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