Nucleic Acids Res, 1989 Sep 25, 17(18), 7333 - 44 In vitro binding of the bacteriophage f1 gene V protein to the gene II RNA-operator and its DNA analog; Michel B et al.; We have investigated the binding of the f1 single-stranded DNA-binding protein (gene V protein) to DNA oligonucleotides and RNA synthesized in vitro . The first 16 nucleotides of the f1 gene II mRNA leader sequence were previously identified as the gene II RNA-operator; the target to which the gene V protein binds to repress gene II translation . Using a gel retardation assay, we find that the preferential binding of gene V protein to an RNA carrying the gene II RNA-operator sequence is affected by mutations which abolish gene II translational repression in vivo . In vitro, gene V protein also binds preferentially to a DNA oligonucleotide whose sequence is the DNA analog of the wild-type gene II RNA-operator . Therefore, the gene V protein recognizes the gene II mRNA operator sequence when present in either an RNA or DNA context. J Biol Chem, 1989 Sep 25, 264(27), 16059 - 66 Structural studies of mutants of T4 lysozyme that alter hydrophobic stabilization; Matsumura M et al.; Multiple replacements at amino acid position 3 of bacteriophage T4 lysozyme have shown that the conformational stability of the protein is directly governed by the hydrophobicity of the residue substituted (Matsumura, M., Becktel, W . J., and Matthews, B . W . (1988) Nature 334, 406-410) . Of the 13 mutant lysozymes made by site-directed mutagenesis, two variants, one with valine (I3V) and the other with tyrosine (I3Y), were crystallized and their structures solved . In this report we describe the crystal structures of these variants at 1.7 A resolution . While the structure of the I3V mutant is essentially the same as that of wild-type lysozyme, the I3Y mutant has substantial changes in its structure . The most significant of these are that the side chain of the tyrosine is not accommodated within the interior of the protein and the amino-terminal polypeptide (residues 1-9) moves 0.6-1.1 A relative to the wild-type structure . Using coordinates based on the wild-type and available mutant structures, solvent accessible surface area of residue 3 as well as the adjacent 9 residues in the folded form were calculated . The free energy of stabilization based on the transfer of these residues from a fully extended form to the interior to the folded protein was found to correlate well with the protein stability determined by thermodynamic analysis . The enhanced thermostability of the variant Ile-3----Leu, relative to wild-type lysozyme, can also be rationalized by surface-area calculations based on a model-built structure . Noncrystallization of most lysozyme variants at position 3 appears to be due to disruption of intermolecular contacts in the crystal . The Ile-3----Val variant is closely isomorphous with wild-type and maintains the same crystal contacts . In the Ile-3----Tyr variant, however, a new set of contacts is made in which direct protein-protein hydrogen bonds are replaced by protein-water-protein hydrogen bonds as well as a novel hydrogen bond involving the phenolic hydroxyl of the substituted tyrosine. FEBS Lett, 1989 Sep 25, 255(2), 435 - 40 Irreversible binding of bacteriophage T5 to its FhuA receptor protein is associated with covalent cross-linking of 3 copies of tail protein pb4; Feucht A et al.; Irreversible binding of bacteriophage T5 to its FhuA receptor protein is characterized by a high activation energy, typical for reactions where covalent bonds are formed {Zarnitz, M.L . and Weidel, W . (1963) Z . Naturforsch . 18b, 276-280} . Upon binding of radiolabeled T5 phages to FhuA formation of a new protein of 250 kDa was observed . Using electrophoretical and Western blotting techniques this protein was shown to be formed by cross-linking of 3 copies of tail protein pb4, rather than by cross-linking of FhuA and the receptor-binding protein. Nature, 1989 Sep 21, 341(6239), 255 - 7 Phasing of protein-induced DNA bends in a recombination complex; Snyder UK et al.; Many of the structures responsible for replication, transcription initiation and recombination arise from complex sets of protein-protein interactions and the folding of DNA in three dimensions, with protein-induced bending of DNA often playing an integral role . The magnitude and orientation of DNA bending induced by various single proteins has been estimated by gel mobility shift methods and by modelling of crystallographic data . The site-specific recombination by which bacteriophage lambda (phage lambda) integrates into the chromosome of its host Escherichia coli requires a host protein, 'integration host factor' (IHF), which is known to be able to bend the DNA to which it binds . To determine the three-dimensional path of DNA within the higher order structure responsible for phage lambda site-specific recombination, we have determined the relative direction of IHF-induced bending at each of the three binding sites within the complex . IHF, which appears to bend DNA by more than 140 degrees, is a major determinant of the DNA path in the recombination complex and is also involved in a wide range of other cellular events. Nature, 1989 Sep 21, 341(6239), 251 - 4 Functional replacement of a protein-induced bend in a DNA recombination site; Goodman SD et al.; In recent years the capacity of proteins to bend DNA by binding to specific sites has become a widely appreciated phenomenon . In many cases, the protein-DNA interaction is known to be functionally significant because destruction of the DNA site or the protein itself results in an altered phenotype . An important question to be answered in these cases is whether bending of DNA is important per se or is merely a consequence of the way a particular protein binds to DNA . Here we report direct evidence from the bacteriophage lambda integration system that a bend introduced by a protein is intrinsically important . We find that a binding site for a specific recombination protein known to bend DNA can be successfully replaced by two other modules that also bend DNA; related modules that fail to bend DNA are ineffective. Biochemistry, 1989 Sep 19, 28(19), 7829 - 42 RNA chain initiation by Escherichia coli RNA polymerase . Structural transitions of the enzyme in early ternary complexes; Krummel B et al.; We have studied the properties and structures of a series of Escherichia coli RNA polymerase ternary complexes formed during the initial steps of RNA chain initiation and elongation . Five different templates were used that contained the bacteriophage T7 A1 promoter or the E . coli Tac or the lac UV5 promoter, as well as variant templates with alterations in the initial transcribed regions . The majority of ternary complexes bearing short transcripts (from two to nine nucleotides) are highly unstable and cannot be easily studied . This includes transcripts from the phage T7 A1 promoter, for which the stability of complexes bearing transcripts as short as four nucleotides has previously been postulated . However, with one Tac promoter template, RNA polymerase forms ternary complexes with transcripts as short as five nucleotides that are stable enough for biochemical study . We describe several approaches to identifying and isolating such stable complexes and show that stringent criteria are needed in carrying out such experiments if the results are to be meaningful . Deoxyribonuclease I (DNase I) footprinting has been used to probe the general structure of the stable ternary complexes formed as the polymerase begins transcription and moves away from the start site . The enzyme undergoes a sequence of structural changes during initiation and transition to an elongating complex . Complexes with five to eight nucleotide transcripts, designated initial transcribing complexes (ITC), have identical footprints; they all retain the sigma factor and have a slightly extended DNase I footprint (-57 to +24) as compared to the open promoter complex (-57 to +20) . ITC complexes all show a region of marked DNase I hypersensitivity in the -25 region that may reflect bending or distortion of the DNA template . Complexes with 10 or 11 nucleotide transcripts, designated initial elongating complexes (IEC), have lost the sigma factor and have a slightly reduced and shifted DNase I footprint (-32 to +30) . However, these IEC have not yet achieved the much smaller footprint (approximately 30 bp) reported as characteristic of elongating ternary complexes bearing longer RNA chains . During the initial phase of transcription, the RNA polymerase does not move monotonically along the DNA template as RNA chains are extended, but instead, the upstream and downstream contacts remain more or less fixed as the nascent transcript is elongated up to about eight nucleotides in length . Only after incorporation of 10 nucleotides is there significant movement of the enzyme away from the promoter region and a commitment to elongation. Biochemistry, 1989 Sep 19, 28(19), 7781 - 8 Release of the sigma subunit from Escherichia coli RNA polymerase transcription complexes is dependent on the promoter sequence; Stackhouse TM et al.; The sigma subunit of bacterial RNA polymerase is required for the specific initiation of transcription at promoter sites . However, sigma is released from the transcription complex shortly after transcription is initiated, and elongation proceeds in the absence of sigma . In order to study the position of sigma release, we have developed a method to quantify the photoaffinity labeling produced by an aryl azide positioned at the leading (5'-) end of nascent RNA, as a function of the transcript length {Stackhouse, T.M., & Meares, C.F . (1988) Biochemistry 27, 3038-3045} . Here we compare photoaffinity labeling of transcription complexes containing three natural bacteriophage promoters (lambda PR, lambda PL, and T7 A1) and two recombinant constructs, A1/PR (T7 A1 promoter with the lambda PR transcribed region) and PR/A1 (lambda PR promoter with the T7 A1 transcribed region) . Significant photoaffinity labeling of the sigma subunit was observed only on the templates containing the lambda PR promoter region, regardless of the sequence of the transcribed region . These results indicate the molecular interactions responsible for the position of sigma release from the transcription complex mainly involve the nucleotide sequence of the promoter region--rather than the transcribed region--of the DNA template . Further studies on transcription complexes containing the A1/PR and the PR/A1 templates were performed, using polyclonal antibodies against the holoenzyme or against the sigma subunit . These experiments corroborate the promoter dependence of sigma release . They also show a correlation between the release of sigma and stable binding of the transcript by the transcription complex. J Biol Chem, 1989 Sep 15, 264(26), 15268 - 73 Cloning and expression of a synthetic gene for Cerebratulus lacteus neurotoxin B-IV; Howell ML et al.; A synthetic gene encoding Cerebratulus lacteus neurotoxin B-IV has been designed, cloned, and expressed in Escherichia coli . Although expression of the toxin alone appears to be incompatible with host viability, large amounts could be synthesized as a fusion protein with either E . coli beta-galactosidase or the gene 9 protein of bacteriophage T7, the latter system being the more efficient . The fusion protein has been purified, and, after Factor Xa-catalyzed hydrolysis at a customized linker site, we have obtained the equivalent of 12 mg of pure toxin B-IV per liter of bacterial culture . The recombinant protein is identical with B-IV isolated from Cerebratulus with respect to high performance liquid chromatography mobility and secondary structure, and its amino acid composition differs only by the presence of an amino-terminal methionine residue and replacement of Hyp10 by Pro . Quantal bioassay indicates that the cloned protein is comparable to the natural toxin in specific toxicity . The small differences observed in comparing the activities of the two proteins are most likely due to the presence of the methionine extension at the amino terminus of the recombinant, although lack of hydroxylation of Pro10 may also contribute. Biochemistry, 1989 Sep 5, 28(18), 7262 - 7 Distamycin paradoxically stimulates the copying of oligo(dA).poly(dT) by DNA polymerases; Levy A et al.; Distamycin A, a polypeptide antibiotic, binds to dA.dT-rich regions in the minor groove of B-DNA . By virtue of its nonintercalating binding, distamycin acts as a potent inhibitor of the synthesis of DNA both in vivo and in vitro . Here we report that distamycin paradoxically stimulates Escherichia coli DNA polymerase I (pol I), its large (Klenow) fragment, and bacteriophage T4 DNA polymerase to copy oligo(dA).poly(dT) in vitro . It is found that distamycin increases the maximum velocity (Vmax) of the extension of the oligo(dA) primer by pol I without affecting the Michaelis constant (Km) of the primer . Gel electrophoresis of the extended primer indicates that the antibiotic specifically increases the rate of addition of the first three dAMP residues . Lastly, in the presence of both distamycin and the oligo(dT)-binding protein factor D, which increases the processivity of pol I, a synergistic stimulation of polymerization is attained . Taken together, these results suggest that distamycin stimulates synthesis by increasing the rate of initiation of oligo(dA) extension . The stimulatory effect of distamycin is inversely related to the stability of the primer-template complex . Thus, maximum stimulation is exerted at elevated temperatures and with shorter oligo(dA) primers . That distamycin increases the thermal stability of {32P}(dA)9.poly(dT) is directly demonstrated by electrophoretic separation of the hybrid from dissociated {32P}(dA)9 primer . It is proposed that by binding to the short primer-template duplex, distamycin stabilizes the oligo(dA).poly(dT) complex and, therefore, increases the rate of productive initiations of synthesis at the primer terminus. Biochemistry, 1989 Sep 5, 28(18), 7139 - 43 Equilibrium dissociation and unfolding of the Arc repressor dimer; Bowie JU et al.; The equilibrium unfolding reaction of Arc repressor, a dimeric DNA binding protein encoded by bacteriophage P22, can be monitored by fluorescence or circular dichroism changes . The stability of Arc is concentration dependent, and the unfolding reaction is well described as a two-state transition from folded dimer to unfolded monomer . The stability of the protein is decreased at low pH and increased by high salt concentration . The salt dependence suggests that two ions bind preferentially to the folded protein . In 10 mM potassium phosphate (pH 7.3) and 100 mM KCl, the unfolding free energy reaches a maximum near room temperature . The results suggest that at the low protein concentrations where operator DNA binding is normally measured, Arc is predominantly monomeric and unfolded. Biochemistry, 1989 Sep 5, 28(18), 7409 - 17 Site-specific mutagenesis of T4 gene 32: the role of tyrosine residues in protein-nucleic acid interactions; Shamoo Y et al.; Bacteriophage T4 gene 32 encodes a single-stranded DNA (ssDNA) binding protein (gp32) required for T4 DNA replication, recombination, and repair . Previous physicochemical studies on gp32 and other ssDNA binding proteins have suggested that binding may involve hydrophobic interactions that result from the close approach of several aromatic amino acid side chains with the nucleic acid bases . In the case of gp32, five tyrosines and two phenylalanines have previously been implicated in gp32.ssDNA complex formation . Site-directed mutagenesis of T4 gene 32 was employed to produce a set of eight gp32 mutant proteins, each of which encoded a single substitution at one of the eight tyrosine residues within gp32 . The mutant gp32 proteins were then subjected to physicochemical analysis to evaluate the role of each tyrosine residue in gp32 structure and function . Oligonucleotide binding studies suggest that tyrosine residues 84, 99, 106, 115, and 186 each contribute from 0.3 to 0.7 kcal/mol to ssDNA binding, which corresponds to 3-7% of the overall binding energy for gp32.ssDNA complex formation . Replacement of tyrosine residues 73 and 92 appears to lead to large structural changes that may be the result of disrupting the zinc binding subdomain within gp32. J Mol Biol, 1989 Sep 5, 209(1), 91 - 100 In vitro packaging of bacteriophage phi 29 DNA restriction fragments and the role of the terminal protein gp3; Grimes S et al.; Restriction fragments of bacteriophage phi 29 DNA-gp3 (DNA-gene product 3 complex) were packaged in a completely defined in vitro system that included purified proheads, the DNA packaging protein gp16 and ATP . Both left and right end DNA-gp3 fragments were packaged in this system, in contrast to the oriented and selective packaging of left end DNA-gp3 fragments in extracts; left ends could be packaged quantitatively in the defined system, while the packaging efficiency of right ends was generally about threefold lower . In addition, certain internal (non-end) DNA fragments were packaged at efficiencies of about 10% to 15% . Digestion of the gp3 with trypsin or proteinase K reduced the packaging of whole-length DNA by a factor of 2 or 4, respectively, and removal of the gp3 from whole-length DNA or end fragments with piperidine reduced packaging to the level of internal fragments . Though the terminal protein gp3 was non-essential for DNA translocation in the defined system, it stimulated packaging of left and right end fragments, and stabilized packaging of the left end . The packaging of end and internal DNA fragments of the related phage M2Y into phi 29 proheads was similar to that of phi 29 DNA fragments, and certain fragments of lambda DNA were packaged at the efficiency of the internal phi 29 DNA fragments . Selective packaging of DNA-gp3 left ends was restored by the addition of bacterial cell extracts or glycerol to the defined system, and these packaging conditions discriminated between phi 29 and M2Y DNAs that have distinct terminal proteins. J Mol Biol, 1989 Sep 5, 209(1), 55 - 64 Repression of the lambda pcin promoter by integrative host factor; Griffo G et al.; The cin-1 mutation creates a new promoter (pcin) in the tR1 region of bacteriophage lambda . The pcin promoter transcribes the cI repressor gene constitutively . lambda cin-1 does not propagate on Escherichia coli mutants lacking the integrative host factor (IHF) . lambda cI- cin-1 grows normally in IHF- mutants, indicating that repressor overproduction from pcin blocks lytic growth . The presence of an IHF binding site which overlaps the pcin promoter led us to the hypothesis that IHF functions as a repressor of pcin transcription . We find that the pcin promoter is fivefold more active in a host lacking IHF than in wild-type cells . In vitro studies show that IHF directly inhibits transcription initiation at pcin . Abortive initiation and gel retardation assays demonstrate that IHF interferes with the binding of RNA polymerase to the pcin promoter . RNA polymerase bound in an open promoter complex is resistant to IHF . We propose that IHF binding to the pcin promoter region blocks the binding of RNA polymerase to the promoter, either by covering specific nucleotides or by distorting DNA structure. J Mol Biol, 1989 Sep 5, 209(1), 101 - 8 Cleaving the prohead RNA of bacteriophage phi 29 alters the in vitro packaging of restriction fragments of DNA-gp3; Grimes S et al.; In vitro packaging of restriction fragments of the bacteriophage phi 29 DNA-gp3 (DNA-gene product 3 complex) in the defined system was dependent on prohead RNA . Truncated prohead RNAs were obtained by in situ RNase A digestion, isolated and sequenced . Proheads having the intact 174 base RNA were compared to proheads having RNAs of 120, 95, 71, 69 or 54 bases for the capacity to package the DNA-gp3 left and right ends and internal (non-end) fragments generated by the restriction enzymes EcoRI, HpaI and BstNI . Proheads with the 174 or 120 base RNAs packaged both left and right ends; internal fragments were packaged more efficiently by proheads with the 120 base RNA . Proheads with the 95 base RNA packaged DNA-gp3 left ends and internal fragments efficiently, but lost the capacity to package right ends . Only internal fragments were packaged by proheads with the 71 base RNA, and proheads having 69 or 54 base RNAs were inactive . RNA-free proheads were effectively reconstituted with purified 174 and 120 base RNAs to produce particles similar in biological activity to the proheads from which the RNAs were isolated . The 95 base RNA was the smallest RNA of the group that could reconstitute the prohead and direct fragment packaging, although packaging was inefficient . Alteration of the specificity of DNA fragment packaging with truncated prohead RNAs has delineated RNA domains that function in DNA-gp3 recognition and prohead binding. Virology, 1989 Sep, 172(1), 293 - 301 In vitro synthesis of biologically active beet necrotic yellow vein virus RNA; Quillet L et al.; Beet necrotic yellow vein virus (BNYVV) has a quadripartite plus-strand RNA genome in which the two smallest genome components, RNA 3 and 4, are not necessary for virus multiplication in leaves . Infectious transcripts of BNYVV RNA 3 and 4 have already been described (V . Ziegler-Graff, S . Bouzoubaa, I . Jupin, H . Guilley, G . Jonard, and K . Richards (1988) J . Gen . Virol . 69, 2347-2357) . In this paper we describe synthesis of a full-length RNA-1 transcript by bacteriophage T7 RNA polymerase-directed run-off transcription of cloned viral cDNA . A recombinant plasmid containing a full-length cDNA insert of RNA 2 could not be maintained in Escherichia coli . Therefore full-length transcript of RNA 2 was produced by transcription of cDNA ligation products without amplification in bacteria . When inoculated together to leaves of Chenopodium quinoa or Tetragonia expansa the RNA 1 and 2 transcripts were infectious; they also supported multiplication of the BNYVV RNA 3 and 4 transcripts, providing a totally synthetic inoculum of the virus . In one recombinant clone of RNA 2 a point mutation causing an arginine to serine substitution at position 119 of the viral coat protein was discovered . The mutation was detected because the resulting coat protein had altered electrophoretic mobility . RNA 2 transcripts containing this mutation were infectious but viral RNA was not encapsidated . The mutation also interfered with long distance movement of the virus in spinach, presumably as a consequence of the packaging deficiency. J Gen Microbiol, 1989 Sep, 135 ( Pt 9), 2387 - 98 Inhibition of biological activities of the aerobactin receptor protein in rough strains of Escherichia coli by polyclonal antiserum raised against native protein; Roberts M et al.; The aerobactin iron-uptake system of plasmid ColV-K30, genetically isolated from other plasmid determinants by molecular cloning, was sufficient to restore full virulence in a mouse peritonitis model to a clinical Escherichia coli isolate, D551 (O78:H-), whose resident aerobactin-encoding ColV plasmid had been lost by curing . Antiserum was raised in rabbits against live E . coli K12 cells expressing the outer-membrane aerobactin receptor protein and absorbed with an isogenic strain lacking the receptor . This antiserum inhibited binding of aerobactin, cloacin DF13 and bacteriophage B74K to the native protein in whole E . coli K12 bacteria expressing the receptor, or in membranes prepared from such organisms . However, it did not react with the native receptor protein in several wild strains unless lipopolysaccharide was first removed by treatment with trichloroacetic acid, nor did it protect mice in experimental infections with strain D551 . Antisera raised in rabbits against partially or fully denatured forms of the aerobactin receptor reacted only in assays involving denatured protein; they showed no inhibition of the biological activities of the native receptor. Curr Genet, 1989 Sep, 16(3), 131 - 7 Molecular cloning of chromosome I DNA from Saccharomyces cerevisiae: localization of a repeated sequence containing an acid phosphatase gene near a telomere of chromosome I and chromosome VIII; de Steensma HY et al.; A 17 kb region from near the right end of chromosome I of Saccharomyces cerevisiae was isolated on recombinant lambda bacteriophages . This region contained the PHO11 gene which was located only 3.4 kb from the right end of the chromosome . We found that this region also was repeated approximately 13 kb from the end of the chromosome VIII DNA molecule . The chromosome VIII sequence appears to be a previously unnamed acid phosphatase gene that we propose to call PHO12 . Thus, similar to the repeated SUC, MAL, X and Y' sequences, some members of the repeated acid phosphatase gene family also appear near the termini of yeast chromosomes. Science, 1989 Sep 1, 245(4921), 952 - 8 Enhancement of bacteriophage T4 late transcription by components of the T4 DNA replication apparatus; Herendeen DR et al.; The expression of the late genes in bacteriophage T4 development is closely connected to viral DNA replication . Three T4-encoded DNA polymerase accessory proteins are shown to stimulate transcription at T4 late promoters in an adenosine triphosphate (ATP) hydrolysis-requiring process . The properties of the activation resemble those found for enhancers of eukaryotic transcription . However, the nature of the enhancer of T4 late transcription is novel in that it is a structure--a break in the nontranscribed DNA stand--to which the three replication proteins bind, rather than a sequence . Since the three DNA polymerase accessory proteins are carried on the moving replication fork as part of the replisome, we postulate that viral DNA replication forks act, in vivo, as the mobile enhancers of T4 late gene transcription . Whereas Escherichia coli RNA polymerase bearing the T4 gene 55 protein can selectively recognize T4 late promoters, it is only capable of responding to the transcription-enhancing activity of the three replication proteins on acquiring an additional T4-specific modification. Mutat Res, 1989 Sep, 218(2), 49 - 65 Structure-function studies of the T4 endonuclease V repair enzyme; Dodson ML et al.; Published data on the structure and mechanism of endonuclease V from bacteriophage T4 are reviewed with the objective of developing a working mechanistic model of this enzyme . Endonuclease V is an interesting and important candidate to be the first DNA-repair enzyme to have its structure determined by crystallography, and a more detailed model of the reaction process is needed to mechanistically interpret such a structure . Such a model should be sufficiently detailed to support future investigations of structure/function relationships between the enzyme and the DNA damage repair pathway it initiates, as probed by site-directed mutagenesis techniques and other methods . The early literature is presented in an historical perspective, followed by a description of prior models and biochemical investigations . The biochemical phenotypes of mutants in the enzyme structural gene are discussed . The results of computer analyses aimed at structural interpretations of the protein sequence are given, together with a brief discussion of the strengths and weaknesses of such experiments. J Bacteriol, 1989 Sep, 171(9), 5229 - 31 Characterization of the pleiotropic phenotypes of rifampin-resistant rpoB mutants of Escherichia coli; Jin DJ et al.; We used our collection of 17 sequenced rifampin resistance alleles in rpoB to perform a systematic analysis of the phenotypes historically reported with this class of mutants, including growth phenotype, ability to support the growth of different bacteriophages, ability to maintain the F' episome, interaction with mutant alleles at other loci, sensitivity to uracil, inhibition by 5-fluorouridine, and dominance . We found that mutational changes leading to the same phenotype were often located together and that certain phenotypes were associated with one another. J Bacteriol, 1989 Sep, 171(9), 5222 - 5 Cloning and sequencing of an Escherichia coli gene, nlp, highly homologous to the ner genes of bacteriophages Mu and D108; Choi YL et al.; An nlp (Ner-like protein) gene was isolated from Escherichia coli . The nucleotide sequence of a 1,342-base-pair chromosomal DNA fragment containing the nlp gene was analyzed . It contained two open reading frames; one encoded 91 amino acid residues with an Mr of 10,361, and the other (ORFX) encoded 131 amino acid residues of the carboxyl-terminal region of a truncated polypeptide . The amino acid sequence deduced from the DNA sequence of nlp was highly homologous (62 to 63%) to the Ner proteins of bacteriophages Mu and D108 . The amino-terminal region of Nlp deduced from the complete open reading frame contained a presumed DNA-binding region . The nlp gene was located at 69.3 min on the E . coli genetic map. J Bacteriol, 1989 Sep, 171(9), 5218 - 21 In vitro construction of gshB::kan in Escherichia coli and use of gshB::kan in mapping the gshB locus; Daws T et al.; The Escherichia coli structural gene for glutathione synthetase, gshB, was cloned into pBR322 . Plasmids containing gshB were able to complement the glutathione requirement of a trxA gshB double mutant, and cells containing the plasmids were found to have elevated levels of glutathione synthetase . A mutant gshB allele was constructed by inserting the kan gene from pUC4K into a unique HpaI site located within gshB . The resulting plasmid-encoded allele was used to replace a genomic gshB+ by homologous recombination . The resulting strain had no detectable glutathione synthetase activity . The gshB allele containing the kan insertion was used to map gshB on the E . coli chromosome by P1 transduction . The results indicated that gshB is located at 63.4 min, between metK and speC . The allele was further localized to a region of 3,100 to 3,120 kilobase pairs on the physical map (restriction map) of E . coli by DNA-DNA hybridization to a series of lambda bacteriophages (Y . Kohara, K . Akiyama, and K . Isono, Cell 50:495-508, 1987). J Bacteriol, 1989 Sep, 171(9), 5117 - 26 Import of biopolymers into Escherichia coli: nucleotide sequences of the exbB and exbD genes are homologous to those of the tolQ and tolR genes, respectively; Eick-Helmerich K et al.; Escherichia coli with mutations in the exb region are impaired in outer membrane receptor-dependent uptake processes . They are resistant to the antibiotic albomycin and exhibit reduced sensitivity to group B colicins . A 2.2-kilobase-pair DNA fragment of the exb locus was sequenced . It contained two open reading frames, designated exbB and exbD, which encoded polypeptides of 244 and 141 amino acids, respectively . Both proteins were found in the cytoplasmic membrane . They showed strong homologies to the TolQ and TolR proteins, respectively, which are involved in uptake of group A colicins and infection by filamentous bacteriophages . exbB and exbD were required to complement exb mutations . Osmotic shock treatment rendered exb mutants sensitive to colicin M, which was taken as evidence that the ExbB and ExbD proteins are involved in transport processes across the outer membrane . It is concluded that the exb- and tol-dependent systems originate from a common uptake system for biopolymers. J Bacteriol, 1989 Sep, 171(9), 4785 - 91 Participation of the lytic replicon in bacteriophage P1 plasmid maintenance; Yarmolinsky MB et al.; P1 bacteriophage carries at least two replicons: a plasmid replicon and a viral lytic replicon . Since the isolated plasmid replicon can maintain itself stably at the low copy number characteristic of intact P1 prophage, it has been assumed that this replicon is responsible for driving prophage replication . We provide evidence that when replication from the plasmid replicon is prevented, prophage replication continues, albeit at a reduced rate . The residual plasmid replication is due to incomplete repression of the lytic replicon by the c1 immunity repressor . Incomplete repression was particularly evident in lysogens of the thermoinducible P1 c1.100 prophage, whose replication at 32 degrees C remained almost unaffected when use of the plasmid replicon was prevented . Moreover, the average plasmid copy number of P1 in a P1 c1.100 lysogen was elevated with respect to the copy number of P1 c1+ . The capacity of the lytic replicon to act as an auxiliary in plasmid maintenance may contribute to the extraordinary stability of P1 plasmid prophage. J Bacteriol, 1989 Sep, 171(9), 4595 - 602 Genetic analysis of bacteriophage N4 adsorption; Kiino DR et al.; We isolated six mutants of Escherichia coli K-12 that were defective in bacteriophage N4 adsorption . We mapped the mutations to four loci designated nfrA through nfrD (N four resistance) . nfrA and nfrB were tightly linked to each other and were mapped to min 12 of the E . coli linkage map . nfrC was mapped to min 85, and nfrD was mapped between min 44 and 58 . We isolated a clone carrying both nfrA and nfrB and identified its gene products through maxicell analysis of plasmid subclones . The nfrA gene product was an outer membrane protein of 96,000 apparent molecular weight, whereas nfrB encoded an inner-membrane protein of 69,500 apparent molecular weight . The nfrB1 mutation did not affect the export of the nfrA gene product to the outer membrane and did not affect the alkaline phosphatase activity of an nfrA-phoA fusion . We propose that nfrA encodes the structural receptor for N4 and that the nfrB gene product may be required for irreversible adsorption and injection of the phage genome and virion-encapsulated RNA polymerase through the inner membrane. Infect Immun, 1989 Sep, 57(9), 2612 - 23 Overproduction and purification of Treponema pallidum recombinant-DNA-derived proteins TmpA and TmpB and their potential use in serodiagnosis of syphilis; Schouls LM et al.; We report the construction of expression plasmids carrying two Treponema pallidum genes encoding for the 42-kilodalton membrane protein TmpA (treponemal membrane protein A) and the 34-kilodalton membrane protein TmpB . Using the leftward promoter of bacteriophage lambda, which is controlled by a thermosensitive repressor, we obtained a high level of heat-inducible synthesis of TmpA and TmpB in Escherichia coli K-12 . Both proteins were purified to near homogeneity, and the presence of antibodies to TmpA and TmpB in human sera was determined by an enzyme-linked immunosorbent assay . Whereas in all 44 serum samples from untreated patients in the secondary and early latent stages of syphilis, high levels of anti-TmpA antibodies were detected, only 34 serum samples contained anti-TmpB antibodies . As has been previously observed for TmpA, a correlation was found between the presence of anti-TmpB antibodies and anti-cardiolipin antibodies, suggesting that the level of antibodies to TmpB drops soon after successful antibiotic treatment . We concluded that, in contrast to TmpA, TmpB is not suitable for serodiagnostic purposes as a single antigen, because a significant fraction of sera from syphilitic patients was nonreactive with TmpB. Biotechniques, 1989 Sep, 7(8), 856 - 65 Construction of mRNA genes for the synthesis and translation of duck alpha globin mRNA; Paddock GV; In studies of the effects of changes in mRNA structure and sequence on the initiation of protein synthesis, we used a generally applicable approach to transcribe reconstructed genes for duck alpha A globin by the bacteriophage SP6 RNA polymerase promoter in a pGEM-2 plasmid vector . The genes were reconstructed such that the first nucleotide to be transcribed, the 5' adenosine, was placed directly adjacent to the SP6 promoter sequence . The 3' ends of the genes were constructed such that cleavage with Ssp 1 endonuclease yielded a template that directed the synthesis of mRNA terminating in a poly A tail containing 56 adenosines and a single 3' uridine . Special conditions using a Mn++ buffer were developed to enable the SP6 RNA polymerase to initiate at the 5' adenosine and synthesize the A-start transcription product . The mRNA could be capped and was subsequently used as an effective template for in vitro translation and synthesis of duck alpha A globin. Microbiologia, 1989 Sep, 5(2), 89 - 94 {Ultrastructure of the intracellular development of bacteriophage phi C 31}; Rodriguez A et al.; The intracellular development of the bacteriophage phi C31 in thermally induced cultures of the lysogen Streptomyces coelicolor 01 changes remarkably its cell structure . At 10 min post-induction, a big number of mesosomes are shown by the cells . At 30 min post-induction, the cytoplasm contains capsids which are still empty . At the end of the latent period mature virions are shown and immediately after, cell lysis occurs through the tip of germinative tubes . In old cultures (10 h or more) no viral progeny is detected . However, when the amino acid glycine is added to the culture medium, new virions are seen, but in smaller number than in germinating cultures . These results seem to indicate that the lysis happens at the tip of the germinative tubes probably because this is an area weakened by the preferential growth that takes place on it. Mol Gen Mikrobiol Virusol, 1989 Sep, (9), 20 - 2 {Preparation and characteristics of monoclonal antibodies to DNA-dependent RNA-polymerase of bacteriophage T7}; Degtiarev IL et al.; The monoclonal antibodies to DNA-dependent RNA-polymerase of bacteriophage T7 have been obtained . Twenty of the obtained 500 clones have inhibited the enzyme activity . Three specificity groups were identified for seven of the clones supporting their affinity for different antigenic determinants. Mol Biol (Mosk), 1989 Sep-Oct, 23(5), 1355 - 63 {The family of env genes of avian retroviruses: molecular analysis of Rous sarcoma virus adapted to duck cells}; Ryndich AV et al.; For the elucidation of the molecular basis of RSV adaptation to conditionally permissive host from the genome library of duck embryo fibroblasts, transformed by Rous sarcoma virus in 30 passages on these cells, recombinant bacteriophages that include provirus sequences, were obtained . Complete and transformation-defective proviruses were characterized, nucleotide sequences of their env-genes were compared with their counterparts the original RSV (Pr-RSV-C) and with viruses of other subgroups (A, B, D and E) . The possible relation of the revealed changes in domains coding gp85 and gp37, with the changes of chicken RSV characteristics during adaptation to duck cells is discussed. Virus Res, 1989 Sep, 14(1), 49 - 55 Episomal HPV 16 DNA isolated from a cervical carcinoma presents a partial duplication of the early region; Di Luca D et al.; An invasive cervical carcinoma was found to harbor an episomal variant of human papillomavirus (HPV) type 16 DNA, with a size of about 10.1 kb . A genomic library of the tumor was constructed in bacteriophage lambda and a recombinant phage clone was isolated by screening with HPV 16 probe . Analysis by restriction mapping and Southern hybridization showed that the isolate contained a 2.2 kb duplication of the early region, which included part of E6, all E7 and part of E1 open reading frames . Possible consequences of this duplication for oncogenesis are discussed. Mol Microbiol, 1989 Sep, 3(9), 1159 - 71 Functional domains of bacteriophage Mu transposase: properties of C-terminal deletions; Betermier M et al.; We have generated a series of 3' deletions of a cloned copy of the bacteriophage Mu transposase (A) gene . The corresponding truncated proteins, expressed under the control of the lambda PI promoter, were analysed in vivo for their capacity to complement a super-infecting MuAam phage, both for lytic growth and lysogeny, and for their effect on growth of wild-type Mu following infection or induction of a lysogen . Using crude cell extracts, we have also examined binding properties of these proteins to the ends of Mu . The results allow us to further define regions of the protein important in replicative transposition, establishment of lysogeny and DNA binding. Mol Microbiol, 1989 Sep, 3(9), 1145 - 58 Characterization of amber mutations in bacteriophage Mu transposase: a functional analysis of the protein; Desmet L et al.; We have characterized a series of amber mutations in the A gene of bacteriophage Mu encoding the phage transposase . We tested different activities of these mutant proteins either in a sup0 strain or in different sup bacteria . In conjunction with the results described in the accompanying paper by Betermier et al . (1989) we find that the C-terminus of the protein is not absolutely essential for global transposase function, but is essential for phage growth . Specific binding to Mu ends is defined by a more central domain . Our results also reinforce the previous findings (Betermier et al., 1987) that more than one protein may be specified by the A gene. J Virol, 1989 Sep, 63(9), 3792 - 800 Identification and characterization of a human cytomegalovirus gene coding for a membrane protein that is conserved among human herpesviruses; Lehner R et al.; A rabbit antiserum was raised against envelope material from purified human cytomegalovirus strain AD169 . The serum recognized polypeptides 200, 170, 160, 75, 58, and 45 kilodaltons in size . It was used to screen a cDNA library constructed from poly(A)+ RNA from human cytomegalovirus-infected cells in the expression vector lambda gt11 . A recombinant bacteriophage expressing cytomegalovirus-specific sequences was identified, and the corresponding gene was mapped to the HindIII R fragment . The gene is transcribed into a late 1.5-kilobase RNA . The nucleotide sequence of the coding region was determined . Computer analysis of the gene product revealed a polypeptide containing multiple potential membrane-spanning domains, representing a type of protein not identified in the envelope of herpesviruses before . The protein shows homology on the amino acid level to hypothetical proteins from reading frames BBRF3 of Epstein-Barr virus, UL10 of herpes simplex virus type 1, and ORF50 of varicella-zoster virus . By using an antiserum raised against procaryote-expressed parts of the cytomegalovirus membrane protein, a 45-kilodalton structural component of the virus was identified as the gene product. Gene, 1989 Sep 1, 81(1), 17 - 24 The rex genes of bacteriophage lambda can inhibit cell function without phage superinfection; Snyder L et al.; The rexA and rexB genes of bacteriophage lambda are expressed from the prophage and cause the exclusion of many superinfecting mutant phages . We cloned the rexA and rexB genes into a multicopy plasmid so that they were overexpressed from the inducible tac promoter . No obvious phenotypes were associated with overexpressing both rexA and rexB or overexpressing rexA in the absence of rexB expression . However, induction of rexA in the presence of limiting rexB activity caused an immediate cessation of cell growth . All macromolecular synthesis abruptly ceased and amino acid transport was severely inhibited . Intracellular levels of adenosine 5'-triphosphate also dropped . These phenotypes are similar to those observed after phage superinfection, leading us to propose that at least some of the exclusion caused by the Rex proteins could be due to a change in their ratio following superinfection. J Bacteriol, 1989 Sep, 171(9), 4938 - 44 Translesion synthesis is the main component of SOS repair in bacteriophage lambda DNA; Defais M et al.; Agents that interfere with DNA replication in Escherichia coli induce physiological adaptations that increase the probability of survival after DNA damage and the frequency of mutants among the survivors (the SOS response) . Such agents also increase the survival rate and mutation frequency of irradiated bacteriophage after infection of treated bacteria, a phenomenon known as Weigle reactivation . In UV-irradiated single-stranded DNA phage, Weigle reactivation is thought to occur via induced, error-prone replication through template lesions (translesion synthesis {P . Caillet-Fauquet, M: Defais, and M . Radman, J . Mol . Biol . 117:95-112, 1977}) . Weigle reactivation occurs with higher efficiency in double-stranded DNA phages such as lambda, and we therefore asked if another process, recombination between partially replicated daughter molecules, plays a major role in this case . To distinguish between translesion synthesis and recombinational repair, we studied the early replication of UV-irradiated bacteriophage lambda in SOS-induced and uninduced bacteria . To avoid complications arising from excision of UV lesions, we used bacterial uvrA mutants, in which such excision does not occur . Our evidence suggests that translesion synthesis is the primary component of Weigle reactivation of lambda phage in the absence of excision repair . The greater efficiency in Weigle reactivation of double-stranded DNA phage could thus be attributed to some inducible excision repair unable to occur on single-stranded DNA . In addition, after irradiation, lambda phage replication seems to switch prematurely from the theta mode to the rolling circle mode. Nucleic Acids Res, 1989 Aug 25, 17(16), 6485 - 97 Efficient initiation of mammalian mRNA translation at a CUG codon; Dasso MC et al.; Nucleotide substitutions were made at the initiation codon of an influenza virus NS cDNA clone in a vector carrying the bacteriophage T7 promoter . When capped mRNA transcripts of these constructs were translated in the rabbit reticulocyte lysate, a change in the initiation codon from...AUAAUGG...to...AUACUGG...reduced the in vitro translational efficiency by only 50-60%, and resulted in only a small increase in the yield of short products presumed to be initiated at downstream sites . Synthesis of the full-length product was initiated exclusively at the mutated codon, with negligible use either of in-frame upstream CUG or GUG codons, or of an in-frame downstream GUG codon . We conclude that CUG has the potential to function as an efficient initiation codon in mammalian systems, at least in certain contexts. J Biol Chem, 1989 Aug 25, 264(24), 14440 - 6 Altered expression of the bacteriophage T4 gene 41 (primase-helicase) in an Escherichia coli rho mutant; Hinton DM; Bacteriophage T4 gene 41 protein is an essential replication protein, part of the primase-helicase required for lagging strand DNA synthesis . In a T4+ infection, 41 RNA is first expressed as a polycistronic transcript attached to the upstream RNA of genes uvsX (recombination protein) and 40 (stimulates head formation (Hinton, D . M . (1989) J . Biol . Chem . 264, 14432-14439) . As infection proceeds, less of the upstream RNA extends into gene 41 due to an RNA 3' end, approximately equal to 60 bases downstream of uvsX . DNA sequence analysis of this region positions this end within gene 40, immediately after a GC-rich hairpin . This end probably arises from host factor-dependent transcription termination or RNA processing since it is observed in RNA expressed by a uvsX-40-41 plasmid in vivo, but is not seen after in vitro transcription with purified Escherichia coli RNA polymerase . The E . coli transcription termination (rho) mutant rho026 has been characterized as a rho mutation whose terminating activity is not effectively overcome by phage lambda antitermination (Das, A., Gottesman, M . E., Wardwell, J., Trisler, P., and Gottesman, S . (1983) Proc . Natl . Acad . Sci . U . S . A . 80, 5530-5534) . During a T4+ abortive infection of rho026, the levels of some phage proteins, including 41, are depressed; a T4 phage mutant in goF gives wild type protein patterns in rho026 (Stitt, B . L., and Mosig, G . (1989) J . Bacteriol., in press) . The RNA analyses presented here demonstrate that the severalfold decrease in 41 protein in rho026 is accompanied by a similar decrease in 41 RNA . There is both a general reduction in polycistronic uvsX-40-41 RNA and a 2-2.5-fold increase in the proportion of uvsX RNA ending at the 3' end . Infection of rho026 by T4 goF1 returns the relative amount of RNA reading into 41 versus that stopped to near a wild type level . These results suggest that host rho and the T4 goF are involved in the expression of T4 41 RNA. J Biol Chem, 1989 Aug 25, 264(24), 14432 - 9 Transcript analyses of the uvsX-40-41 region of bacteriophage T4 . Changes in the RNA as infection proceeds; Hinton DM; The bacteriophage T4 genes uvsX (recombination protein), 40 (stimulates head formation), and 41 (DNA replication protein, part of the primase-helicase) are located together on the T4 genome (5'----3' uvsX-40-41) . Previous analyses have indicated that all three proteins are expressed within 5 min after infection and that the level of 41 protein is less than that of uvsX . The mapping of transcripts from this region (reported here) shows that this expression arises from polycistronic messages detected between 2-4 min after infection, a time when phage-encoded factors are beginning to alter the host transcriptional apparatus . Major RNA 5' ends, 900 and 200 bases upstream of uvsX, show homology with previously deduced T4 transcription sites dependent on the T4 transcription factor motA (Guild, N., Gayle, M., Sweeney, R., Hollingsworth, T., Modeer, T., and Gold, L . (1988) J . Mol . Biol . 199, 241-258) . Analysis of the 3' end of uvsX RNAs shows that initially most transcripts extend through gene 40 and 41, although approximately equal to one-fourth end just past uvsX (within gene 40) . Later, more of the uvsX messages are monocistronic, having 5' ends close to the gene (200 and 55 bases upstream) and having the 3' end within gene 40 . Thus, during infection the level of 41 RNA is lowered relative to uvsX message . Mapping of RNA expressed from an uvsX-40-41 plasmid in an uninfected cell gives 5' ends 700, 450, and 55 bases upstream of uvsX, i.e . positions different from those during T4 infection . This indicates that infection significantly changes the 5' ends for uvsX RNA, either by altering transcription initiation or RNA processing sites . In contrast, the majority of the uvsX RNAs expressed by plasmid in the uninfected cell do end at the stop mapped during infection . Thus, the host alone can produce this 3' end. J Biol Chem, 1989 Aug 25, 264(24), 14246 - 55 Relative efficiency of utilization of promoter and termination sites by bacteriophage T3 RNA polymerase; Sengupta D et al.; Bacteriophage T3 RNA polymerase promoters have been classified as class II and class III on the basis of their relative location in T3 DNA as well as on the function of the protein products encoded by the messages transcribed from them . In the present work, the efficiency of utilization of several class II and class III promoters by bacteriophage T3 RNA polymerase was compared with regard to (a) rate of initiation of transcription as determined by {32P}PPi exchange with GTP; (b) complex formation between polymerase and promoters in the presence of GTP; and (c) competition between different promoters for T3 RNA polymerase in a standard transcription assay . The results of these experiments indicated that the class II promoters at 1.05 and 22.8 T3 map units, whose promoter sequences are remarkably similar to the consensus class III promoter sequences, are nearly as strong as typical class III promoters . In contrast, the class II promoter at 14.3 T3 map units, whose promoter sequence differs from the consensus class III promoter sequence by having a C:G base pair instead of a usual A:T base pair at the -1 position, was considerably weaker than the class III promoter . When the C:G base pair at this position was changed to A:T using site-directed mutagenesis, the rate of initiation of RNA synthesis from the mutant promoter was similar to that of a typical class III promoter . In agreement with this observation, it was observed that changing the A:T base pair at the -1 position of a strong class II promoter, at 1.05 T3 map units, to C:G decreased the rate of RNA synthesis from this promoter by about 65% . These observations indicate that the nucleotide residues at the -1 position play a critical role in determining the efficiency of promoter utilization by T3 RNA polymerase . The two termination sites recognized in vitro by bacteriophage T3 RNA polymerase on the T3 genome have been cloned, sequenced, and mapped . Analysis of the DNA nucleotide sequence surrounding the termination site at 59.7 map units indicated that the putative RNA transcript arising from this region can be arranged into a GC-rich stem-loop structure followed by a U-rich 3' tail . However, a major fraction of T3 RNA polymerase molecules read through this terminator in vitro to transcribe regions of T3 DNA beyond this terminator . In contrast to termination at 59.7 map units, termination of transcription at 100 T3 map units does not occur in response to any putative terminator structure or sequence.(ABSTRACT TRUNCATED AT 400 WORDS) J Biol Chem, 1989 Aug 25, 264(24), 14415 - 23 Nearest neighbor influences on DNA polymerase insertion fidelity; Mendelman LV et al.; The kinetics of forming all possible single base substitution errors are measured for Drosophila melanogaster DNA polymerase alpha and avian myeloblastosis virus reverse transcriptase . Seventeen sites along bacteriophage M13 DNA are investigated so that effects of nearest neighbor base stacking on misinsertion kinetics can be evaluated . Polymerase alpha appears to be more error prone than reverse transcriptase . Polymerase alpha forms transversion mispairs at rates comparable to transition mispairs with two exceptions; A.A and C.C are formed with significantly higher and lower efficiencies, respectively . Reverse transcriptase forms transversions with lower efficiencies than transitions, especially low being A.G, G.G, and C.C . For both enzymes, misinsertion frequencies vary typically by 10-fold for the same mispair in different locations . Misinsertion frequency can be expressed as a product of two components, one based on Km and the other on Vmax . DNA polymerase alpha appears to use primarily Km discrimination (100-5000-fold) to achieve insertion fidelity while reverse transcriptase shows a greater balance between Km and Vmax discrimination . Nearest-neighbor base stacking interactions appear to have opposite effects on the two discrimination components . The 5'-nearest neighbor influence on Km is greater for correct insertions than for incorrect, while the influence on Vmax is greater for the incorrect base . Target sites that have pyrimidine as the 5'-nearest neighbor to incoming nucleotides show a higher than average misinsertion component based on Km, but a lower than average component based on Vmax . Conversely, target sites with nearest neighbor purines have a higher than average Vmax component . These results imply that nucleotide misinsertion "hot spots" will occur next to pyrimidines when Km discrimination is dominant and next to purines when Vmax discrimination is dominant . When Vmax and Km discrimination components have similar magnitudes, nearest neighbor effects tend to cancel thereby reducing the effects of base stacking on insertion error rates. J Mol Biol, 1989 Aug 20, 208(4), 615 - 22 Bacteriophage P1 tail-fibre and dar operons are expressed from homologous phage-specific late promoter sequences; Guidolin A et al.; Two plasmid systems, containing the easily assayable galK and lacZ functions, were employed to study the regulation of the bacteriophage P1 tail-fibre and dar operons . Various P1 DNA fragments carrying either the 5' end of lydA (the 1st gene in the dar operon) or the tail-fibre gene 19 precede the promoterless coding region of galK or were fused, in-frame, to the lacZ gene . In the presence of an induced P1 prophage, GalK and LacZ activities were both detected after a 20 to 30 minute lag period, indicating that the dar and tail-fibre operons are expressed from positively regulated, late promoters . The corresponding DNA operons are expressed from positively regulated, late promoters . The corresponding DNA region of the closely related p15B plasmid exhibits comparable promoter properties . Deletion analysis mapped the promoter of a gene 19-lacZ fusion to a DNA region upstream from gene R, an open reading frame that precedes the coding frame of gene 19 . The tail-fibre gene thus forms the second gene in a three gene operon (genes R, 19 (S) and U) . Sequence comparison between this promoter region, upstream sequences of the lydA gene and the corresponding portions of the p15B genome allowed the identification of a highly conserved 38 base-pair sequence, which most likely represents a P1-specific late promoter . This was confirmed by 5' mapping of P1 mRNA . Transcription of both the tail-fibre and dar operons is initiated at sites five and six base-pairs, respectively, downstream from the first conserved nucleotide of this sequence . The conserved motif consists of a standard Escherichia coli -10 region followed by a nine base-pair palindromic sequence located centrally about position -22. J Mol Biol, 1989 Aug 20, 208(4), 517 - 36 Bacteriophage T4 early promoter regions . Consensus sequences of promoters and ribosome-binding sites; Liebig HD et al.; Twenty-nine early promoters from bacteriophage T4 and 14 early promoters from bacteriophage T6 were isolated using vector M13HDL17, a promoterless derivative of M13mp8 carrying a linker sequence, the bacteriophage lambda-terminator tR1, and the lacZ' gene including part of its ribosome-binding site . The consensus sequence for the T4 promoters is: (sequence; see text) . Ribosome-binding sites of T4 share the sequence: 5'...g.GGAga..aA.ATGAa.a...3' The consensus sequence of the T4 early promoter regions is significantly different in sequence and length from that of Escherichia coli promoters . Only one of the promoters detected with vector M13HDL17 resembled a typical bacterial promoter . The high information content raises the possibility that additional proteins recognize and contact nucleotides within the promoter region . All T4 early promoters also carry DNA sequences that could support DNA curving, a structural feature that might contribute to promoter recognition. Nature, 1989 Aug 17, 340(6234), 575 - 6 Exon shuffling by recombination between self-splicing introns of bacteriophage T4; Hall DH et al.; The organization of genes into exons separated by introns may permit rapid evolution of protein-coding sequences by exon shuffling . Introns could provide non-coding targets for recombination, which would then give rise to novel combinations of exons . Evidence to support this theory is indirect and consists of examples of homologous domains of protein structure encoded in different genes, with introns in conserved positions at the boundaries of these domains . Here, we report the first direct evidence for exon shuffling . Two spontaneous deletion mutations of phage T4 have been characterized by sequencing, and they are clearly the result of recombination between homologous regions of two self-splicing group I introns . As a result of the recombination, exons of different genes are transcribed together, with a hybrid intron between them . One of these introns is proficient in self-splicing. Gene, 1989 Aug 15, 80(2), 369 - 74 Inducible high expression of the Escherichia coli infC gene subcloned behind a bacteriophage T7 promoter; Muralikrishna P et al.; The gene for Escherichia coli translational initiation factor 3 (infC) has been inserted into an overexpression plasmid under the control of the bacteriophage T7 promoter . The infC plasmid was then used to transform a host with a chromosomal T7 RNA polymerase gene controlled by the lacUV5 promoter . Induction of T7 RNA polymerase expression in the host cells resulted in a 200-fold overexpression of infC mRNA and a 100-fold overproduction of initiation factor 3 . Rapid batch purification of biologically active IF3 yielded predominantly the long form of IF3, implying that the short form is an artifact of purification by traditional methods. Anal Biochem, 1989 Aug 15, 181(1), 12 - 7 Rapid isolation of high-molecular-weight DNA from agarose gels; Pollman MJ et al.; We have developed a simple, reliable, and rapid method for recovering DNA from agarose gels . While many methods for DNA extraction have already been described, few provide quantitative recovery of large DNA molecules . These procedures generally require costly apparatus, extended elution times, or considerable handling of the sample after elution . Our method employs a novel electroelution chamber constructed from acrylic plastic . Gel slices containing DNA are placed in the chamber between platinum electrodes . Voltage is applied and a continuous flow of buffer sweeps the eluted DNA from the chamber into an external receptacle . Elution is complete in 7 min . Concentrated DNA is obtained by butanol extraction and alcohol precipitation in 1 h . Recoveries, quantitated by counting radiolabeled DNA or by densitometry of analytical gels, were 94 to 100% for fragments of 4 to 50 kb . The eluted DNA was undegraded and could be digested with restriction enzymes, ligated, end-labeled, or used to transform cells as efficiently as noneluted DNA . Complete elution of a 100-kb plasmid, a 194-kb concatemer of bacteriophage lambda, and of 440- and 550- chromosomes of Saccharomyces cerevisiae was also achieved using the same process . This method is suitable for routine use in a wide range of cloning applications, including the electrophoretic isolation of large DNA molecules. Int J Cancer, 1989 Aug 15, 44(2), 367 - 72 Molecular cloning and characterization of endogenous SV40 DNA from human HBL-100 cells; Saint-Ruf C et al.; The human HBL-100 cell line harbours SV40 DNA integrated in tandem at a unique site . The SV40 T-antigen expressed in these cells is defective in a function essential to the replication of the viral genome . The integrated SV40 sequences were molecularly cloned in a bacteriophage, and a subclone (plasmid pSVHBI) containing a complete SV40 DNA was isolated . As compared to SV40 wild-type strain 776, sequence analysis of pSVHBI early region revealed the presence of several DNA alterations . Among these, a point mutation at position 3199, predicting a change at amino-acid 540 of arginine to isoleucine, was shown by marker rescue to be responsible for the deficiency of T-antigen . This novel mutation further delimits one of the T-antigen domains involved in SV40 DNA replication . Transfection experiments demonstrated that the transforming activity of the SV40 genome from HBL-100 cells is still preserved . Moreover, several transformed human cell clones thus obtained could be permanently established in culture. Nucleic Acids Res, 1989 Aug 11, 17(15), 6043 - 53 The recombinational enhancer for DNA inversion functions independent of its orientation as a consequence of dyad symmetry in the Fis-DNA complex; Kanaar R et al.; The Escherichia coli Fis protein binds to specific DNA sequences whose base composition varies enormously . One known function of Fis is to stimulate site-specific DNA recombination . We used the Gin-mediated DNA inversion system of bacteriophage Mu to analyze Fis-DNA interaction . Efficient inversion requires an enhancer which consists of two Fis binding sites at a fixed distance from each other . Using mutant enhancers in which one of the Fis binding sites is replaced we show that Fis binds symmetrically to the DNA and we locate the center of symmetry . Furthermore, we show that one of the Fis binding sites can be replaced by a Fis binding site that normally functions in a process other than site-specific recombination. Nucleic Acids Res, 1989 Aug 11, 17(15), 6017 - 27 Translational repression by bacteriophage MS2 coat protein does not require cysteine residues; Peabody DS; Previous studies implicated cysteine residues in the translational repressor (i.e . RNA binding) activity of the coat protein of bacteriophage MS2 . It has been proposed that a protein sulfhydryl forms a transient covalent bond with an essential pyrimidine in the translational operator by a Michael addition reaction . We have utilized codon-directed mutagenesis methods to determine the importance of each of the two coat protein cysteines for repressor function in vivo . The results indicate that cys46 can be replaced by a variety of amino acids without loss of repressor function . Cys101, on the other hand, is more sensitive to substitution . Most position 101 substitutions inactivate the repressor, but one (arginine) results in normal repressor activity . Although the possibility of a transient covalent contact between cys101 and RNA is not categorically ruled out, construction of double mutants demonstrates that cysteines are not absolutely required for translational repression by coat protein. Nature, 1989 Aug 10, 340(6233), 467 - 8 High abundance of viruses found in aquatic environments; Bergh O et al.; The concentration of bacteriophages in natural unpolluted waters is in general believed to be low, and they have therefore been considered ecologically unimportant . Using a new method for quantitative enumeration, we have found up to 2.5 x 10(8) virus particles per millilitre in natural waters . These concentrations indicate that virus infection may be an important factor in the ecological control of planktonic micro-organisms, and that viruses might mediate genetic exchange among bacteria in natural aquatic environments. J Biol Chem, 1989 Aug 5, 264(22), 13066 - 73 Characterization of the helicase and primase activities of the 63-kDa component of the bacteriophage T7 gene 4 protein; Bernstein JA et al.; Leading and lagging strand DNA synthesis at the replication fork of bacteriophage T7 DNA requires the helicase and primase activities of the gene 4 protein . Gene 4 protein consists of two colinear polypeptides of 56- and 63-kDa molecular mass . We have demonstrated previously that the 56-kDa protein possesses helicase but lacks primase activity (Bernstein, J . A., and Richardson, C . C . (1988) Proc . Natl . Acad . Sci . U.S.A . 85, 396-400) . The 63-kDa gene 4 protein has now been purified from extracts of T7-infected cells . The preparation contains 5-10% contaminating 56-kDa protein, as shown by Western analysis using polyclonal antibodies to the purified 56-kDa protein . The 63-kDa protein catalyzes DNA-dependent dTTP hydrolysis and has helicase activity; both specific activities are similar to those determined for the 56-kDa protein . The 63-kDa protein efficiently synthesizes sequence-specific di-, tri-, and tetraribonucleotides and stimulates the elongation of tetraribonucleotides by T7 DNA polymerase . Although the 56-kDa protein alone lacks primase activity, it enhances the primase activity of the 63-kDa protein 4-fold . This stimulation can be accounted for by a similar increase in the amount of primers synthesized by the 63-kDa protein in the presence of the 56-kDa protein. J Biol Chem, 1989 Aug 5, 264(22), 12785 - 90 Common sites for recombination and cleavage mediated by bacteriophage T4 DNA topoisomerase in vitro; Chiba M et al.; We have previously shown that purified T4 DNA topoisomerase promotes illegitimate recombination between two lambda DNA molecules, or between lambda and plasmid DNA in vitro (Ikeda, H . (1986) Proc . Natl . Acad . Sci . U . S . A . 83, 922-926) . Since the recombinant DNA contains a duplication or deletion, it is inferred that the cross-overs take place between nonhomologous sequences of lambda DNA . In this paper, we have examined the sequences of the recombination junctions produced by the recombination between two lambda DNA molecules mediated by T4 DNA topoisomerase . We have shown that there is either no homology or there are 1-5-base pair homologies between the parental DNAs in seven combinations of lambda recombination sites, indicating that homology is not essential for the recombination . Next, we have shown an association of the recombination sites with the topoisomerase cleavage sites, indicating that a capacity of the topoisomerase to make a transient double-stranded break in DNA plays a role in the illegitimate recombination . A consensus sequence for T4 topoisomerase cleavage sites, RNAY decreases NNNNRTNY, was deduced . The cleavage experiment showed that T4 topoisomerase-mediated cleavage takes place in a 4-base pair staggered fashion and produces 5'-protruding ends. J Mol Biol, 1989 Aug 5, 208(3), 417 - 28 DNA base changes and RNA levels in N-acetoxy-2-acetylaminofluorene-induced dihydrofolate reductase mutants of Chinese hamster ovary cells; Carothers AM et al.; Formerly, we isolated a series of dihydrofolate reductase-deficient Chinese hamster ovary cell mutants that were induced by N-acetoxy-2-acetylaminofluorene . Deletions and complex gene rearrangements were detected in 28% of these mutants; 72% contained putative point mutations . In the present study, we have localized the putative point mutations in the 25,000 base dhfr gene by RNase heteroduplex mapping . Assignment of a position for each mutation was successful in 16 of 19 mutants studied . We cloned DNA fragments containing the mapped mutations from nine mutants into a bacteriophage lambda vector . In the case of 11 other mutants, DNA was amplified by the polymerase chain reaction procedure . Sequence analysis of cloned and amplified DNA confirmed the presence of point mutations . Most mutants (90%) carried base substitutions; the rest contained frameshift mutations . Of the point mutations, 75% were G.C to T.A transversions in either the dhfr coding sequence or at splice sites; transition G.C to A.T mutations were found in two mutants (10%) . In one of these transition mutants, the base substitution occurred at the fifth base of the third intron . Of the frameshift mutations, one was a deletion of G.C pair and the other was an insertion of an A.T pair . Of the mapped mutants, 38% exhibited greatly reduced (approximately 10-fold) steady-state levels of dhfr mRNA . All eight sequenced mutants displaying this phenotype contained premature chain termination codons . Normal levels of dhfr mRNA were observed in five missense mutants and in five mutants carrying nonsense codons in the translated portion of exon VI . Taken together with the results of other mutagens at this locus, we conclude that the low dhfr mRNA phenotype is correlated with the presence of nonsense codons in exons II to V but not in the last exon of the dhfr gene. Virology, 1989 Aug, 171(2), 588 - 98 Nucleotide sequence of the bacteriophage P22 gene 19 to 3 region: identification of a new gene required for lysis; Casjens S et al.; The nucleotide sequence of a 2558-bp region of bacteriophage P22 at the right end of the genetic map between genes 19 and 3 was determined . A new gene that is partially required for lytic growth, named gene 15, was noted . P22 mutants were constructed which lack gene 15 function, and the gene 15 product was found to be required for lysis in the presence of some divalent cations . It has extensive amino acid sequence similarity with the phage lambda Rz gene, which has a similar function, and weak similarity to the phage T7 18.5 gene which previously had no known function . A hybrid P22 phage, in which the T7 18.5 gene replaces the P22 gene 15, exhibits the plating properties of wild-type P22, strongly suggesting that the two genes have similar functions . In addition, deletions were constructed which show that phage P22 has no additional genes required for lytic growth of lysogeny between genes 19 and 3. Virology, 1989 Aug, 171(2), 475 - 83 Bacteriophage T4 late gene expression: overlapping promoters direct divergent transcription of the base plate gene cluster; Scarlato V et al.; Eight 5' ends of RNA molecules which encompass the bacteriophage T4 base plate late genes 51 to 26 region have been mapped by S1 nuclease protection and reverse transcription within a 246-bp DNA segment . Two of eight 5' ends are initiated at two absolutely conserved late promoter sites, P51 and P26a, that direct RNA synthesis on opposite strands . These two promoters share four of eight promoter sequence base pairs . A third 5' end arises from another promoter, P26b, which shows one base pair mismatch with respect to the absolutely conserved -10 sequence . All the other 5' ends arise from RNA processing and/or degradation . Since no other late transcription promoter sites were found within the base plate cluster sequence, we propose that the two overlapping late promoters, P51 and P26a, direct the expression of the T4 base plate gene cluster, included between map coordinates 114,000 and 121,038: P51 directs the transcription of genes 51, 27, 28, 29, 48, and 54 on the rDNA strand and P26a the transcription of genes 26 and 25 on the /DNA strand . This peculiar promoter configuration might account for the low level of transcription of these late genes. Virology, 1989 Aug, 171(2), 350 - 5 The superimmunity gene sim of bacteriophage P1 causes superinfection exclusion; Kliem M et al.; Previous work has shown that the sim gene of bacteriophage P1, if cloned into a multicopy vector, confers immunity against P1 infection to cells . We show that a 1.85-kb DNA fragment from the sim region of P1 (in the multicopy plasmid pMK4) expresses immunity and encodes three proteins with molecular weights of about 25, 24, and 15 kDa . Deletion of 650 bp from the sim region abolished synthesis of all three proteins and of the sim phenotype . Expression of sim did not prevent adsorption of P1 to cells . Successful transfection with linear P1 DNA suggests that the recombinational circularization of P1 DNA is not inhibited in the presence of sim . Plasmid pMK4 and a P1 prophage can be stably maintained in the cell indicating that replication of the prophage is not disturbed by sim . The prophage can be induced in the presence of sim . This shows that the sim phenotype is not caused by preventing lytic replication or phage maturation . In cells with pMK4 there is no expression of genes from infecting phages and transduction frequency is drastically reduced . We suggest that sim functions as a superinfection exclusion system by preventing transfer of DNA from the adsorbed phages into the cytoplasm. Proc Natl Acad Sci U S A, 1989 Aug, 86(15), 5743 - 7 Nucleotide sequence and genomic organization of feline immunodeficiency virus; Talbott RL et al.; An infectious molecular clone of the Petaluma strain of feline immunodeficiency virus (FIV) was isolated from a recombinant bacteriophage library containing genomic DNA prepared from FIV-infected Crandall feline kidney (CRFK) cells . The integrated provirus has a total length of 9472 base pairs . Three long open reading frames corresponding to GAG, POL, and ENV gene coding frames are evident . In addition, an open reading frame overlaps the 3' end of POL, in the region that encodes viral infectivity factor in the primate viruses . Several short open reading frames are present in the intergenic region between POL and ENV and within ENV, which may serve as exons for production of TAT and REV equivalents in FIV . Alignment of the predicted amino acid sequences of the FIV proteins with those of other lentiviruses indicates that FIV did not arise recently from any other characterized lentivirus. J Virol, 1989 Aug, 63(8), 3472 - 8 The immunity (imm) gene of Escherichia coli bacteriophage T4; Lu MJ et al.; The immunity (imm) gene of the Escherichia coli bacteriophage T4 effects exclusion of phage superinfecting cells already infected with T4 . A candidate for this gene was placed under the control of the lac regulatory elements in a pUC plasmid . DNA sequencing revealed the presence of an open reading frame encoding a very lipophilic 83-residue (or 73-residue, depending on the unknown site of translation initiation) polypeptide which most likely represents a plasma membrane protein . This gene could be identified as the imm gene because expression from the plasmid caused exclusion of T4 and because interruption of the gene in the phage genome resulted in a phage no longer effecting superinfection immunity . It was found that the fraction of phage which was excluded upon infection of cells possessing the plasmid-encoded Imm protein ejected only about one-half of their DNA . Therefore, the Imm protein inhibited, directly or indirectly, DNA ejection. J Virol, 1989 Aug, 63(8), 3284 - 95 Genetic analysis of the filamentous bacteriophage packaging signal and of the proteins that interact with it; Russel M et al.; The single-stranded DNA of filamentous phages (f1, fd, M13, Ike) contains a region that can fold into a hairpin structure that serves to earmark the DNA for encapsidation . Second-site suppressor mutants of f1 that can compensate for deletion of this packaging signal have been isolated and characterized . The mutations lie in three genes, two that encode virion proteins located at the end of the particle that is first to emerge from the cell, the end at which the packaging signal is located, and the third in a gene whose product is required for assembly but which is not itself a part of the virion . Analysis of base substitution and deletion mutations in the packaging signal suggests that both structural and sequence elements are important to its proper function. Gene, 1989 Aug 1, 80(1), 1 - 11 Control of prophage integration and excision in bacteriophage P2: nucleotide sequences of the int gene and att sites; Yu A et al.; Integration of bacteriophage P2 into the Escherichia coli host genome involves recombination between two specific attachment sites, attP and attB, one on the phage and the other on the host genome, respectively . The reaction is controlled by the product of the phage int gene, a basic polypeptide of about 37 kDa {Ljungquist and Bertani, Mol . Gen . Genet . 192 (1983) 87-94} . The int gene appears to be expressed differently by an infecting phage, as opposed to a prophage {Bertani, Proc . Natl . Acad . Sci . USA 65 (1970) 331-336} . A 1200-bp region of P2 DNA containing the int gene and attP, the prophage hybrid ends attL and attR, and one bacterial attachment site, the preferred site locI from E . coli strain C, have all been sequenced . An open reading frame coding for a polypeptide of 337 amino acids corresponds to the int gene . The gene has no obvious promoter sequence preceding it . The int gene transcript seems to continue past the attP site downstream from it, suggesting a possible explanation for the previously observed difference in integration and excision . A comparison of the four attachment sites reveals a common 'core' sequence of 27 bp: 5'-AAAAAATAAGCCCGTGTAAGGGAGATT-3' . The P2 nip1 mutation, which increases prophage excision {Calendar et al., Virology 47 (1972) 68-75}, was found to lie within the int gene itself . The P2 saf variant, which has altered site preference {Six, Virology 29 (1966) 106-125}, has a bp substitution within the core sequence . Three deletion/substitution mutants, vir22, vir94 and del3, also have altered core sequences. J Bacteriol, 1989 Aug, 171(8), 4334 - 41 Lysis of Escherichia coli by the bacteriophage phi X174 E protein: inhibition of lysis by heat shock proteins; Young KD et al.; Lysis of Escherichia coli by the cloned E protein of bacteriophage phi X174 was more rapid than expected when bacteria were shifted from 30 to 42 degrees C at the time of E induction . Since such treatment also induces the heat shock response, we investigated the effect of heat shock proteins on lysis . An rpoH mutant was more sensitive to lysis by E, but a secondary suppressor mutation restored lysis resistance to parental levels, which suggests that the sigma 32 subunit itself did not directly increase lysis resistance . At 30 degrees C, mutants in five heat shock genes (dnaK, dnaJ, groEL, groES, and grpE) were more sensitive to lysis than were their wild-type parents . The magnitude of lysis sensitivity varied with mutation and strain background, with dnaK, dnaJ, and groES mutants consistently exhibiting the greatest sensitivities . Extended protection against lysis occurred when overproduction of heat shock proteins was induced artificially in cells that contained a plasmid with the rpoH+ gene under control of the tac promoter . This protective effect was completely abolished by mutations in dnaK, dnaJ, or groES but not by grpE or groEL mutations . Altered membrane behavior probably explains the contradiction whereby an actual temperature shift sensitized cells to lysis, but production of heat shock proteins exhibited protective effects . The results demonstrate that E-induced lysis can be divided into two distinct operations which may now be studied separately . They also emphasize a role for heat shock proteins under non-heat-shock conditions and suggest cautious interpretation of lysis phenomena in systems where E protein production is under control of a temperature-sensitive repressor. J Biochem Biophys Methods, 1989 Aug-Sep, 19(2-3), 155 - 67 New bacteriophage vectors for the large scale production of single stranded insert DNA; Biernat J et al.; SSEV 18 and SSEV 19, derivatives of the bacteriophages M13mp18/19, are new versatile cloning vectors allowing the large scale preparation of single stranded (ss) insert DNA . Replacing the original multiple cloning site by a synthetic 96 bp DNA fragment, a new polylinker region has been introduced containing complementary sequences designed to form a stem structure where single stranded insert fragments can be excised via a 'master restriction' site . The usefulness of such a vector has been demonstrated by the cloning of a 900 bp HindIII fragment derived from the Mycoplasma hyorhinis 23 S rRNA gene . After excision the single stranded insert was labeled isotopically and tested for sensitivity and specificity in detecting homologous sequences in pure DNA or cellular material immobilized on filters. Proc Natl Acad Sci U S A, 1989 Aug, 86(16), 6126 - 30 Cap-independent translation of mRNA conferred by encephalomyocarditis virus 5' sequence improves the performance of the vaccinia virus/bacteriophage T7 hybrid expression system; Elroy-Stein O et al.; A recombinant vaccinia virus that directs the synthesis of bacteriophage T7 RNA polymerase provides the basis for the expression of genes that are regulated by T7 promoters in mammalian cells . The T7 transcripts, which account for as much as 30% of the total cytoplasmic RNA at 24 hr after infection, are largely uncapped . To improve the translatability of the uncapped RNA, the encephalomyocarditis virus (EMCV) untranslated region (UTR) was inserted between the T7 promoter and the chloramphenicol acetyltransferase (CAT) gene . Experiments with a reticulocyte extract demonstrated that the EMCV UTR conferred efficient and cap-independent translatability to CAT RNA synthesized in vitro by T7 RNA polymerase . In cells infected with recombinant vaccinia viruses containing the T7 promoter-regulated CAT gene, the EMCV UTR increased the amount of CAT RNA on polyribosomes . The polyribosome-derived CAT RNA, which contained the EMCV UTR, was translated in vitro in a cap-independent fashion as well . Use of the EMCV UTR significantly enhanced the vaccinia/T7 hybrid expression system as it resulted in a 4- to 7-fold increase in total CAT activity . A further approximately 2-fold improvement was achieved by incubating the cells in hypertonic medium, which favors the translation of uncapped picornavirus RNA over cellular mRNAs . With this newly modified expression system, CAT was the predominant protein synthesized by infected cells and within 24 hr accounted for greater than 10% of the total cell protein. Mutat Res, 1989 Aug, 226(4), 245 - 52 Mutational specificity studies of endogenous mammalian cell loci: methodological aspects; Glickman BW et al.; We report here the details of a modified cloning method first described by Seed et al . (1983) for use in an extensive study of mutational specificity at the aprt locus of Chinese hamster ovary cells . The technology depends on homologous recombination between a suppressor probe plasmid and the desired insert in a genomic library constructed in a double amber mutant bacteriophage lambda vector . Recombinant phage form blue plaques on a non-suppressor, lacZ amber host . We have determined the sensitivity of the method . Homologous recombination frequency is in the order of 10(-3), 6-7 orders of magnitude above non-homologous events . Recombination efficiency is unaffected when the target phage is diluted 100,000-fold with parental vector . A background of plaques is observed along with the desired blue plaques . Although the color discrimination permits us to easily avoid these background plaques, we have characterized the frequency and the nature of these events. Mol Microbiol, 1989 Aug, 3(8), 995 - 1002 The terminus of the Escherichia coli chromosome is flanked by several polar replication pause sites; Francois V et al.; Replication of two small 'constrained' regions of the Escherichia coli chromosome, one bordered by replication terminator T1 and the other by T2, displays normal velocity in the normal direction whereas it is much slower in the opposite direction (de Massy et al., 1987) . The presence of multiple polar terminators has been investigated, using a bacteriophage lambda derivative which provides a replication origin movable to predetermined loci and inducible on demand . The amount of DNA made from this induced origin was determined by in vivo labelling and hybridization to probes of the surrounding region . A redundancy of terminator-like sequences, or pause sites, has been disclosed . So far, two polar pause sites, in the same orientation and separated by 50 or 80 kb, have been localized on each side of the terminus region . The results are discussed in relation to previously observations indicating that these regions are refractory to genomic inversions. EMBO J, 1989 Aug, 8(8), 2393 - 402 Transcriptional activation of bacteriophage lambda DNA replication in vitro: regulatory role of histone-like protein HU of Escherichia coli; Mensa-Wilmot K et al.; Initiation of bacteriophage lambda DNA replication in vivo and in crude in vitro systems is strongly dependent on transcription at or near the lambda replication origin (ori lambda) . Through its capacity to prevent RNA polymerase-mediated 'transcriptional activation' of lambda DNA replication, the lambda cI repressor is capable of negatively regulating initiation of lambda DNA replication, even when all required replication proteins are present . Surprisingly, the strict requirement for transcriptional activation of lambda DNA replication was lost when lambda replication was initiated in an in vitro system composed of nine purified replication proteins {Mensa-Wilmot et al . (1989) J . Biol . Chem., 264, 2853-2861} . We have found that crude extracts of Escherichia coli contain proteins that are capable of restoring the physiological linkage between transcription and ori lambda-dependent replication when they are added to the nine-protein replication system . The protein primarily responsible for this effect has been purified and identified as protein HU, a histone-like protein that is a major constituent of the bacterial nucleoid . HU, when present at a 1:1 weight ratio with supercoiled ori lambda plasmid, is a potent inhibitor of lambda DNA replication in the nine-protein replication system . However, when the ori lambda template is transcribed by E . coli RNA polymerase, the HU-mediated inhibition of lambda DNA replication is abolished . HU does not inhibit propagation of lambda replication forks . Instead, HU apparently interferes with the assembly or function of nucleoprotein structures containing the E . coli DnaB helicase that are formed at ori lambda prior to priming and DNA synthesis . We suggest that the chromatin structure of the template DNA in the region surrounding ori lambda plays a central role in the negative regulation of the initiation of lambda DNA replication in vivo. Virology, 1989 Aug, 171(2), 516 - 24 Nucleotide sequence and expression of the small (S) RNA segment of Maguari bunyavirus; Elliott RM et al.; The small (S) RNA segment of the Maguari bunyavirus genome has been cloned as cDNA and its nucleotide sequence determined . The nucleocapsid protein, N, (Mr 26K) and a nonstructural protein, NSs, (Mr 11K), are encoded in overlapping reading frames, similar to other bunyavirus S RNA segments . In addition, a third AUG-initiated open reading frame encoding a 9.3K protein was observed . All three polypeptides were translated in cell free systems programmed with RNA transcribed in vitro from the cDNA subcloned downstream of a bacteriophage T7 promoter . The effects on expression of subcloning parts of the cDNA and by site-specific mutagenesis are discussed in relation to the scanning model of initiation of translation . A recombinant baculovirus has been constructed to express the Maguari virus S segment gene products . The N protein was efficiently expressed in infected cells, and a significant amount was in a soluble form . We could not detect the synthesis of NSs nor the 9.3K protein, and the reasons for this are discussed . The 9.3K protein has not been found in Maguari virus-infected cells and so the question of its functional significance remains open. Genetics, 1989 Aug, 122(4), 727 - 36 The effect of attachment site mutations on strand exchange in bacteriophage lambda site-specific recombination; Bauer CE et al.; Recombination of phage lambda attachment sites occurs by sequential exchange of the DNA strands at two specific locations . The first exchange produces a Holliday structure, and the second resolves it to recombinant products . Heterology for base substitution mutations in the region between the two strand exchange points (the overlap region) reduces recombination; some mutations inhibit the accumulation of Holliday structures, others inhibit their resolution to recombinant products . To see if heterology also alters the location of the strand exchange points, we determined the segregation pattern of three single and one multiple base pair substitution mutations of the overlap region in crosses with wild type sites . The mutations are known to differ in the severity of their recombination defect and in the stage of strand exchange they affect . The three single mutations behaved similarly: each segregated into both products of recombination, and the two products of a single crossover were frequently nonreciprocal in the overlap region . In contrast, the multiple mutation preferentially segregated into one of the two recombinant products, and the two products of a single crossover appeared to be fully reciprocal . The simplest explanation of the segregation pattern of the single mutations is that strand exchanges occur at the normal locations to produce recombinants with mismatched base pairs that are frequently repaired . The segregation pattern of the multiple mutation is consistent with the view that both strand exchanges usually occur to one side of the mutant site . We suggest that the segregation pattern of a particular mutation is determined by which stage of strand exchange it inhibits and by the severity of the inhibition. J Bacteriol, 1989 Aug, 171(8), 4378 - 84 A component of the side tail fiber of Escherichia coli bacteriophage lambda can functionally replace the receptor-recognizing part of a long tail fiber protein of the unrelated bacteriophage T4; Montag D et al.; The distal part of the long tail fiber of Escherichia coli bacteriophage T4 consists of a dimer of protein 37 . Dimerization requires the catalytic action of protein 38, which is encoded by T4 and is not present in the virion . It had previously been shown that gene tfa of the otherwise entirely unrelated phage lambda can functionally replace gene 38 . Open reading frame (ORF) 314, which encodes a protein that exhibits homology to a COOH-terminal area of protein 37, is located immediately upstream of tfa . The gene was cloned and expressed in E . coli . An antiserum against the corresponding polypeptide showed that it was present in phage lambda . The serum also reacted with the long tail fibers of phage T4 near their free ends . An area of the gene encoding a COOH-terminal region of ORF 314 was recombined, together with tfa, into the genome of T4, thus replacing gene 38 and a part of gene 37 that codes for a COOH-terminal part of protein 37 . Such T4-lambda hybrids, unlike T4, required the presence of outer membrane protein OmpC for infection of E . coli B . An ompC missense mutant of E . coli K-12, which was still sensitive to T4, was resistant to these hybrids . We conclude that the ORF 314 protein represents a subunit of the side tail fibers of phage lambda which probably recognize the OmpC protein . ORF 314 was designated stf (side tail fiber) . The results also offer an explanation for the very unusual fact that, despite identical genomic organizations, T4 and T2 produce totally different proteins 38 . An ancestor of T4 from the T2 lineage may have picked up tfa and stf from a lambdoid phase, thus possibly demonstrating horizontal gene transfer between unrelated phage species. J Photochem Photobiol B, 1989 Aug, 3(4), 497 - 507 Action spectra for photoinduced inactivation of bacteriophage T7 sensitized by 8-methoxypsoralen and angelicin; Ronto G et al.; The action spectrum (240-300 nm) for photoinactivation of unsensitized phage T7 and the action spectra (310-380 nm) for photoinactivation of phage T7 sensitized with 8-methoxypsoralen (8-MOP) and angelicin were measured by an automated method . For unsensitized phage T7 the action spectrum is in good agreement with the absorption spectrum . For sensitization with angelicin the action spectrum is similar to the absorption spectrum, but for sensitization with 8-MOP the spectra are different . The agreement between the T7 absorption and action spectra in the far-UV region is due to photodamage of DNA, leading to phage inactivation . The similarity in the action and absorption spectra in the near-UV region for sensitization with angelicin seems to be in accordance with the monofunctional photobinding of angelicin to DNA . The action spectrum for sensitization with 8-MOP has a maximum at about 320 nm and this suggests that, in addition to the monoadducts, the biadducts play a role in the inactivation of phage T7 . Taking the number of bound furocoumarin molecules into consideration, the quantum efficiencies were estimated . Furocoumarin increases the quantum efficiency in the near-UV region and the values are similar to those obtained in far-UV light without psoralens. Infect Immun, 1989 Aug, 57(8), 2331 - 8 Interaction of drug resistance plasmids and bacteriophages with diarrheagenic strains of Escherichia coli; Bradley DE; Seven transfer-derepressed plasmids from different incompatibility groups in Escherichia coli K-12 were tested for their ability to enter 43 strains of diarrheagenic E . coli (mostly enteropathogenic E . coli clinical isolates) representing 12 serogroups and including rough and semirough mutants (characterized by sodium dodecyl sulfate-polyacrylamide gel electrophoresis) . Strains in some serogroups were more competent as recipients of plasmids than were those in others . Five test plasmids in an E . coli K-12 (rough) donor transferred significantly less efficiently to two smooth strains than to their rough or semirough isogenic derivatives . When the same smooth and rough strains were used as donors, the plasmids transferred to E . coli K-12 equally well . These results suggested that the O-antigenic lipopolysaccharide side chains of diarrheagenic E . coli isolates shielded the outer membrane receptors for conjugative pili, thus preventing plasmid entry . The different receptors for eight bacteriophages were also covered by O side chains . In addition, a limited survey of clinical isolates for drug resistance markers and resident plasmids was carried out. FEBS Lett, 1989 Jul 31, 252(1-2), 42 - 6 Tentative identification of RNA-dependent RNA polymerases of dsRNA viruses and their relationship to positive strand RNA viral polymerases; Koonin EV et al.; Amino acid sequence stretches similar to the four most conserved segments of positive strand RNA viral RNA-dependent RNA polymerases have been identified in proteins of four dsRNA viruses belonging to three families, i.e . P2 protein of bacteriophage phi 6 (Cystoviridae), RNA 2 product of infectious bursa disease virus (Birnaviridae), lambda 3 protein of reovirus, and VP1 of bluetongue virus (Reoviridae) . High statistical significance of the observed similarity was demonstrated, allowing identification of these proteins as likely candidates for RNA-dependent RNA polymerases . Based on these observations, and on the previously reported sequence similarity between the RNA polymerases of a yeast dsRNA virus and those of positive strand RNA viruses, a possible evolutionary relationship between the two virus classes is discussed. FEBS Lett, 1989 Jul 31, 252(1-2), 47 - 52 Two early genes of bacteriophage T5 encode proteins containing an NTP-binding sequence motif and probably involved in DNA replication, recombination and repair; Blinov VM et al.; It is demonstrated, by computer-assisted analysis, that T5 bacteriophage early genes D10 and D13 encode proteins containing the purine NTP-binding sequence motif . The D10 gene product is shown to be a member of a recently characterized superfamily of (putative) DNA and RNA helicases . The D13 gene product is related at a statistically significant level, to the gene 46 product of bacteriophage T4 which is a component of an exonuclease involved in phage DNA replication, recombination and repair . A lower but also significant degree of sequence similarity was detected between the gene D12 product of T5 and the gene 47 product of T4, the second component of the same nuclease . It is hypothesized that both D10 and D13 gene products of T5 might be NTPases, possibly DNA-dependent, mediating NTP-consuming steps during phage DNA replication, recombination and/or repair. Biochemistry, 1989 Jul 25, 28(15), 6392 - 400 Environmental modulation of M13 coat protein tryptophan fluorescence dynamics; Johnson ID et al.; The effects of detergent {deoxycholate (DOC) and phospholipid {dimyristoylphosphatidylcholine (DMPC)} environments on the rotational dynamics of the single tryptophan residue 26 of bacteriophage M13 coat protein have been investigated by using time-resolved single photon counting measurements of the fluorescence intensity and anisotropy decay . The total fluorescence decay of tryptophan-26 is complex but rather similar in DOC as compared to DMPC when analyzed in terms of a lifetime distribution (exponential series method) . This similarity, in conjunction with the almost identical steady-state fluorescence spectra, indicates only minor differences between the tryptophan environments in DOC and DMPC . The reorientational dynamics of tryptophan-26 are dominated by slow rotation of the entire protein in both detergent and phospholipid environments . The resolved anisotropy decay in DOC can be approximated by a simple hydrodynamic model of protein/detergent micelle rotational diffusion, although the data indicative slightly greater complexity in the rotational motion . The tryptophan fluorescence anisotropy is not sensitive to protein conformational changes in DOC detected by nuclear magnetic resonance on the basis of pH independence in the range 7.5-9.1 . In DMPC bilayers, restricted tryptophan motion with a correlation time of approximately 2 ns is observed together with a second very slow reorientational component . Resolution of the time constant for this slow rotation is obscured by the tryptophan fluorescence time window being too short to clearly locate its anisotropic limit . The possible contribution made by axial rotational diffusion of the protein to this slow rotational process is discussed.(ABSTRACT TRUNCATED AT 250 WORDS) J Biol Chem, 1989 Jul 25, 264(21), 12709 - 16 Structural and enzymatic studies of the T4 DNA replication system . I . Physical characterization of the polymerase accessory protein complex; Jarvis TC et al.; In this study, we have investigated the structural and physical properties of the bacteriophage T4 DNA polymerase accessory proteins . We find that T4 gene 44 and 62 proteins associate to form a tight, highly homogeneous complex, containing four gene 44 protein subunits and one gene 62 protein subunit . The molecular mass of the complex is 163,700 daltons . Sedimentation results suggest that the complex is quite asymmetric, with a prolate ellipsoid axial ratio of about 5:1 . This protein complex is known to carry a DNA-dependent ATPase activity; we show by photoaffinity labeling that the ATP-binding sites reside in the gene 44 protein subunits of the complex . Equilibrium sedimentation and chemical cross-linking studies indicate that the T4 gene 45 protein self-associates to form a trimer in solution . This trimer species also appears to be quite asymmetric, showing an axial ratio for a prolate ellipsoid of about 6:1, assuming normal hydration. J Biol Chem, 1989 Jul 25, 264(21), 12627 - 32 Double-strand cleavage and strand joining by the replication initiator protein of filamentous phage f1; Greenstein D et al.; The replication initiator protein (gene II protein (gpII} of bacteriophage f1 is a multifunctional protein that plays central roles in initiation and termination of phage DNA replication . It introduces a nick at a specific site on the (+)-strand of supercoiled replicative form DNA . The 3'-hydroxyl end of the nick serves as the primer for (+)-strand rolling-circle replication . Upon completion of a round of synthesis, gpII cleaves and circulaizes the displaced single strand . When Mn2+ is included in the buffer instead of Mg2+, gpII cleaves both strands . In this paper, we investigate the mechanism of the Mn2+-dependent double-strand cleavage activity of gpII . This reaction, unlike nicking in the presence of Mg2+, does not require superhelicity . The reaction proceeds in two kinetic steps: first nicking of the (+)-strand, and then cleavage of the (-)-strand . The nucleotide sequence requirement for nicking is reduced compared to that in the presence of Mg2+ . The product of the double-strand cleavage has an unusual structure . The left end is a telomere-like hairpin since the (+)- and (-)-strands are joined, as demonstrated by base sequencing . The right end has a onebase 3'-overhang . This reaction probably reflects the cleavage-joining activity of gpII in the termination event. Nucleic Acids Res, 1989 Jul 25, 17(14), 5623 - 32 Gene transfer of truncated NGF receptor clones leads to cell surface expression in mouse fibroblasts; Sehgal A et al.; Transfection of recombinant bacteriophage clones encoding human NGF receptor sequences resulted in cell surface expression in mouse fibroblasts . Unexpectedly, receptors were expressed even after transfection with phage clones which lack 5' gene sequences . Stable transformants were purified and analyzed in detail . S1 nuclease protection and primer extension analysis revealed that an initiation site lies within an intron sequence in the middle of the receptor gene . A truncated mRNA transcript was detected that allowed for the expression of NGF receptors capable of binding to NGF . Since the original phage clones lacked the first two exons, these results suggest that the normal N-terminal sequences may not be necessary for cell surface expression and binding to NGF. J Biol Chem, 1989 Jul 25, 264(21), 12220 - 5 The bacteriophage T4 DNA replication fork . Only DNA helicase is required for leading strand DNA synthesis by the DNA polymerase holoenzyme; Cha TA et al.; Seven bacteriophage T4-encoded proteins reconstitute a DNA replication apparatus that catalyzes coupled leading and lagging strand DNA synthesis at a replication fork in vitro . The proteins involved are the T4 DNA polymerase holoenzyme (the products of T4 genes 43, 44/62, and 45), a helix-destabilizing (SSB) protein (gene 32 protein), and the T4 primosome which is composed of a DNA helicase (gene 41 protein) and a primase (gene 61 protein) . We show here that the presence of 41 protein on the lagging strand of the fork enables the polymerase holoenzyme to catalyze leading strand DNA synthesis at a maximum rate and with high processivity . This leading strand synthesis is unaffected by the addition of either the gene 32 or the gene 61 protein; the 41 protein cannot be replaced by the dda protein, a second T4-encoded DNA helicase . When the 61 protein is added to the 41 protein to complete the primosome, Okazaki fragment synthesis on the lagging strand accompanies leading strand DNA synthesis in this system even in the absence of the 32 protein . However, the addition of 32 protein decreases the size of the Okazaki fragments made, as expected for an increase in the lagging strand polymerization rate at a fork that has coupled leading and lagging strand DNA polymerase molecules. Nucleic Acids Res, 1989 Jul 25, 17(14), 5565 - 77 Effects of all single base substitutions in the loop of boxB on antitermination of transcription by bacteriophage lambda's N protein; Doelling JH et al.; The 'N' antitermination proteins of lambdoid bacteriophages are essential for overcoming multiple transcription terminators located within the major early operons of these phages (1) . In order for N proteins to function, a genome sequence specifying N utilization, nut, must be located within an operon, between the promoter and the terminators (2) . Two components have been identified within nut: 8-base boxA, conserved among different phages and implicated in the recognition of host NusA protein, required for N function (3); 15-base boxB, an interrupted palindrome (4), diverged in sequence among different lambdoid phages and hypothesized to be the site of recognition for different N proteins, also diverged in sequence (5) . Here we apply a plasmid for testing termination and antitermination of transcription (6) to identify mutations at all positions in the 5-7 base loop of lambda's boxB . Almost every base change at any position within the 5-7 base boxB loop was found to constrain antitermination of transcription by the N protein of bacteriophage lambda . These observations extend previous mutational knowledge of nut (7) and are consistant with the hypothesis that the boxB loop is the direct site of recognition for N protein . Variations among the effects of different base changes suggest differential contacts between N protein and bases of the boxB loop, whether in DNA or RNA. J Biol Chem, 1989 Jul 15, 264(20), 11546 - 9 Overproduction and preliminary crystallographic study of ribonuclease H from Escherichia coli; Kanaya S et al.; To facilitate the preparation of ribonuclease H from Escherichia coli in an amount sufficient for crystallographic studies, we have constructed an overproduction system for the enzyme . The structural gene for the enzyme was subcloned from pSK750 (Kanaya, S., and Crouch, R . J . (1983) J . Biol . Chem . 258, 1276-1281) to make a plasmid vector pPL801, in which the gene was under the control of bacteriophage lambda PL promoter . Thermal induction of the gene accumulated the enzyme in E . coli N4830-1 to approximately 8% of the total cytosolic protein . The level of production of the enzyme in N4830-1 harboring pPL801 was 14 mg/liter culture, which was 3000 times as high as that in the host cell . The enzyme was purified with a yield of more than 80% and crystallized by utilizing the property that the solubility of the enzyme decreased at pH values close to its isoelectric point (pI = 9) . Crystals were grown by successive seeding (hanging drop method) for x-ray crystallographic analysis . The crystals belong to space group P212121 with unit cell dimensions of alpha = 44.1 A, b = 87.0 A, c = 35.5 A and contain one molecule in an asymmetric unit . They diffracted x-rays beyond 2.5 A resolution. Gene, 1989 Jul 15, 79(2), 381 - 3 The Escherichia coli MutH protein is not the repressor of the bacteriophage Mu mom operon; Hattman S; Expression of the bacteriophage Mu mom-operon is under tight regulatory control . One of the factors required for transcription of the operon is the host Escherichia coli Dam activity . It was proposed that DNA methylation by this enzyme prevents the binding of a cellular repressor to an operator site containing three 5'-GATC-3' sequences, the known target site of Dam methylation . Support for this model came from the observation of others that the requirement for Dam was almost completely suppressed in a mutH-lysA deletion mutant, suggesting that the MutH protein is the postulated transcriptional repressor . In this communication, however, I show that the Dam requirement is not effectively relieved in this deletion mutant; therefore, the MutH protein alone is not the mom repressor. Cell, 1989 Jul 14, 58(1), 147 - 59 Gin-mediated recombination of catenated and knotted DNA substrates: implications for the mechanism of interaction between cis-acting sites; Kanaar R et al.; The Gin DNA-inversion system of bacteriophage Mu normally requires a substrate containing two inverted recombination sites (gix) and an enhancer sequence on the same supercoiled DNA molecule . The reaction mechanism was investigated by separating these sites on catenated rings . Catenanes with the gix sites o