, and GrpE heat shock proteins participate in the initiation of bacteriophage lambda DNA replication by mediating the required disassembly of a preinitiation nucleoprotein structure that is formed at the phage replication origin . To gain some understanding in a simpler system of how the DnaJ and GrpE cochaperonins influence the activity of DnaK, we have examined the effect of the cochaperonins on the weak intrinsic ATPase activity of the molecular chaperone DnaK in the presence and absence of peptide effectors . We have found that random sequence peptide chains of 8 or 9 amino acid residues in length yield optimal (10-fold) activation of the DnaK ATPase, whereas peptides with 5 or fewer residues fail to stimulate the ATPase of this bacterial hsp70 homologue . Furthermore, we have discovered that those peptides that interact best with DnaK, as judged by their KA as activators of ATP hydrolysis by DnaK, also act as strong inhibitors of lambda DNA replication in vitro . The inhibitory effect of peptides on lambda DNA replication was overcome by increasing the concentration of DnaK in the replication system . Diminished inhibition was also found when the replication system was supplemented with GrpE cochaperonin, a protein known to increase the effectiveness of DnaK action in lambda DNA replication . These and other results suggest that the peptide-binding site of DnaK is required for its function in lambda DNA replication . Apparently, peptides sequester free DnaK protein and block lambda DNA replication by reducing the amount of DnaK that is free to mediate disassembly of nucleoprotein preinitiation structures . In related studies, we have found that DnaJ, like short peptides, activates the intrinsic ATPase activity of DnaK . DnaJ, however, is substantially more potent in this regard, since it activates DnaK at concentrations 1000-fold below those required for a peptide of random sequence . By itself, the GrpE cochaperonin has no effect on the peptide-independent ATPase activity of DnaK, but GrpE does vigorously stimulate the peptide-dependent ATPase of the DnaK chaperone . Under steady-state conditions, the Vmax of ATP hydrolysis by DnaK was elevated approximately 40-fold by the presence of GrpE and saturating levels of peptides.

EMBO J, 1995 Mar 1, 14(5), 1043 - 55
The rpoE gene encoding the sigma E (sigma 24) heat shock sigma factor of Escherichia coli; Raina S et al.; Previous work has established that the transcription factor sigma E (sigma 24) is necessary for maintaining the induction of the heat shock response of Escherichia coli at high temperatures . We have identified the gene encoding sigma E using a genetic screen designed to isolate trans-acting mutations that abolish expression from either htrA or rpoHP3, two promoters recognized uniquely by sigma E-containing RNA polymerase . Such a screen was achieved by transducing strains carrying a single copy of either phtrA-lacZ or rpoHP3-lacZ fusions with mutagenized bacteriophage P1 lysates and screening for Lac- mutant colonies at 22 degrees C . Lac- mutants were subsequently tested for inability to grow at 43 degrees C (Ts- phenotype) . Only those Lac- Ts- mutants that were unable to accumulate heat shock proteins at 50 degrees C were retained for further characterization . In a complementary approach, those genes which when cloned on a multicopy plasmid led to higher constitutive expression of the sigma E regulon were characterized and mapped . Both approaches identified the same gene, rpoE, mapping at 55.5 min on the E.coli genetic map and encoding a polypeptide of 191 amino acid residues . The wild-type and a mutant rpoE gene products were over-expressed and purified . It was found that the purified wild-type sigma E protein, when used in in vitro run-off transcription assays in combination with core RNA polymerase, was able to direct transcription from the htrA and rpoHP3 promoters, but not from known sigma 70-dependent promoters . In vivo and in vitro analyses of rpoE transcriptional regulation showed that the rpoE gene is transcribed from two major promoters, one of which is positively regulated by sigma E itself.

J Bacteriol, 1995 Mar, 177(6), 1589 - 94
Streptomycin- and rifampin-resistant mutants of Escherichia coli perturb F exclusion of bacteriophage T7 by affecting synthesis of the F plasmid protein PifA; Schmidt CK et al.; Certain alleles of rpsL that confer resistance to the antibiotic streptomycin almost completely relieve F exclusion of bacteriophage T7 . Introduction of a specific rpoB allele conferring resistance to rifampin into the rpsL strain restores the ability of the F-containing strain to exclude T7 . This variation in the severity of F exclusion is reflected in the levels of the F-encoded inhibitor protein PifA: F'-containing cells that harbor specific rpsL alleles are phenotypically Pif-, but become Pif+ by the further acquisition of a specific rpoB allele . F-containing cells harboring the gyrA43(Ts) mutation also appear phenotypically Pif-, possibly because repression of the pif operon is enhanced by an altered DNA conformation in the gyrase mutant strain.

J Bacteriol, 1995 Mar, 177(6), 1425 - 34
Control of transcription termination by an RNA factor in bacteriophage P4 immunity: identification of the target sites; Sabbattini P et al.; Prophage P4 immunity is elicited by a short, 69-nucleotide RNA (CI RNA) coded for within the untranslated leader region of the same operon it controls . CI RNA causes termination of transcription that starts at the promoter PLE and prevents the expression of the distal part of the operon that codes for P4 replication functions (alpha operon) . In this work, we identify two sequences in the untranslated leader region of the alpha operon, seqA and seqC, that are the targets of the P4 immunity factor . seqA and seqC exhibit complementarity to a sequence internal to the CI RNA (seqB) . Mutations in either seqA or seqC that alter its complementarity to seqB abolished or reduced P4 lysogenization proficiency and delayed the shutoff of the long transcripts originating from PLE that cover the entire operon . Both seqA and seqC single mutants were still sensitive to P4 prophage immunity, whereas P4 seqA seqC double mutants showed a virulent phenotype . Thus, both functional sites are necessary to establish immunity upon infection, whereas a single site appears to be sufficient to prevent lytic gene expression when immunity is established . A mutation in seqB that restored complementarity to both seqA and seqC mutations also restored premature termination of PLE transcripts, thus suggesting an important role for RNA-RNA interactions between seqB and seqA or seqC in P4 immunity.

Mutat Res, 1995 Mar, 327(1-2), 121 - 9
Inhalation of benzene leads to an increase in the mutant frequencies of a lacI transgene in lung and spleen tissues of mice; Mullin AH et al.; The goal of this study was to determine if inhalation of benzene leads to an increase in the mutant frequencies in the tissues of male C57BL/6 mice . Mutant frequencies were measured using a previously described assay in which bacteriophage lambda lacI transgenes are rescued from mouse genomic DNA as infectious phage and scored for their LacI phenotype . Eight experimental mice were exposed to a target concentration of 300 ppm of benzene for 6 h/day x 5 days/week x 12 weeks, and eight control mice were treated similarly except that they were not exposed to benzene . Mutant frequencies were calculated as the ratio of LacI-/total phage recovered from organs of interest . The mean mutant frequency measured in lung tissues of mice exposed to benzene was (10.6 +/- 1.4) x 10(-5), which is about 1.7-fold higher than that of the unexposed controls . In spleen tissues from benzene-exposed mice the mean mutation frequency was (12.6 +/- 4.1) x 10(-5), which is about 1.5-fold higher than that of spleen tissues from unexposed controls . The differences in mean mutant frequencies between benzene-exposed and unexposed lung and spleen tissues are statistically significant . In liver tissues, however, the mean mutant frequencies of benzene-exposed mice and unexposed mice are not significantly different . These results demonstrate that inhaled benzene results in a statistically significant increase in the mutant frequencies in lung and spleen, but not in liver tissues of mice.

J Bacteriol, 1995 Mar, 177(5), 1159 - 68
Cin-mediated recombination at secondary crossover sites on the Escherichia coli chromosome; Rozsa FW et al.; The Cin recombinase is known to mediate DNA inversion between two wild-type cix sites flanking genetic determinants for the host range of bacteriophage P1 . Cin can also act with low frequency at secondary (or quasi) sites (designated cixQ) that have lower homology to either wild-type site . An inversion tester sequence able to reveal novel operon fusions was integrated into the Escherichia coli chromosome, and the Cin recombinase was provided in trans . Among a total of 13 Cin-mediated inversions studied, three different cixQ sites had been used . In two rearranged chromosomes, the breakpoints of the inversions were mapped to cixQ sites in supB and ompA, representing inversions of 109 and 210 kb, respectively . In the third case, a 2.1-kb inversion was identified at a cixQ site within the integrated sequences . This derivative itself was a substrate for a second inversion of 1.5 kb between the remaining wild-type cix and still another cixQ site, thus resembling a reversion . In analogy to that which is known from DNA inversion on plasmids, homology of secondary cix sites to wild-type recombination sites is not a strict requirement for inversion to occur on the chromosome . The chromosomal rearrangements which resulted from these Cin-mediated inversions were quite stable and suffered no growth disadvantage compared with the noninverted parental strain . The mechanistic implications and evolutionary relevance of these findings are discussed.

Appl Environ Microbiol, 1995 Mar, 61(3), 1068 - 72
Construction and characterization of a DNA probe for distinguishing strains of Aspergillus flavus; McAlpin CE et al.; Repetitive DNA sequences have proven useful and reliable characters in evaluating genetic relatedness of strains at different levels of taxonomic classification . A DNA probe was constructed to distinguish among strains of Aspergillus flavus by DNA fingerprinting techniques . Chromosomal DNA of A . flavus var . flavus NRRL 6541 was partially digested with EcoRI and ligated to a Lambda Dash bacteriophage vector . Four lambda clones were identified which displayed multiple and distinct bands when hybridized with chromosomal DNA from seven strains of A . flavus var . flavus digested with either EcoRI or PstI . One of these clones was chosen for further analysis and was subcloned into pUC19 . The subclone, pAF28, contained a 6.2-kb chromosomal DNA insert and was able to distinguish among strains characterized by K . E . Papa (Mycologia 78:98-101, 1986) as belonging to 22 different vegetative compatibility groups . The subclone identified unique banding patterns when hybridized to genomic DNA digested with PstI . The cloned probe may be species specific as it hybridized with the DNA of all isolates of A . flavus tested in addition to strains recognized as varieties of A . flavus (e.g., A . flavus var . oryzae, A . flavus var . parasiticus, and A . flavus var . sojae) . pAF28 hybridized to a single band on a Southern blot with Aspergillus nomius DNA but did not hybridize with the DNA of other fungal species tested including Aspergillus ochraceus, Aspergillus auricomus, Aspergillus alliaceus, Fusarium moniliforme, and Penicillium thomii.

Biophys J, 1995 Mar, 68(3), 1128 - 36
2D exchange 31P NMR spectroscopy of bacteriophage M13 and tobacco mosaic virus; Magusin PC et al.; Two-dimensional (2D) exchange 31P nuclear magnetic resonance spectroscopy is used to study the slow overall motion of the rod-shaped viruses M13 and tobacco mosaic virus in concentrated gels . Even for short mixing times, observed diagonal spectra differ remarkably from projection spectra and one-dimensional spectra . Our model readily explains this to be a consequence of the T2e anisotropy caused by slow overall rotation of the viruses about their length axis . 2D exchange spectra recorded for 30% (w/w) tobacco mosaic virus with mixing times < 1 s do not show any off-diagonal broadening, indicating that its overall motion occurs in the sub-Hz frequency range . In contrast, the exchange spectra obtained for 30% M13 show significant off-diagonal intensity for mixing times of 0.01 s and higher . A log-gaussian distribution around 25 Hz of overall diffusion coefficients mainly spread between 1 and 10(3) Hz faithfully reproduces the 2D exchange spectra of 30% M13 recorded at various mixing times in a consistent way . A small but notable change in diagonal spectra at increasing mixing time is not well accounted for by our model and is probably caused by 31P spin diffusion.

Bull Math Biol, 1995 Mar, 57(2), 277 - 97
Dynamical behaviour of biological regulatory networks--II . Immunity control in bacteriophage lambda; Thieffry D et al.; A number of bacterial and viral genes take part in the decision between lysis and lysogenization in temperate bacteriophages . In the lambda case, at least five viral genes (cI, cro, cII, N and cIII) and several bacterial genes are involved . Several attempts have been made to model this complex regulatory network . Our approach is based on a logical method described in the first paper of the series which formalizes the interactions between the elements of a regulatory network in terms of discrete variables, functions and parameters . In this paper two models are described and discussed, the first (two-variable model) focused on cI and cro interactions, the second (four-variable model) considering, in addition, genes cII and N . The treatment presented emphasizes the roles of positive and negative feedback loops and their interactions in the development of the phage . The role of the loops between cI and cro, and of cI on itself (which both have to be positive loops) was discovered earlier; this group's contribution to this aspect mainly deals with the possibility of treating these loops as parts of a more extended network . In contrast, the role of the negative loop of cro on itself had apparently remained unexplained . We realized that this loop buffers the expression of genes cro itself . cII, O and P against the inflation due to the rapid replication of the phage . More generally, negative auto-control of a gene appears an efficient way to render its expression insensitive (or less sensitive) to gene dosage, whereas a simple negative control would not provide this result.

Mol Microbiol, 1995 Mar, 15(6), 1055 - 67
L5 luciferase reporter mycobacteriophages: a sensitive tool for the detection and assay of live mycobacteria; Sarkis GJ et al.; Recombinant bacteriophages provide efficient delivery systems for introducing reporter genes into specific bacterial hosts . We have constructed mycobacteriophage L5 recombinants carrying the firefly luciferase gene inserted into the tRNA region of the phage genome . Infection of Mycobacterium smegmatis by these phages results in expression of the luciferase gene and light emission . Fortuitously, the luciferase gene is expressed continuously in lysogens surviving infection . Synthesis of luciferase from a mycobacterial promoter created by cloning enables the detection of extremely small numbers of M . smegmatis cells . These reporter phages can be used to discriminate between drug-sensitive and drug-resistant strains of M . smegmatis, and may provide tools for the rapid identification and classification of antimycobacterial agents.

Mikrobiologiia, 1995 Mar-Apr, 64(2), 211 - 5
{Inhibition by chitosan of productive infection of T-series bacteriophages in the Escherichia coli culture}; Kochkina ZM et al.; The possibility of the use of chitosan aminopolysaccharide (poly-D-glucosamine) and its two salts--acetate and hydrochloride--to prevent phase infection of the Escherichia coli culture, strain B1, was studied . It was shown that chitosan inhibited productive infection caused by the bacteriophages T2 and T7, the efficiency of inhibition of both bacteriophages depending directly on the final concentration of chitosan in a medium . Neither chitosan nor its salts significantly prevented the growth of the bacterial culture.

Mol Microbiol, 1995 Mar, 15(5), 977 - 84
The Escherichia coli DnaK chaperone machine and bacteriophage Mu late transcription; Sand O et al.; Bacteriophage Mu does not grow on temperature-sensitive E . coli dnaK mutants at elevated temperatures because of a defect in late transcription . As the Mu-encoded C protein is required for activation of transcription from the phage late promoters, we attempted to determine if DnaK and its accessory proteins DnaJ and GrpE are required for synthesis of C protein or at a later step . We found that the chaperones act in Mu late transcription beyond C-protein synthesis, and that C-protein stability is decreased in the mutant hosts . This suggests that the DnaK chaperone machine may be required for the proper folding and/or multimerization of C protein.

J Clin Microbiol, 1995 Mar, 33(3), 631 - 5
Virulence markers of Shiga-like toxin-producing Escherichia coli strains originating from healthy domestic animals of different species; Beutin L et al.; Shiga-like toxin (verotoxin)-producing strains of Escherichia coli (SLTEC) originating from healthy cattle, sheep, goats, pigs, cats, and dogs were investigated for properties which are related to virulence of E . coli for humans . The slt-II (Shiga-like toxin II) and slt-IIc genes were frequent in SLTEC from healthy cattle and dogs but were rarely found in SLTEC from other animals . The slt-IIe gene was detected only in porcine SLTEC . SLTEC from goats and SLTEC from sheep were found to carry different SLT-II determinants which were not further characterized genetically . Sixty (28.8%) of 208 SLTEC from healthy animals showed diffuse adherence to HEp-2 cells . However, none of the strains was positive for genes specific for the local adherence (eaf), diffuse adherence (daa), or enteroaggregative (EAggEC) E . coli type . Only 3 (1.4%) of the 208 SLTEC were positive for attaching and effacing E . coli (eae) sequences . The enterohemolytic phenotype was present in 128 of the 208 SLTEC . Almost all enterohemolytic animal SLTEC were found to carry DNA sequences specific for the plasmid-encoded enterohemorrhagic E . coli hemolysin of E . coli O157 . Bacteriophage-associated enterohemolysin (Ehly1 and Ehly2)-specific sequences were detected only in 14.4% of the 208 SLTEC and were linked with certain serotypes . The SLTEC from healthy animals constitute a very heterogeneous group of E . coli, and many of these strains appeared to be specific for their hosts . The absence of eae sequences in most animal SLTEC could indicate that these strains are less virulent for humans than the classical eae-positive enterohemorrhagic E . coli types.

Proc Natl Acad Sci U S A, 1995 Feb 28, 92(5), 1609 - 13
Display of peptides and proteins on the surface of bacteriophage lambda; Sternberg N et al.; The display of peptides or proteins on the surface of viruses is an important technology for studying peptides or proteins and their interaction with other molecules . Here we describe a display vehicle based on bacteriophage lambda that incorporates a number of features distinct from other currently used display systems . Fusions of peptides or protein domains have been made to the amino terminus of the 11-kDa D protein of the lambda capsid . These fusions assemble onto the viral capsid and appear to be accessible to ligand interactions, based on the ability of a monoclonal antibody to recognize an epitope fused to the D protein on phage heads . To produce large D fusion display libraries and yet avoid the cumbersome task of cloning many fragments into lambda DNA, we have used the Cre-loxP site-specific recombination system in vivo to incorporate plasmids encoding the D fusions into the phage genome . Finally, we show that D fusion proteins can be added in vitro to phage lacking D protein and be assembled onto the viral capsid.

Proc Natl Acad Sci U S A, 1995 Feb 28, 92(5), 1451 - 5
Bacteriophage T4 MotA and AsiA proteins suffice to direct Escherichia coli RNA polymerase to initiate transcription at T4 middle promoters; Ouhammouch M et al.; Development of bacteriophage T4 in Escherichia coli requires the sequential recognition of three classes of promoters: early, middle, and late . Recognition of middle promoters is known to require the motA gene product, a protein that binds specifically to the "Mot box" located at the -30 region of these promoters . In vivo, the asiA gene product is as critical for middle mode RNA synthesis as is that of the motA gene . In vitro, AsiA protein is known to loosen the sigma 70-core RNA polymerase interactions and to inhibit some sigma 70-dependent transcription, presumably through binding to the sigma 70 subunit . Here we show that, in vitro, purified MotA and AsiA proteins are both necessary and sufficient to activate transcription initiation at T4 middle promoters by the E . coli RNA polymerase in a sigma 70-dependent manner . AsiA is also shown to inhibit recognition of T4 early promoters and may play a pivotal role in the recognition of all three classes of phage promoters.

Gene, 1995 Feb 27, 154(1), 51 - 4
Positive-selection vector with enhanced lytic potential based on a variant of phi X174 phage gene E; Henrich B et al.; A cloning vector, pUH89, allowing positive selection of recombinant Escherichia coli clones by insertional inactivation of the modified lysis gene E of bacteriophage phi X174, was developed . Ten unique cloning sites were introduced into gene E by site-directed mutagenesis . To achieve efficient expression of the mutagenized gene, the combined lac and tac promoters were used . Additional restriction sites in the flanking sequences allow screening for transcription terminators and the excision of several cartridges suitable for vector construction.

J Mol Biol, 1995 Feb 24, 246(3), 418 - 28
In vitro packaging of the single-stranded RNA genomic precursors of the segmented double-stranded RNA bacteriophage phi 6: the three segments modulate each other's packaging efficiency; Frilander M et al.; Bacteriophage phi 6 is a double-stranded RNA (dsRNA) virus that has a genome composed of three linear dsRNA segments (l, m, s) . These are encapsidated into a dodecahedral procapsid particle consisting of proteins P1, P2, P4 and P7 . Expression of the cDNA copy of the L segment in Escherichia coli leads to the formation of empty procapsid particles . These particles are able to package the plus-sense single-stranded RNA (ssRNA)s of each genome segment in vitro . We have used this in vitro system for a detailed study of phi 6 RNA packaging . The reaction conditions for RNA packaging were optimized using a RNase protection assay . The RNA packaging reaction is dependent on divalent cations (either Mg2+ or Mn2+) and requires a nucleoside triphosphate (NTP) as an energy source . Any one of the rNTPs, dNTPs or ddNTPs can support the RNA packaging . Purine nucleotides support packaging better than pyrimidine nucleotides, GTP being preferred to ATP . The plus-sense ssRNA of each the three genome segments can be packaged independently into the procapsid . However, when two or three segments are packaged simultaneously, regulatory effects modulating the packaging efficiency can be detected between the segments . The packaging of the s and m segments is more efficient when they are packaged alone, compared to a situation in which they are packaged with the other segments . In contrast, the packaging of the l segment is very inefficient alone, but is enhanced when packaged together with the m segment . We propose that each segment has a preferred high-affinity binding site in the procapsid particle and packaging of the m segment creates the high-affinity binding site for the l segment . If any of the segments is missing from the packaging reaction the other segments can occupy its binding site.

Science, 1995 Feb 24, 267(5201), 1131 - 7
Head-on collision between a DNA replication apparatus and RNA polymerase transcription complex; Liu B et al.; An in vitro system reconstituted from purified proteins has been used to examine what happens when the DNA replication apparatus of bacteriophage T4 collides with an Escherichia coli RNA polymerase ternary transcription complex that is poised to move in the direction opposite to that of the moving replication fork . In the absence of a DNA helicase, the replication fork stalls for many minutes after its encounter with the RNA polymerase . However, when the T4 gene 41 DNA helicase is present, the replication fork passes the RNA polymerase after a pause of a few seconds . This brief pause is longer than the pause observed for a codirectional collision between the same two polymerases, suggesting that there is an inherent disadvantage to having replication and transcription directions oriented head to head . As for a codirectional collision, the RNA polymerase remains competent to resume faithful RNA chain elongation after the DNA replication fork passes; most strikingly, the RNA polymerase has switched from its original template strand to use the newly synthesized daughter DNA strand as the template.

Biochim Biophys Acta, 1995 Feb 22, 1247(1), 149 - 58
Investigations of the interactions of saccharides with the lysozyme from bacteriophage lambda; Duewel HS et al.; The bacteriophage lambda R gene has been isolated into an Escherichia coli expression system and the R gene product, a lysozyme, has been overexpressed and purified to homogeneity using an efficient purification procedure . A turbidimetric assay utilizing chloroform-treated E . coli cells has been optimized to assess the bacteriolytic activity of the purified enzyme . Using this assay, oligomers of beta (1 --> 4) N-acetyl-D-glucosamine at high concentrations were shown to inhibit lysozyme but were not cleaved by the enzyme . Differential scanning calorimetry revealed that the thermal denaturation of lysozyme was found to increase in the presence of (GlcNAc)3 and (GlcNAc)5 . The lysozyme was also expressed in an E . coli strain auxotrophic for methionine, allowing for the incorporation of {methyl-13C}methionine into the enzyme . An alteration of the {1H-13C}HMQC NMR spectra of the labelled enzyme was observed in the presence of (GlcNAc)5 . Commercially available nitrophenyl glycosides did not act as substrates for lambda lysozyme . The results indicate that lambda lysozyme has specific interactions with oligosaccharides of N-acetylglucosamine, but is incapable of hydrolyzing these sugars . The relevance of the structure of peptidoglycan to the activity of lambda lysozyme is discussed.

Nucleic Acids Res, 1995 Feb 11, 23(3), 485 - 90
Intra-chromosomal rearrangements generated by Cre-lox site-specific recombination; Medberry SL et al.; Chromosomal rearrangements are useful genetic and breeding tools but are often difficult to detect and characterize . To more easily identify and define chromosome deletions and inversions, we have used the bacteriophage P1 Cre-lox site-specific recombination system to generate these events in plants . This involves three steps: (i) the introduction of two lox sites into one locus in a plant genome, including one site within a modified Ds transposon; (ii) Ac transposase-mediated transposition of the Ds-lox element to a new locus on the same chromosome; (iii) Cre-mediated site-specific recombination between the two lox sites that bracket a chromosome segment . We report the production of a deletion and three inversion events in tobacco . The utility of chromosomal segments bracketed by lox sites for targeted manipulation and cloning is discussed.

J Mol Biol, 1995 Feb 10, 246(1), 95 - 107
Binding of the junction-resolving enzyme bacteriophage T7 endonuclease I to DNA: separation of binding and catalysis by mutation; Duckett DR et al.; Bacteriophage T7 endonuclease I is a resolving enzyme that selectively cleaves four-way DNA junctions, and related branched species . We have isolated mutants of this protein that retain full structural selectivity of binding to four-way junctions, but which are completely inactive as nucleases . This is consistent with a divisibility of structure-selective binding and catalysis . The mutations that inactivate endonuclease I as a nuclease are clustered into the second quarter of the primary sequence, a region that displays some sequence similarity with the related junction-resolving enzyme endonuclease VII from bacteriophage T4 . This suggests that these residues may form the active site of these enzymes . The configuration of the helical arms of the junction bound by mutant endonuclease I has been investigated by gel electrophoretic methods . We find that the junction is bound in the presence or absence of magnesium ions, and that the global structure of the bound form is apparently identical with or without cations . The patterns of mobilities suggest that the structure of the junction becomes perturbed by the binding of the protein.

J Biol Chem, 1995 Feb 10, 270(6), 2652 - 61
Involvement of glutamic acid 23 in the catalytic mechanism of T4 endonuclease V; Manuel RC et al.; Bacteriophage T4 endonuclease V has both pyrimidine dimer-specific DNA glycosylase and abasic (AP) lyase activities, which are sequential yet biochemically separable functions . Previous studies using chemical modification and site-directed mutagenesis techniques have shown that the catalytic activities are mediated through the alpha-amino group of the enzyme forming a covalent (imino) intermediate . However, in addition to the amino-terminal active site residue, examination of the x-ray crystal structure of endonuclease V reveals the presence of Glu-23 near the active site, and this residue has been strongly implicated in the reaction chemistry . In order to understand the role of Glu-23 in the reaction mechanism, four different mutations (E23Q, E23C, E23H, E23D) were constructed, and the mutant proteins were evaluated for DNA glycosylase and AP lyase activities using defined substrates and specific in vitro and in vivo assays . Replacement of Glu-23 with Gln, Cys, or His completely abolished DNA glycosylase and AP lyase activities, while replacement with Asp retained negligible amounts of glycosylase activity, but retained near wild type levels of AP lyase activity . Gel shift assays revealed that all four mutant proteins can recognize and bind to thymine dimers . The results indicate that Glu-23 is the candidate for stabilizing the charge of the imino intermediate that is likely to require an acidic group in the active site of the enzyme.

J Biol Chem, 1995 Feb 10, 270(6), 2614 - 9
Functional interactions of gene 32, 41, and 59 proteins of bacteriophage T4; Tarumi K et al.; Genes 41 and 59 of bacteriophage T4 are involved in DNA recombination as well as in DNA replication . The 41 protein has a DNA helicase activity . The 59 protein has been recently purified and found to have a specific affinity for both 32 protein (single-stranded DNA-binding protein) and 41 protein (Yonesaki 1994, J . Biol . Chem . 269, 1284-1289) . We examined the effects of 59 protein on ssDNA-dependent ATPase activity and DNA helicase activity of 41 protein in the presence or absence of 32 protein . The ATPase activity of 41 protein was strongly inhibited by 32 protein over a wide range of amounts from subsaturation to oversaturation of ssDNA . The 32 protein was also inhibitory toward DNA helicase activity . Addition of 59 protein effectively eliminated these inhibitory effects of 32 protein . Moreover, 59 protein facilitated 41 protein to overcome the barrier to initiate the unwinding reaction with a duplex flanking a single-stranded DNA gap . Intriguingly, 32 protein at an amount optimal for saturation of ssDNA stimulated the overcoming of the barrier when 59 protein was present . For the best circumvention of this initiation barrier, only eight monomers of 59 protein/one DNA substrate molecule containing 2900 nucleotides of ssDNA were required . These results strongly suggest that 59 protein modulates 41 protein activities by forming a complex with 41 protein and that 41 protein can produce recombinogenic ssDNA with the aid of 32 and 59 proteins.

Anal Biochem, 1995 Feb 10, 225(1), 172 - 4
Transformation of Escherichia coli increases 260-fold upon inactivation of T4 DNA ligase; Michelsen BK; It was possible to obtain high-efficiency transformation of E . coli MC1061 by the following modifications of the standard procedure: cells were harvested at A600 of 550-650, washed with 1, 1/2, and 1/40, and were resuspended in 1/500 culture vol of 1 mM Hepes, pH 7.0, to a cell concentration of 6 x 10(10)-6 x 10(11) cells/ml . Electrocompetent cells were used immediately for electroporation to yield 1.3 +/- 0.5 x 10(9) (mean +/- SD) transformants micrograms of plasmid DNA, which is comparable to the efficiency of bacteriophage lambda infection . Alternatively, cells can be stored frozen in 10% glycerol, although glycerol reduced transformation efficiency to approximately 30% (data not shown) . Freezing and thawing of glycerol-treated cells did not result in any further loss of transformation efficiency (data not shown) . This study showed that it is crucial to inactivate the T4 DNA ligase prior to electrotransformation of ligated DNA, which can be ensured by the introduction of a simple heat inactivation step, increasing the number of transformants by 260-fold . Although this paper focuses on the use of E . coli MC1061/p3, the experiments were repeated with a different plasmid in the parental strain E . coli MC1061 and showed the same result (data not shown.(ABSTRACT TRUNCATED AT 250 WORDS)

Biochemistry, 1995 Feb 7, 34(5), 1779 - 86
Gin invertase of bacteriophage Mu is a dimer in solution, with the domain for dimerization in the N-terminal part of the protein; Spaeny-Dekking L et al.; The Gin protein of bacteriophage Mu mediates recombination between two inverted repeat sequences . Gin binds as a dimer to each of these recombination sites . We show that Gin is a dimer in solution also, and that the dimerization is probably stabilized by hydrophobic interactions between the subunits . The subunits of the dimer could efficiently be cross-linked with the 4-A cross-linker diepoxybutane . Spontaneous oxidation of Cys(24) and/or Cys(27) also resulted in intersubunit cross-linking . One or both cysteine residues are located at the interface of the Gin dimer, which maps the dimerization domain in the N-terminal part of the protein . Binding of the disulfide-bonded dimers of Gin to a recombination site was strongly reduced, suggesting that the subunits need to reorient in order to form a stable protein-DNA complex . In the protein-DNA complex, however, oxidation of cysteine residues still seems to be possible, indicating that the N-terminal parts of two Gin subunits are also in close proximity when bound to DNA.

Biochem Biophys Res Commun, 1995 Feb 6, 207(1), 111 - 9
Genomic structure of the mouse delta opioid receptor gene; Augustin LB et al.; Using mouse delta opioid receptor (DOR) cDNA sequence to probe genomic libraries in bacteriophage lambda and P1 vectors, clones traversing the entire DOR coding sequence and 5' and 3' flanking regions were isolate . Genomic sequence encoding mature DOR message, including 5' and 3' untranslated sequence, is divided by two introns of 26 kb and 3 kb, resulting in the gene occupying 32 kb of chromosomal DNA . Multiple putative transcription initiation sites were located, by RNase protection assay, in TATA-less G+C rich sequence between 390 and 140 nucleotides upstream from the ATG translation start codon . A polyadenylation site was located 1.24 kb downstream from the TGA translation stop codon . Examination of 1.3 kb of 5'flanking sequence revealed potential binding sites for several known transcription factors including: Sp1, Ap-2, NF-kappa B, NF-IL6, and NGFI-B.

Gene, 1995 Feb 3, 153(1), 57 - 62
The pCLIP plasmids: versatile cloning vectors based on the bacteriophage lambda origin of replication; Boyd AC et al.; A series of general-purpose plasmid vectors based on the phage lambda origin of replication (ori) has been constructed . Each vector consists of a backbone plasmid encoding chloramphenicol resistance (CmR) and containing a unique HaeII site into which the lacZ alpha-complementing multiple cloning site (MCS) region of an established vector was inserted . To increase the cloning potential of the inserted MCS, superfluous restriction sites in the backbone were removed by a variety of techniques . The vectors, designated pCLIP (for CmR lambda ori integration proficient) plasmids, are of medium copy number and are compatible with most other vectors in common use . A total of 17 unique restriction sites in pCLIP8, pCLIP9, pCLIP18, pCLIP19 and pCLIP23 are available for cloning . As well as possessing the usual properties of vectors, the pCLIP plasmids are able to integrate reversibly into lambda prophage by homologous recombination . Thus, cloned DNA can be maintained in single or multiple copy at will . By integrating recombinant plasmids into appropriate deletion prophages followed by induction, phage::plasmid hybrids are produced which can be manipulated as phage . These properties are demonstrated using a test recombinant plasmid, pCLIPLEU2 . The pCLIP vectors are therefore useful for routine plasmid cloning, complementation analysis and applications where the ability to manipulate recombinants in plasmid, phage or prophage forms is advantageous.

J Mol Biol, 1995 Feb 3, 245(5), 635 - 44
Structural and functional domains of the large subunit of the bacteriophage T3 DNA packaging enzyme: importance of the C-terminal region in prohead binding; Morita M et al.; During head assembly of phage T3, DNA is packaged into a preformed protein shell, called the prohead, with the aid of non-capsid packaging proteins, the products of genes 18 and 19 (gp18 and gp19) . We have developed a defined system, composed of purified gp18,gp19 and proheads for in vitro packaging of T3 DNA . Our previous results using the defined in vitro system indicate the sequential events in DNA packaging: the packaging proteins, gp18 and gp19, bind DNA and proheads, respectively . These complexes associate to form a direct precursor complexes for DNA translocation into the head . The formation of the precursor complexes requires ATP as an allosteric effector . Subsequent DNA translocation is driven by ATP hydrolysis . gp19 is an ATP binding protein that plays multiple roles in DNA packaging through interaction with ATP . gp19 changes its conformation by binding to ATP, as judged from the analysis of limited proteolysis . Sites cleaved by limited proteolysis were determined and mapped on the gp19 polypeptide (586 amino acid residues) to image the conformational change of gp19 induced by ATP . C-Terminal fragments generated by trypsin digestion bound the prohead and inhibited DNA packaging by intact gp19 in a competitive manner . On the other hand, N-terminal fragments did not bind the prohead nor did they inhibit DNA packaging . These results define a prohead binding domain at the C terminus of gp19 . To identify the prohead binding domain more precisely, deletion mutants lacking the last 10 and 15 amino acids (gp19-delta C10 and gp19-delta C15, respectively) of the extreme C terminus of gp19 were constructed . Limited tryptic digestion patterns of these mutant proteins in the presence or absence of ATP were basically the same as those of gp19-wt, indicating that the conformation and its ATP response were not changed by these deletions . gp19-delta C15 lacked prohead binding activity and, therefore, DNA packaging activity . gp19-delta C10 had significant DNA packaging activity although it was reduced to one-tenth of that of gp19-wt . These results indicate that a C-terminal region of residues L571 to D576 of gp19 is crucial for prohead binding and that the last ten residues D577 to W586 of the C terminus seems to be important in stable binding of gp19 to the prohead.

J Biol Chem, 1995 Feb 3, 270(5), 2139 - 44
The conserved G/F motif of the DnaJ chaperone is necessary for the activation of the substrate binding properties of the DnaK chaperone; Wall D et al.; The universally conserved DnaK and DnaJ molecular chaperone proteins bind in a coordinate manner to protein substrates to prevent aggregation, to disaggregate proteins, or to regulate proper protein function . To further examine their synergistic mechanism of action, we constructed and characterized two DnaJ deletion proteins . One has an 11-amino-acid internal deletion that spans amino acid residues 77-87 (DnaJ delta 77-87) and the other amino acids 77-107 (DnaJ delta 77-107) . The DnaJ delta 77-87 mutant protein, was normal in all respects analyzed . The DnaJ delta 77-107 mutant protein has its entire G/F (Gly/Phe) motif deleted . This motif is found in most, but not all DnaJ family members . In vivo, DnaJ delta 77-107 supported bacteriophage lambda growth, albeit at reduced levels, demonstrating that at least some protein function was retained . However, DnaJ delta 77-107 did not exhibit other wild type properties, such as proper down-regulation of the heat-shock response, and had an overall poisoning effect of cell growth . The purified DnaJ delta 77-107 protein was shown to physically interact and stimulate DnaK's ATPase activity at wild type levels, unlike the previously characterized DnaJ259 point mutant (DnaJH33Q) . Moreover, both DnaJ delta 77-107 and DnaJ259 bound to substrate proteins, such as sigma 32, at similar affinities as DnaJ+ . However, DnaJ delta 77-107 was found to be largely defective in activating the ATP-dependent substrate binding mode of DnaK . In vivo, the ability of the mutant DnaJ proteins to down-regulate the heat-shock response was correlated only with their in vitro ability to activate DnaK to bind sigma 32, in an ATP-dependent manner, and not with their ability to bind sigma 32 . We conclude, that although the G/F motif of DnaJ does not directly participate in the stimulation of DnaK's ATPase activity, nevertheless, it is involved in an important manner in modulating DnaK's substrate binding activity.

Curr Opin Biotechnol, 1995 Feb, 6(1), 37 - 43
Techniques in mammalian genome mapping; Schalkwyk LC et al.; Increasing emphasis is being given to genomic cloning using Escherichia coli vectors of intermediate insert capacity, such as bacteriophage P1, P1-derived artificial chromosomes and the F factor based bacterial artificial chromosomes . These vectors are being used in addition to yeast artifical chromosomes (YACs) in recognition of the difficulties encountered with YAC stability and with handling of YAC DNAs (problems that will not easily be overcome) . Nonetheless, YACs remain the most practical cloning system for global contig building . Efforts are currently under way to produce YAC contigs that represent the human and mouse genomes, and these will increasingly exploit extensive anchoring to detailed genetic maps . Intermediate capacity clone collections based on YAC contigs will follow, enabling the compilation of mapped gene catalogues . In this way, the era of big gene hunts will draw to a close.

J Bacteriol, 1995 Feb, 177(3), 660 - 6
Very short patch repair of T:G mismatches in vivo: importance of context and accessory proteins; Lieb M et al.; In Escherichia coli, T:G mismatches in specific contexts are corrected by a very short patch (VSP) repair system . Previous studies have shown that the product of gene vsr mediates correction of T:G to C:G in the 5'CTAGG/3'GGTCC context and in some related contexts . Amber mutations that arose in CAG sequences in gene cI of bacteriophage lambda were used to determine the effect of flanking bases on the repair of T:G mispairs arising during phage recombination . The experimental findings were combined with published data on mismatch repair of mutations in lambda gene P and E . coli gene lacI . While VSP repair was most efficient in the context 5'CTAGG, there was very significant correction when either the 5'C or the 3' G was replaced by another base . Some mismatch repair of TAG to CAG occurred in all contexts tested . Reduction in VSP repair caused by the lack of MutL or MutS was fully complemented by the addition of vsr+ plasmids when the T:G mispair was in the 5'CTAGG/3'GGTCC context . VSP repair was decreased in bacteria containing mutS+ on a multicopy plasmid . It is suggested that VSP repair maintains sequences such as the repetitive extragenic palindromic (REP) and Chi sequences, which have important roles in E . coli and closely related bacteria.

J Bacteriol, 1995 Feb, 177(3), 497 - 501
The old exonuclease of bacteriophage P2; Myung H et al.; The Old protein of bacteriophage P2 is responsible for interference with the growth of phage lambda and for killing of recBC mutant Escherichia coli . We have purified Old fused to the maltose-binding protein to 95% purity and characterized its enzymatic properties . The Old protein fused to maltose-binding protein has exonuclease activity on double-stranded DNA as well as nuclease activity on single-stranded DNA and RNA . The direction of digestion of double-stranded DNA is from 5' to 3', and digestion initiates at either the 5'-phosphoryl or 5'-hydroxyl terminus . The nuclease is active on nicked circular DNA, degrades DNA in a processive manner, and releases 5'-phosphoryl mononucleotides.

Blood, 1995 Feb 1, 85(3), 727 - 33
Substitution of Val for Met at residue 239 of platelet glycoprotein Ib alpha in Japanese patients with platelet-type von Willebrand disease; Takahashi H et al.; Genomic DNA was studied from four patients with platelet-type von Willebrand disease (vWD) from two Japanese families previously reported . The entire coding region of platelet glycoprotein (GP) Ib alpha, a component of the platelet receptor for von Willebrand factor (vWF), was examined by polymerase chain reaction (PCR) followed by direct DNA sequence analysis . A single point mutation was found in all patients resulting in substitution of Val (GTG) for Met (ATG) at residue 239 of GPIb alpha . All patients were heterozygous for the mutation, whereas none of the unaffected family members had an amino acid substitution at residue 239 . Because the nucleotide substitution destroys an NIa III restriction site on GPIb alpha, PCR products were subjected to digestion with this enzyme; DNA fragments from both normal and mutant alleles were detected in all affected individuals . In allele-specific PCR, DNA was amplified from patients' genomic DNA using either adenine- or guanine-containing primers, whereas only adenine-containing primer successfully amplified DNA from normal individuals . Cloning of amplified DNA into bacteriophage M13mp19 and subsequent DNA sequence analysis confirmed the mutation in these families . The absence of the amino acid substitution at residue 239 of GPIb alpha in the normal individuals tested, together with the linkage of this substitution to the phenotypic expression of disease in these two families and in a family recently described suggest that this amino acid change is a molecular basis for platelet-type vWD, and the substitution may produce a quite similar phenotype to the one reported previously (Gly to Val at residue 233 of GPIb alpha).

Transfusion, 1995 Feb, 35(2), 137 - 44
Construction of bacteriophage expressing mouse monoclonal Fab fragments directed against the human MN glycophorin blood group antigens; Czerwinski M et al.; BACKGROUND: The MN human blood group antigens are complex glycopeptide antigens at the amino terminus of glycophorin A . Many different mouse monoclonal antibodies to these antigens have been produced and characterized . The construction of combinatorial immunoglobulin libraries displaying antibody Fab fragments on the surface of bacteriophage (Fab-phage) represents a novel approach for developing monoclonal reagents, for exploring the diversity of the immune response to specific antigens, and for understanding the molecular basis of the interaction of an antibody with its antigen . However, it is necessary to determine whether Fab fragments displayed on bacteriophage surfaces retain immunologic characteristics similar to the intact antibodies . STUDY DESIGN AND METHODS: Fab-phage were constructed from three anti-N (AH7, N61, and N92) and two anti-M (425/2B and M2A1) murine hybridomas . The Fab-phage and parental hybridomas were compared by enzyme-linked immunosorbent assay, Western blotting, and flow cytometry . RESULTS: In each case, the Fab-phage and its parental hybridoma antibody had similar immunologic characteristics . In particular, their dependence on the pH of the buffer and on sialylation of the target antigen was similar . CONCLUSION: These results suggest that Fab-phage may provide novel reagents with applications in immunohematology and may be useful in the study of the immune response to human blood group antigens.

Mol Microbiol, 1995 Feb, 15(4), 649 - 60
The bacteriophage T4 middle promoter PuvsX: analysis of regions important for binding of the T4 transcriptional activator MotA and for activation of transcription; March-Amegadzie R et al.; Bacteriophage T4 middle promoters, which are transcribed using phage-modified host RNA polymerase and the T4 transcriptional activator, MotA, match the host sigma 70 consensus sequence at -10, but they have a different consensus ((t/a)(t/a)TGCTT(t/c)A) (a MotA box) at -30 . While the T4 middle promoter PuvsX has these -10 and -30 motifs, it also has matches to the MotA box at -35, -51, -70, and -87 . We show that MotA binds to PuvsX DNA, footprinting a region that includes the MotA boxes at -30, -35, and -51 . Very high levels of MotA are required for footprinting and gel-shift experiments, and protein-DNA complexes formed in the presence of both phage-modified polymerase and MotA are more resistant to HindIII cleavage than those formed with either protein alone . These results suggest that MotA-DNA interactions may be stabilized by phage-modified polymerase . Sequences between -18 and -38 are absolutely required for MotA activation of transcription, but sequences upstream of -38 are stimulatory, particularly when chloride instead of glutamate is the major anion . Our results dissect PuvsX into a core promoter, downstream of -38, which is required for MotA activation, and an upstream region that enhances transcription especially under conditions less favourable for protein-DNA interactions.

Farmaco, 1995 Feb, 50(2), 91 - 8
Antiproliferative activity of some unsubstituted angular analogoues of ellipticine; Ferlin MG et al.; With the aim of obtaining further knowledge on the antiproliferative activity of pyrroloquinolines and isoquinolines, we prepared four unsubstituted angular pyridotetrahydrocarbazoles having a fourth non-aromatic ring, via modified Fischer synthesis . These compounds may be considered as simpler analogues of ellipticine . They induced evident antiproliferative effects in Ehrlich ascites and in CHO cells in vitro, but were ineffective on T2 bacteriophage . These compounds formed molecular complexes with DNA in vitro, while in CHO cells in vivo, they induced double-strand breaks in DNA and DNA-protein cross-links . These data suggest that these ellipticine analogoues are capable of inhibiting topoisomerase II, as the parent compound does . The most active derivative was 2N-5H-6,7,8,9-tetrahydropyrido{2,3-a}carbazole, which represents an interesting model for the study of new antitumor drugs.

Bioorg Khim, 1995 Feb, 21(2), 83 - 111
{NMR spectroscopy in the study of the spatial structure of membrane peptides and proteins}; Pervushin KV et al.; The review covers the field of the spatial structure determination of membrane-associated peptides and proteins by the High-Resolution NMR Spectroscopy . The membrane-bound conformations of several hormones, neuropeptides, lipopeptides, peptide antibiotics, bacteriophage coat proteins, transmembrane domains of receptors and others are considered . To mimic the biomembrane environment the appropriate artificial media (organic solvents, micelles of detergents of lipid vesicles) must be adjusted . In that case NMR spectroscopy is a powerful tool for the spatial structure and dynamics investigations of membrane associated peptides and proteins constituting the bases for unraveling of their structure-function relationships.

Curr Biol, 1995 Feb 1, 5(2), 149 - 57
Accessory proteins function as matchmakers in the assembly of the T4 DNA polymerase holoenzyme; Kaboord BF et al.; BACKGROUND: During bacteriophage T4 DNA replication, the 44/62 and 45 accessory proteins combine with the DNA polymerase to form a processive holoenzyme complex . Formation of this complex is dependent upon ATP hydrolysis by the 44/62 protein . It is uncertain, however, whether the 44/62 protein remains with the 45 protein as part of this protein 'sliding clamp' during DNA synthesis, or whether it is required only for complex assembly . RESULTS: To address this tissue, we have stoichiometrically assembled a processive T4 DNA polymerase holoenzyme complex, capable of strand-displacement synthesis, on a forked primer/template . By titrating the 44/62 protein to substoichiometric concentrations, we have shown that it can act catalytically to load on to the primer/template the 45 protein, which, in turn, combines with the DNA polymerase to form a processive complex . Two distinct complex species are formed: most of the complexes are highly stable, with a half life of 7 minutes, whereas the remainder have a half-life of 0.4 minutes . Precipitation of the protein-DNA complexes, followed by western blot analysis, verified that the complexes contain the DNA polymerase and 45 proteins, but not the 44/62 protein . CONCLUSION: Using physiological protein concentrations, we have shown that the composition of the T4 protein sliding clamp consists solely of the 45 protein . The role of the 44/62 protein is that of a molecular matchmaker, in that it serves to load the 45 protein onto the DNA but does not remain an essential component of the processive complex.

Curr Biol, 1995 Feb 1, 5(2), 139 - 48
Swapping DNA strands and sensing homology without branch migration in lambda site-specific recombination; Nunes-Duby SE et al.; BACKGROUND: Many site-specific recombinases act by forming and resolving branched Holliday junction intermediates . Previous findings have been consistent with models involving branch migration across the 'overlap region' of obligate homology, located between the staggered sites where the two single-strand exchanges occur . We have investigated the validity of such models in the case of bacteriophage lambda site-specific recombination . RESULTS: By using synthetic lambda att-site Holliday junctions, incorporating sequence heterologies that impose constraints on branch migration, we have found that the optimal position of the junction for either top-strand or bottom-strand resolution by lambda integrase (Int) is not at the ends, but close to the middle of the seven base-pair overlap region . A minor shift of the branch point around the central base pair caused a remarkable switch in resolution bias . Our findings suggest that branch migration is limited to the central one to three base pairs of the overlap region . They lead to a new model for lambda site-specific recombination, in which there are two symmetrical swaps of two to three nucleotides each, linked by a central isomerization step that causes a change of the stacking interactions between the four junction arms . On the basis of isolated strand-joining reactions carried out by Int in the presence or absence of base complementarity, we propose that sequence homology is sensed during the annealing step prior to strand joining . The new model eliminates mechanistic complications associated with large helical rotations required by branch-migration models . CONCLUSIONS: The results reported here suggest that the recognition of sequence homology in Int-dependent site-specific recombination does not rely primarily on branch migration . The property of cleaving Holliday junctions a few base pairs away from the crossover puts lambda Int into the same category as endonucleases that cleave Holliday junctions in homologous recombination.

Antimicrob Agents Chemother, 1995 Feb, 39(2), 308 - 13
Development of test panel of beta-lactamases expressed in a common Escherichia coli host background for evaluation of new beta-lactam antibiotics; Bradford PA et al.; A test panel of 35 different beta-lactamases expressed in a common Escherichia coli host was created to compare the effect that each beta-lactamase had on susceptibility to various beta-lactam antibiotics . A comparison of the MICs obtained with this panel generally reflected differences in the substrate profiles of the various beta-lactamases examined . In addition, several strains of the panel were subjected to selection with porin-specific bacteriophages to obtain mutants lacking either the OmpC or OmpF porin protein . A mutation in either OmpC or OmpF did change the susceptibilities of certain strains expressing beta-lactamase to certain beta-lactam antibiotics . However, the loss of a single porin did not predictably alter susceptibility to any given beta-lactam drug . This panel of strains producing various beta-lactamases was found to be a useful tool for comparing the effects of different beta-lactamases and outer membrane permeability upon susceptibility to beta-lactam drugs.

Nat Genet, 1995 Feb, 9(2), 177 - 83
Detection of mutations by cleavage of DNA heteroduplexes with bacteriophage resolvases; Mashal RD et al.; We have explored the application of the bacteriophage resolvases T4 endonuclease VII and T7 endonuclease I for detecting mutations in genomic DNA . Heteroduplex DNA fragments prepared by amplification from DNA containing known mutations were cleaved by one or both enzymes at nucleotide mismatches created by 3 of 3 short deletions and 13 of 14 point mutations in fragments as large as 940 basepairs . Heteroduplexes representing all four classes of possible single nucleotide mismatches were cleaved, and the sizes of the cleavage products generated correlated with the location of the mutation . We conclude that bacteriophage resolvases may be useful reagents for the rapid screening of DNA for mutations.

Anim Genet, 1995 Feb, 26(1), 27 - 30
Features of the DNA fingerprinting probe pITZ1; Huebscher KJ et al.; Stringently controlled plasmids generate DNA fingerprint patterns in mammals when used at low hybridization temperatures . In order to develop a probe for use in paternity testing in cattle we screened a bovine, partial genomic plasmid library with the PCR-amplified ori region of plasmid P1 . Of eight isolated clones one generated strong band patterns at high stringency in various mammalian species (data not shown) . Sequence analysis revealed an imperfect, compound dinucleotide repeat region, which was PCR-amplified and cloned into the plasmid vector pUC19 . Fingerprint results generated by this probe (termed pITZ1) in cattle are compared with the results generated by VNTR-probe pV47, which itself was developed by screening a human chromosome 16 library with tandem repeats of bacteriophage M13 . Probe pITZ1 is useful in conjunction with other VNTR-probes for DNA fingerprinting in cattle and donkey populations . The dinucleotide repeat region responsible for the band patterns generated with pITZ1 is close to an Alu-like sequence, which may be involved in eukaryotic replication mechanisms.

Can J Microbiol, 1995 Feb, 41(2), 163 - 9
Contractile-tailed bacteriophages adsorb to Escherichia coli O128ab lipopolysaccharide that is altered by large plasmids to provide receptors and lipopolysaccharide heterogeneity within the serogroup; Kalmokoff ML et al.; The verotoxigenic Escherichia coli strain H.I.8 (originally O128:B12, now not typeable) contained a ColB+M plasmid and two morphologically identical temperate bacteriophages (H18A and H18B) . Both phages were O128ab specific, using the lipopolysaccharide O side chains of susceptible clinical isolates as receptors . SDS polyacrylamide gel electrophoresis with silver staining of O128ab lipopolysaccharide revealed four distinct types of ladder with different interband spacings . No specificity was found between ladder type and sensitivity to either phage . One of the numerous large plasmids present in O128ab isolates was found to modify the structure of the lipopolysaccharide O side chains to provide phage receptors.

Biochemistry, 1995 Jan 31, 34(4), 1120 - 6
Role of entropic interactions in viral capsids: single amino acid substitutions in P22 bacteriophage coat protein resulting in loss of capsid stability; Foguel D et al.; Bacteriophage P22 is a double-stranded DNA containing phage . Its morphogenetic pathway requires the formation of a precursor procapsid that subsequently matures to the capsid . The stability of bacteriophage P22 coat protein in both monomeric and polymeric forms under hydrostatic pressure has been examined previously {Prevelige, P . E., King, J., & Silva, J . L . (1994) Biophys . J . 66, 1631-1641} . The monomeric protein is very unstable to pressure and undergoes denaturation at pressures below 1.5 kbar, whereas the procapsid shell is very stable to applied pressure and does not dissociate with pressure to 2.5 kbar . However, under applied pressure the procapsid shells are cold labile, suggesting they are entropically stabilized . We have analyzed the pressure stability of mutant procapsid shells having either of two single amino acid substitutions in the coat protein (G232D and W48Q) using light-scattering and fluorescence emission methods . While the wild-type shells were stable under 2.2 kbar of pressure at room temperature (22 degrees C), the G232D mutant shells showed time-dependent dissociation under these conditions . Decreasing the temperature to 1 degree C dramatically accelerated the dissociation of G232D mutant under applied pressure . On the other hand, the W48Q mutant shells could be dissociated easily by pressure at room temperature and displayed little dependence on temperature, suggesting a smaller entropic contribution to the stability of this mutant . The unpolymerized mutant subunits displayed a pressure stability similar to that of the wild type.(ABSTRACT TRUNCATED AT 250 WORDS)

Biochim Biophys Acta, 1995 Jan 25, 1260(2), 132 - 8
Histones associated with single-stranded DNA do not preclude the formation of double-helical DNA; Fernandez-Busquets X et al.; The effect of histones on the reaction of reassociation of the two complementary strands of DNA from different sources has been investigated . The reassociation rate of denatured linear DNA from bacteriophage M13 monitored spectrophotometrically and using nuclease S1 is roughly the same in the presence and absence of core histones at physiological ionic strength . Electron microscopy reveals that in the samples containing histones a large network of duplex DNA is produced . Nevertheless, closed circular M13 DNA and a cloned DNA fragment (158 bp) from nucleosomal origin are entirely renatured in the presence of histones as demonstrated by the well-defined double-stranded DNA bands seen in electrophoretic gels . Various experiments performed using the purified (+) and (-) strands of the cloned nucleosome DNA fragment at low ionic strength indicate that core histones initially bound to one or even to the two strands allow the formation of duplex DNA . These findings and the results obtained with partially denatured closed circular M13 DNA allow us to conclude that core histones neither prevent the nucleation nor inhibit the rapid zippering reactions leading to the formation of double-stranded DNA . The mechanism that allows the renaturation of DNA in the presence of histones may also participate in biological processes involving the pairing of complementary nucleotides.

J Biol Chem, 1995 Jan 20, 270(3), 1205 - 12
How 434 repressor discriminates between OR1 and OR3 . The influence of contacted and noncontacted base pairs; Bell AC et al.; The sequence of the bacteriophage 434 OR1 (ACAAAACTTTCTTGT) differs from its OR3 (ACAGTTTTCTTGT) at positions 4-6 . X-ray analysis shows that the side chain of Gln33 of the 434 repressor makes van der Waals' and H-bond contacts with the T at position 4' in complex with OR1, but no specific contact is observed at this position in 434 repressor-OR3 complexes . No contacts are made by repressor to the bases at positions 5 or 6 in either binding site . The significance of the sequence differences between OR1 and OR3 in determining the operator affinity for repressor were examined by constructing synthetic variants of these operators . Measurements of the affinity of these operators for repressor as a function of ionic strength revealed that although base pairs 5 and 6 are not contacted by 434 repressor, they can nonetheless influence operator affinity for repressor by modulating the degree to which ionic interactions contribute to the overall binding energy . Both the magnitude and direction of their effect depends on the status of repressor's contacts to the bases at position 4 . The role of contact made by Gln33 to position 4 was examined by mutating this amino acid to Ala and by examining the affinity of wild type repressor for an operator bearing a 5-methylcytosine at position 4' in an OR1-4G mutant . These experiments showed that repressor's preferences at operator positions 5 and 6 are linked to its position 4 preference via a van der Waals' contact between amino acid 33 and a methyl group on the base at operator position 4' . Together, the results of the experiments shown here reveal that bases that do not contact the protein alter its preferences for bases at the contacted operator position 4.

Genomics, 1995 Jan 20, 25(2), 492 - 500
Cloning, characterization, and chromosomal localization to 4p16 of the human gene (LRPAP1) coding for the alpha 2-macroglobulin receptor-associated protein and structural comparison with the murine gene coding for the 44-kDa heparin-binding protein; Van Leuven F et al.; We report the molecular cloning of the human gene (symbol LRPAP1) coding for the alpha 2-macroglobulin receptor-associated protein (A2MRAP), as well as the gene coding for the 44-kDa heparin-binding protein (HBP-44), its murine counterpart . For both, genomic cosmid clones were isolated, and for the human gene a bacteriophage P1 clone containing the entire A2MRAP gene was also retrieved . The genes were characterized after subcloning: in both species, the known coding part of the cDNA is encoded by eight exons, and the position of the boundaries of the exons was conserved . The human LRPAP1 locus was assigned to chromosome 4 by PCR of human-hamster hybrid cell lines and by fluorescence in situ hybridization to band 4p16.3 . This maps closely to the variable constitutional deletions of the short arm of chromosome 4, observed cytogenetically in patients with the Wolf-Hirschhorn syndrome . Metaphase spreads of two such patients were analyzed by fluorescence in situ hybridization with an LRPAP1 genomic probe . The first patient, with karyotype 46,XY,del4(p14-p16.1), had retained both copies of the LRPAP1 gene . In contrast, the other patient, with karyotype 46,XY,del4(p15.3-pter), displayed no signal for LRPAP1 on the deleted chromosome.

J Mol Biol, 1995 Jan 13, 245(2), 86 - 91
Matching electrostatic charge between DNA and coat protein in filamentous bacteriophage . Fibre diffraction of charge-deletion mutants; Symmons MF et al.; The virion of Ff (fd, f1, M13) filamentous bacteriophage consists of a long tube of coat protein subunits in a shingled, helical array, surrounding a genome of circular single-stranded DNA . Modified fd virions have been generated by a mutation (K48A) that removes one positive charge from each coat protein subunit in the C-terminal region of the polypeptide chain facing the DNA . The number of nucleotides in the mutant DNA is unchanged, but the K48A virions are 35% longer than wild-type . We have measured the X-ray diffraction attributable to single virions in hydrated gels of wild-type and K48A bacteriophages . Most of the diffraction pattern shows no significant difference between wild-type and K48A . Since the DNA is only about 12% by weight of the wild-type virion, the diffraction pattern is dominated by the protein contribution, and the absence of significant differences indicates that there are no significant changes in the symmetry or structure of the protein coat . But there is a change in the diffraction pattern in a region where the DNA and protein contributions are comparable . The diffraction pattern of the K48A mutant shows an increase in intensity of one of the weaker equatorial peaks, relative to wild-type, in a region where the protein contribution has negative sign but the DNA contribution has positive sign . This is consistent with a decrease in the ratio of DNA:protein per unit length of the K48A mutant . The results support the view that the protein forms a sheath lined with positive charges interacting electrostatically and non-specifically with a negatively charged DNA core of matching charge density . The lower positive charge density lining the capsid in the K48A mutant means that correspondingly fewer nucleotides can be packaged per coat protein subunit, which in turn requires an elongation of the DNA inside the virion . A longer virion is thus required to package the same amount of DNA . Within the error of measurement, the number of positive charges on the protein interacting with the DNA is the same in K48A as in the wild-type, despite the fact that the mutant is 35% longer than the wild-type.

J Mol Biol, 1995 Jan 13, 245(2), 141 - 50
Specific interaction of terminase, the DNA packaging enzyme of bacteriophage lambda, with the portal protein of the prohead; Yeo A et al.; Terminase, the bacteriophage lambda DNA packaging protein, is a heteromultimer of two subunits, gpNu1 and gpA, the products of genes Nu1 and A, resp . Phage 21 is a lambdoid phage that produces a terminase similar to that of lambda terminase, the subunits of 21 terminase, gp1 and gp2, have the same domain structures of their lambda analog, gpNu1 and gpA, respectively . The lambda and 21 terminases have different DNA binding and prohead binding specificities . When the C-terminal 32 amino residues of gpA replace the C-terminal 32 residues of gp2, the resulting chimeric terminase specifically uses lambda proheads, indicating that the C-terminal 32 residues of gpA are a specificity domain for prohead binding . A second chimeric terminase, in which the C-terminal six residues of gpA are replaced by the C-terminal six residues of gp2, is unable to utilize lambda proheads, and a lambda phage producing this terminase, lambda Are636, is unable to form plaques . In the present work, a pseudorevertant of lambda Are636 was isolated that contained a mutation Bms8, affecting the prohead . The B gene encodes the portal protein of lambda proheads, which forms the special vertex that is thought to serve as (1) the site of DNA entry into the prohead during packaging, (2) the site for DNA exit during DNA injection, and (3) the site of tail attachment during virion assembly . Bms8 is predicted to change residue 331 of gpB from proline to serine . Burst size measurements and in vitro DNA packaging experiments demonstrated allele-specific interactions between the Are636 terminase and Bms8 proheads . That is, wild-type terminase interacted more efficiently with wild-type proheads than with Bms8 proheads, and Are636 terminase interacted with Bms8 proheads more efficiently than with wild-type proheads . Prohead binding by lambda terminase is stimulated by an assembly catalyst, gpFI . In vitro packaging extracts lacking gpFI were used under conditions in which packaging was gpFI-independent . In the absence of gpFI, Are636 terminase interacted most efficiently with Bms8 proheads, and wild-type terminase interacted most efficiently with wild-type proheads . The allele-specific interactions in the absence of gpFI indicate that the Are636 and Bms8 mutations affect direct interactions between terminase and the portal protein, rather than acting indirectly by altering the interactions of terminase and gpB and gpFI.

J Mol Biol, 1995 Jan 13, 245(2), 126 - 40
Mutational analysis of the prohead binding domain of the large subunit of terminase, the bacteriophage lambda DNA packaging enzyme; Yeo A et al.; Terminase, the DNA packaging enzyme of bacteriophage lambda, is made up of two subunits, gpNul and gpA, the products of the Nu1 and A genes . The activities of terminase include DNA binding, cos cleavage and prohead binding . Specificity domains within the structure of terminase have previously been defined by genetic studies of lambda-21 hybrids . The prohead binding domain of terminase is localized to the last 32 amino acid residues of gpA . Mutations in the prohead binding domain of gpA were constructed by introducing the corresponding amino acids from gp2, the gpA analog of bacteriophage 21 . The last five residues of gpA can be replaced with little effect on the burst size of lambda . A phage with a replacement of the last six residues of gpA with the corresponding residues of gp2 was unable to form plaques, indicating that the sixth-to-last residues of gpA is crucial for prohead binding . Site-specific mutagenesis of the sixth-to-last position of gpA indicated that the sixth-to-last residue of gpA must be hydrophobic, of the seven amino acids tested, only isoleucine and valine can substitute for leucine at this position . Although the last five residues of gp2 were functional when they replaced the last five residues of gpA, two results indicated that the last five residues of gpA functioned better than the corresponding residues of gp2 . First, the presence of a valine residue at the sixth-to-last position of gpA allowed plaque formation, whereas replacement of the last six residues of gpA with those of gp2, which substitutes a valine residue at the sixth-to-last position, was lethal . The second set of results indicating that the last five residues of gpA function better than the gp2 residues were obtained by study of revertants of lethal substitution mutations . In constructing the replacement mutations, a short linker was inserted into the C terminus of the A gene; this insertion created a short duplication of the end of the A gene, so that the normal C-terminal codons were located downstream of the stop codon of the A gene in the substitution mutants . Revertants of the lethal substitution mutations were obtained in which a mutation in the stop codon resulted in addition of the last five residues of gpA to the end of the substitution terminase.(ABSTRACT TRUNCATED AT 400 WORDS)

Gene, 1995 Jan 11, 152(1), 99 - 102
Cloning, sequencing and expression of the dnaJ gene of Coxiella burnetii; Zuber M et al.; A 6-kb EcoRI genomic DNA fragment of Coxiella burnetii, isolated from a recombinant bacteriophage lambda ZapII library, allowed heterologous genetic complementation of Escherichia coli deleted for its dnaJ gene . The C . burnetii dnaJ gene was expressed in E . coli and identified by Western blot analysis using polyclonal antibodies raised against purified E . coli DnaJ protein . Deletion mapping and genetic complementation demonstrated that C . burnetii dnaJ is present on a 2-kb EcoRI-HindIII genomic DNA fragment, from which the nt sequence of the C . burnetii dnaJ gene was determined.

Gene, 1995 Jan 11, 152(1), 35 - 9
Functional phage display of ciliary neurotrophic factor; Saggio I et al.; We report the display of human ciliary neurotrophic factor (hCNTF), a survival factor for neuronal cells belonging to the alpha-helical cytokine superfamily, on the surface of the filamentous bacteriophage fd . The hCNTF cDNA was fused to a DNA sequence encoding the C-terminal domain of pIII, a minor coat protein exposed at one end of fd . Gene fusions were cloned into a plasmid containing the ColE1 plasmid and fd origins of replication, and were packaged into phagemid particles upon superinfection with M13KO7 helper phage . The resulting fusion phage bound specifically to anti-CNTF antibodies and to the recombinant soluble CNTF alpha-receptor . Moreover, phage-displayed hCNTF was found to possess biological activity at concentrations comparable to those of the soluble cytokine . These results demonstrate that CNTF can be displayed on phage in a correctly folded and functionally active form . Binding of fusion phage to immobilized CNTF alpha-receptor and subsequent elution at low pH resulted in affinity purification of CNTF-displaying virions . Utilization of this technology should enable the selection of high-affinity variants from libraries of CNTF mutants displayed on phage.

Virology, 1995 Jan 10, 206(1), 339 - 52
Analysis of 45 kb of DNA located at the left end of the chlorella virus PBCV-1 genome; Lu Z et al.; Forty-five kilobases of DNA, including the previously sequenced 2.2-kb inverted repeat region, located at the left termini of the 330-kb Chlorella virus PBCV-1 genome were sequenced and analyzed . Eighty-five complete open reading frames (ORFs) larger than 195 nucleotides were identified . Thirty-seven of the 85 ORFs, which are densely packed on both strands of the DNA, were considered major ORFs . Fifteen of the major ORFs have similarity to genes in the databases, including bacterial glycerophosphoryl diester phosphodiesterase, bacteriophage T4 endonuclease V, D-isomer specific 2-hydroxyacid dehydrogenases, and beta-alanine synthetase and bacterial nitrilases . Two major ORFs resemble the virus major capsid protein . Three major ORFs contain three or more ankyrin-like repeat elements and four ORFs encode proline-rich proteins.

Virology, 1995 Jan 10, 206(1), 108 - 15
Synthesis of an infectious full-length cDNA clone of rice yellow mottle virus and mutagenesis of the coat protein; Brugidou C et al.; A full-length cDNA clone of rice yellow mottle sobemovirus (RYMV) was synthesized and placed adjacent to a bacteriophage T7 RNA polymerase promoter sequence . Capped-RNA transcripts produced in vitro were infectious when mechanically inoculated onto rice plants (Oryza sativa L) . Individual full-length clones varied in their degree of infectivity but all were less infectious than native viral RNA . A representative clone, designated RYMV-FL5, caused a disease phenotype identical to that produced by viral RNA except that symptoms were somewhat slower to appear than those induced by viral RNA . The infectivity of RYMV-FL5 was verified by ELISA, Western blot analysis, Northern blot hybridization, RT-PCR, and Southern blot hybridization . Frameshift and deletion mutations introduced into the coat protein cistron demonstrated that the coat protein was dispensable for RNA replication in rice protoplasts . However, the coat protein was required for full infectivity in rice plants, presumably by playing a role in phloem-mediated long-distance movement and possibly in cell-to-cell movement.

Biochem Biophys Res Commun, 1995 Jan 5, 206(1), 64 - 76
Induction of membrane proliferation by poliovirus proteins 2C and 2BC; Aldabe R et al.; Poliovirus infection leads to the appearance of a number of cytoplasmic vacuoles involved in the replication of virus genomes . To characterize the viral proteins involved in membrane proliferation different poliovirus proteins have been expressed in HeLa cells . Two recombinant vaccinia viruses have been obtained that express poliovirus protein 2C, one under the 5' untranslated (UTR) sequence of poliovirus and another under the leader region of EMC virus . Expression of 2C was very efficient in both cases, although better results were obtained when poliovirus 2C was expressed under the 5'UTR sequence of EMC virus . Transient expression of poliovirus proteins 2B, 2C or 2BC placed under a T7 promoter was analyzed using a recombinant vaccinia virus that contains the bacteriophage T7 RNA polymerase . The expression of 2C, or 2BC, contrary to 2B, was able to induce the proliferation of vacuoles morphologically similar to those found during poliovirus infection . These findings indicate that poliovirus protein 2C, in addition to its NTPase and RNA binding activities, is also endowed with the capacity to induce the formation of cytoplasmic vacuoles.

Biochem Biophys Res Commun, 1995 Jan 5, 206(1), 230 - 7
An engineered disulfide bridge in the transmembrane region of phage M13 coat protein stabilizes the alpha-helical dimer; Khan AR et al.; A single Cys-residue (Cys24) was introduced into the 50-amino acid major coat protein of M13 bacteriophage as part of a two-site substitution (Y24C-V31A) within the effective transmembrane (TM) segment (Tyr21 to Ile39) of the coat protein . Mutant Y24C-V31A was able to complete the phage life cycle and was shown to contain free sulfhydryls in the intact virus, as evidenced by susceptibility of Y24C-V31A phage to alkylation by Cys-specific 14C-iodoacetamide (14C-IAN) . In contrast, the protein solubilized in deoxycholate micelles was resistant to 14C-IAN modification and was virtually inert to a transition from a characteristic alpha-helical oligomeric state to an aggregated beta-sheet structure relative to WT and V31A coat proteins, as shown by circular dichroism spectroscopy and SDS-PAGE . Reduction of mainly dimeric Y24C-V31A protein using beta-mercaptoethanol (beta-ME) generated monomeric species and resulted in a loss of helical thermostability . The overall results indicated that solubilization of Y24C-V31A coat protein into micelles resulted in formation of thermostable disulfide-bridged helical dimers . The disulfide bridge is deduced to be positioned along the stripe of residues involved in hydrophobic packing of TM parallel helical dimers.

Proc Natl Acad Sci U S A, 1995 Jan 3, 92(1), 200 - 4
Structure, characterization, and expression of the rat oxytocin receptor gene; Rozen F et al.; The multiple hormonal and neurotransmitter functions of the nonapeptide oxytocin are mediated by specific oxytocin receptors (OTRs) . In most target tissues, the number of OTRs is strongly regulated . Specifically, in the uterus, a dramatic OTR upregulation precedes the onset of parturition . To study the molecular mechanisms underlying OTR regulation, we have isolated and characterized recombinant bacteriophage lambda EMBL3 genomic clones containing the rat OTR gene, using sequence information derived from a human myometrial OTR cDNA . The rat OTR gene spans > 20 kb and contains three exons . A 97-bp intron is in the 5' untranslated region and a > 12-kb intron interrupts the coding region between transmembrane domains 6 and 7 . The promoter region lacks an apparent TATA or CCAAT box but contains multiple putative interleukin-response elements {six NF-IL6 (C/EBP beta) and four APRF (STAT3) binding motifs}, supporting the notion that interleukins may mediate labor induction via transcriptional activation of the OTR gene . The predicted amino acid sequence is 93% identical to the human OTR sequence but only 48% and 38% identical to the rat V1 and V2 vasopressin receptor sequences, respectively . At parturition, the OTR gene is highly expressed in the rat uterus and gives rise to at least three transcripts (2.9, 4.8, and 6.7 kb) which differ in the length of their 3' untranslated regions.

Biochim Biophys Acta, 1995 Jan 2, 1260(1), 79 - 84
Site-directed mutagenesis of the M13 gene 5 protein: the role of Arg-21, Tyr-26 and Tyr-41; Turner GP et al.; The gene 5 protein of bacteriophage M13 is a single stranded DNA binding protein essential for phage replication . We have generated the mutations R21A, Y26F and Y41A in the gene 5 protein and purified the mutant proteins for functional characterisation in vitro . The complex of Y26F with single-stranded DNA is disrupted at 0.8 M NaCl, the same salt concentration as that required to dissociate the native complex . However, the mutant proteins R21A and Y41A are considerably less stable and dissociate from single-stranded DNA at at 0.4 M NaCl . The fluorescence of the mutant proteins and the DNA-protein complexes they form has been compared with the wild-type protein to allow an assessment of the contribution from individual residues . We conclude that the fluorescence of Tyr-26 is 50% quenched in the complex with DNA, whereas that of Tyr-41 is fully quenched . Fluorescence titrations of the mutant proteins with poly(dT) show that all three mutant proteins can bind DNA but, in the case of Y41A, with a change of stoichiometry suggesting a loss of cooperativity . Gel retardation analysis of Y41A also shows anomalous behaviour in binding to oligonucleotides, consistent with the proposed involvement of Tyr-41 in dimer-dimer contacts in the nucleoprotein complex.

Med Dosw Mikrobiol, 1995, 47(3-4), 149 - 54
{Bacteriophages of serologic group F converting synthesis of fibrinolysin and beta toxin in S . aureus}; Mlynarczyk G et al.; The properties of the 18 S . aureus bacteriophages of the serogroup F were investigated . The chosen bacteriophages were able to convert production of fibrinolysine inhibited synthesis of beta toxin . All of the investigated bacteriophages were classified as morphological group II of the family Styloviridae on the basis of the electron microscope analysis . The size of capsids of the examined bacteriophages was 54.10 +/- 0.80 nm and the tail length was from 206.1 nm to 311.9 nm . Most of them (15 bacteriophages) had the tail terminated in the basal plate . The lytic properties of the investigated bacteriophages were not identical . 11 of them showed features of lytic group III and 7 of lytic group V (miscellaneous).

Med Dosw Mikrobiol, 1995, 47(3-4), 141 - 7
{Bacteriophages of serologic group B converting synthesis of fibrinolysin in S . aureus}; Mlynarczyk G et al.; On the basis of the length of tail, two morphological types of S . aureus bacteriophages of the serogroup B converting fibrinolysine were shown . The properties of the 22 S . aureus bacteriophages of the serologic group B were investigated . All of the investigated bacteriophages were classified as the morphological group II of the family Styloviridae on the basis of electron microscope analysis . The size of capsids of the examined bacteriophages was 53.85 +/- 1.05 nm and the tail length varied from 135.4 nm to 170.5 nm . All of them had the tail terminated in the basal plate . The lytic properties of the investigated bacteriophages were not identical . 7 of them showed features of lytic group I and 15 of lytic group III . The members of lytic group III had apparently a longer tail than bacteriophages of lytic group I.

Adv Exp Med Biol, 1995, 395, 295 - 300
Structure and organization of the bovine oxytocin receptor gene; Ivell R et al.; A DNA probe specific for the V and VI transmembrane domains of the bovine oxytocin receptor was initially prepared by reverse transcription PCR, and its structure and specificity confirmed by DNA sequencing . This probe was then used to screen a bovine genomic DNA library in bacteriophage lambda, and three positive clones were purified, subjected to restriction analysis and relevant fragments sequenced . Parallel to this, a cDNA library prepared using bovine endometrial RNA at the time of ovulation was screened by PCR employing the same primers as above . The longest cDNA clone was also fully sequenced . This clone still lacked, however, a substantial stretch of 5'sequence . The full transcript structure, and hence also the exon-intron organization, was then obtained by RT-PCR using primer oligonucleotides deduced from the cloned genomic sequence . All nucleotide sequence information was derived from a combination of two independent genomic clones, a cDNA clone and several independent RT-PCR reactions programmed by myometrial RNA, all in both strand orientations . The structural organization of the bovine oxytocin gene essentially conforms to that described for the human gene . Unlike the human gene, however, the 5'non-coding region of the primary transcript is interrupted by only a single intron, with a further intron in the coding region separating the sequences encoding the transmembrane domains VI and VII . The difference between this structure and that for the human gene suggests the existence of a differential splicing of 5' non-coding sequences.

Intervirology, 1995, 38(1-2), 47 - 62
Assembly and antigenicity of hepatitis B virus core particles; Seifer M et al.; Recent studies in Xenopus oocytes and other systems have led to an understanding of the HBV capsid, or core particle, assembly process . Nascent HBV core polypeptides rapidly dimerize . Accumulation of free dimers to a signature concentration (approximately 0.8 microM) then triggers a highly cooperative capsid assembly reaction . This dimer-to-capsid transition is accompanied by a switch from HBe to HBc antigenicity and appears to be nucleated by interaction between core protein and RNA: deletion of a protamine-like RNA binding domain at the C-terminus of the core protein markedly increases the concentration of dimers needed to drive capsid assembly . The simple assembly pathway seen for HBV capsids mirrors that of R17 bacteriophage.

Postepy Hig Med Dosw, 1995, 49(1), 23 - 31
{Studies on diversification and restriction of immune response against glycopeptide antigens}; Czerwinski M; The M and N human blood group antigens are complex glycopeptide determinants at the amino terminus of the red blood cell membrane glycoprotein, glycophorin A . The heavy and light chain variable region cDNA sequences were determined for eight murine monoclonal antibodies recognizing the M or N blood group determinants . Among anti-N, but not anti-M, hybridomas, apparent restriction was found: all four anti-N VH regions of the heavy chains were derived from the same family, VH2(Q52) and all used the same J4 gene segment . To determine whether the Fab fragments displayed on the bacteriophage surface retain the immunological characteristics of intact antibodies, Fab-phages were constructed from five murine monoclonal antibodies recognizing glycophorin A . In each case, the Fab-phage had similar immunological characteristics as the respective antibodies . These results suggest that Fab-phages may be useful in studying the immune response to human glycosylation-dependent peptidic epitopes.

Nucleic Acids Symp Ser, 1995, (33), 256 - 7
RNA-protein interactions of the bacteriophage RB69 RegA translational repressor protein; Jozwik CE et al.; We report the RNA-protein interactions of phage RB69 RegA and RegA691 proteins . In RNase protection assays, RB69 RegA protects from 10 (44 mRNA) to >70 (rpbA) nucleotides; for rpbA in particular, the length of the protected region is RegA-concentration dependent . Quantitative fluorescence quenching measurements with RB69 wildtype RegA and RegA691 show that both proteins bind poly(U) similarly, but RegA691 binds with lower affinity to both 44 and rpbA RNA . The results indicate that both RB69 RegA RNA-protein and protein-protein interactions contribute to a translational repression hierarchy.

Nucleic Acids Symp Ser, 1995, (33), 240 - 3
The isolation of U1 RNA-binding antibody fragments from autoimmune human-derived bacteriophage display libraries; Powers JE et al.; Almost seventy percent of systemic lupus erythematosus (SLE) patients produce autoantibodies specific for U1 RNA, the RNA component of the U1snRNP complex involved in pre-mRNA splicing . Human anti-U1 RNA antibodies from SLE patients provide an ideal model for studying protein-RNA interactions . In addition, a more in depth look at the mechanism by which an antibody interacts with U1 RNA will lend insight into immune disregulation and may have therapeutic potential in the treatment of autoimmune disease . The possibility of cloning an anti-U1 RNA antibody has been investigated using "phage Fab display," a method involving the display of Fab fragments on the outer surface of filamentous phage . Human Fab cDNA libraries were constructed using total RNA collected from leukophoresed anti-U1 RNA antibody-positive SLE patient sera . The resulting Fab genes were subcloned into a phagemid expression vector which produced phage displaying Fab fragments in E.coli1 . Libraries were enriched for U1 RNA-binding clones after several rounds of affinity selection against purified, in vitro transcribed U1 RNA . Putative U1 RNA-binding clones were identified by colony lift and the corresponding Fab genes were expressed and purified from E.coli for binding studies . Results demonstrate that RNA-binding Fab can be isolated from combinatorial phage display libraries.

Nucleic Acids Symp Ser, 1995, (33), 120 - 2
Autoimmune derived combinatorial phage display libraries: methods in construction of and affinity selection for anti-RNA Fabs; Marchbank MT et al.; Display of antibody fragments (Fab) on the surface of filamentous bacteriophage and selection of phage that bind to a particular antigen has enabled the isolation of Fab with numerous specificities . We have examined the possibility of isolating RNA-binding Fab by constructing and screening combinatorial libraries of phage displaying Fab derived from the antibody repertoires of autoimmune humans . The genes and corresponding Fabs will allow for examination of the mechanism by which antibodies recognize RNA . Patients selected exhibit mixed connective tissue disease (MCTD), which is a subset of systemic lupus erythematosus (SLE), MCTD patients contain antibodies reactive against various nucleic acid, protein, and nucleoprotein complexes, most notably those involved in RNA processing . The cDNA libraries were constructed from total RNA derived from leukophoresed patient samples and the resulting Fab genes were expressed in E . oli using the pComb system1 . Affinity selection procedures have been designed to isolate anti-RNA Fabs from these libraries . Results demonstrate the ability to enrich for anti-RNA Fabs using biotinylated RNA-streptavidin capture methodologies.

Methods Enzymol, 1995, 262, 323 - 31
Use of genetic analyses to probe structure, function, and dynamics of bacteriophage T4 DNA polymerase; Reha-Krantz LJ; Functionally distinct mutant DNA polymerases have been isolated by the genetic selection strategies described here . These methods can be supplemented by the use of targeted mutagenesis procedures to enhance mutagenesis of DNA polymerase genes and to direct mutagenesis to specific sites in cloned DNA polymerases (see {22-24, 28}, this volume) . The power of genetic selection is in the ability to identify amino acid residues that are critical for protein structure and function that may not be obvious from studies of structural data alone . For the study of DNA polymerases, it is essential to identify residues involved in the movement of the DNA polymerase along the DNA template and in shuttling the DNA between the polymerase and exonuclease active centers . Ongoing studies are directed toward these goals.

Acta Biochim Pol, 1995, 42(2), 275 - 9
Cloning and expression of the genes coding for tube associated proteins of bacteriophage T4; Nieradko J; Genes 29, 48 and 54 of bacteriophage T4, coding for specific tube associated proteins, were cloned to the expression vector pT7-5 . The molecular mass of the products of these genes was estimated to be 64, 39 and 36 kDa, respectively . The examined genes are cotranscribed with genes 51, 27 and 28 from the same DNA strand and a common late promoter sequence located downstream of gene 51.

Acta Biochim Pol, 1995, 42(2), 233 - 9
Inhibition of transcription starting from bacteriophage lambda pR promoter during the stringent response in Escherichia coli: implications for lambda DNA replication; Szalewska-Palasz A et al.; Replication of lambda plasmid DNA is halted in amino acid-starved wild type (stringent) strains whereas it proceeds in relA (relaxed) mutants . The only transcription which could be important in lambda plasmid DNA replication in amino acid-starved Escherichia coli cells is that starting from the pR promoter . Using a fusion which consists of the lacZ gene under the control of bacteriophage lambda pR promoter we found that transcription starting from this promoter was inhibited during the stringent, but not the relaxed, response in E . coli . We confirmed our conclusion by estimating the relative level of the pR transcript by RNA-DNA hybridization . We propose that decreased transcription from the pR promoter which serves as transcriptional activation of ori lambda is responsible for inhibition of lambda plasmid replication during the stringent response . The results presented in this paper, combined with our recent findings (published elsewhere), indicate that the transcriptional activation of ori lambda may be a main regulatory process controlling lambda DNA replication not only during the relaxed response but also in normal growth conditions.

Virus Genes, 1995, 10(2), 173 - 7
Bacteriophage T4 as a surface display vector; Efimov VP et al.; We describe a method for construction of hymeric bacteriophage T4 particles displaying foreign polypeptides on their surface . The method is based on our finding that minor T4 fibrous protein fibritin encoded by gene wac (whisker's antigen control) could be lengthened at the C terminus without impairing its folding or binding to the phage particle . The lengthened fibritin gene could easily be transferred into the T4 genome by homologous recombination with a plasmid containing the modified gene wac . The modified gene wac is expressed properly during phage reproduction, and the lengthened fibritin is bound to phage particles . As an example of this type of method, we have obtained the hymeric T4 particles carrying a polypeptide of 53 residues, 45 of which are from the pre-S2 region of hepatitis B virus . The T4 display vector extends currently available display systems.

Plant Mol Biol, 1995 Jan, 27(2), 249 - 61
Cloning and developmental expression of pea ribulose-1,5-bisphosphate carboxylase/oxygenase large subunit N-methyltransferase; Klein RR et al.; Ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) large subunit (LS) N-methyltransferase (protein methylase III, Rubisco LSMT, EC 2.1.1.43) catalyzes methylation of the epsilon-amino group of Lys-14 in the LS of Rubisco . With limited internal amino acid sequence information obtained from HPLC-purified peptic polypeptides from Rubisco LSMT, a full-length cDNA clone was isolated utilizing polymerase chain reaction-based technology and conventional bacteriophage library screening . The 1802 bp cDNA of Rubisco LSMT encodes a 489 amino acid polypeptide with a predicted molecular mass of ca . 55 kDa . A derived N-terminal amino acid sequence with features common to chloroplast transit peptides was identified . The deduced sequence of Rubisco LSMT did not exhibit regions of significant homology with other protein methyltransferases . Southern blot analysis of pea genomic DNA indicated a low gene copy number of Rubisco LSMT in pea . Northern analysis revealed a single mRNA species of about 1.8 kb encoding for Rubisco LSMT which was predominately located in leaf tissue . Illumination of etiolated pea seedlings showed that the accumulation of Rubisco LSMT mRNA is light-dependent . Maximum accumulation of Rubisco LSMT transcripts occurred during the initial phase of light-induced leaf development which preceded the maximum accumulation of rbcS and rbcL mRNA . Transcript levels of Rubisco LSMT in mature light-grown tissue were similar to transcript levels in etiolated tissues indicating that the light-dependent accumulation of Rubisco LSMT mRNA is transient . This is the first reported DNA and amino acid sequence for a protein methylase III enzyme.

J Gen Virol, 1995 Jan, 76 ( Pt 1), 181 - 7
Sequence of host-range determinants in the env gene of a full-length, infectious proviral clone of exogenous avian leukosis virus HPRS-103 confirms that it represents a new subgroup (designated J); Bai J et al.; A genomic DNA library was constructed, in a bacteriophage lambda vector, from line 0 chick embryo fibroblasts (CEFs) infected with HPRS-103, an exogenous avian leukosis virus (ALV; envelope subgroup J) recently isolated from meat-type chickens . The library was screened at high stringency using a full length RAV-1 (subgroup A) proviral probe . From 10(6) plaques, two clones which hybridized strongly to the RAV-1 probe were isolated; one contained a full-length copy of the proviral genome of HPRS-103 and the other contained a copy lacking the 5'-long terminal repeat (LTR) and part of gag . The relative strength of hybridization of RAV-1 and HPRS-103 clones, to RAV-1 probes representing different parts of the proviral genome, indicated that the gag and pol genes of HPRS-103 share a high level of identity with those of RAV-1 but that the env gene and the LTRs are considerably less well conserved . Infectious virus was recovered from CEFs transfected with the full-length clone, as detected by ELISA . The recovered virus appeared to be identical to HPRS-103 by electron microscopy and by Southern blotting of proviral DNA . The recovered virus was shown to be of the same subgroup as HPRS-103 by serum neutralization and receptor interference assays . Sequence analysis of the env gene of HPRS-103 shows that it differs considerably from the env genes of other ALV subgroups, particularly in the host range determinants, consistent with the finding that HPRS-103 represents a new subgroup (designated J).

J Bacteriol, 1995 Jan, 177(1), 222 - 8
Gin mutants that can be suppressed by a Fis-independent mutation; Spaeny-Dekking L et al.; The Gin invertase of bacteriophage Mu mediates recombination between two inverted gix sites . Recombination requires the presence of a second protein, Fis, which binds to an enhancer sequence . We have isolated 24 different mutants of Gin that are impaired in DNA inversion but proficient in DNA binding . Six of these mutants could be suppressed for inversion by introduction of a second mutation, which when present in the wild-type gin gene causes a Fis-independent phenotype . Only one of the six resulting double mutants shows an inversion efficiency which is comparable to that of the wild-type Gin and which is independent of Fis . The corresponding mutation, M to I at position 108 (M108I), is located in a putative alpha-helical structure, which in the homologous gamma delta resolvase has been implicated in dimerization . The properties of the M108I mutant suggest that in Gin this dimerization helix might also be the target for Fis interaction . The five other mutants that show a restored inversion after introduction of a Fis-independent mutation appear to be completely dependent on Fis for this inversion . The corresponding mutations are located in different domains of the protein . The properties of these mutants in connection with the role of Fis in inversion will be discussed.

Micron, 1995, 26(3), 213 - 45
Electron microscopic studies on intracellular phage development--history and perspectives; Kellenberger E et al.; This review is centered on the applications of thin sections to the study of intracellular precursors of bacteriophage heads . Results obtained with other preparation methods are included in so far as they are essential for the comprehension of the biological problems . This type of work was pioneered with phage T4, which contributed much to today's understanding of morphogenesis and form determination . The T4 story is rich in successes, but also in many fallacies . Due to its large size, T4 is obviously prone to preparation artefacts such as emptying, flattening and others . Many of these artefacts were first encountered in T4 . Artefacts are mostly found in lysates, however, experience shows that they are not completely absent from thin sections . This can be explained by the fact that permeability changes induced by fixatives occur . The information gained from T4 was profitably used for the study of other phages . They are included in this review as far as electron microscopic studies played a major role in the elucidation of their morphogenetic pathways . Research on phage assembly pathways and form determination is a beautiful illustration for the power of the integrated approach which combines electron microscopy with biochemistry, genetics and biophysics . As a consequence, we did not restrict ourselves to the review of electron microscopic work but tried to integrate pertinent data which contribute to the understanding of the molecular mechanisms acting in determining the form of supramolecular structures.

Gene Expr, 1995, 4(4-5), 253 - 64
Overexpression, purification, and characterization of the ADP-ribosyltransferase (gpAlt) of bacteriophage T4: ADP-ribosylation of E . coli RNA polymerase modulates T4 "early" transcription; Koch T et al.; The bacteriophage T4 Alt gene product is a component of the phage head and enters the host cell in the process of infection together with the phage DNA . It immediately ADP-ribosylates host RNA polymerase, presumably at only one of the two alpha-subunits . Transcription from T4 "early" promoters, therefore, might be catalyzed, at least in part, by an altered RNA polymerase . The T4 alt gene was cloned into the expression vector pBluescript . E . coli cells, transformed with this recombinant vector, overexpressed the 76 kDa Alt gene product, which was purified to homogeneity . The purified enzyme not only ADP-ribosylates the alpha-subunit of RNA polymerase, but also subunits beta and beta', as well as the sigma 70-factor . The recombinant enzyme behaved like the native enzyme isolated from mature phage particles . The effect of the ribosylation reaction on the transcription activity of host RNA polymerase was investigated in vivo . It results in a modulation of T4 "early" promoter strengths, presumably, in a number of cases, leading to an overexpression of T4 "early" genes . The degree of overexpression, in some cases, should reach 50%, and seems to be well dosed for each promoter, controlling an individual transcription unit.

Mol Microbiol, 1995 Jan, 15(2), 381 - 93
Structure-function analysis of the vitamin B12 receptor of Escherichia coli by means of informational suppression; Hufton SE et al.; We describe a genetic analysis of the vitamin B12 receptor of Escherichia coli . Through the use of informational suppression, we have been able to generate a family of receptor variants, each identical save for a single, known substitution (Ser, Gln, Lys, Tyr, Leu, Cys, Phe) at a known site . We have studied 22 different mutants, 14 in detail, distributed throughout the length of the btuB gene . Most amino acid substitutions have a pleiotropic effect with respect to all ligands tested, the two colicins E1 and E3, the T5-like bacteriophage BF23, and vitamin B12 . (The dramatic effect of a single amino acid substitution is also well exemplified by the G142A missense change which renders the receptor completely non-functional.) In some instances, however, we have been able to modify a subset of receptor functions (viz . Q62, Q150 and Q299 and the response to phage BF23) . These data are summarized on a two-dimensional folding model for the BtuB protein in the outer membrane (devised using both amphipathic beta-strand analysis and sequence conservation amongst the TonB-dependent receptors) . In addition, we report that the extreme C-terminus of BtuB is vital for receptor localization and provide evidence for it being a membrane-spanning beta-sheet with residue L588 situated on its hydrophobic surface . Two of the C-terminal btuB mutations are located within the region of overlap with the recently identified dga (murl) gene.

Mol Microbiol, 1995 Jan, 15(2), 333 - 9
Coevolution of RNA helix stability and Shine-Dalgarno complementarity in a translational start region; Olsthoorn RC et al.; The initiation region of the coat-protein gene of RNA bacteriophage MS2 adopts a well-defined hairpin structure with the start codon occupying the loop position, while the Shine-Dalgarno (SD) sequence is part of the stem . In a previous study, we introduced mutations in this hairpin that changed its thermodynamic stability . The resulting phages evolved to regain the wild-type stability by second-site compensatory substitutions . Neither the original nor the suppressor mutations were in the SD region . In the present analysis, we have made changes in the SD region that shorten or extend its complementarity to the 3' end of 16S rRNA and monitored their evolution to a stable pseudorevertant species . Phages in which the SD complementarity was decreased evolved an initiator hairpin of lower stability than wild type while those in which the complementarity was extended evolved a hairpin with an increased stability . We conclude that weaker SD sequences still allow maximal translation if the secondary structure of the ribosome-landing site is destabilized accordingly . Alternatively, translation-initiation regions with a stronger secondary structure still allow maximal expression, if the SD complementarity is extended . These findings support a previously published model in which the SD interaction helps the ribosome to melt the structure in a translation-initiation region.

Environ Mol Mutagen, 1995, 25(3), 256 - 62
Software package for the management of sequencing projects using lacI transgenic animals; de Boer JG; The bacterial lacI gene has been used for many years as a mutational target for the study of the mechanisms of mutation . A wealth of information has been collected for many mutagenic treatments and in strains with diverse DNA repair backgrounds . Recently this gene has been used in the construction of a transgenic mouse, named Big Blue, and a transgenic rat, as well as a rat cell line . The lacI gene in these animals and cells can conveniently be recovered and analyzed in bacteria . This makes it possible to study mutagenic potential of chemical compounds in vivo using a mammal . Tissue, strain, and gender specificity can be addressed . In addition, mutations recovered from tumour tissues or from animals with specific genetic backgrounds can be analyzed conveniently . The mammalian systems can produce large numbers of mutants that require computer assistance to manage the samples and the resulting DNA sequence data . Accordingly, a computer software system was developed . The system maintains an inventory of bacteriophage lambda lacI mutants and allows entry of mutant sequences while performing accuracy checks on the data . The software features several options for displaying lists of mutants . The system can perform several analyses, including mutant class compilations, mutational spectra comparisons, and clonal expansions analysis . An extensive database obtained from the bacterial lacI system is included with the software and can be analyzed along with mutants derived from transgenic animals.

Biophys J, 1995 Jan, 68(1), 157 - 63
Structural changes in Escherichia coli membranes induced by bacteriophage T4 at different temperatures; Tarahovsky YS et al.; This paper presents some further evidence for our model of DNA translocation into Escherichia coli cells by bacteriophage T4 (see Tarahovsky, Y . S., Khusainov, A . A., Deev, A . A., Kim, Y . V . 1991 . FEBS Lett . 289:18-22) . When lowering the temperature, we succeeded in slowing down the infection process and in observing a few separate stages by electron microscopy . Also, potassium leakage at different temperatures was measured . At 0-6 degrees C the phage was found to be irreversibly adsorbed on the cell surface, its tail to be contracted, and the outer membrane to be invaginated . Membrane fusion and formation of broad intermembrane bridges with a hole for potassium leakage were shown to start above 7 degrees C . At about 17-20 degrees C the diameter of the bridge decreased considerably, which could correspond to the sealing of the membrane.

Anal Biochem, 1995 Jan 1, 224(1), 413 - 9
Selection procedures for nonmatured phage antibodies: a quantitative comparison and optimization strategies; Kretzschmar T et al.; Libraries of peptides and proteins can be displayed on the surface of filamentous bacteriophage . The efficient capturing of phage recognizing a defined target molecule remains a serious obstacle, in particular when the phage are present at a low frequency or have a reduced affinity like nonmatured phage antibodies and when the availability of target molecules is limited . We present theoretical considerations and experimental data which allowed us to substantially improve microselection under these conditions . We used a model phage displaying an anti-(2-phenyl-5-oxazolone) single-chain Fv antibody fragment . Following standard protocols and aiming at a low nonspecific binding, only 3.6 x 10(-3)% of the phage input could be recovered from a single round of selection performed in the wells of a microtiter plate . Our results explain why this often employed panning in wells is not efficient, especially with high-molecular-weight target molecules . We devised a procedure which increased the probability of microselection by a factor of 34 . An alternative capturing method using immunotubes with a new protocol decreased the amount of required work by a factor of 30 . In the case of a nonlimited supply of target molecules, column-affinity chromatography is recommended.

Genetics, 1995 Jan, 139(1), 5 - 17
Evidence for conservative (two-progeny) DNA double-strand break repair; Yokochi T et al.; The double-strand break repair models for homologous recombination propose that a double-strand break in a duplex DNA segment is repaired by gene conversion copying a homologous DNA segment . This is a type of conservative recombination, or two-progeny recombination, which generates two duplex DNA segments from two duplex DNA segments . Transformation with a plasmid carrying a double-strand gap and an intact homologous DNA segment resulted in products expected from such conservative (two-progeny) repair in Escherichia coli cells with active E . coli RecE pathway (recBC sbcA) or with active bacteriophage lambda Red pathway . Apparently conservative double-strand break repair, however, might result from successive events of nonconservative recombination, or one-progeny recombination, which generates only one recombinant duplex DNA segment from two segments, involving multiple plasmid molecules . Contribution of such intermolecular recombination was evaluated by transformation with a mixture of two isogenic parental plasmids marked with a restriction site polymorphism . Most of the gap repair products were from intramolecular and, therefore, conservative (two-progeny) reaction under the conditions chosen . Most were conservative even in the absence of RecA protein . The double-strand gap repair reaction was not affected by inversion of the unidirectional replication origin on the plasmid . These results demonstrate the presence of the conservative (two-progeny) double-strand break repair mechanism . These experiments do not rule out the occurrence of nonconservative (one-progeny) recombination since we set up experimental conditions that should favor detection of conservative (two-progeny) recombination.

Genetics, 1995 Jan, 139(1), 35 - 43
Transduction, restriction and recombination patterns in Escherichia coli; McKane M et al.; Chromosomal DNA from several Escherichia coli reference (ECOR) strains was transduced by bacteriophage P1 into E . coli strain K12 W3110 trpA33 . Recombination patterns of the transductants were determined by restriction fragment length polymorphism over a 40-kb region centering on a single marker (trpA+) in the tryptophan operon . These experiments demonstrate that transduction between different strains of E . coli can result in recombinational replacements that are small in comparison to the entrant molecule (replacements average 8-14 kb, whereas P1 packages approximately 100 kb) often in a series of discrete segments . The transduction patterns generated resemble the natural mosaic sequence patterns of the ECOR strains described in previous work . Extensive polymorphisms in the restriction-modification systems of the ECOR strains are a possible explanation for the sequence patterns in nature . To test this possibility two transductants were back-transduced into strain K12 W3110 trpA33 . The resulting patterns were strikingly different from the original transductions . The size of the replacements was greater, and no multiple replacements were observed, suggesting a role for restriction-modification systems in the transduction patterns and perhaps for the mosaic sequence patterns in nature.

Biotechniques, 1995 Jan, 18(1), 84 - 6, 88-90
Priming efficiency in PCR; Rychlik W; Taq and Pfu DNA polymerases were tested for their propensity to prime from mismatched primers . Two series of bacteriophage lambda primers were designed with progressively longer mismatched 5' termini . Effects of the primer concentration, annealing temperature, salt and solvent concentrations on PCR yield were tested . At the standard PCR conditions, priming was detectable when the 3'-terminal portion of the partially mismatched primer formed a continuous duplex more stable than -11 kcal/mol with the target DNA . In the presence of low magnesium ion concentrations, priming was significantly reduced, but glycerol (5%) and formamide (2.5%) had only a slight effect (Taq DNA polymerase) . Although priming specificities of Taq and Pfu DNA polymerases were similar, the solvents had no effect on Pfu DNA polymerase-directed PCR . Oligonucleotides that are GC rich at their 3' ends exhibit high priming efficiency but are also prone to false priming, since the shorter fragments of their 3' ends are stable enough to serve as primers.

Biotechniques . 1995 Jan;18(1):152-7,
pTRIPLEscript: a novel cloning vector for generating in vitro transcripts from tandem promoters for SP6, T7 and T3 RNA polymerase; Meador JW 3rd et al.; We have constructed a family of novel in vitro transcription vectors in which functional T3, T7 and SP6 RNA polymerase promoters are arranged in tandem and directed towards a multiple cloning site . This prototype vector, named pTRIPLEscript, permits the transcription of one strand of a DNA insert by any of the three commonly used bacteriophage RNA polymerases with no apparent cross talk, i.e., use of the wrong promoter sequence . The vector has two main uses: (i) to clone probe sequences that will be distributed to many laboratories, allowing the use of the most convenient RNA polymerase; and (ii) to circumvent the problem of RNA polymerase-dependent premature termination.

Acta Biochim Pol, 1995, 42(1), 103 - 8
Binding of Escherichia coli integration host factor (IHF) to the origin segment of p15A plasmid; Hiszczynska-Sawicka E et al.; The integration host factor (IHF) is a sequence-specific, histone-like, multi-functional DNA-binding and -bending protein of Escherichia coli . Characterization and functional analysis of this protein has been carried out mainly in bacteriophage lambda and other mobile genetic elements . In this paper we report data concerning the binding of IHF protein to the plasmid orip15A region . IHF binds to the single site of the DNA fragment containing the orip15A, as shown by the gel mobility shift assays and footprinting experiment . On the basis of the ihf consensus sequences published, we have been able to identify one sequence of putative ihf site into the orip15A sequence with two mismatches in relation to the consensus sequence of Kur et al., 1989, Gene 81, 1-15 . One ihf binding site was also found in the oriColE1 region sequence with three mismatches in relation to this consensus sequence.

DNA Seq, 1995, 5(3), 199 - 201
Bacteriophage T4 gene 28; Bova R et al.; The complete nucleotide sequence of bacteriophage T4D gene 28 has been determined . Gene 28 product is a structural component of the viral baseplate for which an enzymatic activity has also been proposed.

Arch Virol, 1995, 140(6), 1033 - 47
Analysis of the open reading frames of the main capsid proteins of actinophage VWB; Anne J et al.; The nucleotide sequence of a 6 kb fragment encoding the main late proteins (p14, p38 and p24) of actinophage VWB was obtained . Sequence comparison of the encoded proteins with those filed in databases indicated that the phage VWB main late proteins were all novel . A search for special motifs revealed that p14 (13.3 kDa) has a P-loop sequence commonly found in ATP- and GTP-binding proteins . This observation might indicate that p14 is important for ATP-driven DNA translocation during encapsidation of VWB phage DNA into the phage head . Furthermore, the polypeptide ORF2 (26.9 kDa) has an unusual primary structure consisting of 3 stretches of acidic amino acid residues and a glycine/arginine rich C-terminal end . From comparison with other proteins including the bacteriophage T4 prohead core component and from the data of special motif analysis the ORF2 gene product is probably involved in prohead core formation.

Immunogenetics, 1995, 42(2), 101 - 11
Cloning and characterization of a novel NK cell-specific serine protease gene and its functional 5'-flanking sequences; Smyth MJ et al.; Rat natural killer cell Met-ase-1 (RNK-Met-1) is a 30,000 M(r) serine protease (granzyme) found in the cytolytic granules of CD3- large granular lymphocytes (LGL) with natural killer (NK) activity . To characterize the genomic sequences responsible for the CD3- LGL-restricted expression of this gene, we screened a rat genomic library with RNK-Met-1 cDNA, and obtained bacteriophage clones that contained the RNK-Met-1 gene . The RNK-Met-1 gene comprises 5 exons and spans approximately 5.2 kilobases (kb), exhibiting a similar structural organization to a class of CTL-serine proteases with protease catalytic residues encoded near the borders of exons 2, 3, and 5 . The 5'-flanking region of the RNK-Met-1 gene contains a number of putative promoter and enhancer regulatory elements and shares several regions of homology with the 5'-flanking region of the mouse perforin gene . We have prepared nested deletions from approximately 3.3 kb of the 5'-flanking region of the RNK-Met-1 gene, and inserted these upstream of the chloramphenicol acetyltransferase (CAT) reporter gene . These 5'-flanking RNK-Met-1-CAT constructs were transiently transfected into rat LGL leukemia, T-lymphoma, and basophilic leukemia cell lines . The transcriptional activity of the RNK-Met-1 5'-flanking region was strong, restricted to the RNK-16 LGL leukemia and controlled by several positive cis-acting regions spread over at least 3.3 kb . The longest and most active 5'-flanking region (-3341 to -33) was also used to drive specific expression of beta-galactosidase in RNK-16 . These data are consistent with the NK cell-specific expression of RNK-Met-1 and suggest the potential utility of this gene promoter in the development of transgene models of NK cell biology in vivo.

Appl Theor Electrophor, 1995, 4(4), 211 - 7
Gel electrophoretic analysis of bacteriophage assembly intermediates in bacteriophage plaques; Serwer P et al.; To increase the efficiency with which the phenotype of bacteriophage mutants is determined by gel electrophoresis, procedures are developed here for the preparation of the contents of bacteriophage plaques for gel electrophoresis . During the formation of plaques, the plaque-supporting upper layer gel is changed from the traditional agar gel to a gel made of a mixture of low-melt agaroses; the lower layer gel is eliminated . To extract particles from plaques, the plaque-supporting gel is disintegrated by both shaking and raising the temperature to 39-43 degrees C . During shaking, the gel is broken to domains that are 5-30 microns in diameter . After extraction, the contents of plaques are subjected to two electrophoretic analyses: (1) Nondenaturing agarose gel electrophoresis is performed after treatment with DNase . This procedure reveals both mature bacteriophage and immature capsids . (2) Nondenaturing agarose gel electrophoresis is performed after release of DNA from DNase-treated capsids . This latter procedure reveals both completely packaged (mature length) DNA and incompletely packaged (shorter than mature length) DNA . The amount of mature length DNA released per 2-3 mm plaque is 10-60 ng . In agreement with results previously obtained in liquid culture, most incompletely packaged DNA has the right, but not the left, mature T7 DNA end.

Thromb Haemost, 1995 Jan, 73(1), 144 - 50
Identification of novel peptide antagonists for von Willebrand factor binding to the platelet glycoprotein Ib receptor from a phage epitope library; South V et al.; We have constructed a fusion phage epitope library in the filamentous bacteriophage fuse5 . The library was made by inserting a degenerate oligonucleotide which encodes 15 variable amino acids into the NH2-terminal region of the phage gene III protein . This library, containing over 10(7) different epitope bearing phage, has been used in an attempt to identify inhibitors of the von Willebrand factor (vWF)-platelet Glycoprotein Ib interaction . The library was screened with a monoclonal antibody (RG46) that recognizes the GPIb binding domain of vWF (amino acids 445-733) . A total of 30 clones falling into 8 classes have been identified that react with the RG46 antibody . Isolates from all 8 classes are positive by immunoblot analysis . The amino acid sequence of the gene III fusion protein from positive clones showed a strong homology to the known RG46 epitope . Peptides identified from the screen were synthesized and used to demonstrate that some of the synthetic peptides exhibited inhibitory activity towards ristocetin induced binding of vWF to the GPIb receptor . Thus, we have demonstrated that screening a fusion phage epitope library with a monoclonal antibody that inhibits vWF binding to the GPIb receptor can be a useful tool not only for mapping antibody recognizing determinants, but also can serve as a source for identifying novel peptides that are antagonists for vWF binding to the platelet GPIb receptor.

J Biol Chem, 1994 Dec 30, 269(52), 33069 - 81
The gene 59 protein of bacteriophage T4 modulates the intrinsic and single-stranded DNA-stimulated ATPase activities of gene 41 protein, the T4 replicative DNA helicase; Morrical SW et al.; The T4 gene 59 protein (gp59) serves as an accessory protein to the essential T4-encoded DNA helicase, the gene 41 protein (gp41) . gp59 stimulates gp41-dependent DNA synthesis reactions by promoting the assembly of gp41 onto single-stranded DNA (ssDNA), where the enzyme is activated to perform its DNA helicase functions . To better understand the mechanism of helicase-ssDNA assembly, we have studied the effects of gp59 on the intrinsic and ssDNA-stimulated ATPase activities of gp41 . Our results indicate that gp59 exerts a direct effect upon the conformation and ATPase activity of gp41, by increasing the affinity of gp41 for ATP . In addition, we find that gp59 is nearly essential for promoting the assembly of gp41 onto ssDNA molecules that are covered with saturating amounts of the T4-encoded helix-destabilizing protein, gene 32 protein (gp32) . Results of protein affinity chromatography experiments suggest that gp59 contains distinct binding sites for gp41 and gp32 and may therefore act as a molecular adapter between the helicase and helix-destabilizing proteins . Together, the data indicate that specific gp59-gp41 and gp59-gp32 protein-protein interactions both play important roles in the assembly of the helicase onto single-stranded DNA.

J Biol Chem, 1994 Dec 30, 269(52), 33063 - 8
A role for two DNA helicases in the replication of T4 bacteriophage DNA; Barry J et al.; The T4 bacteriophage gene 41 protein is the highly processive DNA helicase of the T4 primosome, a central part of the protein machinery that moves the T4 DNA replication fork . The T4 gene 59 protein accelerates the loading of 41 protein onto DNA covered with 32 protein (the T4 single strand binding protein), and it makes the 41 protein DNA helicase activity rapidly available to catalyze replication fork movement through a DNA double helix (Barry, J., and Alberts, B.M . (1994) J . Biol . Chem . 269, 33049-33062) . With the aid of the 59 protein, we show that the T4 primosome (the T4 gene 41 and 61 proteins) can move rapidly through a promoter-bound RNA polymerase molecule that would otherwise stop replication fork movement . A second, very different DNA helicase, the T4 dda protein, provides an alternative pathway for replication past this DNA-bound RNA polymerase (Bedinger, P., Hochstrasser, M., Jongeneel, C . V., and Alberts, B.M . (1983) Cell 34, 115-123) . Combined with other data, these in vitro experiments allow us to propose a model that explains why either the 59 protein or the dda protein, but not both, are required to begin efficient DNA replication inside the T4 bacteriophage-infected cell.

J Biol Chem, 1994 Dec 30, 269(52), 33049 - 62
Purification and characterization of bacteriophage T4 gene 59 protein . A DNA helicase assembly protein involved in DNA replication; Barry J et al.; The T4 bacteriophage gene 59 protein is required for normal T4 DNA replication . We have purified this protein to homogeneity in two steps and show that it binds both to single-stranded DNA and to the T4 gene 32 protein, a DNA single strand binding protein . In in vitro assays, covering DNA with 32 protein makes this DNA inaccessible to the 41 protein, the highly processive DNA helicase, that associates with the T4 DNA primase (gene 61 protein) to form an active primosome . However, the 59 protein brings about the rapid assembly of 41 protein onto single-stranded DNA, even if this DNA is covered with 32 protein . The 59 protein is therefore a DNA helicase assembly protein . The observed requirements for the 59 protein in the vivo T4 DNA replication are explained by there being two alternative pathways for loading the 41 protein onto a replication fork at early times of T4 DNA synthesis, with only a 59 protein-mediated pathway remaining operative for the recombination-mediated replication that dominates later in infection (Barry, J., and Alberts, B . M . (1994) J . Biol . Chem . 269, 33063-33068).

J Biol Chem, 1994 Dec 30, 269(52), 33082 - 90
Structural organization of the human neuronal nitric oxide synthase gene (NOS1); Hall AV et al.; Neuronal nitric oxide (NO) synthase, localized to human chromosome 12, uniquely participates in diverse biologic processes; neurotransmission, the regulation of body fluid homeostasis, neuroendocrine physiology, control of smooth muscle motility, sexual function, and myocyte/myoblast biology, among others . Restriction enzyme mapping, subcloning, and DNA sequence analysis of bacteriophage- and yeast artificial chromosome-derived human genomic DNA indicated that the mRNA for neuronal NO synthase is dispersed over a minimum of 160 kilobases of human genomic DNA . Analysis of intron-exon splice junctions predicted that the open reading frame is encoded by 28 exons, with translation initiation and termination in exon 2 and exon 29, respectively . Determination of transcription initiation sites in brain poly(A) RNA with primer extension analysis and RNase protection revealed a major start site 28 nucleotides downstream from a TATA box . Sequence inspection of 5'-flanking regions revealed potential cis-acting DNA elements: AP-2, TEF-1/MCBF, CREB/ATF/c-Fos, NRF-1, Ets, NF-1, and NF-kappa B-like sequences . Diversity appears to represent a major theme apparent upon analysis of human neuronal NO synthase mRNA transcripts . A microsatellite of the dinucleotide variety was detected within the 3'-untranslated region of exon 29 . Multiple alleles were evident in normal individuals indicating the existence of allelic mRNA sequence variation . Characterization of variant human neuronal NO synthase cDNAs indicated the existence of casette exon 9/10 and exon 10 deletions as examples of structural mRNA diversity due to alternative splicing . The latter deletion of a 175-nucleotide exon introduces a frame-shift and premature stop codon indicating the potential existence of a novel NH2 terminus protein . In summary, analysis of the human neuronal NO synthase locus reveals a complex genomic organization and mRNA diversity that is both allelic and structural.

Nucleic Acids Res, 1994 Dec 25, 22(25), 5571 - 5
In vivo decay kinetic parameters of hammerhead ribozymes; Sioud M et al.; Ribozymes offer a potentially important way to inactivate intracellular RNA from almost any gene whose nucleotide sequence is known . Recently, we found that hammerhead ribozymes directed against mRNA of tumour necrosis factor alpha (TNF alpha) and its derivatives, preferentially bind to a cellular protein(s) . To better understand the effect of different 3'-terminal hairpins on ribozyme stability as well as their effect on the protein binding to the ribozyme, a mathematical treatment of the decay of three TNF alpha ribozymes that differed at their 3' ends was performed . One ribozyme contained a 3'-terminal hairpin derived from a transcription terminator of bacteriophage T7, another contained the same hairpin but modified to be highly enriched for G+C nucleotides, and a third lacked a hairpin . The TNF alpha ribozyme decay had two kinetic components . The slow component exhibited exponential decay with a half life of approximately 250 h in all cases . The 3'-terminal hairpin has no significant effect on this component . This slow phase accounted for 60-80% of ribozyme decay . The rapid phase also exhibited exponential decay . For this phase, a 3'-terminal hairpin roughly doubled the half-life (1.7-3.4) . The slow phase of degradation was about three times faster for a ribozyme directed at the integrase mRNA of human immunodeficiency virus-1 than that seen with the TNF alpha ribozyme . Taken together, these results suggest that the ribozyme population is initially sensitive to degradation, with the presence of a hairpin provides some protection, and indicate that the addition of the hairpin to the ribozyme did not prevent the in vivo additional stabilizing effect of the protein(s).

Proc Natl Acad Sci U S A, 1994 Dec 20, 91(26), 12525 - 9
A mutation in ribosomal protein L9 affects ribosomal hopping during translation of gene 60 from bacteriophage T4; Herbst KL et al.; Ribosomes hop over a 50-nt coding gap during translation of gene 60 mRNA from bacteriophage T4 . This event occurs with near-unitary efficiency when gene 60-lacZ fusions are expressed in Escherichia coli . One of the components necessary for this hop is an RNA hairpin structure containing the 5' junction of the 50-nt coding gap . A mutant E . coli was isolated and found to significantly increase hopping when carrying gene 60-lacZ constructs with altered hairpins . The mutation, hop-1, changed Ser93 to Phe in rplI, the gene coding for ribosomal large-subunit protein L9 . Ribosomal hopping on a synthetic sequence in the absence of a hairpin was also increased by this mutation . These data suggest that hop-1 may substitute for the function of the hairpin during ribosomal hopping.

J Mol Biol, 1994 Dec 16, 244(5), 494 - 510
DNA conformational changes associated with the cooperative binding of cI-repressor of bacteriophage lambda to OR; Strahs D et al.; The cI repressor protein (cI) maintains bacteriophage lambda in the lysogenic state in infected Escherichia coli cells by binding cooperatively to three tandemly repeated sequences comprising the right operator (OR) . Cooperative interactions occur between alternate pairs of cI dimers bound to adjacent sites . Although crystallographic studies have revealed the structure of the DNA in the 92 amino acid residue amino-terminal fragment-OL1 complex, the structure of the DNA within the OR-cI complex with intact, cooperatively bound cI has not been described . In this study, the structure of the DNA within OR was quantitatively examined using sequence and structure-dependent nuclease cleavage patterns as a function of cI binding . The cooperative binding of cI to OR1 and OR2 induces a conformational change in the DNA of OR3 that is detectable by both DNase I and 5-phenyl-1,10-phenanthroline . Hydroxyl radical footprinting indicates the presence of an "A-tract" between OR1 and OR2 at the site of a run of four adenine-thymine base-pairs, implying a stable bend between the sites of approximately 18 degrees . 5-Phenyl-1,10-phenanthroline footprinting reports conformational changes within the central base-pairs of all three sites that is dependent upon the sequence-specific binding of cI . The observed conformational changes are more extensive within OR2 and OR3 compared with OR1, consistent with an "induced-fit" model of sequence-specific recognition . A number of changes in nuclease reactivity within the individual binding sites were quantitatively correlated with cI binding at the other sites within OR . These results demonstrate that changes in the DNA structure are propagated among the sites in response to the binding of cI and imply a role for DNA sequence-dependent conformational changes in the mechanisms of both the intrinsic and cooperative binding reactions of cI to OR.

J Biol Chem, 1994 Dec 16, 269(50), 31885 - 90
C1 repressor-mediated DNA looping is involved in C1 autoregulation of bacteriophage P1; Heinzel T et al.; C1 repressor is required to repress the lytic functions of a P1 prophage in vivo . Transcription of the c1 gene is autoregulated via the C1-controlled operator Op99a,b which overlaps the promoter of the c1 gene . It is negatively affected by Lxc corepressor and the DNA region upstream of c1, which contains the additional operators Op99c, d, and e . We have explored these effects by constructing a set of lacZ reporter plasmids with Op99a,b and varying parts of the upstream DNA region . Transcription levels were measured in vivo with a two-plasmid system containing the lacZ reporter and a c1+ lxc+ or c1+ lxc- plasmid . Compared to the C1+Lxc-repressed lacZ reporter with all operators present, the basal level of beta-galactosidase activity increases successively when (i) upstream operators were deleted or inactivated, (ii) Lxc corepressor was removed, and (iii) C1 and Lxc were absent . By that means a 2 x 2 x 15-fold stepwise increase in enzyme activity was found . Using electron microscopy to visualize the interaction of C1 repressor with the operators in vitro, looped DNA molecules were observed . Although all operators can participate in C1-mediated DNA looping, loops between Op99a,b and Op99d occurred predominantly . Lxc is not required but increases drastically the frequency of loop formation . The results indicate that C1-mediated DNA looping may be a second element besides Lxc for fine-tuning the autoregulation of c1 transcription.

Eur J Biochem, 1994 Dec 15, 226(3), 831 - 9
Inhibition of T7 RNA polymerase transcription by phosphate and phosphorothioate triplex-forming oligonucleotides targeted to a R.Y site downstream from the promoter; Alunni-Fabbroni M et al.; The effect of triplex-forming oligonucleotides (TFO) on the transcription activity of T7 RNA polymerase has been investigated by an in vitro assay . The TFOs, either containing only phosphate (PO2) or phosphate and phosphorothioate (POS) internucleotide linkages, were targeted to a 30-bp homopurine . homopyrimidine (R.Y) site cloned in plasmid Bluescript KS+ about four helical turns downstream from the T7 RNA promoter . Band-shift and ultraviolet absorption melting experiments showed that the designed pyrimidine PO2 and POS TFOs form stable triple-helical complexes with the R.Y target duplex (the delta GTFO values of triplex formation vary from -42 to -63 kJ/mol) . The triple-helical complexes resulting from POS oligonucleotides were less stable (by 4-12 kJ/mol) than those obtained with PO2 analogues, the magnitude of destabilization being dependent on the number of POS groups present in the third strand . The designed TFOs were shown to efficiently repress bacteriophage T7 RNA polymerase transcription under different experimental conditions . The repression depended on pH, TFO concentration and temperature . When the TFO/template ratio was fixed to 100, a strong repressive effect was observed with normal and phosphorothioate pyrimidine TFOs, also under physiological conditions . In contrast, a purine-rich oligonucleotide containing 44% of guanine residues promoted only a weak transcription inhibition, even at a TFO/template ratio as high as 750 . Both PO2- and POS-containing pyrimidine TFOs produced their strong repressive effect on T7 RNA polymerase transcription even when they were added to the reaction mixture simultaneously with the polymerase . A mechanism of transcription repression is discussed . The data reported in this paper are useful for designing oligonucleotides acting as artificial repressors in the antigene strategy and indicate that the R.Y target need not to be precisely confined to the promoter.

Biochemistry, 1994 Dec 13, 33(49), 14908 - 17
Mutants affecting nucleotide recognition by T7 DNA polymerase; Donlin MJ et al.; Analysis of two mutations affecting nucleotide selection by the DNA polymerase from bacteriophage T7 is reported here . Two conserved residues (Glu480 and Tyr530) in the polymerase active site of an exonuclease deficient (exo-) T7 DNA polymerase were mutated using site-directed mutagenesis (Glu480-Asp and Tyr530-Phe) . The kinetic and equilibrium constants governing DNA binding, nucleotide incorporation, and pyrophosphorolysis were measured with the mutants E480D(exo-) and Y530F(exo-) in single-turnover experiments using rapid chemical quench-flow methods . Both mutants have slightly lower Kd values for DNA binding compared to that of wild-type(exo-) . With Y530F(exo-) the ground state nucleotide binding affinity was unchanged from wild-type for dGTP and dCTP, was 2-fold lower for dATP and 8-10-fold lower for dTTP binding . With E480D(exo-), the binding constants were 5-6-fold lower for dATP, dGTP, and dCTP and 40-fold lower for dTTP binding compared to those constants for wild-type(exo-) . The significance of a specific destabilization of dTTP binding by these amino acids was examined using a dGTP analog, deoxyinosine triphosphate, which mimics the placement and number of hydrogen bonds of an A:T base pair . The Kd for dCTP opposite inosine was unchanged with wild-type(exo-) (197 microM) but higher with Y530F(exo-) (454 microM) and with E480D(exo-) (1 mM) . The Kd for dITP was the same with wild-type(exo-) (180 microM) and Y530F(exo-) (229 microM), but significantly higher with E480D(exo-) (3.2 mM) . These data support the suggestion that E480 selectively stabilizes dTTP in the wild-type enzyme, perhaps by hydrogen bonding to the unbonded carbonyl . Data on the incorporation of dideoxynucleotide analogs were consistent with the observation of a selective stabilization of dTTP by both residues . Pyrophosphorolysis experiments revealed that neither mutation had a significant effect on the chemistry of polymerization . The fidelity of the mutants were examined in misincorporation assays . Both E480D(exo-) and Y530F(exo-) showed saturation kinetics with the wrong nucleotide, with binding constants of 1-3 mM compared to the estimated binding affinity of 6-8 mM with wild-type(exo-) . Accordingly, both mutants showed slightly lower selectivity against misincorporation . Taken together, these results indicate that E480 and Y530 each contribute to ground state nucleotide binding and suggest that the E480 may serve to specifically stabilize the incoming dTTP of A:T base pairs to compensate for the fewer hydrogen bonds compared to G:C base pairs.

Nucleic Acids Res, 1994 Dec 11, 22(24), 5204 - 10
Studies of bacteriophage P2 DNA replication: localization of the cleavage site of the A protein; Liu Y et al.; Bacteriophage P2 replicates via a modified rolling circle-type of mechanism, where the P2 A protein acts as an initiator of the replication by inducing a single-stranded cut at the origin of replication (ori) . The exact location of the cut induced by the A protein in vivo is determined in this report by: (i) restriction analysis; (ii) DNA sequence analysis; and (iii) primer extensions . It is located 89.2% from the left end of the P2 genome, which is within the coding part of the A gene, in a region devoid of secondary structures . The A gene has been cloned into an expression vector, and the A protein has been purified . The purified A protein does not bind to double-stranded ori containing DNA, but it cleaves single-stranded ori containing DNA, which indicates that a special DNA structure and/or protein is required to make the ori accessible for the A protein.

Nucleic Acids Res, 1994 Dec 11, 22(24), 5177 - 83
Family A and family B DNA polymerases are structurally related: evolutionary implications; Zhu W et al.; In order to establish the evolutionary relationship between the family A and B DNA polymerases, we have closely compared the 3'-->5' exonuclease domains between the Klenow fragment of E.coli DNA polymerase I (a family A DNA polymerase) and the bacteriophage PRD1 DNA polymerase, the smallest member of the DNA polymerase family B . Although the PRD1 DNA polymerase has a smaller 3'-->5' exonuclease domain, its active sites appear to be very similar to those of the Klenow fragment . Site-directed mutagenesis studies revealed that the residues important for the 3'-->5' exonuclease activity, particularly metal binding ligands for the Klenow fragment, are all conserved in the PRD1 DNA polymerase as well . The metal binding ligands are also essential for the strand-displacement activity of the PRD1 DNA polymerase . Based on these results and the studies by others in various systems, we conclude that family A and B DNA polymerases, at least in the 3'-->5' exonuclease domain, are structurally as well as evolutionarily related.

J Biol Chem, 1994 Dec 9, 269(49), 30999 - 1005
Allosteric effectors are required for subunit association in T4 phage ribonucleotide reductase; Hanson E et al.; Bacteriophage T4 encodes its own aerobic ribonucleotide reductase (RNR), which reduces ribonucleoside diphosphates to the corresponding deoxyribonucleoside diphosphates . T4 RNR is composed of homodimeric large (R1) and small (R2) subunits . Intricate regulation of enzymatic activity is accomplished by the binding of nucleotide effectors to R1 . Berglund (Berglund, O . (1972) J . Biol . Chem . 247, 7270-7275) described similarities between T4 RNR and the corresponding enzyme from aerobic Escherichia coli . An important difference, however, is that T4 RNR forms a tight R1.R2 complex, while the E . coli R1 and R2 more readily dissociate . In this study we purified the phage R2 subunit from an overexpression vector constructed by Tseng et al . (Tseng, M., Hilfinger, J., He, P., and Greenberg, R . (1992) J . Bacteriol . 174, 5740-5744) and used this as an immunogen to generate polyclonal antiserum . Using co-immunoprecipitation techniques, we probed in vitro for interactions between the phage-induced R1 and R2 subunits . Our studies indicate that tight binding of the phage RNR subunits is completely dependent upon the known allosteric effectors of the enzyme . Once the R1.R2 holoenzyme has been formed it appears to be remarkably stable when in the presence of dATP . However, if dATP is removed, the R1.R2 complex readily dissociates.

Proc Natl Acad Sci U S A, 1994 Dec 6, 91(25), 12327 - 31
A functional chimeric DNA primase: the Cys4 zinc-binding domain of bacteriophage T3 primase fused to the helicase of bacteriophage T7; Hine AV et al.; Two colinear bacteriophage T7 gene 4 proteins provide helicase and primase functions in vivo . T7 primase differs from T7 helicase by an additional 63 residues at the amino terminus . This terminal domain contains a zinc-binding motif which mediates an interaction with the basic primase recognition sequence 3'-CTG-5' . We have generated a chimeric primase in which the 81 amino-terminal residues are derived from the primase of phage T3 and the 484 carboxyl-terminal residues are those of phage T7 helicase . The amino-terminal domain of T3 primase is 50% homologous with that of T7 primase . The resulting T3/T7 chimeric protein is a functional primase in vivo . While the primase activity of the purified protein is about one-third that of T7 primase, the recognition sites used and the oligoribonucleotides synthesized from these sites are identical . We conclude that the residues responsible for the interaction with the sequence 3'-CTG-5' are conserved between the chimeric and T7 proteins.

Proc Natl Acad Sci U S A, 1994 Dec 6, 91(25), 12198 - 202
Terminal protein-primed DNA amplification; Blanco L et al.; By using appropriate amounts of four bacteriophage phi 29 DNA replication proteins--terminal protein, DNA polymerase, protein p6 (double-stranded DNA-binding protein), and protein p5 (single-stranded DNA-binding protein)--it has been possible to amplify limited amounts of the 19,285-bp-long phi 29 DNA molecule by three orders of magnitude after 1 hr of incubation at 30 degrees C . Moreover, the quality of the amplified material was demonstrated by transfection experiments, in which infectivity of the synthetic (amplified) phi 29 DNA, measured as the ability to produce phage particles, was identical to that of the natural phi 29 DNA obtained from virions . The results presented in this paper establish some of the requisites for the development of isothermal DNA amplification strategies based on the bacteriophage phi 29 DNA replication machinery that are suitable for the amplification of very large (> 70 kb) segments of DNA.

J Mol Biol, 1994 Dec 2, 244(3), 291 - 300
Calcium ion-induced structural changes in bacteriophage phi X174; Ilag LL et al.; Monoclinic P2(1) crystals of the bacteriophage phi X174 have been incubated with calcium ions (Ca2+) and the induced structural conformational changes studied to 3 A resolution with X-ray crystallographic methods . Three different types of Ca2+ binding sites have been located within the asymmetric unit of the virion . Two sets of sites are associated with the F capsid protein . One set of sites associated with the F protein is in a general position near the icosahedral 3-fold axes of the virus, with the main-chain carbonyl oxygen atoms of residues Gly1321, Asp1421, Met1424 and Ser1426, and the side-chains of Gln1004 and Asp1421 as ligands . The other set of sites associated with the F protein is on the icosahedral 3-fold axes, with the symmetry-related main-chain carbonyl oxygen atoms of Ser1001 and the side-chains of Asn1002 as ligands . The bound Ca2+ induce a conformational change of the amino-terminal residues of the F proteins . A third set of sites, consisting of a pair of Ca2+ on the icosahedral 5-fold axes, are associated with the G spike protein and are concurrently liganded by the symmetry-related carbonyl oxygen side-chains of Asp2117 . Concomitant with the binding of Ca2+ to the phage is the rotation of the Asp1209 side-chain of the F protein towards some additional electron density that was not observed in the absence of Ca2+ . This density is situated in a shallow depression near the icosahedral 2-fold axes of the virus, and has been tentatively interpreted as a bound glucose molecule that is ordered only in the presence of Ca2+ . The putative glucose binding site may be related to the attachment of the virus to cell surface lipopolysaccharides in the initial stages of Escherichia coli infection.

J Mol Biol, 1994 Dec 2, 244(3), 279 - 90
Crystal structure of bacteriophage fr capsids at 3.5 A resolution; Liljas L et al.; The structure of recombinant capsids of the bacterial virus fr has been determined by X-ray crystallography at 3.5 A resolution . The capsids were produced by expressing the fr coat protein in Escherichia coli, the natural host of the virus, and are probably essentially identical to the protein shell of the native virus . The structure was determined using molecular replacement with the protein shell of the related MS2 virus, and refined to a crystallographic R-factor of 0.228 . A comparison of the protein shells of the viruses shows that they are very similar, and indicates that they may have a similar regulation of the assembly of the quasi-symmetrical protein shell.

Gene, 1994 Dec 2, 150(1), 159 - 62
A cosmid with a HyR marker for fungal library construction and screening; Orbach MJ; The construction of a double-cos-site cosmid vector, pMOcosX, for use in making filamentous fungal genomic DNA libraries, is described . The vector has features that allow for selection of clones introduced into fungi by transformation and for efficient chromosome walking experiments . These features include (i) two cos sites allowing for easy construction of libraries without requiring size selection of insert DNA; (ii) an XhoI site for insertion of Sau3AI or MboI partially digested genomic DNA inserts that allows usage of a half-site fill-in method which minimizes the possibility of producing clones containing chimeric inserts; (iii) a bacterial hygromycin phosphotransferase-encoding gene fused to a modified cpc-1 promoter of Neurospora crassa for direct selection of cosmid clones upon introduction into fungal cells; and (iv) T7 and T3 bacteriophage promoters and EcoRI, NotI and BamHI restriction sites flanking the cloning site that allow for synthesis of, or isolation of, end-specific probes for chromosome walking . The combination of features in this vector allows for the easy construction and use of high-quality fungal DNA libraries from small amounts of genomic DNA.

J Bacteriol, 1994 Dec, 176(24), 7430 - 8
Mutations affecting two adjacent amino acid residues in the alpha subunit of RNA polymerase block transcriptional activation by the bacteriophage P2 Ogr protein; Ayers DJ et al.; The bacteriophage P2 ogr gene product is a positive regulator of transcription from P2 late promoters . The ogr gene was originally defined by compensatory mutations that overcame the block to P2 growth imposed by a host mutation, rpoA109, in the gene encoding the alpha subunit of RNA polymerase . DNA sequence analysis has confirmed that this mutation affects the C-terminal region of the alpha subunit, changing a leucine residue at position 290 to a histidine (rpoAL290H) . We have employed a reporter plasmid system to screen other, previously described, rpoA mutants for effects on activation of a P2 late promoter and have identified a second allele, rpoA155, that blocks P2 late transcription . This mutation lies just upstream of rpoAL290H, changing the leucine residue at position 289 to a phenylalanine (rpoAL289F) . The effect of the rpoAL289F mutation is not suppressed by the rpoAL290H-compensatory P2 ogr mutation . P2 ogr mutants that overcome the block imposed by rpoAL289F were isolated and characterized . Our results are consistent with a direct interaction between Ogr and the alpha subunit of RNA polymerase and support a model in which transcription factor contact sites within the C terminus of alpha are discrete and tightly clustered.

J Gen Virol, 1994 Dec, 75 ( Pt 12), 3441 - 51
Localization of Bunyamwera bunyavirus G1 glycoprotein to the Golgi requires association with G2 but not with NSm; Lappin DF et al.; The Bunyamwera bunyavirus (BUN) M RNA genome segment encodes three proteins, two glycoproteins termed G1 and G2 and a non-structural protein called NSm, in the form of a polyprotein precursor that is co-translationally cleaved to give the mature proteins . Indirect immunofluorescence experiments have shown that these proteins localize to the Golgi complex in BUN-infected cells . We have used a recombinant vaccinia virus (vTF7-3), which expresses bacteriophage T7 RNA polymerase, to drive the expression of plasmids containing either the entire BUN M segment cDNA or fragments that encode the G1, G2 and NSm proteins separately under control of the T7 promoter . After transfection of these plasmids into vTF7-3-infected cells, correctly sized and processed proteins were detected by immunoprecipitation with BUN-specific antibodies . Immunofluorescence experiments showed that G1, G2 and NSm localized to the Golgi when transiently expressed from the full-length cDNA . When G2 or NSm were expressed separately they also localized to the Golgi, but when G1 was expressed alone a staining pattern typical for the endoplasmic reticulum was obtained . However coexpression of G2 and G1 from independent plasmids resulted in G1 localizing to the Golgi . In contrast translocation of G1 to the Golgi was not observed when G1 was coexpressed with NSm, although NSm itself was still detected in the Golgi . Similar results were obtained when the proteins were expressed from transfected plasmids containing the G2-, NSm- or G1-coding sequences under control of the cytomegalovirus immediate-early promoter . The localization of G1 to the Golgi when coexpressed with G2 was confirmed by the loss of endoglycosidase H (endo H) sensitivity of G1 after approximately 60 min in a pulse-chase experiment; G1 remained sensitive to endo H when expressed either alone or in combination with NSm . These results suggest that G2 contains the Golgi targeting and/or retention signals and that G1 has to interact with this protein to localize to this cellular compartment.

Clin Exp Immunol, 1994 Dec, 98(3), 520 - 5
Characterization of naturally occurring autoantibodies against tumour necrosis factor-alpha (TNF-alpha): in vitro function and precise epitope mapping by phage epitope library; Sioud M et al.; Naturally occurring autoantibodies against cytokines exist in the sera of patients with autoimmune diseases as well as in the sera of normal individuals . We report here that affinity-purified autoantibodies against human TNF-alpha from one rheumatoid arthritis (RA) patient inhibited the cytotoxic effect of TNF-alpha on the mouse fibrosarcoma cell line WEHI 164, by 50% . In an attempt to predict the autoantibodies' recognition site on TNF-alpha protein we screened a random nanopeptide phage library with the affinity-purified TNF-alpha autoantibodies . Among 63 random selected clones, 46 clones carried the sequence ASSLLASSP, NSSPYLNTK or PQSPGSSFP . Frequency analysis of the relative occurrence of the 20 amino acids in the nanopeptides displayed by 50 random bacteriophages picked before selection and 63 after selection to bind to TNF-alpha autoantibodies indicated that proline (P < 0.0003) and serine (P < 0.04) are involved in the binding of the autoantibodies to the phages . Furthermore, we demonstrated that three synthetic peptides (ASSLLASSP, NSSPYLNTK and PPLKPVIDE) displayed by the selected phages reduced the binding of the autoantibodies to TNF-alpha protein by 50% . Interestingly, the sera of mice (BALB/c) immunized with phages displaying ASSLLASSP and NSSPYLNTK peptide showed an anti-TNF-alpha response as detected by ELISA . This response was not found in mice immunized with the wild type phage . Thus, the recombinant phages selected from the phage libraries could be used as carrier for immunization, and therefore as a tool for vaccine development . This work sets the stage for experiments designed to isolate ligands for protective antibodies.

J Virol, 1994 Dec, 68(12), 8217 - 22
Optimal lengths for DNAs encapsidated by Epstein-Barr virus; Bloss TA et al.; We measured the efficiency of DNA packaging by Epstein-Barr virus (EBV) as a function of the length of the DNA being packaged . Plasmids that contain oriP (the origin of latent EBV DNA replication), oriLyt (the origin of lytic EBV DNA replication), the viral terminal repeats (necessary for cleavage and packaging by EBV), and various lengths of bacteriophage lambda DNA were introduced into EBV-positive cells . Upon induction of the resident EBV's lytic phase, introduced plasmids replicated as concatemers and were packaged . Plasmid-derived concatemers of DNA with certain lengths were found to predominate in isolated virion particles . We measured the distribution of lengths of plasmid concatemers found within cells supporting the lytic phase of the viral life cycle and found that this distribution differed from the distribution of lengths of concatemers found in mature virion particles . This finding indicates that the DNA packaged into mature virions represents a selected subset of those present in the cell during packaging . These observations together indicate that the length of DNA affects the efficiency with which that DNA is packaged by EBV . Finally, we measured the length of the packaged B95-8 viral DNA and found it to be approximately 165 kbp, or 10 kbp shorter than the originally predicted size for B95-8 based on its sequence . Together with the results of other studies, these findings indicate that the packaging of DNAs by EBV is dependent on two imprecisely recognized elements: the viral terminal repeats and the length of the DNA being packaged by the virus.

J Bacteriol, 1994 Dec, 176(23), 7280 - 90
Cloning, sequencing, and expression of bacteriophage BF23 late genes 24 and 25 encoding tail proteins; Nakayama S et al.; Two bacteriophage BF23 late genes, genes 24 and 25, were isolated on a 7.4-kb PstI fragment from the phage DNA, and their nucleotide sequences were determined . Gene 24 encodes a minor tail protein with the expected M(r) of 34,309, and gene 25 located 4 bp upstream of gene 24 encodes a major tail protein with the expected M(r) of 50,329 . When total cellular RNA isolated from either phage-infected cells or cells bearing the cloned genes was analyzed by the primer extension method using the primers specific to either gene 25 or gene 24, we identified a possible late gene promoter, designated P25, in the 5'-flanking region of gene 25 . This promoter was similar in structure to Escherichia coli promoters for sigma 70 . Studies of the translational gene 25- and gene 24-lacZ fusions in the cloned gene system revealed that the promoter P25 was responsible for the expression of both genes 25 and 24 even in the absence of the regulatory genes which were absolutely required for late gene expression in the normal phage-infected cells . These results indicate that the two genes constitute an operon under the control of P25 and that the regulatory gene products of BF23 do not participate directly in specifying the late gene promoter.

Genetics, 1994 Dec, 138(4), 983 - 92
Integration of plasmids into the bacteriophage T4 genome; Kreuzer HW et al.; We have analyzed the integration of plasmids into the bacteriophage T4 genome via homologous recombination . As judged by genetic selection for a plasmid-borne marker, a mutation in phage gene uvsX or uvsY essentially blocked the integration of a plasmid with homology to the T4 genome but no phage replication origin (non-origin plasmid) . The strict requirement for these two proteins suggests that plasmid integration can proceed via a strand-invasion reaction similar to that catalyzed in vitro by the T4-encoded strand-exchange protein (UvsX) in concert with UvsY and gp32 . In contrast to the results with the non-origin plasmid, a mutation in uvsX or uvsY reduced the integration of a T4 replication origin-containing plasmid by only 3-10-fold . These results suggest that the origin-containing plasmid integrates by both the UvsXY-dependent pathway used by the non-origin plasmid and by a UvsXY-independent pathway . The origin-containing plasmid integrated into the phage genome during a uvsX- or uvsY-mutant infection of a recA-mutant host, and therefore origin-dependent integration can occur in the absence of both phage- and host-encoded strand-exchange proteins (UvsX and RecA, respectively).

Genetika, 1994 Dec, 30(12), 1621 - 5
{Analysis of DNA polymorphism detected by genomic fingerprinting based on phage M13 DNA, in populations of Bashkir and Komi}; Khusnutdinova EK et al.; Hyperpolymorphism of minisatellite DNA, detected using the M13 bacteriophage DNA hybridization probe, was studied in three ethnographic groups of Bashkirs and in the Komi population . Bands from 2 to 20 kb were analyzed in hybridization patterns . A significant population difference was detected both in evaluation of the average number of hybridization fragments per individuals and in the distribution of frequency of some fractions . Thus, it seems possible to describe a set of so-called characteristic fractions for each population . According to the hybridization fragment frequency for the populations investigated, an index of genetic similarity was calculated . The possibility of using this kind of multiple polymorphism of DNA in population genetic investigations at the level of ethnic groups and peoples is discussed, and a conclusion is made about the necessity of searching for the most informative methods of analyzing the data obtained.

J Clin Microbiol, 1994 Dec, 32(12), 3013 - 7
Laboratory investigation of a multistate food-borne outbreak of Escherichia coli O157:H7 by using pulsed-field gel electrophoresis and phage typing; Barrett TJ et al.; Two hundred thirty-three isolates of Escherichia coli O157:H7 were analyzed by both pulsed-field gel electrophoresis (PFGE) and bacteriophage typing . All 26 isolates from persons whose illness was associated with a recent multistate outbreak of E . coli O157:H7 infections linked to the consumption of undercooked hamburgers and all 27 isolates from incriminated lots of hamburger meat had the same phage type and the same PFGE pattern . Twenty-five of 74 E . coli O157:H7 isolates from Washington State and 10 of 27 isolates from other states obtained during the 6 months before the outbreak had the same phage type as the outbreak strain, but only 1 isolate had the same PFGE pattern . PFGE thus appeared to be a more sensitive method than bacteriophage typing for distinguishing outbreak and non-outbreak-related strains . The PFGE patterns of seven preoutbreak sporadic isolates and five sporadic isolates from the outbreak period differed from that of the outbreak strain by a single band, making it difficult to identify these isolates as outbreak or non-outbreak related . Phage typing and PFGE with additional enzymes were helpful in resolving this problem . While not as sensitive as PFGE, phage typing was helpful in interpreting PFGE data and could have been used as a simple, rapid screen to eliminate the need for performing PFGE on unrelated isolates.

Mol Biotechnol, 1994 Dec, 2(3), 295 - 8
Enhancing PCR amplification and sequencing using DNA-binding proteins; Rapley R; The polymerase chain reaction (PCR) is a powerful core molecular biology technique, which when coupled to chain termination sequencing allows gene and DNA sequence information to be derived rapidly . A number of modifications to the basic PCR format have been developed in an attempt to increase amplification efficiency and the specificity of the reaction . We have applied the use of DNA-binding protein, gene 32 protein from bacteriophage T4 (T4gp32) to increase amplification efficiency with a number of diverse templates . In addition, we have found that using single-stranded DNA-binding protein (SSB) or recA protein in DNA sequencing reactions dramatically increases the resolution of sequencing runs . The use of DNA-binding proteins in amplification and sequencing may prove to be generally applicable in improving the yield and quality of a number of templates from various sources.

Protein Expr Purif, 1994 Dec, 5(6), 604 - 13
Overexpression and rapid purification of rabbit fast skeletal troponin I from Escherichia coli: effect of different promoters, host strains, and culture conditions; Jha PK et al.; Rabbit fast skeletal troponin I (TnIf) cDNA was expressed using two Escherichia coli expression vectors, pRE1 containing the bacteriophage lambda pL promoter and pAED4, a T7 RNA polymerase-based vector . Although both vectors expressed TnIf, overexpression of the target protein was achieved with pAED4 . The effect of several parameters such as culture condition, compatible host strain, and inhibition of protein synthesis by rifampicin on the expression of TnIf was investigated . The overexpressed target protein synthesized during a brief induction period of only 2 h was conveniently purified from inclusion bodies by a simple and rapid procedure involving extraction with urea, ultracentrifugation, DE-52 column chromatography, and gel filtration . About 50-75 mg of highly purified TnIf was obtained per liter E . coli culture by this method, which does not involve time-consuming multistep procedures such as affinity and ion exchange chromatography as previously reported in the literature . The isolated unfused protein is stable and is indistinguishable from native protein in all biological parameters examined . The parameters optimized in this report for overexpression of TnIf may also be applicable for other eukaryotic proteins.

Anal Chem, 1994 Dec 1, 66(23), 4382 - 3
Labeling of double-stranded DNA by ROX-dideoxycytosine triphosphate using terminal deoxynucleotidyl transferase and separation by capillary electrophoresis; Figeys D et al.; Terminal transferase is used to add a single fluorescently labeled dideoxynucleotide to double-stranded DNA prepared by restriction endonuclease action on a bacteriophage . The product is separated by capillary electrophoresis with both hydroxypropylmethylcellulose and non-cross-linked polyacrylamide . The reaction products generate single peaks for each fragment with hydroxypropylmethylcellulose . However, the higher resolution separation produced by non-cross-linked polyacrylamide shows that the product contains two components for each restriction digest fragment . This labeling technique should be useful in restriction fragment length polymorphism studies.

Bioessays, 1994 Dec, 16(12), 865 - 8
TKO'ed: lox, stock and barrel; Chambers CA; The generation of panels of mutant mice by homologous recombination has greatly increased the ability to assess the function of particular gene products in vivo . The ability to control the developmental stage, the tissue and the nature of the mutation would be an important innovation . A recent report demonstrates that the conservative site-specific recombination of bacteriophage P1, namely Cre-lox, can be used successfully in combination with homologous recombination to generate temporal- and cell-restricted mutations in vivo . This technical advance allows a greater flexibility in gene targeting and will have a significant impact on how complex gene functions are studied in vivo.

Mol Gen Genet, 1994 Dec 1, 245(5), 623 - 7
Position and direction of strand exchange in bacteriophage HK022 integration; Kolot M et al.; The positions of the endonucleolytic cleavages promoted by the integrase protein (Int) of coliphage HK022 within its attB site were determined . The protein catalyses a staggered cut, which defines an overlap sequence of 7 bp within the core site . The overlap region is at the center of symmetry of a palindromic sequence which appears in all four putative att core binding sites for Int . We confirm that the order of strand exchange is similar to that in phage lambda.

Eur J Immunol, 1994 Dec, 24(12), 3188 - 93
Identification of the core residues of the epitope of a monoclonal antibody raised against glycoprotein D of herpes simplex virus type 1 by screening of a random peptide library; Schellekens GA et al.; Random peptide libraries (RPL) displayed on the surface of a filamentous bacteriophage can be used to identify peptide ligands that interact with target molecules . We have used a 15-amino acid residue RPL displayed on bacteriophage M13 to identify the core residues within the epitope of a monoclonal antibody (mAb) A16 which interacts with a continuous epitope restricted to amino acid residues 9 to 19 in the N-terminal region of glycoprotein D of herpes simplex virus type 1 (gD-1) . The single peptide sequence obtained after three rounds of selection contained identical residues at three positions compared to the authentic gD-1 sequence . Synthetic peptides were prepared based on the sequence of the original epitope and the phage-derived epitope . The binding constants (Ka) with mAb A16 were determined using surface plasmon resonance (SPR) biosensor technology . The RPL-derived peptide and peptide 9-19 of gD-1 had approximately the same affinity for mAb A16 . This suggests that those residues within the epitope that are essential for binding were identified . The synthesis of shorter versions of the RPL-derived peptide restricted the binding region to seven amino acid residues . These results show that minimal information retrieved from the screening of an RPL combined with peptide synthesis can characterize the epitope of an mAb with high resolution . Immunization of mice with the phage-derived peptide protected against a challenge with a lethal dose of herpes simplex virus type 1 equally well as the gD-1 derived peptide.

Biol Chem Hoppe Seyler, 1994 Dec, 375(12), 833 - 6
Expression in Escherichia coli of rat neurotensin receptor fused to membrane proteins from the membrane-containing bacteriophage PRD1; Hanninen AL et al.; Bacteriophage PRD1 is a membrane-containing phage which could be used for expression of foreign membrane proteins such as neurotensin receptor (NTR), a seven-helix G-protein coupled receptor . To ensure recognition of NTR by the phage system six different fusion genes were constructed, each encoding a different phage integral membrane protein fused to the N-terminus of NTR, and expression of the fusion proteins in Escherichia coli was analysed . Here we report the identification of two fusion constructs that retained the function of NTR in E . coli . This provides the basis to develop the phage system as a heterologous expression system for seven-helix receptors.

Curr Biol, 1994 Dec 1, 4(12), 1099 - 103
Cre-loxP-mediated gene replacement: a mouse strain producing humanized antibodies; Zou YR et al.; BACKGROUND: The bacteriophage-derived Cre-loxP recombination system operates efficiently in mammalian cells . This system is particularly useful in gene-targeting experiments in the mouse, and has already been used to generate 'clean' deletions of target genes in the germ line, as well as to inactivate target genes in a conditional manner (based on regulated expression of the Cre recombinase) . In principle, Cre-loxP-mediated recombination should also allow gene replacement, and thus the introduction of virtually any kind of mutation into the genome . RESULTS: We used the Cre-loxP system, in mouse embryonic stem cells, to replace the mouse gene C gamma 1, which encodes the constant region of the heavy chain of IgG1 antibodies, with its human counterpart . The mutation was transmitted through the mouse germ line, and the resulting mutant mice were crossed with mice expressing kappa light chains with a human, instead of a mouse, constant region . Mice homozygous for both mutations produce humanized, kappa-chain-bearing IgG1 antibodies at the same level and efficiency as wild-type mice produce murine IgG1 antibodies . These animals should enable the ex vivo production of humanized, chimeric monoclonal antibodies specific for any antigen to which the mouse can respond . CONCLUSIONS: Cre-loxP-mediated gene replacement is a simple and efficient general method of targeted mutagenesis in the mouse.

Biophys J, 1994 Dec, 67(6), 2205 - 22
An electrostatic spatial resonance model for coaxial helical structures with applications to the filamentous bacteriophages; Marzec CJ et al.; A model is presented that treats the symmetry matching problem in structures made of two interacting coaxial helices of point charges . The charges are sources of a potential field that mediates a non-specific attractive interaction between the helices . The problem is represented in Fourier space, which affords the most generality . It is found that coaxial helices with optimally mated symmetries can lock into spatial resonance configurations that maximize their interaction . The resonances are represented as vectors in a discrete three-dimensional space . Two algebraic relations are given for the four symmetry parameters of two helices in resonance . One-start inner helices interacting with coaxial one-start or NR-start outer helices are considered . Applications are made to the filamentous bacteriophages Ff, Pf1, Xf, and Pf3 . The interaction given by the linearized Poisson-Boltzmann equation is calculated in this formalism to allow comparison of the electrostatic free energy of interaction of different resonance structures . Experimental nucleotide/subunit ratios are accounted for, and models for the DNA-protein interfaces are presented, with particular emphasis on Pf1.

Cancer Gene Ther, 1994 Dec, 1(4), 267 - 77
Ribozyme-mediated in vitro cleavage of transcripts arising from the major transforming genes of human papillomavirus type 16; Lu D et al.; Human papillomaviruses (HPV) have been strongly implicated as important cofactors in the development of several human malignancies, particularly anogenital carcinomas . Products arising from the E6 and E7 open reading frames (ORFs) from HPV-16, a type commonly associated with human cervical carcinoma, are essential for viral transformation . Unfortunately, a highly effective treatment for this infection is not available . To develop a novel treatment for this disease, ribozymes were designed to cleave all transcripts encoding HPV-16 E6 and E7 ORFs in proximity to their translational start sites ("AUG") . Cleavage sites for Rz110 and Rz558 occur immediately 3' to nucleotides 110 and 558 of the viral genomic DNA, respectively . Oligonucleotides corresponding to these ribozymes were synthesized and inserted into a eucaryotic viral vector derived from the nonpathogenic parvovirus, adeno-associated virus . Ribozyme transcription from this vector, termed CWRT7:SVN, is under control of both the highly active Rous sarcoma virus long terminal repeat and bacteriophage T7 promoters . T7 transcripts of the E6 and E7 ribozymes efficiently cleaved their cognate targets in vitro under a variety of conditions, including physiological temperature . These results may provide the basis for the development of a ribozyme-based, gene therapeutic treatment for HPV-associated diseases.

Anal Biochem, 1994 Dec, 223(2), 291 - 8
A mammalian expression vector for the expression of GAL4 fusion proteins with an epitope tag and histidine tail; Witzgall R et al.; Expression of newly cloned cDNAs in mammalian cell lines is an essential tool for the functional analysis of the proteins encoded by these cDNAs . In many instances, however, evaluation of the protein is difficult because of the difficulty in purification of the expressed protein and/or the lack of specific antibodies which react with the proteins on Western blots or for immunocytochemistry or immunoprecipitation . A number of gene fusion systems have been employed in which a known peptide is fused to the expression product of interest and the fusion protein is purified using affinity chromatography and identified in extracts or by immunocytochemistry using antibodies directed against the affinity handle peptide . The DNA-binding domain of the yeast transcription factor GAL4 is widely used to construct fusion proteins with putative transcription factors to evaluate potential trans-acting domains . Because of the lack of commercially available anti-GAL4 antibodies, the further biochemical characterization of these fusion proteins has remained difficult . We describe the construction of two mammalian expression vectors, pMFH/GAL4 and pMFH2/GAL4 (where pMFH stands for pM2, Flag, Histidine tail), which encode the DNA-binding domain of the yeast transcription factor GAL4 with a Flag peptide (consisting of the 11-amino-acid leader peptide of the gene 10 product from bacteriophage T7) at the NH2-terminus and a tail of six histidines at the COOH-terminus . Unique restriction sites allow both the construction of fusion proteins with the GAL4 DNA-binding domain and the replacement of the GAL4 fragment with another insert.(ABSTRACT TRUNCATED AT 250 WORDS)

Mutat Res, 1994 Dec 1, 311(2), 199 - 208
Damage and mutagenesis of E . coli and bacteriophage lambda induced by oxathiolane and aziridinyl steroids; Qadri SA et al.; lambda-Escherichia coli complexes exhibited remarkable sensitivity to the treatment with test steroidal derivatives in the presence of Cu(II) . The decline in plaque-forming units after steroid treatment was more pronounced in complexes with some of the radiation repair-defective mutants of E . coli K-12, i.e., recA, lexA and polA, as compared to uvrA and wild-type strains . The red gene of lambda phage and recA gene of E . coli seem to have a complementary effect on the steroid-induced lesions . An enhanced level of mutagenesis was observed when steroid-treated E . coli cells were transformed with steroid-treated pBR322 plasmid DNA . A remarkable degree of c mutation was also observed when steroid I-treated phage particles were allowed to adsorb on steroid-treated wild-type bacteria . Moreover, the oxathiolane steroid treatment of lambda cI857-E . coli lysogen resulted in prophage induction in nutrient broth even at 32 degrees C . Thus on the basis of these results, the role of SOS repair system in steroid-induced mutagenesis and repair of DNA lesions in E . coli and bacteriophage lambda has been suggested.

J Mol Biol, 1994 Nov 25, 244(2), 144 - 50
Control of translation by mRNA secondary structure in Escherichia coli . A quantitative analysis of literature data; de Smit MH et al.; Translational efficiency in Escherichia coli is strongly controlled by the secondary structure of the mRNA in the translational initiation region . We have previously shown that protein production from the coat-protein gene of RNA bacteriophage MS2 is directly related to the fraction of mRNA molecules in which the ribosome binding site is unfolded . This fraction is dictated by the free energy (delta Gf0) of the local secondary structure . We now present a similar analysis of published data on four other ribosome binding sites . The results conform quantitatively to the same relationship as found for the MS2 coat-protein gene . The efficiency of translation is determined by the overall stability of the structure at the ribosome binding site, whether the initiation codon itself is base-paired or not . Structures weaker than -6 kcal/mol usually do not reduce translational efficiency . Below this threshold, all systems show a tenfold decrease in expression for every -1.4 kcal/mol, as predicted from theory.

J Biol Chem, 1994 Nov 25, 269(47), 30056 - 64
The role of 3-hydroxyethyldeoxyuridine in mutagenesis by ethylene oxide; Bhanot OS et al.; Ethylene oxide, a direct-acting mutagen and carcinogen, produces 3-hydroxyethyldeoxyuridine (3-HE-dU) after initial alkylation at N3 of dC, followed by rapid hydrolytic deamination . The significance of formation of 3-HE-dU in DNA was investigated by in vitro DNA replication of 3-HE-dU . A 55-nucleotide DNA template, containing 3-HE-dU at a single site, was constructed . DNA products, synthesized on the site-modified template, were analyzed and mutagenic bypass at 3-HE-dU estimated . The 3-HE-dU lesion blocked DNA replication by the Klenow fragment of Escherichia coli polymerase I (Kf Pol I) and bacteriophage T7 polymerase (T7 Pol) 3' to 3-HE-dU and after incorporating a nucleotide opposite 3-HE-dU . DNA synthesis past 3-HE-dU was negligible (< 3%) . Substitution of Kf Pol I (exo-) and T7 Pol (exo-), polymerases lacking 3'-->5' exonuclease proofreading activity, for Kf Pol I and T7 Pol, respectively, facilitated DNA synthesis past 3-HE-dU . The bypass synthesis by Kf Pol I (exo-) was 60% and 90% by T7 Pol (exo-) . These results suggest that the 3-HE-dU lesion could be bypassed, but that the extension at 3-HE-dU is rate-limiting . In the absence of proofreading, the nucleotide incorporated opposite 3-HE-dU is not excised and remains in position long enough for extension to occur . During post-lesion synthesis, both dA and dT were incorporated opposite 3-HE-dU . Since 3-HE-dU is derived from dC alkylation by ethylene oxide, incorporation of dA and dT opposite 3-HE-dU implicates this lesion in G.C-->A.T and G.C-->T.A mutagenesis.

Nucleic Acids Res, 1994 Nov 25, 22(23), 4947 - 52
An RNA-protein contact determined by 5-bromouridine substitution, photocrosslinking and sequencing; Willis MC et al.; An analogue of the replicase translational operator of bacteriophage R17, that contains a 5-bromouridine at position -5 (RNA 1), complexes with a dimer of the coat protein and photocrosslinks to the coat protein in high yield upon excitation at 308 nm with a xenon chloride excimer laser . Tryptic digestion of the crosslinked nucleoprotein complex followed by Edman degradation of the tryptic fragment bearing the RNA indicates crosslinking to tyrosine 85 of the coat protein . A control experiment with a Tyr 85 to Ser 85 variant coat protein showed binding but no photocrosslinking at saturating protein concentration . This is consistent with the observation from model compound studies of preferential photocrosslinking of BrU to the electron rich aromatic amino acids tryptophan, tyrosine, and histidine with 308 nm excitation.

Gene, 1994 Nov 18, 149(2), 389 - 90
A new hybrid promoter directs transcription at identical start points in mammalian cells and in vitro; Dirks W et al.; A hybrid promoter which generates large amounts of mRNAs with transcription start points (tsp) differing in one nt, both in mammalian cells and in vitro, was constructed by integrating the promoter of bacteriophage T7 in between the TATA box and the tsp of the retroviral myeloproliferative sarcoma virus (MPSV) long terminal repeat (LTR) . This promoter was designed for sequence and secondary structure studies of 5' untranslated regions (UTR) with respect to mRNA stability and translatability.

Virology, 1994 Nov 15, 205(1), 51 - 65
Bacteriophage P2 and P4 morphogenesis: assembly precedes proteolytic processing of the capsid proteins; Marvik OJ et al.; Several of the structural proteins of phage P2 and its satellite P4 undergo proteolytic processing during development of mature phage particles . Here, we report that uncleaved shell protein, gpN, is present in immature capsids of both P2 and P4, showing that assembly precedes processing . This excludes the possibility that processing of gpN is involved in capsid size determination . We also find that N*, the fully processed version of gpN, produced from a plasmid, can assemble into both P2- and P4-sized particles, implying that the amino-terminal end of gpN is not required for assembly initiation nor for the formation of a T = 4 shell . As may be expected for a scaffolding protein, we find that gpO coexists with gpN in immature P2, as well as P4, capsids . This result supports the conclusion that gpO is required for both phages and strongly suggests that the O derivative, h7 (found in mature capsids), results from proteolytic cleavage after gpN/gpO coassembly.

Virology, 1994 Nov 15, 205(1), 188 - 97
Binding of scaffolding subunits within the P22 procapsid lattice; Greene B et al.; The capsid assembly pathways of the dsDNA bacteriophages, herpesviruses, and adenoviruses all proceed through a precursor shell lacking DNA . These procapsids contain scaffolding proteins required for assembly but absent from mature virions . The bacteriophage P22 procapsid contains approximately 300 molecules of the 33-kDa gene 8 scaffolding protein, in addition to the 420 molecules of gene 5 coat protein . During the process of DNA packaging and phage maturation, all 300 scaffolding molecules are released intact to participate in subsequent rounds of procapsid assembly . Low concentrations of guanidine hydrochloride (GuHCl) reproduce the release of scaffolding from procapsids in vitro, in the absence of DNA . The release was reversible; when the GuHCl was removed by dialysis, the scaffolding subunits reentered the extracted capsids to regenerate morphologically normal procapsids . The subunits presumably exited and reentered through the channels recently observed at the centers of the pentamers and hexamers (Prasad, B . V . V., Prevelige, P . E., Marietta, E., Chen, R . O., Thomas, D., King, J., and Chiu, W . (1993) . J . Mol . Biol . 231 65-74) . We have utilized this reaction to investigate the binding of scaffolding within normal procapsids and to other large structures of coat protein . Procapsids contained two classes of scaffolding subunits, which may represent binding of scaffolding to different specific positions within the T = 7 procapsid lattice . These sites became lost or inaccessible upon phage maturation.

Virology, 1994 Nov 15, 205(1), 170 - 8
NTP binding induces conformational changes in the double-stranded RNA bacteriophage ø6 subviral particles; Ojala PM et al.; Bacteriophage o6 is a double-stranded RNA virus consisting of a nucleocapsid (NC) surrounded by a membrane . Beneath the NC major coat protein, P8, resides the o6 RNA polymerase complex which is composed of four early proteins P1, P2, P4, and P7 . Protein P1 forms the dodecahedral framework with which the other three proteins are associated . We have developed a new method for the isolation of stable polymerase complex particles which retain their structural integrity and polymerase activity for several days . Purine nucleotides, especially GTP, dGTP, ddGTP, and GDP, stabilized the particle efficiently . Furthermore, binding of any NTP was shown to induce conformational changes in the NC structure, as detected by alterations in the binding properties of NC-specific monoclonal antibodies . In the presence of NTPs, most of the epitopes in protein P4 become more exposed than without NTPs, while the epitopes in protein P8 were either masked or unmasked due to NTP binding . Based on the accessibility of the epitopes of protein P1 on the NC, we postulate that at least part of this protein is also accessible on the NC surface.

Eur J Biochem, 1994 Nov 15, 226(1), 53 - 8
Display of expression products of cDNA libraries on phage surfaces . A versatile screening system for selective isolation of genes by specific gene-product/ligand interaction; Crameri R et al.; Techniques for cloning cDNAs from bacteriophage libraries immobilised on solid supports are well established . However, these techniques do not allow selective enrichment of clones expressing proteins of interest . Screening of cDNA libraries would be simplified if the proteins encoded by cDNAs could be expressed on the surface of phage . Phage carrying genes encoding proteins for which a ligand is available can be selected directly by affinity interaction {Crameri, R . & Suter, M . (1993) Gene (Amst.) 137, 69-75} . The expression products from a cDNA library from Aspergillus fumigatus have been displayed on the surface of the filamentous phage M13 and screened for gene products binding to human serum IgE . The physical linkage of cDNA-encoded proteins to the genetic information required for their production, achieved by exploiting the high-affinity interaction of the Jun and Fos leucine zippers, allows screening of up to 1 x 10(10) independent clones in 50-microliters aliquots applied to a well of a microtiter plate coated with the ligand . Phage displaying IgE-binding proteins were selectively enriched 10(5)-10(6)-fold over non-specific phage after six rounds of growth and selection . The apparent molecular mass of the proteins selected from the cDNA library was in the range 20-40 kDa . Restriction enzyme analysis and preliminary sequence determination of 12 selected inserts revealed different sequences . The ability of the proteins to bind to human serum IgE was corroborated by enzyme-linked immunosorbent assay and by Western-blot analysis . The developed cloning strategy allows isolation of cDNAs encoding proteins for which a ligand is available and circumvents immobilisation of the libraries on solid-phase supports which hamper selective enrichment of clones expressing the desired protein.

Structure, 1994 Nov 15, 2(11), 1041 - 8
A novel class of winged helix-turn-helix protein: the DNA-binding domain of Mu transposase; Clubb RT et al.; BACKGROUND: Mu transposase (MuA) is a multidomain protein encoded by the bacteriophage Mu genome . It is responsible for translocation of the Mu genome, which is the largest and most efficient transposon known . While the various domains of MuA have been delineated by means of biochemical methods, no data have been obtained to date relating to its tertiary structure . RESULTS: We have solved the three-dimensional solution structure of the DNA-binding domain (residues 1-76; MuA76) of MuA by multidimensional heteronuclear NMR spectroscopy . The structure consists of a three-membered alpha-helical bundle buttressed by a three-stranded antiparallel beta-sheet . Helices H1 and H2 and the seven-residue turn connecting them comprise a helix-turn-helix (HTH) motif . In addition, there is a long nine-residue flexible loop or wing connecting strands B2 and B3 of the sheet . NMR studies of MuA76 complexed with a consensus DNA site from the internal activation region of the Mu genome indicate that the wing and the second helix of the HTH motif are significantly perturbed upon DNA binding . CONCLUSIONS: While the general appearance of the DNA-binding domain of MuA76 is similar to that of other winged HTH proteins, the connectivity of the secondary structure elements is permuted . Hence, the fold of MuA76 represents a novel class of winged HTH DNA-binding domain.

J Mol Biol, 1994 Nov 11, 243(5), 811 - 5
Identification of recognition elements on bacteriophage Q beta minus strand RNA that are essential for template activity with Q beta replicase; Schuppli D et al.; In order to identify the structural elements important for the activity of the Q beta minus strand RNA as a template for Q beta replicase, a series of minus strand RNAs with internal or external deletions were prepared by in vitro transcription from suitable expression plasmids . The template activities of the deletion mutants were determined by single-round replication assays using purified replicase holoenzyme or core enzyme (lacking subunit alpha) in vitro . Two elements of RNA structure and/or sequence important for template activity were found . The first is a segment in the 5'-terminal region (map segment 4078 to 4132) containing a potential stem-loop structure, whose sequence was previously recognized to be highly conserved in the small variant MDV-1 RNA and suggested to be involved in its template recognition . The second element is defined by two partially complementary sequence segments in the 3'-terminal region (map positions 557 to 576 and 24 to 35), that appear to be engaged in long-range base-pairing and may form the stem of a large secondary structure domain, whose branches are not necessary for template recognition . The results obtained with replicase holoenzyme and core enzyme were identical within the accuracy of the method . They confirm the absence of any role of S1 protein in the interaction of replicase with minus strand RNA and further emphasize the profound difference in the interactions of replicase with the plus and minus strand.

J Biol Chem, 1994 Nov 11, 269(45), 27815 - 8
Bacteriophage T4 gene 55.9 encodes an activity required for anaerobic ribonucleotide reduction; Young P et al.; Bacteriophage T4 contains a phage-encoded anaerobic ribonucleoside triphosphate reductase, nrdD, previously named sunY . An open reading frame, 55.9, that resides downstream of the phage reductase was observed to have amino acid sequence similarity with the E . coli pyruvate formate-lyase (Pfl) activating enzyme . A stop codon was engineered into the cloned 55.9 gene and then recombined back into the phage genome . Phage-infected extracts that lack a functional 55.9 product have a 6-fold reduction in anaerobic ribonucleotide reductase activity and are unable to activate overexpressed T4 NrdD . Restoration of reductase activity was possible when 55.9- and nrdD- T4-infected Escherichia coli extracts were conjointly assayed . Comparing the anaerobic burst size of 55.9- infections to that of the parental phage indicates that anaerobic de novo synthesis of deoxyribonucleotides is nearly abolished in phage lacking the 55.9 product . We propose that T4 55.9 encodes an enzyme that activates T4 NrdD by generating a glycyl radical in the phage-encoded reductase . The homology between the Pfl activating enzyme and T4 55.9 product (in this communication renamed NrdG) in function as well as amino acid sequence is presumably a remnant of an ancient heritage between Pfl and the anaerobic ribonucleotide reductases.

Proc Natl Acad Sci U S A, 1994 Nov 8, 91(23), 11163 - 7
Toward a code for the interactions of zinc fingers with DNA: selection of randomized fingers displayed on phage; Choo Y et al.; We have used two selection techniques to study sequence-specific DNA recognition by the zinc finger, a small, modular DNA-binding minidomain . We have chosen zinc fingers because they bind as independent modules and so can be linked together in a peptide designed to bind a predetermined DNA site . In this paper, we describe how a library of zinc fingers displayed on the surface of bacteriophage enables selection of fingers capable of binding to given DNA triplets . The amino acid sequences of selected fingers which bind the same triplet are compared to examine how sequence-specific DNA recognition occurs . Our results can be rationalized in terms of coded interactions between zinc fingers and DNA, involving base contacts from a few alpha-helical positions . In the paper following this one, we describe a complementary technique which confirms the identity of amino acids capable of DNA sequence discrimination from these positions.

Proc Natl Acad Sci U S A, 1994 Nov 8, 91(23), 11007 - 11
Localization of an aminoacridine antitumor agent in a type II topoisomerase-DNA complex; Freudenreich CH et al.; Type II topoisomerases are the targets of several classes of chemotherapeutic agents that stabilize an intermediate of the catalytic cycle with the enzyme covalently linked to cleaved DNA . We have used 3-azido-AMSA {4'-(3-azido-9-acridinylamino)methanesulfon-m-anisidide}, a photo-activatible analog of the inhibitor m-AMSA {4'-(9-acridinylamino)methanesulfon-m-anisidide}, to localize the inhibitor binding site in a cleavage complex consisting of an oligonucleotide substrate and the bacteriophage T4 type II DNA topoisomerase . Upon photoactivation, the inhibitor covalently attached to the substrate only in the presence of topoisomerase . Sites of inhibitor attachment were detected by primer-extension analysis and by piperidine-induced cleavage of the covalently modified substrate . 3-Azido-AMSA reacted with bases immediately adjacent to the two phosphodiester bonds cleaved by the enzyme . Therefore, topoisomerase creates or stabilizes preferential binding sites for the inhibitor precisely at the two sites of DNA cleavage.

Proc Natl Acad Sci U S A, 1994 Nov 8, 91(23), 10972 - 6
The DNA-binding domain of the MotA transcription factor from bacteriophage T4 shows structural similarity to the TATA-binding protein; Finnin MS et al.; The bacteriophage T4 middle-mode transcription factor MotA consists of two domains of approximately equal size . The C-terminal domain has been shown to contain the DNA-binding elements of the molecule, and the N-terminal domain appears to interact with RNA polymerase . A 12.5-kDa fragment of the C-terminal domain (MotCF), comprising residues 105-211 of MotA, was found to be suitable for structural studies by NMR . The 1H and 15N assignments have been made for MotCF by using two-dimensional homonuclear and heteronuclear experiments . A secondary structure has been determined which consists of a six-stranded antiparallel beta-pleated sheet with three alpha-helical segments . The secondary structure of MotCF has a clear similarity to one half of the eukaryotic TATA-binding protein (TBP), which is an intramolecular dimer . Therefore, MotCF may be related to a monomeric ancestral protein of TBP . TBP binds its target DNA in the minor groove by specific interactions with hydrophobic and aromatic residues on the exposed sheet surface of the protein . Similar residues are also present on the beta-sheet surface of MotCF, suggesting that it too binds DNA in the minor groove.

Biochemistry, 1994 Nov 8, 33(44), 13049 - 56
Kinetic and equilibrium alpha-secondary tritium isotope effects on reactions catalyzed by dCMP hydroxymethylase from bacteriophage T4; Graves KL et al.; Deoxycytidylate (dCMP) hydroxymethylase (CH) catalyzes the formation of 5-(hydroxymethyl)-dCMP, essential for DNA synthesis in phage T4, from dCMP and methylenetetrahydrofolate (CH2THF) . The nucleotide analog 5-fluorodeoxuridylate (FdUMP) stoichiometrically inactivates CH by formation of a covalent complex containing enzyme, FdUMP, and CH2THF . Similar FdUMP complexes are formed by dTMP synthase and dUMP hydroxymethylase, enzymes which are homologous to CH . Both the association and the dissociation rate of the FdUMP complex are shown to be increased by the mutation of active site Asp179 to Asn . The mutated enzyme, CH(D179N), has an altered substrate preference, favoring dUMP rather than dCMP {Graves, K . L., et al . (1992) Biochemistry 31, 10315} . A value of 0.8 was determined for the alpha-secondary tritium equilibrium isotope effect on the binding of {6-3H}FdUMP to wild-type CH and to CH(D179N), using a mixture of 2-14C- and 6-3H-labeled FdUMP . These effects, similar to that found for TS, indicate that C6 of the nucleotide is saturated (i.e., sp3 hybridized) in the covalent complex of CH, FDUMP, and CH2THF . This strongly suggests that catalysis by CH proceeds via sequential sp2-->sp3-->sp2 hybridization changes at C6 of substrate nucleotides, and it is consistent with a transient covalent linkage of C6 to the thiol of an essential CH residue, Cys148 . The values of the alpha-secondary 3H kinetic isotope effect (KIE) on kcat/KM for CH-catalyzed formation of Hm5dCMP caused by 6-3H-substitution of dCMP, with both wild-type CH and CH(D179N), were very close to 1.0 . However, the KIE for CH-(D179N) with dUMP was 0.82.(ABSTRACT TRUNCATED AT 250 WORDS)

Biochemistry, 1994 Nov 8, 33(44), 12990 - 7
Location of M13 coat protein in sodium dodecyl sulfate micelles as determined by NMR; Papavoine CH et al.; The major coat protein (gVIIIp) of bacteriophage M13 solubilized in sodium dodecyl sulfate (SDS) detergent micelles was used as a model system to study this protein in the lipid-bound form . In order to probe the position of gVIIIp relative to the SDS micelles, stearate was added, spin-labeled at the 5- or 16-position with a doxyl group containing a stable nitroxide radical . The average position of the spin-labels in the micelles was derived from the line broadening of the resonances in the 13C spectrum of SDS . Subsequently, we derived a model of the relative position of gVIIIp in the SDS micelle from the effect of the spin-labels on the gVIIIp resonances, monitored via 1H-15N HSQC and TOCSY experiments . The results are consistent with the structure of gVIIIp having two helical strands . One strand is a long hydrophobic helix that spans the micelle, and the other is a shorter amphipathic helix on the surface of the micelle . These results are in good agreement with the structure of gVIIIp in membranes proposed by McDonnell et al . on the basis of solid state NMR data {McDonnell, P . A., Shon, K., Kim, Y., & Opella, S . J . (1993) J . Mol . Biol . 233, 447-463} . This study indicates that high-resolution NMR on this membrane protein, solubilized in detergent micelles, is a very suitable technique for mimicking these proteins in their natural environment . Furthermore, the data indicate that the structure of the micelle near the C-terminus of the major coat protein is distorted.(ABSTRACT TRUNCATED AT 250 WORDS)

Plant Mol Biol, 1994 Nov, 26(3), 781 - 90
Reduced ethylene synthesis by transgenic tomatoes expressing S-adenosylmethionine hydrolase; Good X et al.; We have utilized a gene from bacteriophage T3 that encodes the enzyme S-adenosylmethionine hydrolase (SAMase) to generate transgenic tomato plants that produce fruit with a reduced capacity to synthesize ethylene . S-adenosylmethionine (SAM) is the metabolic precursor of 1-aminocyclopropane-1-carboxylic acid, the proximal precursor to ethylene . SAMase catalyzes the conversion of SAM to methylthioadenosine and homoserine . To restrict the presence of SAMase to ripening fruit, the promoter from the tomato E8 gene was used to regulate SAMase gene expression . Transgenic tomato plants containing the 1.1 kb E8 promoter bore fruit that expressed SAMase during the breaker and orange stage of fruit ripening and stopped expression after the fruit fully ripened . Plants containing the 2.3 kb E8 promoter expressed SAMase at higher levels during the post-breaker phases of fruit ripening and had a substantially reduced capacity to synthesize ethylene.

J Bacteriol, 1994 Nov, 176(22), 6907 - 14
Expression, purification, and functional characterization of the carboxyl-terminal domain fragment of bacteriophage 434 repressor; Carlson PA et al.; The repressor protein of bacteriophage 434 binds to DNA as a dimer of identical subunits . Its strong dimerization is mediated by the carboxyl-terminal domain . Cooperative interactions between the C-terminal domains of two repressor dimers bound at adjacent sites can stabilize protein-DNA complexes formed with low-affinity binding sites . We have constructed a plasmid, pCT1, which directs the overproduction of the carboxyl-terminal domain of 434 repressor . The protein encoded by this plasmid is called CT-1 . Cells transformed with pCT1 are unable to be lysogenized by wild-type 434 phage, whereas control cells are lysogenized at an efficiency of 1 to 5% . The CT-1-mediated interference with lysogen formation presumably results from formation of heteromeric complexes between the phage-encoded repressor and the plasmid-encoded carboxyl-terminal domain fragment . These heteromers are unable to bind DNA and thereby inhibit the repressor's activity in promoting lysogen formation . Two lines of evidence support this conclusion . First, DNase I footprinting experiments show that at a 2:1 ratio of CT-1 to intact 434 repressor, purified CT-1 protein prevents the formation of complexes between 434 repressor and its OR1 binding site . Second, cross-linking experiments reveal that only a specific heterodimeric complex forms between CT-1 and intact 434 repressor . This latter observation indicates that CT-1 interferes with 434 repressor-operator complex formation by preventing dimerization and not by altering the conformation of the DNA-bound repressor dimer . Our other evidence is also consistent with this suggestion . We have used deletion analysis in an attempt to define the region which mediates the 434 repressor-CT-1 interaction . CT-1 proteins which have more than the last 14 amino acids removed are unable to interfere with 434 repressor action in vivo.

J Bacteriol, 1994 Nov, 176(21), 6730 - 7
The phage lambda orf gene encodes a trans-acting factor that suppresses Escherichia coli recO, recR, and recF mutations for recombination of lambda but not of E . coli; Sawitzke JA et al.; Bacteriophage lambda can recombine in recBC sbcB sbcC mutant cells by using its own gene orf, the Escherichia coli recO, recR, and recF genes, or both . Expression of an orf-containing plasmid complements the recombination defects of orf mutant phage . However, this clone does not complement a recO mutation for conjugational recombination or recO, recR, or recF mutations for repair of UV-induced DNA damage . A plasmid clone of orf produces a protein with an apparent molecular mass of 15 kDa.

J Bacteriol, 1994 Nov, 176(21), 6439 - 48
Purification and characterization of the SegA protein of bacteriophage T4, an endonuclease related to proteins encoded by group I introns; Sharma M et al.; Although not encoded by an intron, the bacteriophage T4 SegA protein shares common amino acid motifs with a family of proteins found within mobile group I introns present in fungi and phage . Each of these intron-encoded proteins is thought to initiate the homing of its own intron by cleaving the intronless DNA at or near the site of insertion . Previously, we have found that SegA also cleaves DNA . In this report, we have purified the SegA protein and characterized this endonuclease activity extensively . SegA protein cleaved circular and linear plasmids, DNA containing unmodified cytosines, and wild-type T4 DNA containing hydroxymethylated, glucosylated cytosines . In all cases, certain sites on the DNA were highly preferred for cleavage, but with increasing protein concentration or time of incubation, cleavage occurred at many sites . SegA cleaving activity was stimulated by the presence of ATP or ATP gamma S . Sequence analysis of three highly preferred cleavage sites did not reveal a simple consensus sequence, suggesting that even among highly preferred sites, SegA tolerates many different sequences . A T4 segA amber mutant that we constructed had no phenotype, and PCR analyses indicated that several T-even-related phages lack the segA gene . Taken together, our results show that SegA is an endonuclease with a hierarchy of site specificity, and these results are consistent with the insertion of segA DNA into the T4 genome some time after the divergence of the closely consistent with the insertion of segA DNA into the T4 genome some time after the divergence of the closely related T-even phages.

Eur J Biochem, 1994 Nov 1, 225(3), 1097 - 103
Cellular site of synthesis and dynamics of cell surface re-expression of polysialic acid of the neural cell adhesion molecule; Scheidegger P et al.; Homopolymers of alpha-2,8-ketosidically linked sialic acid (polysialic acid) represent a posttranslational modification which, in mammals, appears to be unique for the neural cell adhesion molecule and the alpha subunit of sodium channels in brain . Under steady-state conditions, polysialic acid is detectable in the plasma membrane of different cell types but not in the cytoplasm . We have studied the site of synthesis and the cell surface re-expression of polysialic acid in a clonal subline of small cell lung carcinoma using the monoclonal antibody 735 and bacteriophage endosialidase, both specific reagents for polysialic acid . After enzymic removal, cell surface polysialic acid re-expression reached control levels only after 5 days . When Golgi to plasma membrane transport of endosialidase-treated cells was blocked by culture at 20 degrees C or in the presence of monensin at 37 degrees C, de-novo-synthesized polysialic acid became detectable in the Golgi apparatus . Our data show that synthesis of polysialic acid of the neural cell adhesion molecule with a degree of polymerization of at least nine occurs intracellular in the Golgi apparatus of a human small cell lung carcinoma cell line.

EMBO J, 1994 Nov 1, 13(21), 5240 - 4
The occurrence of heritable Mu excisions in starving cells of Escherichia coli; Foster PL et al.; A strain of Escherichia coli constructed by Shapiro has a segment of Mu bacteriophage DNA inserted between the araC and lacZ genes . Excision events that produce an in-frame fusion of lacZ to araB result in a cell (here designated Ara-Lac+) that can grow on lactose if arabinose is present as an inducer . Whether or not these excision events occur in the absence of selection for the Ara-Lac+ phenotype has figured prominently in the debate of the phenomenon known as 'directed' or 'adaptive' mutation . In an attempt to settle the issue, we have used classic fluctuation tests to show that cells capable of producing a clone of descendants that are phenotypically Ara-Lac+ do, indeed, arise in stationary phase cultures kept starving in depleted minimal medium . We found that Ara-Lac+ progenitors arise rapidly under these conditions, in contrast to the delayed appearance of Ara-Lac+ mutants when cells are incubated on lactose-arabinose minimal plates . Similar results are reported in the accompanying paper by Maenhaut-Michel and Shapiro, who used indirect selection to isolate Ara-Lac+ cells in the absence of selection . However, their sequencing data have introduced a new unexpected complication to the interpretation of all such experiments, and it is no longer clear exactly when the fusions arise.

J Virol, 1994 Nov, 68(11), 7244 - 52
Characterization and molecular basis of heterogeneity of the African swine fever virus envelope protein p54; Rodriguez F et al.; It has been reported that the propagation of African swine fever virus (ASFV) in cell culture generates viral subpopulations differing in protein p54 (C . Alcaraz, A . Brun, F . Ruiz-Gonzalvo, and J . M . Escribano, Virus Res . 23:173-182, 1992) . A recombinant bacteriophage expressing a 328-bp fragment of the p54 gene was selected in a lambda phage expression library of ASFV genomic fragments by immunoscreening with antibodies against p54 protein . The sequence of this recombinant phage allowed the location of the p54 gene in the EcoRI E fragment of the ASFV genome . Nucleotide sequence obtained from this fragment revealed an open reading frame encoding a protein of 183 amino acids with a calculated molecular weight of 19,861 . This protein contains a transmembrane domain and a Gly-Gly-X motif, a recognition sequence for protein processing of several ASFV structural proteins . In addition, two direct tandem repetitions were also found within this open reading frame . Further characterization of the transcription and gene product revealed that the p54 gene is translated from a late mRNA and the protein is incorporated to the external membrane of the virus particle . A comparison of the nucleotide sequence of the p54 gene carried by two virulent ASFV strains (E70 and E75) with that obtained from virus Ba71V showed 100% similarity . However, when p54 genes from viral clones generated by cell culture passage and coding for p54 proteins with different electrophoretic mobility were sequenced, they showed changes in the number of copies of a 12-nucleotide sequence repeat . These changes produce alterations in the number of copies of the amino acid sequence Pro-Ala-Ala-Ala present in p54, resulting in stepwise modifications in the molecular weight of the protein . These duplications and deletions of a tandem repeat sequence array within a protein coding region constitute a novel mechanism of genetic diversification in ASFV.

Curr Biol, 1994 Nov 1, 4(11), 1026 - 9
Phage tailspike protein . A fishy tale of protein folding; Goldenberg DP et al.; The crystal structure of bacteriophage P22 tailspike protein reveals a striking fold with a distinctive, fish-like appearance, and helps explain many of the properties of this unusual molecule and its folding pathway.

Genetics, 1994 Nov, 138(3), 553 - 64
Rates of spontaneous mutation in bacteriophage T4 are independent of host fidelity determinants; Santos ME et al.; Bacteriophage T4 encodes most of the genes whose products are required for its DNA metabolism, and host (Escherichia coli) genes can only infrequently complement mutationally inactivated T4 genes . We screened the following host mutator mutations for effects on spontaneous mutation rates in T4: mutT (destruction of aberrant dGTPs), polA, polB and polC (DNA polymerases), dnaQ (exonucleolytic proofreading), mutH, mutS, mutL and uvrD (methyl-directed DNA mismatch repair), mutM and mutY (excision repair of oxygen-damaged DNA), mutA (function unknown), and topB and osmZ (affecting DNA topology) . None increased T4 spontaneous mutation rates within a resolving power of about twofold (nor did optA, which is not a mutator but overexpresses a host dGTPase) . Previous screens in T4 have revealed strong mutator mutations only in the gene encoding the viral DNA polymerase and proofreading 3'-exonuclease, plus weak mutators in several polymerase accessory proteins or determinants of dNTP pool sizes . T4 maintains a spontaneous mutation rate per base pair about 30-fold greater than that of its host . Thus, the joint high fidelity of insertion by T4 DNA polymerase and proofreading by its associated 3'-exonuclease appear to determine the T4 spontaneous mutation rate, whereas the host requires numerous additional systems to achieve high replication fidelity.

Lijec Vjesn, 1994 Nov-Dec, 116(11-12), 315 - 8
{50 years of molecular biology}; Trgovcevic Z; In 1865, Johann Gregor Mendel laid the mathematical foundation of the science of genetics . His "elements of heredity" (later called genes) were postulated as pure algebraic units . At about the same time (1868), Friedrich Miescher extracted a gelationous substance from the nuclei of white blood cells found in pus . This substance was called nuclein; later it became known as nucleic acid . Fifty years ago, Oswald T . Avery and his colleagues showed that one type of nucleic acid--DNA mediates genetic transformation in pneumococci . This was the first demonstration that Miescher's nuclein is the repository of Mendel's hypothetical elements of heredity . The genes thus "materialized" . Although not recognized by contemporaries, Avery's discovery may be considered as a landmark in the history of biology . The molecular era of genetics and biology has begun . Other events associated with the beginning of this new era (bacteriophage research, work on nutritional mutants in Neurospora) are also described in the present review.

Zentralbl Bakteriol, 1994 Nov, 281(4), 406 - 14
Bacteriophages specifically recognizing the lipopolysaccharide antigens O4, O5, O6, and O7 of Escherichia coli; Nimmich W et al.; Four bacteriophages recognizing the Escherichia coli lipopolysaccharide (LPS) antigens O4, O5, O6, and O7, respectively, were isolated from pooled sewage samples . Electron microscopic investigations revealed icosahedral phage structures . Phages phi O4, phi O5, and phi O7 belonged to Bradley's morphology group C, while phi O6 had a tail and resembles phages of group A of Bradley . The nucleic acid of the phages was identified as double-stranded DNA of different genomic sizes . Host range studies showed that only E . coli strains with homologous O antigens were attacked . No lysis of encapsulated and rough E . coli strains was observed . The phages specifically depolymerized the homologous LPS of their host strains; they may be useful for detecting respective non-capsulated E . coli strains in epidemiological studies as simple alternative to the laborious serological typing . Diagnostic application is restricted, however, as strains carrying K antigens have not been detected . The high specificity of the phage-associated enzymes provides a mild method for the preparation of oligosaccharides from the LPS for structural studies.

Mol Biol (Mosk), 1994 Nov-Dec, 28(6), 1321 - 9
{Features of the structure of transfer RNA coded by bacteriophage T5}; Shliapnikov MG et al.; Nucleotide sequence of 24 genes for the bacteriophage T5 tRNAs specific for all amino acids involved in protein synthesis was determined . All of them, except tRNA(Pro), were shown to differ significantly from the generalized "clover-leaf" structure consisting in the displacement of invariant and semi-invariant residues, the absence of pairing in the stems and some other deviations from the canonical parameters of the model . Basing on the available information on the functional activity of the phage-specific tRNAs, one can put forward a suggestion that, at least for some of them, the above anomalies do not essentially influence their activity . A comparison of phage T5 tRNAs with the Escherichia coli and phage T4 counterparts was carried out . The majority of the known elements determining the specificity of E . coli tRNA aminoacylation was also found in all phage T5 tRNAs, except tRNAPhe).

J Biol Chem, 1994 Oct 28, 269(43), 26655 - 62
The bacteriophage T4 regB ribonuclease . Stimulation of the purified enzyme by ribosomal protein S1; Ruckman J et al.; Infection of Escherichia coli by bacteriophage T4 induces a mRNA ribonuclease activity that shows specificity for cleavage within the sequence GGAG . Substrates of the activity in vivo include a number of phage mRNAs that are cleaved at GGAGs within the Shine/Dalgarno domains of their translation initiation regions . Induction of the ribonuclease depends on the product of the T4 gene regB . We describe here the overproduction and extensive purification of the RegB protein . RegB precisely co-purifies with an activity that cleaves within the sequence GGAG in oligonucleotide and polynucleotide RNAs and is therefore likely to constitute the sequence-specific catalytic component of the observed activity . We further report that the low cleavage rate observed with our preparations of purified RegB is substantially increased (1-2 orders of magnitude) by the addition of E . coli ribosomal protein S1 . We discuss the implications of this observation for the mechanism of action of the RegB ribonuclease in vitro and in vivo.

J Mol Biol, 1994 Oct 21, 243(2), 268 - 82
Faithful cleavage of the P1 packaging site (pac) requires two phage proteins, PacA and PacB, and two Escherichia coli proteins, IHF and HU; Skorupski K et al.; The PacA and PacB subunits of the bacteriophage P1 DNA packaging enzyme (pacase) are necessary for cleavage of the phage packaging site (pac) . In the accompanying paper, we show that the PacA subunit of the enzyme specifically binds to pac in the absence of PacB, but requires factors present in an Escherichia coli extract to do so . We show here that either of two E . coli DNA binding proteins, integration host factor (IHF) or HU, can replace this extract and promote the binding of PacA to pac . IHF binds to pac independently of PacA and DNase I footprinting experiments show that IHF protects approximately 40 bp of DNA around an IHF consensus sequence adjacent to the cleavage site . DNase I footprinting experiments also show that in the presence of either IHF or HU, PacA binds to the hexanucleotide sequences (5'-TGATCA/G) that flank the cleavage site and that have been previously shown to be essential for pac cleavage . The importance of IHF and HU in pac cleavage is further demonstrated by the severe reduction in both the fidelity and efficiency of pac cleavage in vitro with extracts lacking both IHF and HU . Addition of either IHF or HU to the deficient extracts renders them fully proficient for pac cleavage . Finally, we show that IHF bends DNA at the IHF site within pac . Based on these results, we propose a model that can account for the role of the various phage and host proteins, and for DNA bending in the pac cleavage reaction.

J Mol Biol, 1994 Oct 21, 243(2), 258 - 67
Purification and DNA-binding activity of the PacA subunit of the bacteriophage P1 pacase enzyme; Skorupski K et al.; The bacteriophage P1 packaging site (pac) cleavage enzyme (pacase) consists of two phage encoded proteins, PacA and PacB . Both proteins are necessary for the recognition and cleavage of pac and for subsequent packaging of cleaved DNA into phage particles . We have purified PacA to homogeneity from a bacterial strain that overproduces the protein . Purified PacA complements an Escherichia coli extract containing the PacB protein for DNA cleavage at the pac site and recognizes and binds to methylated pac DNA independently of PacB in gel retardation experiments . The latter property of PacA is absolutely dependent on the presence of a wildtype E . coli extract, suggesting that E . coli host proteins play a role in the pac cleavage reaction.

J Biol Chem, 1994 Oct 21, 269(42), 26234 - 8
Mutational analysis of a Ser/Thr phosphatase . Identification of residues important in phosphoesterase substrate binding and catalysis; Zhuo S et al.; The Ser/Thr phosphoprotein phosphatases (PPases) display similarities in amino acid sequence and biochemical properties . Most members of this family require transition metal ions for activity . The smallest family member, the bacteriophage lambda PPase (lambda-PPase), has been successfully overexpressed in Escherichia coli, purified, and characterized (Zhuo, S., Clemens, J.C., Hakes, D.J., Barford, D., and Dixon, J . E . (1993) J . Biol . Chem . 268, 17754-17761) . Site-directed mutagenesis has now been employed to define amino acid residues in lambda-PPase required for metal ion binding and catalysis . Conservative amino acid substitutions at residues Asp20, His22, Asp49, His76, and Glu77 affected lambda-PPase catalysis and metal ion binding, whereas substitutions at residues Arg53 and Arg73 affected catalysis and substrate binding . Each of these residues is invariant in all phosphoprotein phosphatases, suggesting that these residues may play important roles in binding and catalysis in all of the PPases . Computer-assisted sequence alignment further revealed that lambda-PPase residues Asp20, His22, Asp49, His76, Arg53, and Arg73 lie within three larger regions of PPase sequence identity with the consensus sequence (DXH-(approximately 25)-GDXXD-(approximately 25)-GNHD/E) . This motif can be found in a wide variety of phosphoesterases unrelated to the PPases and defines structural and catalytic features utilized by a diverse group of enzymes for the hydrolysis of phosphate esters.

J Mol Biol, 1994 Oct 21, 243(2), 167 - 72
Structural mimicry and enhanced immunogenicity of peptide epitopes displayed on filamentous bacteriophage . The V3 loop of HIV-1 gp120; di Marzo Veronese F et al.; The principal neutralizing determinant of the human immunodeficiency virus type 1 (HIV-1) is an intra-chain disulphide-bridged loop, designated V3, in the third hypervariable region of the surface glycoprotein gp120 . Peptide sequences from the V3 loop of gp120 from HIV-1 strain MN (HIV-1MN) were engineered into the N-terminal region of the major coat protein of filamentous bacteriophage fd, leading to their display in multiple copies on the surface of the bacteriophage virion . Peptides displayed in this way were shown to be remarkably effective structural mimics of the natural epitope . They were recognised by human HIV antisera and evoked high titres of antibodies in mice, which cross-reacted with other strains of HIV and were capable of neutralizing the virus . In addition, antibody production could be stimulated by simultaneous inoculation with T-cell epitopes similarly displayed on filamentous bacteriophage . The bacteriophage display system offers a powerful means of studying the immunological recognition of proteins and is a promising vaccine model.

Biochim Biophys Acta, 1994 Oct 18, 1219(2), 267 - 76
Purification and characterization of PRD1 DNA polymerase; Zhu W et al.; A small lipid-containing bacteriophage PRD1 encodes a DNA polymerase that utilizes a protein primer for the initiation of DNA replication . The purification of the PRD1 DNA polymerase has been hampered by the insolubility of the overexpressed enzyme in Escherichia coli cells . We have developed a simple and rapid procedure for purification of the overexpressed PRD1 DNA polymerase . This method is based on guanidine hydrochloride denaturation and renaturation of the insoluble PRD1 DNA polymerase overexpressed in E . coli containing the recombinant plasmid pEJG . The purified DNA polymerase was extensively characterized and found to be indistinguishable from the normal soluble PRD1 DNA polymerase as judged by enzymatic properties . These properties include: protein-primed initiation of PRD1 DNA replication, strand-displacement DNA synthesis, DNA polymerase processivity, 3' to 5' exonuclease activity and filling-in repair type DNA synthesis . Furthermore, the kinetic parameters determined for dNTPs and primer-terminus were of the same order of magnitude . The availability of a simple purification procedure for the PRD1 DNA polymerase should permit detailed structure-function analysis of this enzyme.

Biochim Biophys Acta, 1994 Oct 18, 1219(2), 551 - 4
Tandem orientation of the inter-alpha-trypsin inhibitor heavy chain H1 and H3 genes; Diarra-Mehrpour M et al.; The inter-alpha-trypsin inhibitor H1 (ITIH1) and inter-alpha-trypsin inhibitor H3 (ITIH3) genes have both previously been mapped to chromosomes 3 and 14 in the human and mouse, respectively . We now present evidence that these genes are physically linked . By using cDNA probes, a recombinant DNA phage has been isolated from a bacteriophage DNA library, which contains sequences flanking the 5' end of the ITIH3 gene and the 3' end of the ITIH1 gene . Restriction endonuclease mapping, PCR analysis and DNA sequence determination of the recombinant phage and comparison to genomic DNA revealed that the genes are in tandem, 2721 base pairs apart, with the ITIH1 gene to the 5' side of the ITIH3 gene . Their respective transcriptional units are thus on the same strand of DNA and most probably arose in evolution as the consequence of a duplication of a common ancestral gene.

FEMS Microbiol Lett, 1994 Oct 15, 123(1-2), 19 - 26
Low-copy-number T7 vectors for selective gene expression and efficient protein overproduction in Escherichia coli; Dersch P et al.; A set of low-copy-number vectors (pPD) has been constructed that permit selective gene expression and high-level protein overproduction in Escherichia coli, based on the bacteriophage T7 RNA polymerase/T7 promoter system . These plasmids carry a chloramphenicol resistance gene (cat) as a selective marker and an extended multiple cloning site for convenient gene cloning . Their replication is mediated by ori sequences derived from the low-copy-number vector pSC101 . The efficient T7 gene 10 promoter present on these vectors allows selective and high-level transcription of cloned genes carrying their own translational initiation signals . In addition, low-copy-number T7 vectors were constructed that permit expression of genes lacking their own transcription and translation initiation elements by providing a ribosome binding site, an ATG start codon and a multiple cloning site devised for the cloning in all three reading frames . The pPD expression vectors were used to achieve high-level overproduction of the E . coli integral outer membrane protein Tsx, and the cytoplasmic enzymes beta-galactosidase (beta Gal) and UTP:alpha-D-glucose-1-phosphate uridylyltransferase (GalU) . The characteristics of these low-copy-number T7 expression vectors should prove very useful for the cloning and high-level overexpression of genes whose gene products are deleterious to the E . coli host.

Eur J Biochem, 1994 Oct 15, 225(2), 747 - 53
An intrinsic-tryptophan-fluorescence study of phage phi 29 connector/nucleic acid interactions; Urbaneja MA et al.; The protein p10 of bacteriophage phi 29 assembled into connectors exhibit an intrinsic fluorescence with an emission peak centered at 335 nm, which suggests a hydrophobic environment of the three tryptohan residues that the protein contains . Upon incubation with linear DNA (but not with circular DNA), a decrease in the connector intrinsic fluorescence is measured which does not show any sequence specificity . The decrease in fluorescence is not observed when DNA is incubated with proteolyzed connectors, which lack the DNA-binding domain, suggesting that the fluorescence quenching is related to the binding of DNA to the phi 29 connectors . Acrylamide quenching studies reveal a higher accessibility of tryptophan residues to the quencher when the connector is bound to DNA . Protein denaturation by guanidine hydrochloride occurs at lower denaturant concentrations in the presence of linear DNA (but not circular DNA) than in its absence, suggesting a conformational change of phi 29 connector upon binding to linear DNA . This hypothesis is supported by the fact that the proteolyzed connectors, which do not bind DNA, are denatured at the same denaturant concentration, regardless of the presence of DNA . phi 29 connectors also bind RNA, but this interaction does not exert any effect on acrylamide quenching or guanidine hydrochloride denaturation . This result, together with that showing that proteolyzed connectors are able to interact with RNA, reinforces the idea that phi 29 connectors have two independent domains for interaction with DNA and RNA.

Nature, 1994 Oct 13, 371(6498), 623 - 6
Crystal structure of an RNA bacteriophage coat protein-operator complex; Valegard K et al.; The RNA bacteriophage MS2 is a convenient model system for the study of protein-RNA interactions . The MS2 coat protein achieves control of two distinct processes--sequence-specific RNA encapsidation and repression of replicase translation--by binding to an RNA stem-loop structure of 19 nucleotides containing the initiation codon of the replicase gene . The binding of a coat protein dimer to this hairpin shuts off synthesis of the viral replicase, switching the viral replication cycle to virion assembly rather than continued replication . The operator fragment alone can trigger self-assembly of the phage capsid at low protein concentrations and a complex of about 90 RNA operator fragments per protein capsid has been described . We report here the crystal structure at 3.0 A resolution of a complex between recombinant MS2 capsids and the 19-nucleotide RNA fragment . It is the first example of a structure at this resolution for a sequence-specific protein-RNA complex apart from the transfer RNA synthetase complexes . The structure shows sequence-specific interactions between conserved residues on the protein and RNA bases essential for binding.

Gene, 1994 Oct 11, 148(1), 75 - 80
A novel Tn10 tetracycline regulon system controlling expression of the bacteriophage T3 gene encoding S-adenosyl-L-methionine hydrolase; Collier GB et al.; To study the effects of in vivo DNA methylation, we have developed an inducible system to control the intracellular concentration of S-adenosyl-L-methionine (AdoMet) . The product of the bacteriophage T3 AdoMet hydrolase-encoding gene (amh), which degrades AdoMet to L-homoserine and 5'-methylthioadenosine, was employed to lower AdoMet concentrations in vivo . The amh gene was placed downstream from the inducible tetA promoter of the Tn10 tetracycline regulon substituting for most of the tetA gene . Unlike in the original isolates {Hughes et al., J . Bacteriol . 169 (1987) 3625-2632}, this promoter allows controlled expression . These constructs are stable and can be induced in a dose-dependent manner . The system is maximally induced 2-3 h after addition of the inducer, autoclaved chlortetracycline (cTc) . DNA methylation in vivo was assessed in this model system by BamHI cleavage of plasmid DNA isolated from cells cotransformed by two compatible plasmids, one carrying the inducible amh gene, the other M.BamHII methyltransferase encoding gene . The induction of amh decreased the intracellular pool of AdoMet which M.BamHII requires as a cofactor . Under these conditions, there is a decrease in DNA methylation . The unmethylated DNA is assayed by BamHI cleavage . This system will be useful for studying transcription, DNA replication, gene repair and other cellular phenomena affected by methylation.

J Biol Chem, 1994 Oct 7, 269(40), 25129 - 36
The thumb subdomain of T7 RNA polymerase functions to stabilize the ternary complex during processive transcription; Bonner G et al.; To examine the function of the thumb subdomain in bacteriophage T7 RNA polymerase we constructed a set of deletion mutants within this subdomain . These mutants exhibited reduced processivity during the processive, but not the abortive, stage of transcription . Reduced processivity was found to be due primarily to an increase in the processive ternary complex dissociation rate (destabilization of the processive ternary complex) . The destabilization of the ternary complex does not appear to be due to a decrease in the affinity of the polymerase for the nascent RNA . These observations support the proposal that the thumb subdomain functions to stabilize the processive ternary complex during the processive (but not the abortive) stage of transcription, probably by wrapping around the template to prevent polymerase dissociation.

Biochemistry, 1994 Oct 4, 33(39), 11980 - 6
Continuous in vitro evolution of bacteriophage RNA polymerase promoters; Breaker RR et al.; Rapid in vitro evolution of bacteriophage T7, T3, and SP6 RNA polymerase promoters was achieved by a method that allows continuous enrichment of DNAs that contain functional promoter elements . This method exploits the ability of a special class of nucleic acid molecules to replicate continuously in the presence of both a reverse transcriptase and a DNA-dependent RNA polymerase . Replication involves the synthesis of both RNA and cDNA intermediates . The cDNA strand contains an embedded promoter sequence, which becomes converted to a functional double-stranded promoter element, leading to the production of RNA transcripts . Synthetic cDNAs, including those that contain randomized promoter sequences, can be used to initiate the amplification cycle . However, only those cDNAs that contain functional promoter sequences are able to produce RNA transcripts . Furthermore, each RNA transcript encodes the RNA polymerase promoter sequence that was responsible for initiation of its own transcription . Thus, the population of amplifying molecules quickly becomes enriched for those templates that encode functional promoters . Optimal promoter sequences for phage T7, T3, and SP6 RNA polymerase were identified after a 2-h amplification reaction, initiated in each case with a pool of synthetic cDNAs encoding greater than 10(10) promoter sequence variants.

Virology, 1994 Oct, 204(1), 251 - 3
Reconstitution of active replicase in procapsids of the segmented dsRNA bacteriophage phi 6; Casini G et al.; Bacteriophage phi 6 has a genome of three segments of double-stranded RNA enclosed in a procapsid composed of four different proteins . The preformed procapsid is capable of packaging plus strand transcripts of the genomic segments in an in vitro reaction . The plus strands then serve as templates for in vitro minus strand synthesis . Procapsids that are missing protein P2 are incapable of minus strand synthesis . In this report we show that incubation with a cell extract containing P2 results in particles with normal amounts of attached P2 and with packaging and replicase activity . Particles lacking P7 have reduced replicase activity which can be augmented by incubation with extracts containing P7, but the amount of attached P7 is small.

J Virol, 1994 Oct, 68(10), 6161 - 9
Localization of the Vpx packaging signal within the C terminus of the human immunodeficiency virus type 2 Gag precursor protein; Wu X et al.; Viral protein X (Vpx) is a human immunodeficiency virus type 2 (HIV-2) and simian immunodeficiency virus accessory protein that is packaged into virions in molar amounts equivalent to Gag proteins . To delineate the processes of virus assembly that mediate Vpx packaging, we used a recombinant vaccinia virus-T7 RNA polymerase system to facilitate Gag protein expression, particle assembly, and extracellular release . HIV genes were placed under control of the bacteriophage T7 promoter and transfected into HeLa cells expressing T7 RNA polymerase . Western immunoblot analysis detected p55gag and its cleavage products p39 and p27 in purified particles derived by expression of gag and gag-pol, respectively . In trans expression of vpx with either HIV-2 gag or gag-pol gave rise to virus-like particles that contained Vpx in amounts similar to that detected in HIV-2 virus produced from productively infected T cells . Using C-terminal deletion and truncation mutants of HIV-2 Gag, we mapped the p15 coding sequence for determinants of Vpx packaging . This analysis revealed a region (residues 439 to 497) downstream of the nucleocapsid protein (NC) required for incorporation of Vpx into virions . HIV-1/HIV-2 gag chimeras were constructed to further characterize the requirements for incorporation of Vpx into virions . Chimeric HIV-1/HIV-2 Gag particles consisting of HIV-1 p17 and p24 fused in frame at the C terminus with HIV-2 p15 effectively incorporate Vpx, while chimeric HIV-2/HIV-1 Gag particles consisting of HIV-2 p17 and p27 fused in frame at the C terminus with HIV-1 p15 do not . Expression of a 68-amino-acid sequence of HIV-2 containing residues 439 to 497 fused to the coding regions of HIV-1 p17 and p24 also produced virus-like particles capable of packaging Vpx in amounts similar to that of full-length HIV-2 Gag . Sucrose gradient analysis confirmed particle association of Vpx and Gag proteins . These results demonstrate that the HIV-2 Gag precursor (p55) regulates incorporation of Vpx into virions and indicates that the packaging signal is located within residues 439 to 497.

Carcinogenesis, 1994 Oct, 15(10), 2331 - 40
Translesional synthesis on DNA templates containing site-specifically placed deoxyadenosine and deoxyguanosine adducts formed by the plant carcinogen aristolochic acid; Broschard TH et al.; Synthetic oligonucleotides (18-mers) containing either a single deoxyadenosine residue or a single deoxyguanosine residue were treated with aristolochic acid I (AAI) or aristolochic acid II (AAII), the main components of the plant carcinogen aristolochic acid (AA) . These reactions resulted in the formation of site-specifically adducted oligonucleotides containing the two known AAI-DNA adducts (dA-AAI, dG-AAI) or the two known AAII-DNA adducts (dA-AAII, dG-AAII) at position 15 from the 3' end . Using HPLC chromatography, the oligonucleotides were purified and subsequently shown to contain the adducts of interest by 32P-postlabelling . The adducted oligonucleotides were used as templates in primer (11-mer) extension reactions catalysed by modified bacteriophage T7 DNA polymerase (Sequenase) . Regardless of the type of DNA adduct examined, DNA synthesis was blocked predominantly (80-90%) at the nucleotide 3' to each adduct, although primer extension to the full length of the template was noted with unmodified control templates . However, 15 nucleotide products, indicating blocking of DNA synthesis after incorporation of a nucleotide opposite the adduct and translesional synthesis products were formed in all cases in different amounts, depending on the adduct structure . When a 14-mer primer together with high dNTP concentrations was used to examine nucleotide incorporation directly across from the four different purine adducts we found that the deoxyadenosine adducts (dA-AAI and dA-AAII) allowed incorporation of dAMP and dTMP equally well, whereas the deoxyguanosine adducts (dG-AAI and dG-AAII) allowed preferential incorporation of dCMP . Molecular dynamic simulations showed that the aristolactam moiety of all adducts exhibit a strong stacking, with the adenine residue at the 3' end of the 14-mer primer . These studies demonstrate that all AA purine adducts provide severe blocks to DNA replication and that the guanine adducts may not be very efficient mutagenic lesions . In contrast, the translesional bypass past adenine adducts of the aristolochic acids suggests a mutagenic potential resulting from dAMP incorporation by polymerase . AT-->TA transversion mutations would be the mutagenic consequences of AA adenine adducts, which are consistent with the activating mutations of c-ras genes found in AA-induced tumours of rodents.

Am J Trop Med Hyg, 1994 Oct, 51(4), 495 - 500
Cloning and characterization of a repetitive DNA sequence specific for Wuchereria bancrofti; Siridewa K et al.; A genomic library constructed in a bacteriophage lambda replacement vector (EMBL3) with Wuchereria bancrofti DNA partially digested with Sau 3A I was screened with 32P-labeled W . bancrofti total DNA, and a strongly reactive recombinant, EMBL3Wb34, was isolated . This clone contained an approximately 16-kb insert that showed some cross-hybridization with Brugia malayi and B . pahangi DNA . However, a 969-bp subclone from EMBL3Wb34, designated pWb12, hybridized only with W . bancrofti DNA and was able to detect as little as 300 pg . Furthermore, pWb12 could detect DNA from a single infective larva or one microfilaria . It has a moderate copy number (450-700) and appears to be interspersed within the parasite genome . The nucleotide sequence contains 66% A+T and 34% G+C and shows no notable internal repeats.

J Bacteriol, 1994 Oct, 176(19), 6059 - 65
A new gene of bacteriophage P4 that controls DNA replication; Terzano S et al.; Bacteriophage P4 replication may result in either a lytic cycle or plasmid maintenance, depending on the presence or absence, respectively, of helper phase P2 genome . Bacteriophage P4 DNA replication depends on the product of gene alpha, which has origin recognition, primase, and helicase activities . An open reading frame with the coding capacity for a protein of 106 amino acids (orf106) is located upstream of the alpha gene . Genes orf106 and alpha are transcriptionally coregulated . Three amber mutations and an internal deletion (del51) were introduced into orf106 . All of the amber mutations exhibited a polar effect on transcription of the downstream alpha gene . The P4 del51 mutant was slightly defective in lytic growth and could not be propagated in the plasmid state . In this latter condition, P4 DNA overreplication was observed . Overexpression of Orf106 severely inhibited P4 DNA replication, preventing P4 lytic growth and plasmid maintenance . The inhibitory effect of Orf106 on P4 replication was not observed when both orf106 and alpha were overexpressed . We suggest that orf106 is involved in P4 replication and that a balanced expression of orf106 relative to alpha may be necessary for proper P4 DNA replication . In particular, orf106 appears to be essential for the control of P4 genome replication in the plasmid state . We propose that orf106 be named cnr, for copy number regulation.

J Bacteriol, 1994 Oct, 176(19), 5904 - 11
Deletion between direct repeats in T7 DNA stimulated by double-strand breaks; Kong D et al.; An in vitro system based on extracts of Escherichia coli infected with bacteriophage T7 was used to study genetic deletions between directly repeated sequences . The frequency of deletion was highest under conditions in which the DNA was actively replicating . Deletion frequency increased markedly with the length of the direct repeat both in vitro and in vivo . When a T7 gene was interrupted by 93 bp of nonsense sequence flanked by 20-bp direct repeats, the region between the repeats was deleted in about 1 out of every 1,600 genomes during each round of replication . Very similar values were found for deletion frequency in vivo and in vitro . The deletion frequency was essentially unaffected by a recA mutation in the host . When a double-strand break was placed between the repeats, repair of this strand break was often accompanied by the deletion of the DNA between the direct repeats, suggesting that break rejoining could contribute to deletion during in vitro DNA replication.

Environ Health Perspect, 1994 Oct, 102 Suppl 6, 217 - 20
Modification of plasmid and bacteriophage DNA by aromatic amines: effects on survival, template activity, and mutagenicity; King CM et al.; The carcinogenic and mutagenic effects of the aromatic amines are believed to depend on their covalent modification of DNA, primarily through the formation of adducts at C8 of guanine . The actual biologic and biochemical responses to these adducts can be envisioned as the consequence of the abilities of the cell to repair the lesions, with or without fidelity, and the introduction of errors through bypass of the adducts by polymerases . A key question is whether changes in DNA sequence arise through the participation of common repair processes that cause mutations independent of adduct structure . Alternatively, do mutations arise through miscoding during polymerase bypass at the site of the adducts and are, therefore, more likely to produce sequence changes that are more characteristic of adduct structure? This question has been approached using single, site-specific, or randomly introduced aromatic amine DNA adducts in bacterial cells, and in vitro studies with DNA polymerases that employ site-specifically modified templates . The results of both approaches demonstrate that these adducts are distinguished readily by virtue of their structures, thus supporting the conclusion that mutagenic effects of the aromatic amines arise from their structures rather than from their triggering a common inaccurate repair response.

Anal Biochem, 1994 Oct, 222(1), 163 - 7
Inhibition of in vivo transcription by actinomycin D treatment of ethylenediaminetetraacetic acid-permeabilized Escherichia coli: effects on bacteriophage proliferation; Fastame IG et al.; Rifampicin, which is able to block DNA-dependent RNA synthesis, has been widely used to selectively inhibit host protein synthesis in RNA bacteriophage-infected Escherichia coli without affecting the viral specific protein synthesis . However, in many cases it is necessary to increase rifampicin levels to 200 micrograms/ml in order to obtain an almost complete suppression of bacterial protein synthesis, and these high antibiotic concentrations cause at the same time a strong inhibition of phage proliferation resulting in a 50- to 100-fold reduction of phage yields . We have partially avoided this difficulty by using actinomycin D after permeabilization of bacteria by a brief incubation with EDTA . To optimize the method the effects of changing EDTA and actinomycin concentrations as well as the duration of the permeabilization period have been studied . With this procedure it has been possible to shut off bacterial RNA and protein synthesis with phage yields about 10 times higher than those observed in the presence of high levels of rifampicin . The usefulness of the described method is particularly evident when working with rifampicin-resistant strains of E . coli.

Biochem Mol Biol Int, 1994 Oct, 34(3), 429 - 35
Degradation of bacteriophage lambda deoxyribonucleic acid in vitro by sulfur mustard; Venkateswaran N et al.; The degradation of bacteriophage lambda (lambda) deoxyribonucleic acid (DNA) by interaction with 0.1, 0.5 and 1 mM concentrations of sulfur mustard (SM) was investigated using agarose gel electrophoresis . Alkaline agarose gel electrophoresis also revealed single strand breaks at 0.5 and 0.1 mM concentrations of SM . The presence of magnesium ions in the reaction mixture prevented DNA degradation . It is proposed that the degradation of lambda DNA by its interaction with an excess of SM may be caused by the breakage of phosphodiester backbone of DNA via the formation of an intermediate phosphotriester bond.

Mol Microbiol, 1994 Oct, 14(2), 291 - 300
Analysis of the rnc locus of Coxiella burnetii; Zuber M et al.; A 3.2 kb EcoRI genomic DNA fragment of Coxiella burnetii was isolated by virtue of its ability to suppress mucoidy in Escherichia coli . Nucleotide sequence analysis revealed the presence of the genes homologous to rnc, era and recO of E . coli . Suppression of capsule synthesis, measured by beta-galactosidase expression in lon- cps-lac fusion strains of E . coli, is caused by gene-dosage effects of the plasmid-borne rnc genes of either C . burnetii or E . coli . The rnc gene of C . burnetii complemented rnc- E . coli hosts for lambda plaque morphology and stimulation of lambda N gene expression . We also demonstrated heterologous complementation of an E . coli strain defective for the expression of Era, an essential protein in E . coli, using the plasmid-borne C . burnetii era . Under the control of the bacteriophage lambda PL promoter, this 3.2 kb EcoRI DNA fragment directed the synthesis in E . coli of three proteins with approximate molecular masses of 35, 27 and 25 kDa . Antibodies against purified E . coli Era protein cross-reacted with the 35 kDa protein of C . burnetii on Western blots.

Genetics, 1994 Oct, 138(2), 247 - 52
Involvement of a replicative DNA helicase of bacteriophage T4 in DNA recombination; Yonesaki T; Bacteriophage T4 gene 41 encodes a replicative DNA helicase that is a subunit of the primosome which is essential for lagging-strand DNA synthesis . A mutation, rrh, was generated and selected in the helicase gene on the basis of limited DNA replication that ceases early . The survival of ultraviolet-irradiated phage and the frequency of genetic recombination are reduced by rrh . In addition, rrh diminishes the production of concatemeric DNA . These results strongly suggest that the gene 41 replicative helicase participates in DNA recombination.

Protein Expr Purif, 1994 Oct, 5(5), 476 - 85
Importance of codon preference for production of human RAP74 and reconstitution of the RAP30/74 complex; Wang BQ et al.; RAP30 and RAP74 are subunits of RAP30/74 (TFIIF, beta gamma), a general initiation and elongation factor for transcription by RNA polymerase II . Methods were previously published for production of human RAP30 and RAP74 in bacterial cells, using a bacteriophage T7 promoter expression system . The vectors described for production of RAP74 were not very efficient and produced significant quantities of RAP74 amino terminal fragments . To improve these vectors, a segment of the human RAP74 cDNA was recoded using a preferred set of codons for translation in Escherichia coli . Recoding dramatically improved protein production and suppressed production of amino-terminal fragments . Improved vectors are reported that produce RAP74 with an LEHHHHHH carboxy-terminal extension (RAP74-H6), for purification on a Ni(2+)-affinity column, and also with the native carboxy terminus (RAP74) . Methods for purification of RAP74-H6 and RAP74 are reported . Using these improved vectors, approximately 30 mg of soluble and active RAP74-H6 or RAP74 can be produced and purified from 1 liter of E . coli culture, representing a 10-fold improvement in protein production . Methods have also been developed for reconstitution of native RAP30/74 complex using recombinant proteins . This complex has indistinguishable activity from human RAP30/74 for accurate transcription in vitro.

Nippon Jinzo Gakkai Shi, 1994 Oct, 36(10), 1103 - 12
{Measurement of antibodies to double-stranded and single-stranded DNA using bacteriophage lambda DNA as the antigen--avidity of antibodies to DNA in patients with lupus nephritis}; Saito M et al.; IgG-class antibodies to double-stranded (ds) and single-stranded (ss) DNA in patients with collagen disease were measured by enzyme-linked immunosorbent assay (ELISA) using bacteriophage lambda DNA as the antigen . Moreover, avidity of IgG-class antibodies to both dsDNA and ssDNA in patients with systemic lupus erythematosus (SLE) was evaluated by ELISA as salt-dependent binding activities . The results are as follows: 1) Elevated IgG-class antibodies to dsDNA were largely restricted to patients with active SLE . Furthermore, the levels of IgG-class antibodies to dsDNA correlated with the disease activity of SLE . 2) Lupus nephritis (LN) patients with nephrotic syndrome (NS) or diffuse proliferative LN as determined by renal biopsy had high levels of IgG-class antibodies to both dsDNA and ssDNA . 3) LN patients with NS showed the highest means of avidity index of IgG-class antibodies to both dsDNA and ssDNA . These findings suggest that the present method of measuring IgG-class antibodies to dsDNA is useful for the diagnosis and management of patients with active SLE and that IgG-class antibodies to both dsDNA and ssDNA with high avidity may be an important factor in the pathogenesis of LN with NS.

J Biomol Struct Dyn, 1994 Oct, 12(2), 457 - 74
Investigation of domain motions in bacteriophage T4 lysozyme; Arnold GE et al.; Hinge-bending in T4 lysozyme has been inferred from single amino acid mutant crystalline allomorphs by Matthews and coworkers . This raises an important question: are the different conformers in the unit cell artifacts of crystal packing forces, or do they represent different solution state structures? The objective of this theoretical study is to determine whether domain motions and hinge-bending could be simulated in T4 lysozyme using molecular dynamics . An analysis of a 400 ps molecular dynamics simulation of the 164 amino acid enzyme T4 lysozyme is presented . Molecular dynamics calculations were computed using the Discover software package (Biosym Technologies) . All hydrogen atoms were modeled explicitly with the inclusion of all 152 crystallographic waters at a temperature of 300 K . The native T4 lysozyme molecular dynamics simulation demonstrated hinge-bending in the protein . Relative domain motions between the N-terminal and C-terminal domains were evident . The enzyme hinge bending sites resulted from small changes in backbone atom conformations over several residues rather than rotation about a single bound . Two hinge foci were found in the simulation . One locus comprises residues 8-14 near the C-terminal of the A helix; the other site, residues 77-83 near the C-terminal of the C helix . Comparison of several snapshot structures from the dynamics trajectory clearly illustrates domain motions between the two lysozyme lobes . Time correlated atomic motions in the protein were analyzed using a dynamical cross-correlation map . We found a high degree of correlated atomic motions in each of the domains and, to a lesser extent, anticorrelated motions between the two domains . We also found that the hairpin loop in the N-terminal lobe (residues 19-24) acted as a mobile 'flap' and exhibited highly correlated dynamic motions across the cleft of the active site, especially with residue 142.

Farmaco, 1994 Oct, 49(10), 607 - 14
Pyrroloquinolinone methylderivatives, furocoumarin analogues: synthesis and biological activity; Rodighiero P et al.; Searching new photochemotherapeutic agents, a series of methylpirroloquinolinones were prepared by a new synthetic pathway, thus univocally obtaining the title compounds . The photobiological activity of some of these compounds was assayed; upon UVA activation, a marked capacity of inhibiting macromolecular synthesis in Ehrlich cells was observed, which appeared to be markedly high testing protein synthesis . Pyrroloquinolinones induced a strong inhibition of the clonal growing capacity of HeLa cells cultivated in vitro . Studying DNA photodamage in HL60 cells high amounts of single strand breaks and DNA-protein cross-links were detected . Pyrroloquinolines inhibited T2 bacteriophage infectivity, but induced no significant amounts of revertants in E . coli WP2 TM9, a strain very sensitive to DNA damage . On the contrary, 8-MOP, tested in the same experimental conditions exhibited an evident photomutagenecity . These data suggest that pyrroloquinolines induced antiproliferative effects by a mechanism in which DNA-photobinding practically does not takes place, and therefore different from that shown by known furocoumarins . Pyrroloquinolinones showed also a moderate antiproliferative activity in the dark.

J Infect Dis, 1994 Oct, 170(4), 867 - 72
A large, antigenically conserved protein on the surface of Moraxella catarrhalis is a target for protective antibodies; Helminen ME et al.; A monoclonal antibody (MAb) to Moraxella catarrhalis O35E bound to a surface-exposed epitope of a proteinaceous antigen of this organism . The antigen, designated UspA, was present in every strain of the pathogen tested in a colony blot RIA . UspA had a molecular mass on SDS-PAGE that varied between 300 and 400 kDa, depending on the individual M . catarrhalis strain . Passive immunization of mice with the UspA-reactive Mab enhanced pulmonary clearance of M . catarrhalis . Use of this Mab to screen a M . catarrhalis genomic DNA library permitted identification of a recombinant bacteriophage expressing the M . catarrhalis UspA protein . The recombinant UspA protein was used in Western blot analysis with sera from patients with M . catarrhalis pneumonia . Convalescent-phase sera but not acute-phase sera from these patients contained antibodies to this M . catarrhalis surface protein, indicating that M . catarrhalis strains growing in vivo express this molecule.

Biotechnology (N Y), 1994 Oct, 12(10), 999 - 1002
Clonal selection and amplification of phage displayed antibodies by linking antigen recognition and phage replication; Duenas M et al.; The immune response generates a tremendous array of antibody specificities by VDJ-gene rearrangements . A similar diversity can be obtained by expressing entire V-gene repertoires on the surface of filamentous bacteriophages creating large antibody libraries . Here we describe how the clonal selection mechanisms of the humoral immune response can also be mimicked in the phage display system by linking antigen-recognition and phage replication . We have achieved this by displaying antibody libraries on engineered, non-infectious phage with gene 3 deletions . Individual, antigen-specific phage are made replication competent by allowing a fusion protein, consisting of the antigen and phage coat protein 3, to bind the displayed antibody fragment . This fusion protein bridges the phage and F-pili of the bacteria and allows infection to be initiated and the phage to be clonally amplified with specific enrichment factors of approximately 10(10) after only two rounds.

J Mol Biol, 1994 Sep 30, 242(4), 547 - 58
Quantification of the effect of excluded volume on double-stranded DNA; Louie D et al.; By steric exclusion of volume, neutral polymers both condense and increase the effective concentration of DNA random coils . Neutral polymers also stimulate the in vitro packaging of DNA in the capsids of some double-stranded DNA bacteriophages . In the present study, the physical effects of neutral polymers on DNA random coils have been quantified by assaying the DNA products at equilibrium of the following two reactions of the 12 nucleotide single-stranded complementary (cohesive) ends of mature bacteriophage lambda DNA: bimolecular joining of half-molecules of mature lambda DNA, and cyclization of intact lambda DNA; cyclization is used as a probe for unimolecular DNA condensation . The smaller neutral molecules, including polyethylene glycol of molecular mass 200 Da (PEG200), shift both reactions towards dissociation; this shift is partially correlated with reduced water activity . The larger PEGs (molecular mass of 1540 or more) shift both reactions towards association . Water activity-corrected equilibrium constants for the larger PEGs are found to increase as a function of PEG concentration . Below 2% to 3% (w/v) PEG, these equilibrium constants are independent of PEG molecular mass; at higher PEG concentrations, these equilibrium constants increase as the molecular mass of the PEG increases . The following conclusions are drawn . (1) Volume exclusion among PEG molecules is the primary cause of the PEG molecular mass-dependence of excluded volume . (2) At the lower PEG concentrations, the PEG radius obtained by quantification of excluded volume is usually equal to the hydrodynamic PEG radius . (3) At any given PEG concentration, the PEG-DNA excluded volume is approximately the same for bimolecular DNA joining as it is for unimolecular DNA cyclization . (4) Polymer-induced stimulation of in vitro bacteriophage DNA packaging is derived primarily from alteration of water activity, not alteration of excluded volume.

J Mol Biol, 1994 Sep 30, 242(4), 470 - 86
Fibritin encoded by bacteriophage T4 gene wac has a parallel triple-stranded alpha-helical coiled-coil structure; Efimov VP et al.; The bacteriophage T4 late gene wac (whisker's antigen control) encodes a fibrous protein which forms a collar/whiskers complex . Whiskers function as a helper protein for the long tail fibres assembly and plays a role in regulating retraction of the long tail fibres in response to environmental conditions . In this work we show that expression of the cloned wac gene in Escherichia coli yields a protein oligomer of 53 nm length which we call fibritin, and which is able to complement gpwac T4 particles in vitro . CD spectroscopy of fibritin indicates a 90% alpha-helical content, and scanning calorimetry shows that the protein has several distinct domains . The analysis of the 486 amino acid sequence of fibritin reveals three structural components: a 408 amino acid region that contains 12 putative coiled-coil segments with a canonical heptad (a-b-c-d-e-f-g)n substructure where the "a" and "d" positions are preferentially occupied by apolar residues, and the N and C-terminal domains (47 and 29 amino acid residues, respectively) have no heptad substructure . The distribution of hydrophobic residues within heptads is more similar to a triple than to a double coiled-coil . The alpha-helical segments are separated by short "linker" regions, variable in length, that have a high proportion of glycine and proline residues . Each coiled-coil segment has, on the borders with linker regions, residues that are common to the N and C-terminal caps of the alpha-helices . Full-length and amino-terminally truncated fibritins can be reassembled in vitro after temperature-induced denaturation . Co-assembly of full-length fibritin and the N-terminal deletion mutant, as well as analytical centrifugation, indicates that the protein is a parallel triple-standard alpha-helical coiled-coil . Deletions of various N-terminal portions of fibritin did not block trimerisation but the mutant trimers are unable to bind to T4 particles . The last 18 C-terminal residues of fibritin are required for correct trimerisation of gpwac monomers in vivo . We propose that fibritin might serve as a convenient model for the investigation of folding and assembly mechanisms of alpha-fibrous proteins.

J Mol Biol, 1994 Sep 30, 242(4), 364 - 77
Isolation and characterization of an Escherichia coli DnaK mutant with impaired ATPase activity; Burkholder WF et al.; A temperature-sensitive mutant of DnaK, the principal Escherichia coli member of the 70 kDa heat shock protein family, has been isolated . The mutation, dnaK25, lies in the putative ATP binding pocket of DnaK . It consists of a C to T transition that changes the highly conserved proline 143 to serine . Mutant strains do not support the propagation of bacteriophage lambda or of plasmids that require DnaA for replication . They are also defective in the utilization of mannose and sorbitol . ATPase activity of the mutant protein is reduced 20-fold relative to wild-type, while autophosphorylation is unaffected . DnaK25 has a fourfold faster rate of nucleotide exchange than wild-type DnaK; nucleotide exchange by both proteins is markedly increased by GrpE . The DnaK25 ATPase is still stimulated by DnaJ and GrpE and by peptide substrates . However, the affinity of most peptides tested for stimulating the DnaK25 ATPase is reduced significantly . These results indicate that a mutation in the N-terminal nucleotide binding domain can alter substrate interactions with the C-terminal substrate binding site . Nucleotide exchange by both wild-type DnaK and DnaK25 proceeds at a much faster rate than ATP hydrolysis, and therefore cannot be the rate limiting step of ATP hydrolysis under the conditions used in these experiments . Consistent with this, peptides, which stimulate ATP hydrolysis, have no effect on nucleotide exchange . Peptides thus appear to stimulate the ATPase by acting at another step, such as increasing the rate of phosphate bond cleavage.

J Biol Chem, 1994 Sep 30, 269(39), 24221 - 8
The rapid dissociation of the T4 DNA polymerase holoenzyme when stopped by a DNA hairpin helix . A model for polymerase release following the termination of each Okazaki fragment; Hacker KJ et al.; We have examined the molecular mechanism that enables the T4 bacteriophage DNA polymerase holoenzyme to synthesize DNA processively on the leading strand of the replication fork for many minutes, while allowing an identical holoenzyme on the lagging strand to recycle from one Okazaki fragment to the next in less than 4 s . We use a perfect hairpin helix of 15 base pairs to mimic the encounter of the polymerase with the end of a previously synthesized Okazaki fragment . Polymerase dissociation is monitored during the stall at the hairpin helix by the addition of excess T4 gene 32 protein (SSB protein), which rapidly melts the helix and allows a stalled polymerase molecule to continue DNA synthesis . In the accompanying paper, we show that polymerase holoenzyme dissociation is slow (half-life of 2.5 min) when this enzyme is stalled by nucleotide omission (Hacker, K . J., and Alberts, B . M . (1994) J . Biol . Chem . 269, 24209-24220) . In contrast, the holoenzyme dissociates with a half-life of 1 s after hitting the hairpin helix, a rate sufficient to allow efficient polymerase recycling on the lagging strand in vivo . We conclude that, upon completing each Okazaki fragment, the holoenzyme senses an encounter with duplex DNA and then switches to a state that rapidly dissociates.

J Biol Chem, 1994 Sep 30, 269(39), 24034 - 9
Identification of a Src SH3 domain binding motif by screening a random phage display library; Cheadle C et al.; A phage display library was constructed in the filamentous bacteriophage fuse5 . The library was made by inserting a degenerate oligonucleotide which encodes 15 variable amino acids into the NH2-terminal region of the phage gene III protein . This library, containing over 10(7) different phage, was screened with a glutathione S-transferase (GST) fusion protein containing the Src homology 3 (Src SH3) domain and a protein kinase A phosphorylation site (GST/PKA/Src SH3) . A family of proline-rich sequences was isolated following four cycles of enrichment and amplification . Phage containing these sequences were shown to specifically bind to the GST/PKA/Src SH3 protein but not to GST/PKA only . A comparison of the inferred amino acid sequence of the different phage clones revealed a consensus sequence, RPLPXXP, which conforms to a Src SH3 domain binding motif identified independently during an affinity screen of a lambda-lox mouse embryo cDNA library using a 32P-labeled Src SH3 protein fragment as the probe (Y . Ivashchenko, manuscript in preparation) . Peptides based upon the 7-amino acid SH3 binding domain core motif displayed strong binding to both the Src and to the Fyn SH3 domains, but failed to bind to the SH3 domain of p21 Ras-GTPase-activating protein (Ras-GAP) and other proteins . We anticipate that further screening of the phage display library will be a useful tool for the rapid identification of additional SH3 domain binding sequences and will also help to establish the essential core motifs that define the specificity of interactions among the diverse proteins containing SH3 domains and those containing SH3 binding motifs.

Gene, 1994 Sep 30, 147(2), 189 - 95
Production in Escherichia coli, purification and immunogenicity of acrosomal protein SP-10, a candidate contraceptive vaccine; Reddi PP et al.; The testis-specific human sperm antigen, SP-10, has been designated a 'primary vaccine candidate' by the World Health Organization Taskforce on Contraceptive Vaccines . Molecular cloning and sequencing of the cDNAs coding for human (h) and baboon (b) SP-10 have been reported . To produce large amounts of pure antigen for ongoing studies of the immunogenicity and anti-fertility effects of SP-10, we used an efficient Escherichia coli expression system . The full-length open reading frames for hSP-10 and bSP-10 were placed under the inducible T7 bacteriophage RNA polymerase/promoter system . An in-frame fusion was made such that a His6 stretch was produced at the C terminus of SP-10 . Upon induction of gene expression, large amounts of hSP-10 or bSP-10 were synthesized and the recombinant (re-) protein segregated into an insoluble fraction . The protein was then solubilized in 6 M guanidine.HCl and purified by immobilized metal affinity chromatography (IMAC) . The yield of purified bSP-10 preparation was approx . 20 micrograms/ml of culture . Immunoreactivity of the purified re-SP-10 with MHS-10, a monoclonal antibody specific to SP-10, and rabbit polyclonal sera raised against SP-10, indicated that the synthesized antigen was suitable for immunization studies . Four female baboons were then immunized with the re-bSP-10 antigen . Immunoblots using pre-immune and immune sera from these animals indicated that all four baboons produced antibodies that reacted with native SP-10 extracted from human sperm in a manner identical to that of MHS-10, the positive control . Immune sera also stained the acrosome region of human and baboon sperm heads by immunofluorescence.(ABSTRACT TRUNCATED AT 250 WORDS)

Science, 1994 Sep 30, 265(5181), 2096 - 8
Alignment and sensitive detection of DNA by a moving interface; Bensimon A et al.; In a process called "molecular combining," DNA molecules attached at one end to a solid surface were extended and aligned by a receding air-water interface and left to dry on the surface . Molecular combing was observed to extend the length of the bacteriophage lambda DNA molecule to 21.5 +/- 0.5 micrometers (unextended length, 16.2 micrometers) . With the combing process, it was possible to (i) extend a chromosomal Escherichia coli DNA fragment (10(6) base pairs) and (ii) detect a minute quantity of DNA (10(3) molecules) . These results open the way for a faster physical mapping of the genome and for the detection of small quantities of target DNA from a population of molecules.

Nucleic Acids Res, 1994 Sep 25, 22(19), 3887 - 94
Gene expression mediated by bacteriophage T3 and T7 RNA polymerases in transgenic trypanosomes; Wirtz E et al.; Messenger RNAs of higher eukaryotes share a functionally essential 5' monomethyl CAP structure generated during a reaction that is linked exclusively to RNA polymerase II transcription . In unicellular parasites belonging to the Kinetoplastida, however, mRNAs acquire their 5' CAP through a trans-splicing reaction which effectively uncouples pol II transcription and capping . Consequently functional mRNAs can be produced by endogenous RNA polymerase I . Here we demonstrate the extension of this flexibility to heterologous bacteriophage polymerases . Transgenic Trypanosoma brucei cell lines stably expressing functional, nuclearly localized T3 or T7 RNA polymerase were established and assayed using reporter plasmids bearing the corresponding phage promoters . In these cell lines the levels of phage promoter-driven gene expression ranges from one half to greater than 5 times that mediated by endogenous pol I . Analysis of 5' ends of transcripts synthesized by the T7 polymerase revealed that they are trans-spliced . Thus the usual eukaryotic link between mRNA production and pol II transcription can be by-passed by the introduced phage polymerases, thereby significantly expanding the critically small panel of promoters currently available for exploitation in reverse genetic approaches in T . brucei.

Biochem Biophys Res Commun, 1994 Sep 15, 203(2), 798 - 804
clpX encoding an alternative ATP-binding subunit of protease Ti (Clp) can be expressed independently from clpP in Escherichia coli; Yoo SJ et al.; ClpX, an alternative ATP-binding subunit for protease Ti (also called Clp), has been shown to support the ATP-dependent hydrolysis of lambda O-protein by ClpP . clpX has also been reported to be in an operon with clpP, and therefore both are co-transcribed in a single mRNA using the promoter proximal to clpP . Here, we show that clpX can be expressed independently from clpP using its own promoter . The cells carrying clpX alone on a multicopy plasmid successively produced the 46-kDa ClpX protein . Moreover, in vitro translation analysis revealed that the recombinant plasmid containing clpX generates the 46-kDa protein that can be immunoprecipitated with anti-ClpX antibody . In addition, it has recently been reported that CipX, but not ClpP, is required for normal replication of bacteriophage Mu . Thus, it appears that clpX can be expressed alone and/or co-expressed with clpP in cells depending on physiological conditions.

Gene, 1994 Sep 15, 147(1), 77 - 9
The complete nucleotide sequence of cosmid vector pTL5: location and origin of its genetic components; Slightom JL et al.; The complete nucleotide sequence (5793 bp) of the cosmid vector pTL5 and the origin of its genetic components has been determined . Cosmid pTL5, a derivative of cosmid vector pHC79, is composed of genetic components from pBR322, bacteriophage lambda and the hybrid lambdoid bacteriophage Charon (Ch) 4A cohesive ends (cos) region . The Ch4A cos region contains genetic components from two bacteriophages, the lambda cos-left arm and the phi 80 cos-right arm regions . The Ch4A cos region has been used in the construction of many other cosmid-type vectors, some of which have been sequenced and entered into the GenBank database.

Gene, 1994 Sep 15, 147(1), 71 - 6
Cloning and expression of genes from the genomic region between genes cd and 30 of bacteriophage T4; Zajanckauskaite A et al.; Ten open reading frames (ORFs) from the cd-30 region of bacteriophage T4, as well as genes 31 and rIII, have been cloned, and all were found to be expressed in a T7 RNA polymerase system . Expression data confirm the existence of genes 31.2, 31.1, 31, rIII, 30.9, 30.8, 30.7, 30.6, 30.5, 30.4, 30.3 and 30.2, which predict peptides of 78, 102, 111, 82, 58, 110, 121, 95, 65, 68, 152 and 278 amino acids (aa), respectively . The major product of gene 30.9 is a basic peptide initiated at the second AUG codon that is found 42 nucleotides downstream and in frame from the first AUG . A plasmid carrying gene 30.3 expresses two different peptides, one of which is of the size predicted for gp30.3 . By site-directed mutagenesis we have shown that the smaller peptide is encoded by gene 30.3' which is completely embedded within gene 30.3, but in a different reading frame . Gene 30.3' encodes a 75-aa basic peptide with its C terminus rich in charged aa.

Carbohydr Res, 1994 Sep 15, 262(2), 311 - 22
Structural investigation of the capsular polysaccharide of Escherichia coli O101 : K103 : H- using bacteriophage degradation and NMR spectroscopy; Grue MR et al.; NMR spectroscopy was performed on the depyruvated capsular antigen of E . coli K103 and on the oligosaccharide obtained by depolymerisation of the native polysaccharide with a viral-borne endoglycanase . This capsular polysaccharide is the only one to be co-expressed with O group 101 and joins a small group of unusual capsular polysaccharides which possess pyruvic acid as the only acidic function . The primary structure was shown to be composed of the repeating unit: {formula: see text}

FEMS Microbiol Lett, 1994 Sep 15, 122(1-2), 195 - 9
Lysis of Escherichia coli cells induced by bacteriophage T4; Tarahovsky YS et al.; Structural changes in the envelope of Escherichia coli cells accompanying their lysis from without by bacteriophage T4 have been studied . The hypothesis concerning the role of collapse of membrane potential and formation of periplasmic vesicles in the process of lysis from without has been advanced.

Biochim Biophys Acta, 1994 Sep 13, 1219(1), 15 - 25
Structure, promoter analysis and chromosomal assignment of the human APEX gene; Akiyama K et al.; APEX nuclease is a mammalian DNA repair enzyme having apurinic/apyrimidinic endonuclease, 3'-5'-exonuclease, DNA 3' repair diesterase and DNA 3'-phosphatase activities . This report describes the organization of the gene (APEX gene) for human APEX nuclease . Human APEX gene was cloned using human APEX cDNA and a human leukocyte genomic library in bacteriophage vector EMBL-3 . We proved that human APEX gene consists of 5 exons spanning 2.64 kilobases and suggested that the gene exists as a single copy in the haploid genome . The boundaries between exon and intron follow the GT/AG rule . The major transcription initiation site was assigned by primer extension analysis to C at 515 nucleotides upstream from the ATG initiation codon . The translation initiation and termination sites locate in the exon II and V, respectively . The 5' flanking region (0.89 kilobase) sequenced lacks typical TATA and CAAT boxes, but contains TATA- and CAAT-like sequences and putative cis-acting regulatory elements such as binding sites for Sp1, AP2 and ATF . A part of the 5' flanking region belongs to a CpG island, which extends to the intron II . The CpG island is thought to be a transcription regulatory region of APEX gene, a housekeeping gene . The promoter activity of the 5' upstream region was analyzed by introducing the region in HeLa cells in an expression construct containing luciferase gene as a reporter gene, and the region from position 130 bp upstream to position 205 bp downstream of the major transcription initiation site was shown to be enough for high promoter activity . Northern hybridization experiments suggested that the gene is expressed ubiquitously in human cells . The locus of APEX gene was mapped to human chromosome 14q11.2-q12 using the in situ hybridization technique.

Nucleic Acids Res, 1994 Sep 11, 22(18), 3748 - 52
Mutations that increase the affinity of a translational repressor for RNA; Lim F et al.; The coat protein of the RNA bacteriophage MS2 is a specific RNA binding protein that represses translation of the viral replicase gene during the infection cycle . As an approach to characterizing the RNA-binding site of coat protein we have isolated a series of coat mutants that suppress the effects of a mutation in the translational operator . Each of the mutants exhibits a super-repressor phenotype, more tightly repressing both the mutant and wild-type operators than does the wild-type protein . The variant coat proteins were purified and subjected to filter binding assays to determine their affinities for the mutant and wild-type operators . Each protein binds the operators from 3 to 7.5-fold more tightly than normal coat protein . The amino acid substitutions seem to extend the normal binding site by introducing new interactions with RNA.

J Mol Biol, 1994 Sep 9, 242(1), 45 - 61
Molecular cloning and expression of a novel hydroxymethylcytosine-specific restriction enzyme (PvuRts1I) modulated by glucosylation of DNA; Janosi L et al.; The kanamycin resistance plasmid Rts1 restricts the growth of bacteriophage T2, T4 and T6 . The DNA of these phage contains hydroxymethylcytosine (HMC) in place of regular cytosine and is modified by glucosylation . When HMC is not glucosylated, as in the DNA of glucosyl transferase-deficient T4 phage, this restriction becomes less apparent, a phenomenon not observed with any other known restriction systems . On the other hand, glucosylation of HMC in T6 phage leads to a less efficient restriction, while restriction of bacteriophage T2 remains unchanged . The modulating effect of glucose cannot be seen when cells contain a large amount of this enzyme, as in the case when multiple copies of its determinant are present in the cells . T-odd phage and bacteriophage lambda are not restricted by Rts1 suggesting that the restriction is specific to DNA containing HMC . The restriction phenotype is due to a single gene coding for a polypeptide of 293 amino acids . This enzyme has been named PvuRts1I . A gene with the sequence motifs similar to modification enzymes was found upstream of the gene coding for PvuRts1I . This gene, however, neither modifies the restriction phenotype of PvuRts1I, nor codes for detectable modification enzyme . T4 mutants with increased resistance to PvuRts1I appear to have deficiency in their beta-glucosyl transferase enzyme.

J Biol Chem, 1994 Sep 9, 269(36), 22712 - 8
Identification of polysialic acid-containing glycoprotein in the jelly coat of sea urchin eggs . Occurrence of a novel type of polysialic acid structure; Kitazume S et al.; Sea urchin eggs are surrounded by a gelatinous layer (called the jelly coat) that consists of mixture of fucoserich polysaccharides and sialic acid-rich glycoproteins . Chemical and 500 MHz 1H NMR spectroscopic studies revealed for the first time the presence of a novel polysialic acid (polySia) structure in the jelly coat glycoproteins isolated from Hemicentrotus pulcherrimus (designated polySia-gp(H)) . The structure of the polySia chains was thoroughly characterized as (-->5-Oglycolyl-Neu5Gc alpha 2-->)n, where n ranges from 4 to more than 40 Neu5Gc residues . The polyNeu5Gc chains were attached to core oligosaccharides that were O-glycosidically linked to threonine residues on a core polypeptide . Each polypeptide contained about 17 O-linked polysialylglycan chains . The apparent molecular weight of polySia-gp(H) was 180,000 . The expression of this new polySia structure in place of alpha 2-->8-linked polySia is the main structural feature that distinguishes polySia-gp from other known polysialylated glycoproteins . The (-->5-Ogly-colyl-Neu5Gc alpha 2-->)n chains were resistant to exo- and endosialidases from Arthrobacter ureafaciens and bacteriophage K1F, respectively . Discovery of these (-->5-Ogly-colyl-Neu5Gc alpha 2-->)n chains adds a new class of naturally occurring polySia to the structurally diverse family of polysialylated glycoproteins . The structure of a poly-Sia-gp from a different sea urchin species, Stronglyocentrotus purpuratus (designated polySia-gp(S)), was also determined to ascertain if there were any species-specific differences . The 500 MHz 1H NMR spectra of the two polySia-gps were identical, indicating that at this level of molecular detail the structures were the same . The molecular weight of polySiagp(S) was larger, however (250,000), and it contained about 25 polySia chains O-glycosidically linked to both threonine (two-thirds) and serine (one-third) residues.

J Biol Chem, 1994 Sep 9, 269(36), 22507 - 16
Biochemical characterization of P22 phage-modified Escherichia coli RecBCD enzyme; Murphy KC; The biochemical properties of phage P22 Abc-modified RecBCD enzyme from Escherichia coli have been examined . RecBCD purified from a cell that expresses Abc (anti-RecBCD) contains all three RecBCD subunits and the 11.6-kDa Abc protein in equal stoichiometric amounts . Abc depresses the rate of RecBCD double-stranded DNA exonuclease, helicase, and ATPase activities about 3-4-fold, yet it has no effect on the rate of the single-stranded DNA exonuclease activity . Abc induces a slight increase in the ATP-independent single-stranded DNA endonuclease activity and does not induce dimerization of the RecBCD trimer . Abc-modified RecBCD helicase activity possesses reduced but significant processivity (10 kilobase pairs) relative to the native enzyme (30 kilobase pairs) . In the absence of ATP, Abc-modified RecBCD shows a 2-4-fold higher affinity for double-stranded DNA ends . The RecBCD-binding Gam protein from bacteriophage lambda inhibits binding of both native and Abc-modified RecBCD to double-stranded DNA ends . Finally, unlike the native enzyme, the nonspecific nuclease activity of Abc-modified RecBCD is not suppressed by Chi sites in vitro . These findings are discussed in terms of the recombination-deficient phenotype of cells expressing Abc in vivo and the relationship between Abc-modified RecBCD and two mutant RecBCD's previously characterized: the RecBCD-K117Q and RecB2109CD mutant enzymes.

Transfusion, 1994 Sep, 34(9), 802 - 10
Quencher-enhanced specificity of psoralen-photosensitized virus inactivation in platelet concentrates; Margolis-Nunno H et al.; BACKGROUND: Treatment with psoralens and UVA (PUVA) has been shown to be efficacious in eliminating the risk of virus transmission by platelet concentrates (PCs) . It has previously been demonstrated that, during the inactivation of cell-free vesicular stomatitis virus (VSV) by aminomethyltrimethylpsoralen (AMT) and UVA in PCs, platelet function could be protected either by oxygen removal before irradiation or by inclusion of a type I free radical quencher, such as mannitol . STUDY DESIGN AND METHODS: Under previous PUVA treatment conditions for PCs (25 micrograms/mL AMT; 30 min UVA at 7 mW/cm2; 2 mM {2 mmol/L} mannitol), more than 6 log10 of added cell-free VSV was completely inactivated . In the current study, various PUVA conditions are evaluated for efficacy in inactivating other viral forms that could be present in PCs . Maintenance of platelet integrity (i.e., platelet number, solution pH, and aggregation response during initial storage after treatment) and kill of cell-associated VSV are examined . RESULTS: While cell-free viruses were inactivated efficiently under previous PUVA conditions, cell-associated VSV and the non-lipid-enveloped bacteriophage M13 were not . Effective inactivation of these viruses was achieved by raising the concentration of AMT to 50 micrograms per mL and extending the period of irradiation to 90 minutes (39 J/cm2) . However, for maintenance of platelet integrity under these conditions, the prior removal of oxygen or the inclusion of compounds known to quench both type I and type II photoreactants (e.g., flavonoids such as rutin) was required . CONCLUSION: These findings suggest that the viral safety of PCs may be enhanced through treatment with AMT and UVA in the presence of flavonoids, and that flavonoid use may prove beneficial in other systems where oxygen-mediated damage occurs.

J Gen Virol, 1994 Sep, 75 ( Pt 9), 2509 - 12
Synthesis of infectious transcripts of blueberry scorch carlavirus in vitro; Lawrence DM et al.; Blueberry scorch carlavirus (BBScV) is a filamentous virus with a polyadenylated, positive-sense RNA genome . A near full-length cDNA clone of BBScV was constructed by assembly of clones from a cDNA library . To generate a full-length cDNA clone, the 5' terminus was mutagenized by PCR to introduce nucleotides present in the wild-type virus and not in the near full-length clone, and then fused directly to the T7 bacteriophage RNA polymerase promoter at the 5' terminus . Capped and uncapped BBScV transcripts were synthesized in vitro from the full-length cDNA clone . Capped transcripts were infectious, producing systemic symptoms identical to those caused by the wild-type virus following inoculation onto Chenopodium quinoa leaves . Uncapped transcripts were substantially less infectious than capped transcripts . This represents the first report of infectious transcripts for a member of the carlavirus group.

EMBO J, 1994 Sep 1, 13(17), 4181 - 92
Genomic polymorphism in the T-even bacteriophages; Repoila F et al.; We have compared the genomes of 49 bacteriophages related to T4 . PCR analysis of six chromosomal regions reveals two types of local sequence variation . In four loci, we found only two alternative configurations in all the genomes that could be analyzed . In contrast, two highly polymorphic loci exhibit variations in the number, the order and the identity of the sequences present . In phage T4, both highly polymorphic loci encode internal proteins (IPs) that are encapsidated in the phage particle and injected with the viral DNA . Among the various T4-related phages, 10 different ORFs have been identified in the IP loci; their amino acid sequences have the characteristics of internal proteins . At the beginning of each of these coding sequences is a highly conserved 11 amino acid leader motif . In addition, both 5' and 3' to most of these ORFs, there is a approximately 70 bp sequence that contains a T4 early promoter sequence with an overlapping inversely repeated sequence . The homologies within these flanking sequences may mediate the recombinational shuffling of the IP sequences within the locus . A role for the new IP-like sequences in determining the phage host range is proposed since such a role has been previously demonstrated for the IP1 gene of T4.

Blood, 1994 Sep 1, 84(5), 1635 - 9
In vivo neutrophil and lymphocyte function studies in a patient with leukocyte adhesion deficiency type II; Price TH et al.; We investigated in vivo neutrophil and lymphocyte function in a patient who lacks Sialyl-Lewis-X, a ligand for the selectin family of leukocyte adhesion molecules (leukocyte adhesion deficiency II, LAD II) . As assessed by skin chamber and skin window techniques, in vivo chemotaxis of neutrophils was markedly impaired (less than 6% of normal values) . A marginal pool was present as determined by an increase in circulating neutrophils after epinephrine injection and calculated recovery of infused radiolabeled autologous neutrophils . Kinetic studies showed a reduced half-life of 3.2 hours (normal 7 hours) and markedly increased turnover rate (cells/kg/d) of approximately eight times the normal value . A normal antibody response to the T-cell-dependent antigen bacteriophage phi X174 showed that T/B-cell interaction is not affected in LAD II . These findings provide direct evidence that the selectin family and its ligands play an important role in neutrophil function.

Mol Cell Biol, 1994 Sep, 14(9), 5898 - 909
Proteins binding to 5' untranslated region sites: a general mechanism for translational regulation of mRNAs in human and yeast cells; Stripecke R et al.; We demonstrate that a bacteriophage protein and a spliceosomal protein can be converted into eukaryotic translational repressor proteins . mRNAs with binding sites for the bacteriophage MS2 coat protein or the spliceosomal human U1A protein were expressed in human HeLa cells and yeast . The presence of the appropriate binding protein resulted in specific, dose-dependent translational repression when the binding sites were located in the 5' untranslated region (UTR) of the reporter mRNAs . Neither mRNA export from the nucleus to the cytoplasm nor mRNA stability was demonstrably affected by the binding proteins . The data thus reveal a general mechanism for translational regulation: formation of mRNA-protein complexes in the 5' UTR controls translation initiation by steric blockage of a sensitive step in the initiation pathway . Moreover, the findings establish the basis for novel strategies to study RNA-protein interactions in vivo and to clone RNA-binding proteins.

J Virol, 1994 Sep, 68(9), 5588 - 95
The human astrovirus RNA-dependent RNA polymerase coding region is expressed by ribosomal frameshifting; Marczinke B et al.; The genomic RNA of human astrovirus serotype 1 (HAst-1) contains three open reading frames (ORFs), 1a, 1b, and 2 . ORF 1b is located downstream of, and overlaps, 1a, and it has been suggested on the basis of sequence analysis that expression of ORF 1b is mediated through -1 ribosomal frameshifting . To examine this possibility, a cDNA fragment containing the 1a-1b overlap region was cloned within a reporter gene and placed under the control of the bacteriophage SP6 promoter in a recombinant plasmid . Synthetic transcripts derived from this plasmid, when translated in the rabbit reticulocyte lysate cell-free system, specified the synthesis of polypeptides whose size and antibody reactivity were consistent with an efficient -1 ribosomal frameshift event at the overlap region . The HAst-1 frameshift signal has two essential components, a heptanucleotide slippery sequence, A6C, and a stem-loop structure in the RNA . The presence of this structure was confirmed by complementary and compensatory mutation analysis and by direct structure probing with single- and double-stranded RNA-specific reagents . The HAst-1 frameshift signal, like that present at the overlap of the gag and pro genes of the retrovirus human T-cell lymphotrophic virus type II, does not involve the formation of an RNA pseudoknot.

J Virol, 1994 Sep, 68(9), 5411 - 22
Fusogenic mechanisms of enveloped-virus glycoproteins analyzed by a novel recombinant vaccinia virus-based assay quantitating cell fusion-dependent reporter gene activation; Nussbaum O et al.; The fusogenic activities of enveloped-virus glycoproteins were analyzed by using a quantitative, sensitive, rapid, and highly versatile recombinant vaccinia virus-based assay measuring activation of a reporter gene upon fusion of two distinct cell populations . One population uniformly expressed vaccinia virus-encoded viral glycoproteins mediating specific binding and fusion activities; the other expressed the corresponding cellular receptor(s) . The cytoplasm of one population also contained vaccinia virus-encoded bacteriophage T7 RNA polymerase; the cytoplasm of the other contained a transfected plasmid with the Escherichia coli lacZ gene linked to the T7 promoter . When the two populations were mixed, cell fusion resulted in activation of the LacZ gene in the cytoplasm of the fused cells; beta-galactosidase activity was assessed by colorimetric assay of detergent cell lysates or by in situ staining . We applied this approach to study the human immunodeficiency virus type 1 envelope glycoprotein (Env)-CD4 interaction . Beta-Galactosidase was detected within 1 h after cell mixing and accumulated over the next several hours . Cell fusion dependence was demonstrated by the strict requirement for both CD4 and functional Env expression and by the inhibitory effects of known fusion-blocking monoclonal antibodies and pharmacological agents . Quantitative measurements indicated much higher sensitivity compared with analysis of syncytium formation . The assay was used to probe mechanisms of the cell type specificity for Env-CD4-mediated fusion . In agreement with known restrictions, cell fusion occurred only when CD4 was expressed on a human cell type . Membrane vesicle transfer experiments indicated that CD4 initially produced in either human or nonhuman cells was functional when delivered to human cells, suggesting that the fusion deficiency with nonhuman cells was not associated with irreversible defects in CD4 . We also demonstrated that the infectivity specificities of different human immunodeficiency virus type 1 isolates for peripheral blood lymphocytes versus continuous CD4+ cell lines were associated with corresponding fusion selectivities of the respective recombinant Env proteins . The assay enabled analysis of the fusogenic activity of the fusion glycoprotein/hemagglutinin-neuraminidase of the paramyxovirus simian virus 5 . This system provides a powerful tool to study fusion mechanisms mediated by enveloped-virus glycoproteins, as well as to screen fusion-blocking antibodies and pharmacological agents.

Virology, 1994 Sep, 203(2), 294 - 8
The ADP-ribosyltransferases (gpAlt) of bacteriophages T2, T4, and T6: sequencing of the genes and comparison of their products; Koch T et al.; The Alt gene product is a component of the T4 phage head . Upon infection of the host cell, approximately 40 copies of the Alt protein enter the cell together with the viral DNA molecule . The Alt protein then ADP-ribosylates one of the two alpha-subunits of host RNA polymerase . A restriction fragment harboring the ADP-ribosyltransferase gene of bacteriophage T4 was cloned into the plasmid vector pBluescript, the nucleotide sequence was determined, and the reading frame was identified . Two M13 clone libraries, established with DNA isolated from bacteriophages T2 and T6, then were screened for the corresponding genes . The nucleotide sequences of the three alt genes and the deduced amino acid sequences were compared . Secondary structure predictions and NAD-binding studies resulted in the location of the substrate-binding site in the NH2-terminal regions of the enzymes.

Mol Biol (Mosk), 1994 Sep-Oct, 28(5), 1176 - 82
{A new method of covalent immobilization of oligodeoxyribonucleotides on nylon membranes for hybridization with nucleic acids}; Ivanovskaia MG et al.; A new method of covalent immobilization of oligodeoxyribonucleotides on nylon membranes which contain surface amino groups was developed . The method consists in condensation between the amino group of the membrane and the carboxyl group of modified oligonucleotide by means of 1-ethyl-3-(3'-dimethylaminopropyl)carbodiimide . The carboxyl group was introduced into the oligonucleotide by means of postsynthetic attachment of peptide (reduced glutathione) at the terminal phosphate group of the oligonucleotide, using the N-hydroxybenzotriazole method of phosphate activation . Membranes containing a covalently immobilized 23-membered oligonucleotide were tested in hybridization with complementary oligonucleotide, and with single-stranded DNA of bacteriophage M13 which has a complementary sequence . The method of covalent immobilization is very simple and convenient . The membranes with covalently immobilized oligonucleotides may be used not only in hybridization analysis, but also for purification of nucleic acids and proteins which recognize nucleotide sequences and in sense biotechnology.

Microbiol Rev, 1994 Sep, 58(3), 293 - 316
Translocation of DNA across bacterial membranes; Dreiseikelmann B; DNA translocation across bacterial membranes occurs during the biological processes of infection by bacteriophages, conjugative DNA transfer of plasmids, T-DNA transfer, and genetic transformation . The mechanism of DNA translocation in these systems is not fully understood, but during the last few years extensive data about genes and gene products involved in the translocation processes have accumulated . One reason for the increasing interest in this topic is the discussion about horizontal gene transfer and transkingdom sex . Analyses of genes and gene products involved in DNA transfer suggest that DNA is transferred through a protein channel spanning the bacterial envelope . No common model exists for DNA translocation during phage infection . Perhaps various mechanisms are necessary as a result of the different morphologies of bacteriophages . The DNA translocation processes during conjugation, T-DNA transfer, and transformation are more consistent and may even be compared to the excretion of some proteins . On the basis of analogies and homologies between the proteins involved in DNA translocation and protein secretion, a common basic model for these processes is presented.

Microbiologia, 1994 Sep, 10(3), 285 - 96
A direct membrane filter method for enumerating somatic coliphages in drinking water; Alonso MC et al.; The application of a simple membrane filter method to enumerate specific somatic bacteriophages of Escherichia coli, using E . coli C as host strain, from drinking water samples was studied . The efficiency of the method using cellulosic membrane filters, samples pretreated with magnesium ions and Tween 80 added to agar medium-host cell lawns ranged from 68.9 to over 112%, depending on the phage content of the sample . To avoid the pre-treatment of the sample with magnesium salts, electropositive-charged filters of cellulosic ester (HA-PEI and HA-Nalco) and Virosorb-1MDS filters were tested in conjunction with the simple membrane filter method . The electropositive filters showed wide bacteriophage recovery rate intervals depending on the sample treatment, ranging between 31.4 and 96.2% for ester-type filters, and a mean recovery lower than 2.2% for Virosorb filters . On the other hand, it was proved that the use of Tween 80 as an eluent improved somatic coliphage recovery rates for all the filters tested . In short, this methodology provides a rapid analysis (6-8 h) of the somatic coliphages from drinking water using the membrane filtration technique.

Mutagenesis, 1994 Sep, 9(5), 451 - 8
Sequence spectra of spontaneous lacZ gene mutations in transgenic mouse somatic and germline tissues; Douglas GR et al.; As a critical step in determining whether transgenic mouse gene mutation systems are suitable models for the detection and quantification of induced gene mutations in vivo, spontaneous mutant frequencies and mutation spectra have been characterized for liver, bone marrow, and male germ cells of the lacZ transgenic mouse strain 40.6 . The lacZ transgene is carried on a recombinant bacteriophage lambda shuttle vector that is recovered from mouse genomic DNA, and analysed in vitro for mutations that occurred in the mouse tissues . Mutations are detected visually as clear or pale blue plaques when X-gal is the substrate for beta-galactosidase; whereas, the wild-type plaques are dark blue . There was no statistical difference in the mutant frequency among the three tissues studied, the pooled mutant frequency being 2.23 +/- 0.41 per 10(5) pfu . The predominant type of mutation was GC-->AT transitions, with most occurring in 5'-CpG dinucleotides, suggesting that the deamination of 5-methylcytosine is the main mechanism of mutagenesis . There was, however, a statistically significant difference in the base pair substitution mutation spectrum for the liver and bone marrow when mutations were grouped according to GC or AT base-pairs . The proportion of transition versus transversion mutations was also statistically different among the three tissues, resulting mainly from the fact that germ cells were different from both bone marrow and liver . A lower number of spontaneous transitions in male germ cells was accompanied by an increase in transversions, with the proportion of GC-->AT transitions in 5'-CpG sites also declining.(ABSTRACT TRUNCATED AT 250 WORDS)

J Eukaryot Microbiol, 1994 Sep-Oct, 41(5), 435 - 41
Cloning of a partial length cDNA encoding the C-terminal portion of the 75-77-kDa antigen of Trypanosoma cruzi; Yang S et al.; It has been suggested that several Trypanosoma cruzi antigens have possible protective epitopes which may be suitable vaccine candidates . We found previously that animals resistant to T . cruzi infection produced antibodies against the 75-77-kDa parasite antigen . To test the ability of the recombinant form of this antigen to protect animals from T . cruzi infection, the cDNA which encodes a portion of the 75-77-kDa antigen was cloned using a cDNA library constructed in an orientation-specific bacteriophage expression vector (lambda gt 11) from poly (A)+ RNA of Brazil strain epimastigotes . One clone, named SFS-40, was selected by screening the library using affinity purified antibodies specific for the 75-77-kDa parasite antigen as probe . The cDNA corresponding to the 1.7-kilobase insert of SFS-40 was subcloned into plasmid vectors and characterized . The cDNA sequence encodes a polypeptide of about 40 kDa . The putative product of the cDNA was homologous to members of the 70-kDa stress protein family . When epimastigotes were shifted from 29 degrees C to 37 degrees C, there was no change in the level of SFS-40 mRNA . In contrast, the 70-kDa heat shock protein mRNA of the parasite was increased about four fold by this treatment.

Mutat Res, 1994 Sep, 325(1), 39 - 45
Effect of abasic sites on bacteriophage T7 protein synthesis; Sanchez G et al.; We have examined protein synthesis directed by bacteriophage T7 which had been alkylated with methyl methanesulfonate so as to produce apurinic sites in its DNA in vivo . Both repair-proficient and repair-deficient (xth nfo mutant) strains of Escherichia coli served as host cells . In repair-proficient cells, all three classes of phage proteins were synthesized, although with significant delays . In mutant cells, only class I proteins were produced and their synthesis was delayed and reduced, demonstrating a perturbation of protein synthesis and providing the first in vivo indication that transcription is inhibited by abasic sites . However, the proposed effects of abasic sites on transcription appear to be weaker than those on replication.

J Biotechnol, 1994 Aug 31, 36(3), 247 - 51
'One for all scales' methods for plasmid, phagemid and bacteriophage DNAs preparation; Chang PK et al.; We have established two simple, reliable, economical and time-saving methods, one for plasmid and phagemid DNAs another for bacteriophage DNAs, that can be applied for any desired scale of vector and its recombinant DNAs preparation . The methods require neither toxic chemicals nor expensive enzymes or chemical reagents . In case of the small-scale preparation, the entire procedure can be done in one microfuge tube . The A260/A280 values for isolated DNAs were constantly between 1.60 and 1.85 . The isolated plasmid or phagemid DNA can be used for restriction digestion, religation, transformation, construction of deletion mutants, sequencing, PCR and in vitro transcription . The double-stranded and single-stranded phage DNAs prepared from the present method have the quality to serve as good templates for PCR and site-directed mutagenesis experiments.

Proc Natl Acad Sci U S A, 1994 Aug 30, 91(18), 8552 - 6
Expression cloning of a gibberellin 20-oxidase, a multifunctional enzyme involved in gibberellin biosynthesis; Lange T et al.; In the biosynthetic pathway to the gibberellins (GAs), carbon-20 is removed by oxidation to give the C19-GAs, which include the biologically active plant hormones . We report the isolation of a cDNA clone encoding a GA 20-oxidase {gibberellin, 2-oxoglutarate:oxygen oxidoreductase (20-hydroxylating, oxidizing) EC 1.14.11.-} by screening a cDNA library from developing cotyledons of pumpkin (Cucurbita maxima L.) for expression of this enzyme . When mRNA from either the cotyledons or the endosperm was translated in vitro using rabbit reticulocyte lysates, the products contained GA12 20-oxidase activity . A polyclonal antiserum was raised against the amino acid sequence of a peptide released by tryptic digestion of purified GA 20-oxidase from the endosperm . A cDNA expression library in lambda gt11 was prepared from cotyledon mRNA and screened with the antiserum . The identity of positive clones was confirmed by the demonstration of GA12 20-oxidase activity in single bacteriophage plaques . Recombinant protein from a selected clone catalyzed the three-step conversions of GA12 to GA25 and of GA53 to GA17, as well as the formation of the C19-GAs, GA1, GA9, and GA20, from their respective aldehyde precursors, GA23, GA24, and GA19 . The nucleotide sequence of the cDNA insert contains an open reading frame of 1158 nt encoding a protein of 386 amino acid residues . The predicted M(r) (43,321) and pI (5.3) are similar to those determined experimentally for the native GA 20-oxidase . Furthermore, the derived amino acid sequence includes sequences obtained from the N terminus and two tryptic peptides from the native enzyme . It also contains regions that are highly conserved in a group of non-heme Fe-containing dioxygenases.

Proc Natl Acad Sci U S A, 1994 Aug 30, 91(18), 8522 - 6
Dimeric structure of a human apolipoprotein B mRNA editing protein and cloning and chromosomal localization of its gene; Lau PP et al.; Apolipoprotein B (apoB) mRNA editing consists of a posttranscriptional C-->U conversion involving the first base of the codon CAA encoding glutamine-2153 to UAA, a stop codon, in apoB mRNA . Using a cloned rat cDNA as a probe, we cloned the cDNA and genomic sequences of the gene for a human apoB mRNA editing protein . Expression of the cDNA in HepG2 cells results in editing of the intracellular apoB mRNA . By fluorescence in situ hybridization, we localized the gene for the editing protein to chromosome band 12p13.1-p13.2 . By Northern blot analysis, it was shown that the human editing protein mRNA is expressed exclusively in the small intestine . The cDNA sequence predicts a translation product of 236-aa residues . By attaching an epitope tag sequence to the C terminus of the editing protein, we examined the polymerization state of the editing protein synthesized in vitro . We found that the editing protein undergoes spontaneous polymerization . The migration of the human apoB mRNA editing protein on an HPLC column and the stoichiometry of polymeric epitope-tagged to untagged protein indicate that the protein exists as a dimer . Dimerization does not require glycosylation of a consensus N-linked glycosylation sequence present in the protein and is not mediated by disulfide bridge formation . The human apoB mRNA editing protein is a cytidine deaminase showing structural homology to some known mammalian and bacteriophage deoxycytidylate deaminases . The latter enzymes exist as homopolymers . The fact that the apoB mRNA editing protein also exists as a homodimer has important implications for the mechanism of apoB mRNA editing in humans.

Biochem Biophys Res Commun, 1994 Aug 30, 203(1), 190 - 9
Characterization of the genomic structure, chromosomal location and promoter of human prostaglandin H synthase-2 gene; Tazawa R et al.; Prostaglandin H synthase (PGHS) is the rate-limiting enzyme in the conversion of arachidonic acid to prostanoids . The human PGHS has two isoforms . PGHS-1 is a house keeping gene whereas PGHS-2 is an inducible gene . We reported here the isolation of the entire PGHS-2 gene and its 5'-flanking region from a human bacteriophage P1 genomic library . The gene containing 10 exons is 7.5 kb in length and located at chromosome 1 . The transcriptional start site was mapped at 134 bases upstream from the ATG start codon . Nucleotide sequence of 1.8 kb promoter region contains a TATA box and a number of potential regulatory elements including CRE, NF-kappa B, Sp1 and AP2 sites . Studies of the promoter activity showed that the first 460 nucleotides of 5'-flanking region efficiently drove transcription of the luciferase reporter gene in human umbilical vein endothelial cells upon stimulation with phorbor ester.

Biochemistry, 1994 Aug 30, 33(34), 10521 - 6
Evidence from 18O exchange studies for an exocyclic methylene intermediate in the reaction catalyzed by T4 deoxycytidylate hydroxymethylase; Butler MM et al.; 18O exchange experiments were designed to identify the final intermediate in the catalytic mechanism of bacteriophage T4 deoxycytidylate (dCMP) hydroxymethylase (CH) . CH catalyzes the formation of 5-(hydroxymethyl)-dCMP (HmdCMP) from dCMP and methylenetetrahydrofolate (CH2-THF) . CH resembles thymidylate synthase (TS), an enzyme of known three-dimensional structure, in both amino acid sequence and the reaction catalyzed . The final intermediate in the reaction catalyzed by TS or CH has been proposed to be the nucleotide with an exocyclic 5-methylene group covalently linked to the enzyme . This intermediate is then hydrated to HmdCMP (by CH) or reduced to deoxythymidylate (by TS) . We report here that CH catalyzes the incorporation of 18O from solvent water into the product, HmdCMP, in the presence of tetrahydrofolate (THF) . The cause of this exchange is a reverse reaction followed by a resynthesis . CH also catalyzes the exchange of 18O from solvent water into HmdCMP in the absence of exogenous THF and in the presence of THF analogues that lack N-5 . N-5 is the nitrogen that is likely to be bound to the methylene as it is transferred to dCMP . A CH variant that lacks the nucleophilic Cys 148 is incapable of promoting these 18O exchange reactions . The THF analogues lacking N-5 do not promote a CH-catalyzed reverse reaction . Rather, we propose that the CH-catalyzed 18O exchange reaction promoted by these THF analogues occurs via 5-methylene-dCMP linked to the enzyme through Cys 148 . We conclude here that enzyme-bound 5-methylene-dCMP is the final intermediate during catalysis by CH, as has also been proposed for TS and dUMP.

Proc Natl Acad Sci U S A, 1994 Aug 30, 91(18), 8680 - 4
A small mitochondrial double-stranded (ds) RNA element associated with a hypovirulent strain of the chestnut blight fungus and ancestrally related to yeast cytoplasmic T and W dsRNAs; Polashock JJ et al.; A small double-stranded (ds) RNA element was isolated from a moderately hypovirulent strain of the chestnut blight fungus Cryphonectria parasitica (Murr.) Barr . from eastern New Jersey . Virulence was somewhat lower in the dsRNA-containing strain than in a virulent dsRNA-free control strain, but colony morphology and sporulation levels were comparable . A library of cDNA clones was constructed, and overlapping clones representing the entire genome were sequenced . The 2728-bp dsRNA was considerably smaller than previously characterized C . parasitica dsRNAs, which are 12-13 kb and ancestrally related to the Potyviridae family of plant viruses . Sequence analysis revealed one large open reading frame, but only if mitochondrial codon usage (UGA = Trp) was invoked . Nuclease assays of purified mitochondria confirmed that the dsRNA was localized within mitochondria . Assuming mitochondrial translation, the deduced amino acid sequence had landmarks typical of RNA-dependent RNA polymerases . Alignments of the conserved regions indicate that this dsRNA is more closely related to yeast T and W dsRNAs and single-stranded RNA bacteriophages such as Q beta than to other hypovirulence-associated dsRNAs.

J Biol Chem, 1994 Aug 26, 269(34), 21870 - 9
UV-catalyzed cross-linking of Escherichia coli uracil-DNA glycosylase to DNA . Identification of amino acid residues in the single-stranded DNA binding site; Bennett SE et al.; Photochemical cross-linking of Escherichia coli uracil-DNA glycosylase (Ung) to oligonucleotide dT20 was performed to identify amino acid residues that reside in or near the DNA-binding site . UV-catalyzed cross-linking reactions produced a covalent Ung x dT20 complex which was resolved from uncross-linked enzyme by SDS-polyacrylamide gel electrophoresis . Cross-link formation required native Ung and was inhibited by increasing concentrations of NaCl in a manner characteristics of NaCl inhibition of Ung catalytic activity . The Ung x dT20 complex was purified to apparent homogeneity, and mass spectrometry revealed that Ung was cross-linked to dT20 in 1:1 stoichiometry as a 31,477 dalton complex . Purified Ung x dT20 lacked detectable uracil-DNA glycosylase activity and failed to bind single-stranded DNA . Recently, we demonstrated that the bacteriophage PBS2 uracil-DNA glycosylase inhibitor (Ugi) binds Ung and prevents further interaction with DNA (Bennett, S . E., Schimerlik, M . I., and Mosbaugh, D . W . (1993) J . Biol . Chem . 268, 26879-26885) . Addition of the Ugi protein to the cross-linking reaction blocked formation of the Ung x dT20 cross-link . Conversely, the Ung x dT20 cross-link was refractory to Ugi binding . Upon trypsin digestion of Ung x dT20, four distinct products were identified as peptide x dT20 cross-links . A combination of amino acid sequence and mass spectrometric analysis revealed that four tryptic peptides (T6, T18, T19, and T18/19) were adducted to dT20 . These observations suggest that dT20 is cross-linked to the Ung DNA-binding site.

J Mol Biol, 1994 Aug 26, 241(4), 507 - 23
Identification of high affinity binding sites for LexA which define new DNA damage-inducible genes in Escherichia coli; Lewis LK et al.; A multi-step screening procedure was devised to identify new operators for the LexA repressor in the sequenced portions of the genomes of Escherichia coli and its plasmids and bacteriophages . Sequence analysis methods were employed initially to distinguish true LexA operators from "operator-like" sequences stored within the GenBank and EMBL databases . The affinity of purified LexA protein for cloned DNA fragments containing several of the prospective new sites was then assessed using quantitative electrophoretic mobility shift assays and site-directed mutagenesis . Calculated binding affinities were compared directly with values determined for known and mutant LexA operators in concurrent experiments . Three E . coli chromosomal segments (near pyrC, hsdS and ntrla) and two bacteriophage sequences (near the P1 cre and lambda oop genes) bound LexA protein specifically . These sites and most others identified in the screening are located immediately upstream of known genes and/or large open reading frames . These results and additional transcription data demonstrate that several of the sequences define new DNA damage-inducible (din) genes and include the previously uncharacterized dinD locus . Furthermore, the search identified an SOS gene within the genome of P1 which encodes a protein that is homologous to UmuD', the RecA-promoted cleavage product of the umuD gene . The success of the combinatorial approach described here suggests that analogous searches for new regulatory sequences within the E . coli genome and the genomes of other organisms will also yield favorable results.

Nucleic Acids Res, 1994 Aug 25, 22(16), 3322 - 30
Pyrimidine phosphorothioate oligonucleotides form triple-stranded helices and promote transcription inhibition; Xodo L et al.; The ability of phosphorothioate (POS) oligonucleotides to recognise and bind to homopurine-homopyrimidine DNA double-stranded sites via triple helix formation has been investigated . It has been found that the homologous pyrimidine POS sequences Y11-Si (i = 0, 1,2,3,4,10), which have been obtained by an increasing sulphur substitution in the sugar-phosphate backbone of d(CTTCCTCCTCT) (Y11), and the target hairpin duplex d(GAAGGAGGAGA-T4-TCTCCTCCTTC) (h26) can form stable triple helices, as indicated by PAGE, CD and UV melting experiments . The thermal stability of the triple helices depends on the number of POS linkages in the third Y11 strand, varying from 48 degrees C (Y11, with only phosphate groups, PO2) to 31 degrees C (Y11-S10 containing exclusively thioate groups) . On average, a Tm depression of about 2 degrees C per POS linkage introduced in Y11 was observed . CD data indicate that the sulphurization of the third strand results in minimal changes of triple-stranded structures . The energetics of the triplex-to-hairpin plus single-strand transition has been determined by van't Hoff analyses of the melting curves . In free energy terms, the POS triplexes h26.Y11-Si are less stable than the normal PO2 h26.Y11 triplex by values between 2.7 and 5.4 kcal/mol, depending on the number of POS linkages contained in the third strand . Phosphorothioate oligonucleotides being resistant towards several nucleases offer an interesting choice as gene blockers in antisense strategy . Thus, their ability to inhibit transcription via triple helix formation has been examined in vitro . We found that triplex-forming POS oligonucleotides of 20 bases in length (with a cytosine contents of 45%), containing either 10% or 26% thioate groups, strongly repress the transcription activity of the bacteriophage T7 RNA polymerase at pH 6.9, when used in excess compared to the target (mol oligo/mol template = 125) . The here reported data are useful for designing phosphorothioate oligonucleotides targeted to genomic DNA in antigene strategy.

J Biol Chem, 1994 Aug 19, 269(33), 21123 - 6
Spatial relationship between polymerase and exonuclease active sites of phage T4 DNA polymerase enzyme; Gopalakrishnan V et al.; The spatial relationship between the polymerase and exonuclease active sites of bacteriophage T4 DNA polymerase enzyme has been examined using a bulky biotin-streptavidin block at a specified position in an oligonucleotide (Fig . 1) . The idea was to monitor the closest distance of approach of the T4 enzyme before being blocked by the bulky biotin-streptavidin complex while performing either of its activities . The results indicated a distance of 4-5 nucleotides between the biotin-streptavidin probe and the exonuclease site and a distance requirement of at least 7 nucleotides between the bulky probe and the 3'-primer terminus for efficient polymerization by the T4 enzyme . The difference in the two distances suggested a separation of 2-3 nucleotides between the two active sites of the T4 enzyme.

Gene, 1994 Aug 19, 146(1), 67 - 72
Structural analysis of DNA cleaved in vivo by bacteriophage T4 terminase; Bhattacharyya SP et al.; Phage terminases are protein complexes that cleave concatemeric phage DNA and generate termini of the packaged DNA molecule . In phage T4, the DNA packaging proteins gp16 and gp17 are supposed to function as terminases . The recombinant T4 terminase proteins, upon expression in vivo from strong promoters, cleaved plasmid DNA in a sequence-independent manner . Resolution of the cleaved DNA by agarose-gel electrophoresis showed a smear throughout the lane including a fraction that was retained in the well {Bhattacharyya and Rao, Virology 196 (1993) 34-44} . The appearance of a smear in the high-M(r) region could not be explained solely on the basis of a simple random-cutting mechanism . Various hypotheses were tested to elucidate the structure of the high-M(r) DNA . The data show that the high-M(r) DNA did not arise either by attachment of protein(s) to DNA, or by covalent linkage of cleaved DNA molecules by a recombinational mechanism . It appears that the high-M(r) DNA arose as a result of non-covalent linkage of plasmid DNA through single strands . A working model for the action of T4 terminase is presented.

Gene, 1994 Aug 19, 146(1), 39 - 45
Conjugal transfer of cosmid DNA from Escherichia coli to Saccharopolyspora spinosa: effects of chromosomal insertions on macrolide A83543 production; Matsushima P et al.; Cosmid pOJ436, containing large inserts of Saccharopolyspora spinosa (Ss) DNA, was transferred by conjugation from Escherichia coli to Ss an integrated into the chromosome, apparently by homologous recombination, at high frequencies (10(-5) to 10(-4) per recipient) . Transfer was mediated by the plasmid RP4 (RK2) transfer functions in E . coli, and the RK2 oriT function located on pOJ436 {Bierman et al., Gene 116 (1992) 43-49} . pOJ436 lacking Ss DNA, or containing a small insert (approx . 2 kb) of Ss DNA, conjugated from E . coli and integrated at either of two bacteriophage phi C31 attB sites at low frequency (approx . 10(-7) per recipient) . Exconjugants containing homologous inserts or inserts at the phi C31 attB sites were stable in the absence of antibiotic selection, and most produced control levels of tetracyclic macrolide A83543 factors . Some exconjugants contained similar kinds of large deletions and were defective in macrolide production.

Biochim Biophys Acta, 1994 Aug 17, 1207(2), 236 - 48
Identification of antigenic sites on the Na+/K(+)-ATPase beta-subunit: their sequences and the effects of thiol reduction upon their structure; Sun Y et al.; In contrast to the catalytic (alpha) subunit of the Na+/K(+)-ATPase holoenzyme, the glycoprotein (beta) subunit has proven to be a poor antigen for monoclonal antibody (Mab) production . However, in this work six Mabs directed against the beta-subunit of the lamb kidney holoenzyme have been isolated . These Mabs all recognize the holoenzyme, but their 'in solution' binding affinities for deglycosylated enzyme or isolated beta are generally at least 10-fold higher . Species specificity mapping, antibody patterns of binding to beta-fragments and competition binding studies indicated that there were only three distinct epitopes, with two antibodies binding in the NH2-terminal half (epitopes I and II) and 4 Mabs binding at the same or overlapping site (III) in the -COOH terminal half of beta . DNA sequence analysis of isolated collections of bacteriophage M13 that contain a 15 amino-acid 'epitope library' insert in the pIII protein, which enables them to bind to the antibodies, revealed the residues KYRDS (amino acids 111-115) and LETYP (amino acids 197-201) to be the deduced sequences for the epitopes of Mabs M19-P7-E5 (II) and M17-P5-F11 (III), respectively . The epitope I site was not, however, identified . Further studies showed that antibody binding to these three determinant sites had no affect on the Na+/K(+)-ATPase and K(+)-stimulated p-nitrophenylphosphatase (pNPPase) activities of either holoenzyme or deglycosylated enzyme, nor any affect on the cation- (Na+, K+ or Mg2+) and ouabain-induced conformational changes monitored with FITC-labeled deglycosylated enzyme . Interestingly, anti-beta Mab access to the three epitopes was increased following beta-mercaptoethanol inactivation of the holoenzyme, but this thiol reduction abolished the binding of two conformation-sensitive anti-alpha Mabs to the enzyme . These results are consistent with the previous suggestion of Kirley ((1990) J . Biol . Chem . 265, 4227-4232) that the beta-disulfide linkages not only maintain beta-structure but they are critical for maintaining alpha-conformation and holoenzyme activity.

EMBO J, 1994 Aug 15, 13(16), 3909 - 16
Requirement for a zinc motif for template recognition by the bacteriophage T7 primase; Mendelman LV et al.; Gene 4 of bacteriophage T7 encodes two proteins, a 63 kDa and a colinear 56 kDa protein . The coding sequence of the 56 kDa protein begins at the residues encoding an internal methionine located 64 amino acids from the N-terminus of the 63 kDa protein . The 56 kDa gene 4 protein is a helicase and the 63 kDa gene 4 protein is a helicase and a primase . The unique 7 kDa N-terminus of the 63 kDa gene 4 protein is essential for primer synthesis and contains sequences with homology to a Cys4 metal binding motif, Cys-X2-Cys-X17-Cys-X2-Cys . The zinc content of the 63 kDa gene 4 protein is 1.1 g-atom/mol protein, while the zinc content of the 56 kDa gene 4 protein is < 0.01, as determined by atomic absorption spectrometry . A bacteriophage deleted for gene 4, T7 delta 4-1, is incapable of growing on Escherichia coli strains that contain plasmids expressing gene 4 proteins with single amino acid substitutions of Ser at each of the four conserved Cys residues (efficiency of plating, 10(-7)) . Primase containing a substitution of the third Cys for Ser has been overexpressed in E . coli and purified to homogeneity . This mutant primase cannot catalyze template-directed synthesis of oligoribonucleotides although it is able to catalyze the synthesis of random diribonucleotides in a template-independent fashion . The mutant primase has reduced helicase activity although it catalyzes single-stranded DNA-dependent hydrolysis of dTTP at rates comparable with wild type primase . The zinc content of the mutant primase is 0.5 g-atom/mol protein.

J Mol Biol, 1994 Aug 12, 241(2), 208 - 13
Structure of a foreign peptide displayed on the surface of bacteriophage M13; Kishchenko G et al.; The use of filamentous bacteriophage M13 as a vehicle for display of foreign peptides and proteins provides a means for the construction of therapeutic, diagnostic and technological tools of broad utility . The usefulness of this technology is dependent on the ability of an inserted peptide to act as a ligand when fused to a structural protein . This, in turn, depends on the configuration in which the fused peptide is presented on the surface of the phage . X-ray diffraction from oriented fibers of three M13 strains with different sequences inserted near the amino terminus of the major coat protein (gp8) has been used to demonstrate that the inserts do not affect the helical symmetry of the phage particles . The structure of one insertion mutant (M13BOM2) was analyzed in detail . This strain contains the pentapeptide GQASG inserted between amino acids 4 and 5 of the major coat protein . Analysis of fiber diffraction from this strain was used to obtain its structure to 7 A resolution . Examination of the resulting electron density map indicated that the insert is presented in an extended conformation in a shallow groove between two alpha-helices on the surface of the virion . This arrangement is reminiscent of the presentation of peptides by major histocompatibility antigens . The extended conformation of the peptide provides substantial surface exposure and puts it in a favorable position to act as a ligand in a biochemical process . This form of presentation may contribute to the high immunogenicity observed for peptides inserted into the gene 8 product of M13 . The length of the groove appears to correspond to the upper length limit observed when foreign peptides are fused to all copies of gp8.

J Biol Chem, 1994 Aug 12, 269(32), 20229 - 32
Intron-containing T4 bacteriophage gene sunY encodes an anaerobic ribonucleotide reductase; Young P et al.; The function of the SunY protein, encoded by an intron-containing gene of bacteriophage T4, has remained hitherto unknown in contrast to the extensively studied self-splicing reaction of the SunY intron . Here we show that anaerobic T4 infections of Escherichia coli induce a ribonucleoside triphosphate reductase activity that is 10-30-fold higher than the bacterial host level of the corresponding enzyme . Inactivation of the T4 sunY gene (in this communication renamed nrdD) significantly decreased both the induced activity and the anaerobic production of phage, confirming the role of the T4 NrdD (SunY) protein as a phage-specific anaerobic ribonucleotide reductase . With the identification of the T4 nrdD (sunY) gene product as an anaerobic ribonucleotide reductase, all known bacteriophage introns are found to share the common and as yet unexplained property of residing within genes of DNA metabolism.

FEBS Lett, 1994 Aug 8, 349(3), 429 - 32
The studies of cooperative regions in T7 RNA polymerase; Protasevich II et al.; The heat denaturation of bacteriophage T7 RNA polymerase (T7RNAP) was studied by scanning microcalorimetry . The thermodynamic parameters of the denaturation were estimated within the pH range 6-9 . The analysis of the denaturation curves showed the presence of two cooperative parts of the T7RNAP molecule melting according to the 'all-or-none' principle . The molecular masses of these parts were determined as 22 and 77 kDa . These values are close to the molecular masses of protein domains obtained from X-ray diffraction and limited trypsinolysis data . The smaller N-terminal domain was shown to increase the thermostability of the 'catalytic' C-terminal domain within the intact T7RNAP molecule.

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