Bacteriophages

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 on one circle and the enhancer on the other recombined efficiently . Thus, the enhancer was fully functional even though it was located in trans to the gix sites . Multiple links between the rings are required for recombination . Multiply linked catenanes with gix sites on separate circles, one of which contained the enhancer, were also efficient substrates . Knotted constructs carrying directly repeated gix sites were recombined . Catenated and knotted substrates must also be supercoiled . These experiments eliminate simple tracking or looping models as explanations for why the enhancer and gix sites must be in cis with standard substrates . Rather, the Gin synaptic complex requires the three sites to be mutually intertwined in a right-handed fashion with a unique polarity of the gix sites . This geometry is achieved by branching of the DNA substrate and requires the energy and structure of supercoiling, catenation, or knotting.

Science, 1989 Jul 7, 245(4913), 54 - 7
Influence of interior packing and hydrophobicity on the stability of a protein; Sandberg WS et al.; Protein interiors contain many tightly packed apolar atoms in a nearly crystalline state . Both shielding of apolar atoms from solvent and efficient interior packing arrangements affect protein stability, but their relative importance is unclear . To separate these effects, the stabilities of wild-type and mutant gene V proteins from bacteriophage fl were studied by measuring resistance to denaturation . The effects of subtle interior packing changes, both separate from and combined with changes in buried side chain hydrophobicity, were measured . For the interior apolar-to-apolar substitutions studied, the two effects were of the same magnitude and alteration of packing without accompanying hydrophobicity changes substantially destabilized the protein.

J Biol Chem, 1989 Jul 5, 264(19), 11497 - 502
Mapping of a putative surface-binding site of human coagulation factor XII; Clarke BJ et al.; We have localized the binding epitope(s) of two murine monoclonal antibodies (B7C9 and P5-2-1) that were shown previously to inhibit the activation of human coagulation factor XII by negatively charged surfaces . A factor XII cDNA expression library in lambda gt11 was screened with antibody B7C9, and 16 immunoreactive bacteriophage were isolated . Fusion proteins from each of the recombinant phage were reactive with both monoclonal antibodies . Two of the phage cDNA inserts were found to code for amino acid residues -6-+31 and +1-+47 of factor XII, respectively, thereby defining the limits of the antigenic peptide to amino acids +1-+31 . Each of the remaining 14 recombinant phage contained longer factor XII cDNA inserts that included sequences coding for the amino-terminal 31 amino acid residues . These results were confirmed by direct binding of antibody B7C9 to synthetic peptides containing amino acids 1-14 and 1-28 of factor XII . Further experiments with a set of nested peptides also indicated that amino acid residues 1-4 were essential but not sufficient for binding of B7C9 to the peptides . Hydrophobicity analysis of the amino-terminal region of plasma factor XII revealed a highly hydrophilic region between amino acid residues 5 and 15 that contained positively charged lysine residues at positions 8, 11, and 13 . We conclude that a major epitope(s) recognized by monoclonal antibodies B7C9 and P5-2-1 is present in the amino-terminal 28 amino acids of factor XII . It is proposed that binding of these antibodies to factor XII blocks interaction of the positively charged region between residues 5 and 15 with negatively charged surfaces, thereby inhibiting activation.

Vet Immunol Immunopathol, 1989 Jul, 21(3-4), 239 - 48
Lymphocyte alterations in zinc-deficient calves with lethal trait A46; Perryman LE et al.; Lymphocyte numbers and activities were evaluated at 2, 4, 8 and 12 weeks of age in two calves with lethal trait A46 (A46), a genetic disorder affecting intestinal zinc absorption . Plasma zinc concentrations declined to subnormal by 3 weeks of age, after which anorexia, diarrhea, alopecia and hyperkeratosis occurred . Lymphocyte response to phytohemagglutinin-P (PHA), concanavalin A (Con A) and pokeweed mitogen (PWM) stimulation was variably reduced . CD4+ T-lymphocytes were subnormal on at least one observation period following onset of zinc deficiency, and relative numbers of B lymphocytes were decreased at 8 weeks . Secondary antibody responses to bacteriophage phi X 174 were significantly reduced . The results demonstrate that calves homozygous for the A46 trait have normal numbers of functional lymphocyte subpopulations at birth, and that the activity of their lymphocytes is altered once the calves become zinc deficient.

Mol Gen Genet, 1989 Jul, 218(1), 137 - 42
Translational regulation of the lysis gene in RNA bacteriophage fr requires a UUG initiation codon; Adhin MR et al.; Single nucleotide substitutions identify a UUG triplet as the initiation codon of the lysis gene in RNA bacteriophage fr . This initiation codon is non-functional in de novo initiation but is activated by translational termination at the overlapping coat gene . The UUG initiation codon is crucial for gene regulation in the phage, as it excludes uncontrolled access of ribosomes to the start of the lysis gene . Replacement of UUG by either GUG or AUG results in the loss of genetic control of the lysis gene . A model is presented in which initiation factor IF3 proofreads de novo initiation at UUG codons.

Genetics, 1989 Jul, 122(3), 471 - 9
Genetic definition of two functional elements in a bacteriophage T4 host-range "cassette"; Snyder M et al.; Gene 37 of T4 encodes the major subunit of the distal half of the tail fiber . The distal tip of the fiber, comprised of the carboxy-terminal ends of two molecules of gene 37 product (gp37), carries the principal determinant of the phage host range . The gp37 carboxyl termini recognize the bacterial surface during infection, and, in addition, include a site required for interaction with the product of gp38 during distal half-fiber assembly . In the absence of interaction with gp38, gp37 polypeptides do not dimerize . Eleven temperature-sensitive mutants with defects located near the promoter-distal end of gene 37 were tested at nonpermissive temperatures for production of an antigen that is diagnostic of distal half-fiber assembly . Six of the mutations prevent distal half-fiber assembly . The other five allow assembly of distal half fibers, which combine with proximal half fibers and attach to phage particles, but the resulting phage do not adsorb to bacteria . These two classes of mutations define two adjacent but separate genetic regions, corresponding to two different functional domains in gp37 . These two regions and the neighboring gene 38 comprise a functional unit that can be considered as a host-range "cassette," with features that are strikingly similar to corresponding functional units in other unrelated as well as related phages.

Proc Natl Acad Sci U S A, 1989 Jul, 86(13), 5030 - 4
Physical mapping of complex genomes by cosmid multiplex analysis; Evans GA et al.; A rapid and powerful approach for linking individual clones of a cosmid library and the assembly of a large physical map is presented, which depends on the simultaneous analysis of many cosmid clones for overlapping regions . This method uses cosmid vectors that contain endogenous bacteriophage T3 and T7 promoters to allow for the identification of overlapping clones through the synthesis of end-specific RNA probes . A genomic library is constructed and organized as an ordered matrix such that each clone is assigned an identifying coordinate . DNA from mixtures of cosmid clones is pooled such that each pool contains only one common member with any other pool, RNA probes are prepared from mixtures of cosmid clones, and groups of clones overlapping with the constituents of the mixtures are determined by hybridization . Pooled probes are most simply prepared by grouping clones according to the rows and columns of the library matrix . The pairwise comparison of data generated by the hybridization of mixed probes can be decoded by using simple algorithms that predict the order and linkage of all clones in the collection and organize them into predicted contigs . To demonstrate the feasibility of multiplexed analysis of cosmids, a genomic library was prepared from a mouse-human somatic cell hybrid that contains a portion of the long arm of human chromosome 11 . Preparation, arrangement on a matrix, and analysis of pooled cosmid clones from this collection resulted in the detection of 1099 linked pairs of cosmids, which could be assembled into 315 contigs . Thus, with a minimal amount of effort, a substantial portion of this genomic region has been linked in multiple overlapping contigs . This method may have practical applications in the large-scale mapping and sequencing of mammalian genomes.

Proc Natl Acad Sci U S A, 1989 Jul, 86(13), 4912 - 6
Structural characterization of the rat malic enzyme gene; Morioka H et al.; We have identified and characterized lambda bacteriophage clones containing genomic DNA encoding rat malic enzyme {(S)-malate:NADP+ oxidoreductase (oxaloacetate-decarboxylating); EC 1.1.1.40} . The malic enzyme gene is unexpectedly large, spanning at least 95 kilobases . It is divided into 14 exons that range in size from 76 to 1513 base pairs . The sizes and boundaries of the exons were determined by Southern blotting and DNA sequencing . The sequences at the 5' and 3' ends of each intron conformed to the consensus sequence for mammalian introns . S1 nuclease and primer-extension assays showed that transcription of the malic enzyme gene initiates at multiple sites, the strongest one at position -31 relative to the ATG . "TATA and CCAAT box" homologies are not present in the proximal promoter region . Analysis of the 3' end of the gene showed that the utilization of alternate polyadenylylation signals in exon 14 results in two mRNAs with 3' untranslated regions of 345 and 1345 nucleotides, respectively.

Carcinogenesis, 1989 Jul, 10(7), 1307 - 14
Transcription-terminating lesions induced by bifunctional alkylating agents in vitro; Pieper RO et al.; The present study was initiated to determine if DNA damage induced by the bifunctional anti-tumor alkylating agents melphalan, nitrogen mustard, a spontaneously activated derivative of cyclophosphamide or chlorambucil inhibits transcription in vitro, and to determine if the potential sites of transcription termination correlate with the sites of N7 guanine adducts predominantly formed by these agents . To assess drug effects on in vitro transcription, linearized plasmid DNA containing the 420-bp PstI fragment of exon two of the human c-myc oncogene was incubated with various concentrations of the drugs . After drug removal and further drug-free incubation, the sense strand of the c-myc insert was transcribed with either of two bacteriophage RNA polymerases in the presence of {32P}UTP . The labeled products of the reaction were electrophoresed next to the labeled products of RNA sequencing reactions, and the location of transcription termination along the DNA template was determined . The sites of transcription termination were then compared with the sites of drug-induced guanine N7 alkylation in the template, as determined by modified Maxam-Gilbert sequencing . At the drug exposures examined, all the drugs were shown to alkylate any guanine in the template . Transcription of this alkylated DNA, however, resulted in RNA molecules truncated not at every alkylated guanine, but at various discrete sites throughout the template . Transcription was terminated at every adenine pair examined in the melphalan-treated template, at selected guanine pairs in the nitrogen-mustard-treated template, and at selected adenine-guanine and guanine-adenine pairs in the chlorambucil-treated template . Transcription of cyclophosphamide-derivative-treated DNA was unaffected . These results suggest that only some bifunctional alkylating agents induce DNA damage capable of terminating transcription in vitro, and that these agents do so in a sequence-specific, drug-specific manner inconsistent with patterns of guanine N7 alkylation.

Trends Genet, 1989 Jul, 5(7), 209 - 13
Bacteriophage introns: parasites within parasites?
Belfort M.
Several bacteriophage introns carry the information necessary to perform a complex series of RNA as well as DNA rearrangements . The RNA splicing reactions allow expression of the intron-containing genes, whereas recombination at the DNA level results in the mobility and invasive potential of the introns . The RNA and DNA transactions are temporally regulated to permit timely splicing early in infection, and expression of intron-encoded DNA endonucleases involved in mobility at late times . This is achieved through efficient use of the viral regulatory machinery, ensuring the perpetuation of the 'parasitic' introns.

Biochem J, 1989 Jul 1, 261(1), 265 - 8
High-level expression of human dihydropteridine reductase (EC 1.6.99.7), without N-terminal amino acid protection, in Escherichia coli; Armarego WL et al.; The cDNA coding for human dihydropteridine reductase {Dahl, Hutchinson, McAdam, Wake, Morgan & Cotton (1987) Nucleic Acids Res . 15, 1921-1936} was inserted downstream of tandem bacteriophage lambda PR and PL promoters in Escherichia coli vector pCE30 . Since pCE30 also expresses the lambda c1857ts gene, transcription may be controlled by variation of temperature . The recombinant plasmid in an E . coli K12 strain grown at 30 degrees C, then at 45 degrees C, directed the synthesis of dihydropteridine reductase to very high levels . The protein was soluble, at least as active as the authentic human enzyme, and lacked the N-terminal amino acid protection.

Res Virol, 1989 Jul-Aug, 140(4), 373 - 88
Genome organization in hybrids between prophage phi 80 and Escherichia coli virus phi gamma; Gratia JP; Prophage phi 80 is used for the detection of "discrete" viruses such as phage phi gamma by genetic recombination . Several genetic events have produced a series of hybrids which are currently being characterized . Characteristic genomic properties of the parent phages are recognized in these hybrids . "lambda"-type phages contain a cos-ended DNA of a single sequence, the size of which may be altered by hybridization . "gamma"-type phages contain DNA molecules of uniform size (about 52 kb) and of variable ends; their genome is able to promote highly efficient transduction (pug type) regardless of the origin of the right arm . In both hybrids, the att site is most often a junction point between the parental genomes due to common int-promoted recombination . New evidence is provided for the formation of viable heterozygous "lambda/gamma"-type bacteriophages.

Proc Natl Acad Sci U S A, 1989 Jul, 86(14), 5400 - 4
Regulated expression of foreign genes in mammalian cells under the control of coliphage T3 RNA polymerase and lac repressor; Deuschle U et al.; Systems that stringently regulate the expression of individual genes within a complex genetic background have contributed greatly to the analysis of gene function . In this report the development of a highly regulated expression system in mammalian cells is described in which transcription of a foreign gene is mediated by the bacteriophage T3 RNA polymerase under the control of the Escherichia coli lac repressor . Rabbit kidney cell lines have been established that constitutively express the phage RNA polymerase and lac repressor . The two bacterial proteins regulate the transcription of the coding sequence of the firefly luciferase, which has been placed under the control of a T3 promoter/lac operator fusion . In the presence of the inducer isopropyl beta-D-thiogalactoside, efficient T3 polymerase-dependent transcription is observed, which is tightly repressed in the absence of inducer . Translation of the T3 transcripts can be mediated by vaccinia virus functions . The demonstration that a specific transcription activity can be regulated over a range of several orders of magnitude in higher eukaryotic cells by using a highly specific and nontoxic inducer has broad implications for a variety of studies.

Infect Immun, 1989 Jul, 57(7), 2014 - 20
Characterization and vaccine potential of a novel recombinant coccidial antigen; Miller GA et al.; A cDNA clone derived from sporulated oocysts of Eimeria tenella and encoding the expression product GX3262 was identified using a monoclonal antibody raised against Eimeria acervulina sporozoites . The cDNA fragment containing the coccidial antigen gene was cloned in bacteriophage lambda gt11, transferred to a plasmid, and introduced into Escherichia coli for analysis of the gene products . The strain carrying the plasmid produced GX3262 as part of a fusion protein consisting of the first 1,006 amino acids of E . coli beta-galactosidase and 112 amino acids of the E . tenella protein of approximately 12 kilodaltons . Partially purified antigen, heat-killed recombinant bacterin, and live E . coli containing the recombinant coccidial antigen were used to immunize 1-week-old or newly hatched broiler chicks . Several immunization protocols were utilized, including boosts with partially purified beta-galactosidase-GX3262, bacterin, or small numbers of live E . tenella oocysts . After challenge with an experimental E . tenella infection, the birds were evaluated by scoring cecal lesions to determine the level of protection . The greatest degree of protection was seen after only a single immunization of 2-day-old birds with a live recombinant E . coli preparation . The results presented here identify GX3262 as a potential candidate coccidial vaccine antigen and provide evidence for the first time that newly hatched chickens can be successfully vaccinated with a recombinant antigen.

J Gen Microbiol, 1989 Jul, 135 ( Pt 7), 1857 - 63
Escherichia coli tolQ mutants are resistant to filamentous bacteriophages that adsorb to the tips, not the shafts, of conjugative pili; Bradley DE et al.; The tolQ (previously fii) mutation in Escherichia coli K12 inhibits infection by filamentous bacteriophages f1 and IKe but not by RNA-containing phage f2 . This work extends these observations to other plasmid-specific bacteriophages including various filamentous . RNA-containing, and lipid-containing isolates . Only tip-adsorbing filamentous phages were affected by tolQ and not shaft-adsorbing ones . Electron microscopy showed that RP4-specific filamentous phage Pf3 was one of the latter kind . Several tip-adsorbing filamentous phages inhibited conjugation between tolQ strains carrying their specific plasmids, implicating the phage receptors (conjugative pili) as mating organelles . tolQ mutant strains were as proficient as their parents in conjugation mediated by a wide range of plasmids.

Biotechniques, 1989 Jul-Aug, 7(7), 756 - 61
A phage-linked immunoabsorbant system for the detection of pathologically relevant antigens; Block T et al.; This report describes a novel system for the immunological detection of immobilized antigen . The detection of herpes simplex virus (HSV) antigen was used as an example . Bacteriophage M13, containing the E . coli lac Z gene, was used as the "reporter" molecule in an immunoassay which is otherwise analogous to the enzyme-linked immunoabsorbant assay (ELISA) . Briefly, HSV infected cells were incubated with a mouse monoclonal antibody specific for HSV antigen, followed by rabbit anti-mouse serum and mouse anti-M13 serum . Immune complexes were incubated with viable M13 phage . M13 binding was due to the presence of M13 antibodies, whose presence ultimately depended on the binding of monoclonal antibody to HSV . Phage was recovered by elution in pH = 11 . Recovered phage was used to infect E . coli . M13 was quantitated by either plaque assay or by an assay for phage-induced beta-galactosidase activity in appropriate E . coli strains . The amount of M13 recovered was proportional to the number of HSV infected cells probed . Therefore, M13 served as a "bio-amplifiable tag" to antibody, as enzymes do in the ELISA . Since M13 is viable, its signal can be amplified by infection of susceptible bacteria, and the promise for an enormously sensitive immunoassay exists . The sensitivity of the assay described here is compared to the ELISA in the detection of HSV infection cells, as an example of the novel assay's potential . Significantly, the novel assay was more sensitive than the ELISA when samples were tested under identical circumstances . This technique is called the phage-linked immunoabsorbant assay (PHALISA), by analogy to the ELISA.

J Bacteriol, 1989 Jul, 171(7), 3872 - 80
Impaired expression of certain prereplicative bacteriophage T4 genes explains impaired T4 DNA synthesis in Escherichia coli rho (nusD) mutants; Stitt BL et al.; The Escherichia coli rho 026 mutation that alters the transcription termination protein Rho prevents growth of wild-type bacteriophage T4 . Among the consequences of this mutation are delayed and reduced T4 DNA replication . We show that these defects can be explained by defective synthesis of certain T4 replication-recombination proteins . Expression of T4 gene 41 (DNA helicase/primase) is drastically reduced, and expression of T4 genes 43 (DNA polymerase), 30 (DNA ligase), 46 (recombination nuclease), and probably 44 (DNA polymerase-associated ATPase) is reduced to a lesser extent . The compensating T4 mutation goF1 partially restores the synthesis of these proteins and, concomitantly, the synthesis of T4 DNA in the E . coli rho mutant . From analyzing DNA synthesis in wild-type and various multiply mutant T4 strains, we infer that defective or reduced synthesis of these proteins in rho 026-infected cells has several major effects on DNA replication . It impairs lagging-strand synthesis during the primary mode of DNA replication; it delays and depresses recombination-dependent (secondary mode) initiation; and it inhibits the use of tertiary origins . All three T4 genes whose expression is reduced in rho 026 cells and whose upstream sequences are known have a palindrome containing a CUUCGG sequence between the promoter(s) and ribosome-binding site . We speculate that these palindromes might be important for factor-dependent transcription termination-antitermination during normal T4 development . Our results are consistent with previous proposals that the altered Rho factor of rho 026 may cause excessive termination because the transcription complex does not interact normally with a T4 antiterminator encoded by the wild-type goF gene and that the T4 goF1 mutation restores this interaction.

Biotechniques, 1989 Jul-Aug, 7(7), 730 - 5
Convenient uses of polymerase chain reaction in analyzing recombinant cDNA clones; Nishikawa BK et al.; We have used polymerase chain reaction to accelerate our analysis of recombinant lambda-cDNA clones . We have amplified the inserts of lambda gt10 or lambda gt11 recombinants starting with bacteriophage in cored plaques or isolated DNA . The amplifications made with simple or complex oligonucleotide primers have allowed convenient sizing, subcloning and translation of the phage inserts into protein.

Biotechniques, 1989 Jul-Aug, 7(7), 674 - 80
Sequencing of cloned DNA using bacteriophage lambda gt11 templates; Steffens DL et al.; A procedure for isolating and directly sequencing recombinant bacteriophage lambda gt11 DNA templates is described . Approximately 250-300 bases of sequence can be obtained directly from the lambda gt11 template, eliminating the need for subcloning prior to dideoxynucleotide sequencing of clones.

Mol Biol (Mosk), 1989 Jul-Aug, 23(4), 1007 - 12
{Thermo-regulated expression of the htpR gene under the control of the PR-promotor of bacteriophage lambda induces supersynthesis of heat shock proteins}; Kiselev VI et al.; The mechanisms of induction of heat shock protein synthesis in E . coli have been studied . For this purpose plasmids in which htpR gene expression is controlled by the PR-promoter of bacteriophage lambda and by the Trp-promoter have been constructed . An effective induction of heat shock proteins requires both an increased content of htpR protein and additional cofactors formed in the cell under heat shock conditions.

Mol Cell Biol, 1989 Jul, 9(7), 3081 - 7
A single essential gene, PRI2, encodes the large subunit of DNA primase in Saccharomyces cerevisiae; Foiani M et al.; DNA primase activity of the yeast DNA polymerase-primase complex is related to two polypeptides, p58 and p48 . The reciprocal role of these protein species has not yet been clarified, although both participate in formation of the active center of the enzyme . The gene encoding the p58 subunit has been cloned by screening of a lambda gt11 yeast genomic DNA library, using specific anti-p58 antiserum . Antibodies that inhibited DNA primase activity could be purified by lysates of Escherichia coli cells infected with a recombinant bacteriophage containing the entire gene, which we designate PR12 . The gene was found to be transcribed in a 1.7-kilobase mRNA whose level appeared to fluctuate during the mitotic cell cycle . Nucleotide sequence determination indicated that PR12 encodes a 528-amino-acid polypeptide with a calculated molecular weight of 62,262 . The gene is unique in the haploid yeast genome, and its product is essential for cell viability, as has been shown for other components of the yeast DNA polymerase-primase complex.

Mol Gen Genet, 1989 Jul, 218(1), 13 - 7
Synchronous division induced in Escherichia coli K12 by gemts mutants of phage Mu; Paolozzi L et al.; Infection with the bacteriophage mutant Mu c+ gemts2 at 42 degrees C induces synchrony in cell division in cultures of Escherichia coli K12 . This synchrony may last for several cycles and is not only due to selection since synchronization is observed even when bacterial survival to the infection is over 80% as in lysogens for Mu c+ gemts2 . The mechanism by which synchrony is induced is not known, but since the product of Mu gene gem (previously called lig) has been shown to interact with the enzymatic system in the bacteria controlling the degree of DNA supercoiling, the phenomenon could be a consequence of this interaction.

Mol Gen Mikrobiol Virusol, 1989 Jul, (7), 29 - 35
{UV-inducibility of the LT-toxin operon}; Tiganova IG et al.; The plasmid elt-operon pVZ14 was constructed by fusing of the eltoperon of the plasmid pVZ357 with the lac-gene of the bacteriophage Mud1 (Amp, Lac) . lacZ gene has been proven to be fused with an elt-promoter by the loss of toxin production coded by pVZ357 and acquiring of Lac+ phenotype by pVZ14 containing cells, as well as by HindIII fragments hybridization of pVZ357 and pVZ14 with the labelled elt-probe . The kinetics of beta-galactosidase synthesis in E . coli cells harboring pVZ14 shows an elt-operon promoter to have expressed constitutive activity and to be activated by a SOS-inducing agent, UV-light.

Biochimie, 1989 Jul, 71(7), 839 - 52
Crosslinking of ribosomal proteins S4, S5, S7, S8, S11, S12 and S18 to domains 1 and 2 of 16S rRNA in the Escherichia coli 30S particle; Chiaruttini C et al.; RNA-protein crosslinks were introduced into Escherichia coli 30S ribosomal subunits by treatment with 1-ethyl-3(3-dimethylaminopropyl)carbodiimide (EDC) . Complexes of 16S rRNA cross-linked to 30S ribosomal proteins were isolated and hybridized with a series of single-stranded bacteriophage M13-rDNA probes . These probes, each carrying an inserted rDNA fragment, were used to select contiguous 16S rRNA sections covering all of domain 1 and the major part of domain 2 (starting at the 5'-P terminus and ending at nucleotide 869) and the proteins covalently linked to each of these sections were identified by 2-dimensional polyacrylamide gel electrophoresis . This procedure identified proteins S4, S5, S7, S8, S11, S12, and S18 as the species most efficiently crosslinked to domains 1 and 2 of 16S rRNA . These results are discussed in the light of current knowledge of the tertiary structure of 16S rRNA in the E . coli 30S ribosomal subunit.

J Clin Invest, 1989 Jul, 84(1), 56 - 61
A monoclonal antibody to von Willebrand factor (vWF) inhibits factor VIII binding . Localization of its antigenic determinant to a nonadecapeptide at the amino terminus of the mature vWF polypeptide; Bahou WF et al.; vWF is a multimeric glycoprotein that serves as the major carrier in plasma of Factor VIII (FVIII) . We have used an anti-human vWF MAb W5-6A to investigate the FVIII binding site on vWF . W5-6A inhibited FVIII binding to vWF-coated polystyrene tubes in a concentration-dependent manner with 90% inhibition of FVIII binding at a concentration of 10 micrograms/ml . The W5-6A epitope was identified by screening a vWF fragment library using the bacteriophage expression vector lambda gt11 . DNA sequence analysis of 29 immunoreactive phage clones localized the W5-6A epitope to a nonadecapeptide spanning amino acid residues threonine 78 to threonine 96 at the amino-terminus of the mature vWF polypeptide . Purified beta-galactosidase/vWF fusion protein from one of these clones, vWF9, was incubated with radiolabeled W5-6A and caused near complete inhibition of W5-6A binding to vWF . Inhibitory activity was lost after vWF9 trypsinization or reduction and alkylation . These data indicate that (a) the antigenic determinant recognized by W5-6A localizes to a nonadecapeptide at the NH2 terminus of the mature vWF polypeptide, (b) disulfide bonds within vWF9 may be necessary to maintain the structure required for immunoreactivity with W5-6A, and (c) W5-6A recognizes an immunogenic region on vWF that may be at (or near) the major FVIII binding domain.

Gene, 1989 Jun 30, 79(1), 9 - 20
High efficiency vectors for cosmid microcloning and genomic analysis; Evans GA et al.; We describe the construction and use of cosmid vectors designed for microcloning, gene isolation and genomic mapping starting from submicrogram amounts of eukaryotic DNA . These vectors contain (1) multiple cos sites to allow for simple and efficient cloning using non size-selected DNA; (2) bacteriophage T3 and T7 promoter sequences flanking the cloning site to allow for the synthesis of end-specific probes for chromosome walking; (3) a selectable gene for immediate gene transfer of cosmid DNA into mammalian cells; (4) recognition sequences for specific oligodeoxyribonucleotides to allow rapid restriction mapping; (5) unique NotI, SacII or SfiI sites flanking the cloning site to allow for removal of the cloned DNA insert from the vector . These cosmid vectors allow the construction of high quality genomic libraries in situations where the quantity of purified DNA is extremely limited, such as when using DNA prepared from purified mammalian chromosomes isolated by fluorescence-activated cell sorting.

Biochem Biophys Res Commun, 1989 Jun 30, 161(3), 1056 - 63
High-level expression of self-processed HIV-1 protease in Escherichia coli using a synthetic gene; Hostomsky Z et al.; A synthetic gene coding for HIV-1 protease (PR) has been constructed and a system for its efficient expression in E . coli has been established: PR is synthesized as a fusion protein with E . coli dihydrofolate reductase under the control of a bacteriophage T7 promoter . The synthetic gene was constructed to enable rapid construction of defined mutants by restriction fragment replacement . A set of mutants has been constructed which may facilitate elucidation of the mechanism of PR self-cleavage from polyprotein precursors . We have demonstrated that the C-terminal residue (Phe99 in the native sequence) of the processing intermediate is absolutely required for subsequent cleavage at the N-terminal cleavage site . The potential structural role of this residue is discussed with reference to the recently published HIV-1 PR structure.

Cell, 1989 Jun 30, 57(7), 1201 - 10
Translation of the bacteriophage Mu mom gene is positively regulated by the phage com gene product; Wulczyn FG et al.; Expression of the bacteriophage Mu mom gene is subject to posttranscriptional regulation by the phage com gene product . We have used mom-lacZ translational fusion genes to define the sequence requirements for stimulation of mom expression by Com . We show that the mom translation initiation region (TIR) is inactive in the absence of Com . We suggest that this repressed state is due to mRNA secondary structure in the TIR, since a deletion that destabilizes a stem-loop structure in the TIR results in high levels of Com-independent translation . We identify sequences on the mRNA, adjacent to the stem and loop, that are required for stimulation by Com . We propose that Com acts to stimulate initiation of translation by relieving the structural repression of the mom TIR . Indirect evidence is presented suggesting that Com binds to a site in the TIR.

Nucleic Acids Res, 1989 Jun 26, 17(12), 4567 - 77
Regions at the carboxyl end of bacteriophage phi 29 protein p6 required for DNA binding and activity in phi 29 DNA replication; Otero MJ et al.; Series of deletions corresponding to the carboxyl end of the phage phi 29 protein p6 have been constructed and their activity in the initiation of phi 29 DNA replication and their capacity to interact with the phi 29 DNA ends have been studied . Determination of the activity of the deletion mutants in phi 29 DNA replication indicated the dispensability of the 14 carboxy-terminal amino acids of the protein . The activity of protein p6 decreased with deletions from 23 to 39 amino acids and was undetectable when 44 amino acids were removed . A similar behaviour was obtained when the interaction of the mutant proteins with the phi 29 DNA ends was analyzed . These results indicate that the stimulation of phi 29 DNA replication by protein p6 requires a specific binding to the phi 29 DNA ends.

J Biol Chem, 1989 Jun 25, 264(18), 10872 - 7
Reconstitution of template-dependent in vitro transcriptase activity of a yeast double-stranded RNA virus; Fujimura T et al.; Isolated mature L-A viral particles from yeast have a transcriptase activity that uses endogenous L-A double-stranded RNA (dsRNA) as template . We have previously demonstrated that empty particles derived from mature L-A viral particles have replicase activity capable of synthesizing minus strand single-stranded RNA (ssRNA) on an added plus strand ssRNA template to form dsRNA . We report here that empty particles also have transcriptase activity that uses added viral dsRNA as template . The newly synthesized ssRNA was the plus strand, and some of these transcripts were converted to the dsRNA form by the replicase activity associated with the empty particles . This transcriptase activity, however, required a much higher concentration of polyethylene glycol than that used previously for the replicase activity . The mode of transcription was conservative . The enzyme transcribed ssRNA from L-A, M1, or X (a deletion mutant of L-A) dsRNAs but not from other yeast dsRNAs (L-BC, T, or W), bacteriophage Phi6 dsRNAs, or animal rotavirus dsRNAs, indicating the same template specificity as that expected for the in vivo reaction . This assay system, and the replicase assay system, will allow us to study in vitro all the enzymatic reactions essential for the viral replication cycle.

J Biol Chem, 1989 Jun 25, 264(18), 10709 - 18
Heat shock protein-mediated disassembly of nucleoprotein structures is required for the initiation of bacteriophage lambda DNA replication; Alfano C et al.; Three Escherichia coli heat shock proteins, DnaJ, DnaK, and GrpE, are required for replication of the bacteriophage lambda chromosome in vivo . We show that the GrpE heat shock protein is not required for initiation of lambda DNA replication in vitro when the concentration of DnaK is sufficiently high . GrpE does, however, greatly potentiate the action of DnaK in the initiation process when the DnaK concentration is reduced to a subsaturating level . We demonstrate in the accompanying articles (Alfano, C . and McMacken, R . (1989) J . Biol . Chem . 264, 10699-10708; Dodson, M., McMacken, R., and Echols, H . (1989) J . Biol . Chem . 264, 10719-10725) that DnaJ and DnaK bind to prepriming nucleoprotein structures that are assembled at the lambda replication origin (ori lambda) . Binding of DnaJ and DnaK completes the ordered assembly of an ori lambda initiation complex that also contains the lambda O and P initiators and the E . coli DnaB helicase . With the addition of ATP, the DnaJ and DnaK heat shock proteins mediate the partial disassembly of the initiation complex, and the P and DnaJ proteins are largely removed from the template . Concomitantly, on supercoiled ori lambda plasmid templates, the intrinsic helicase activity of DnaB is activated and DnaB initiates localized unwinding of the DNA duplex, thereby preparing the template for priming and DNA chain elongation . We infer from our results that DnaK and DnaJ function in normal E . coli metabolism to promote ATP-dependent protein unfolding and disassembly reactions . We also provide evidence that neither the lambda O and P initiators nor the E . coli DnaJ and DnaK heat shock proteins play a direct role in the propagation of lambda replication forks in vitro.

J Biol Chem, 1989 Jun 25, 264(18), 10719 - 25
Specialized nucleoprotein structures at the origin of replication of bacteriophage lambda . Protein association and disassociation reactions responsible for localized initiation of replication; Dodson M et al.; Binding of the O protein of phage lambda to the replication origin (ori lambda) results in the formation of an organized nucleoprotein structure termed the O-some . The O-some serves to localize and initiate a six-protein sequential reaction that provides for localized unwinding of the origin region, the critical prepriming step for precise initiation of DNA replication . By the use of electron microscopy of gold-tagged antibody complexes, we have defined four stages of protein association and dissociation reactions that are involved in the prepriming pathway . First, as defined previously, O protein binds to multiple DNA sites and self-associates to form the O-some . Second, lambda P and host DnaB proteins add to the O-some to generate an O.P.DnaB.ori lambda complex . Addition of the DnaK and DnaJ proteins yields a third stage complex containing DnaK, DnaJ, O, P, and DnaB . With the addition of ATP and single-strand binding protein (SSB), the P protein is largely removed, and the DnaB acts as a helicase to generate locally unwound, SSB-coated single strand DNA . Thus, the initiation of lambda DNA replication requires ordered assembly and partial disassembly of specialized nucleoprotein structures . The disassembly activity of DnaK and DnaJ may be their general role in the heat shock response.

J Biol Chem, 1989 Jun 25, 264(18), 10699 - 708
Ordered assembly of nucleoprotein structures at the bacteriophage lambda replication origin during the initiation of DNA replication; Alfano C et al.; Replication of the chromosome of bacteriophage lambda depends on the cooperative action of two phage-coded proteins and seven replication and heat shock proteins from its Escherichia coli host . As previously described, the first stage in this process is the binding of multiple copies of the lambda O initiator to the lambda replication origin (ori lambda) to form the nucleosomelike O-some . The O-some serves to localize subsequent protein-protein and protein-DNA interactions involved in the initiation of lambda DNA replication to ori lambda . To study these interactions, we have developed a sensitive immunoblotting protocol that permits the protein constituents of complex nucleoprotein structures to be identified . Using this approach, we have defined a series of sequential protein assembly and protein disassembly events that occur at ori lambda during the initiation of lambda DNA replication . A second-stage ori lambda.O (lambda O protein).P (lambda P protein).DnaB nucleoprotein structure is formed when O, P, and E . coli DnaB helicase are incubated with ori lambda DNA . In a third-stage reaction the E . coli DnaJ heat shock protein specifically binds to the second-stage structure to form an ori lambda.O.P.DnaB.DnaJ complex . Each of the nucleoprotein structures formed in the first three stages was isolated and shown to be a physiological intermediate in the initiation of lambda DNA replication . The E . coli DnaK heat shock protein can bind to any of these early stage nucleoprotein structures, and in a fourth-stage reaction a complete ori lambda.O.P.DnaB.DnaJ.DnaK initiation complex is assembled . Addition of ATP to the reaction enables the DnaK and DnaJ heat shock proteins to mediate a partial disassembly of the fourth-stage complex . These protein disassembly reactions activate the intrinsic helicase activity of DnaB and result in localized unwinding of the ori lambda template . The protein disassembly reactions are described in the accompanying articles.

Science, 1989 Jun 23, 244(4911), 1457 - 61
DNA looping generated by DNA bending protein IHF and the two domains of lambda integrase; Moitoso de Vargas L et al.; The multiprotein-DNA complexes that participate in bacteriophage lambda site-specific recombination were used to study the combined effect of protein-induced bending and protein-mediated looping of DNA . The protein integrase (Int) is a monomer with two autonomous DNA binding domains of different sequence specificity . Stimulation of Int binding and cleavage at the low affinity core-type DNA sites required interactions with the high affinity arm-type sites and depended on simultaneous binding of the sequence-specific DNA bending protein IHF (integration host factor) . The bivalent DNA binding protein is positioned at high affinity sites and directed, by a DNA bending protein, to interactions with distant lower affinity sites . Assembly of this complex is independent of protein-protein interactions.

J Mol Biol, 1989 Jun 20, 207(4), 695 - 717
Determinants of site-specific recombination in the lambdoid coliphage HK022 . An evolutionary change in specificity; Yagil E et al.; The temperate bacteriophage HK022, like its relative lambda, inserts its chromosome into a specific site in the bacterial chromosome during lysogenization and excises it after induction . However, we find that the recombinational specificities of the two phages differ: they use different bacterial sites, and neither promotes efficient insertion or excision of the other phage chromosome . In order to determine the basis for this difference in specificity, we sequenced the HK022 elements that are involved in insertion and excision, and compared them to the corresponding lambda elements . The location, orientation, size and overall arrangement of the int and xis genes and the phage attachment sites are nearly identical in the two genomes, as is common for other functionally related elements in lambdoid phages . The Xis proteins of the two phages are functionally interchangeable, and their predicted amino acid sequences differ by but one residue . In contrast, the two Int proteins are not functionally interchangeable, and their sequences, although similar, differ at many positions . These sequence differences are not uniformly distributed: the amino-terminal 55 residues are completely conserved, but the remaining 302 show a pattern of differences interspersed with identities and conservative changes . These findings imply that the specificity difference between HK022 and lambda site-specific recombination is a consequence of the inability of the respective Int proteins to recognize pairs of heterologous attachment sites . The two phage attachment sites are remarkably similar, especially the two "arm" segments, which in lambda contain binding sites for Int, Xis and integration host factor . They are less similar in the segment between the two arms, which in lambda contains the points of recombinational strand exchange and a second class of binding site for Int protein (the "core-type" sites) . The two bacterial attachment sites are quite different, although both have a short stretch of perfect homology with their respective phage partners at the points of strand exchange . We propose that the two Int proteins recognize similar or identical sites in the arms of their cognate attachment sites, and that differences in binding or action at the core-type sites is responsible for the divergent specificities . Genetic experiments and sequence comparisons suggest that both proteins recognize different but overlapping families of core-type sites, and that divergence in specificity has been achieved by an alternating succession of small, mutually compatible changes in protein and site.

FEBS Lett, 1989 Jun 19, 250(1), 91 - 8
Structure of the alpha 1 subunit of horse Na,K-ATPase gene; Kano I et al.; Genomic DNA for Na,K-ATPase alpha 1 subunit was obtained from libraries of horse kidney genomic DNA in Charon 4A and in EMBL3 bacteriophages by screening with the full sized cDNA probe of the alpha 1 subunit of rat Na,K-ATPase as probe . The gene spans 30 kb and consists of 23 exons and 22 intervening sequences . Intron-exon boundaries were analyzed . The protein-coding nucleotide sequence encodes 1016 amino acids with an Mr of 112,264 . The putative amino acid sequence of horse alpha 1 is 96-97% homologous to those of other mammalian species.

Biochem J, 1989 Jun 15, 260(3), 931 - 4
Discovery of a protein phosphatase activity encoded in the genome of bacteriophage lambda . Probable identity with open reading frame 221; Cohen PT et al.; Infection of Escherichia coli with phage lambda gt10 resulted in the appearance of a protein phosphatase with activity towards 32P-labelled casein . Activity reached a maximum near the point of cell lysis and declined thereafter . The phosphatase was stimulated 30-fold by Mn2+, while Mg2+ and Ca2+ were much less effective . Activity was unaffected by inhibitors 1 and 2, okadaic acid, calmodulin and trifluoperazine, distinguishing it from the major serine/threonine-specific protein phosphatases of eukaryotic cells . The lambda phosphatase was also capable of dephosphorylating other substrates in the presence of Mn2+, although activity towards 32P-labelled phosphorylase was 10-fold lower, and activity towards phosphorylase kinase and glycogen synthase 25 50-fold lower than with casein . No casein phosphatase activity was present in either uninfected cells, or in E . coli infected with phage lambda gt11 . Since lambda gt11 lacks part of the open reading frame (orf) 221, previously shown to encode a protein with sequence similarity to protein phosphatase-1 and protein phosphatase-2A of mammalian cells {Cohen, Collins, Coulson, Berndt & da Cruz e Silva (1988) Gene 69, 131-134}, the results indicate that ORF221 is the protein phosphatase detected in cells infected with lambda gt10 . Comparison of the sequence of ORF221 with other mammalian protein phosphatases defines three highly conserved regions which are likely to be essential for function . The first of these is deleted in lambda gt11.

Mol Biochem Parasitol, 1989 Jun 15, 35(2), 119 - 25
An oligonucleotide probe specific for Onchocerca volvulus; Harnett W et al.; A genomic DNA library of a Liberian strain of Onchocerca volvulus was prepared in the vector bacteriophage lambda gt10 . The library was differentially screened by hybridisation with radiolabelled total DNA from the homologous parasite, two heterologous Onchocerca parasites (Onchocerca gibsoni and Onchocerca gutturosa) and human liver cells . A clone (C1A1) was isolated whose binding to O . volvulus DNA was at least 50 times stronger than to the other parasite DNA samples . No binding was observed with human DNA . The insert of C1A1 was subcloned into the filamentous phage vector M13 mp18 and sequenced . Two oligonucleotides, each corresponding to a unique region of 60 nucleotides (out of a total of 154) were synthesised and examined for hybridisation with three different geographical isolates of O . volvulus (including forest and savannah strains) and six other Onchocerca spp . One of the oligonucleotides (C1A1-2) was found to hybridise to the three O . volvulus isolates with an intensity in the region of 300 times greater than to any other Onchocerca spp . Since the other species include the two which may be most closely related to O . volvulus, i.e., O . gibsoni and Onchocerca ochengi, it is concluded that C1A1-2 is likely to represent a truly species-specific probe.

J Biol Chem, 1989 Jun 15, 264(17), 10139 - 47
Large-scale purification and characterization of the Escherichia coli rep gene product; Lohman TM et al.; We report a procedure for the large-scale purification of the Escherichia coli Rep protein, a helicase that is involved in the replication of the E . coli chromosome as well as a number of single-stranded bacteriophages . The procedure starts with E . coli cells harboring an overproducing plasmid, pRepO, in which the E . coli rep gene is under transcriptional control of the inducible lambda PL promoter (Colasanti, J., and Denhardt, D . T . (1987) Mol . Gen . Genet . 209, 382-390) . The purification procedure results in greater than 98% pure Rep protein, which is free of contaminating nuclease activity, with yields of 40-50 mg of Rep protein/50 g of induced MZ-1/pRepO cells . We also show that cell death occurs upon inducing such a large overproduction of the E . coli Rep protein in MZ-1/pRepO . The Rep protein purified by this procedure has high specific single-stranded DNA-dependent ATPase activity, as well as helicase activity, with an apparent 3' to 5' directionality . The extinction coefficient of purified E . coli Rep protein is epsilon 280 = 1.16 +/- 0.04 ml mg-1 cm-1 (8.47 +/- 0.28 X 10(4) M-1 cm-1) in 10 mM Tris (pH 7.5), 20% (v/v) glycerol, 0.10 M NaCl at 25 degrees C . The solubility properties of the purified Rep protein have been examined as a function of glycerol, NaCl, MgCl2, ATP, and ADP concentrations at 25 and 37 degrees C (pH 7.5) . Rep protein solubility decreases significantly with decreasing concentrations of glycerol and monovalent salt and increasing temperature; however, the presence of 1.5 mM ATP or ADP or MgCl2 at low NaCl concentrations increases the solubility . At 4 degrees C, in the presence of 20% glycerol and greater than or equal to 50 mM NaCl, the free Rep protein exists as a stable monomer under all conditions examined (+/- ATP and +/- MgCl2) . The single-stranded DNA-dependent ATPase activity decreases with increasing glycerol concentration, such that in 25% (v/v) glycerol it has approximately 40% of its activity as compared to solutions that contain no glycerol . The dependence of the single-stranded DNA-dependent ATPase activity on salt concentration for a series of monovalent salts indicates the presence of both cation and anion effects, with decreasing activity in the order glutamate greater than acetate greater than chloride . The ability to obtain highly purified E . coli Rep protein in large quantities with relative ease will greatly facilitate physical characterizations of the protein and its interactions with DNA.

Biochemistry, 1989 Jun 13, 28(12), 5210 - 8
Terminator-distal sequences determine the in vitro efficiency of the early terminators of bacteriophages T3 and T7; Telesnitsky A et al.; Bacteriophages T3 and T7 contain homologous terminators for Escherichia coli RNA polymerase that restrict early phage transcription to the leftmost 20% of the linear phage genomes . These two terminators serve equally well as p-independent terminators in vivo, but their in vitro efficiencies and sensitivity to salt and nucleotide concentrations differ dramatically . Sequence analysis shows that the T7 and T3 terminators differ at only two sites in the region normally accepted as defining terminator function . In order to determine which structural features of these two terminators are responsible for their functional differences, a series of hybrid terminators were constructed in which structural features of the two terminators were systematically interchanged . Transcription of hybrid terminator templates revealed that sequences downstream of the termination release sites are responsible for the differences in efficiency of in vitro termination . These sequences also determine the sensitivity of these terminators to elevated salt concentrations and to alterations of substrate concentrations . Alteration of the sequences in the region between three and seven nucleotides downstream of the final T7Te release site is sufficient to reduce termination efficiency to that of T3Te, and point mutations in this region yield terminators with intermediate efficiency . Hence, the determinants of p-independent terminator efficiency in vitro must include elements of the transcription complex other than the structure of the 3' end of the transcript . The termination differences between T7Te, T3Te, and their hybrid derivatives are overcome in vivo; all of these sites become very efficient . This finding further supports the hypothesis that protein factors or other cellular features enhance the efficiency and specificity of p-independent terminators in vivo.

Biochemistry, 1989 Jun 13, 28(12), 4993 - 9
Expression of a human liver cytochrome P-450 protein with tolbutamide hydroxylase activity in Saccharomyces cerevisiae; Brian WR et al.; The human liver cytochrome P-450 (P-450) proteins responsible for catalyzing the oxidation of mephenytoin, tolbutamide, and hexobarbital are encoded by a multigene family (CYP2C) . Although several cDNA clones and proteins related to this "P-450MP" family have been isolated, assignment of specific catalytic activities remains uncertain . Sulfaphenazole was found to inhibit tolbutamide hydroxylation to a greater extent than mephenytoin or hexobarbital hydroxylation . The inhibition by sulfaphenazole was competitive for tolbutamide and hexobarbital hydroxylation but with much different Ki values (5 vs 480 microM, respectively) . Inhibition of mephenytoin hydroxylase was not competitive . The results suggest that different P-450 proteins in the P450MP family may be involved in the metabolism of these compounds . A cDNA clone (MP-8) related to the P-450MP family, isolated from a bacteriophage lambda gt11 human liver library, was expressed in Saccharomyces cerevisiae by using the pAAH5 expression vector . Yeast transformed with pAAH5 containing the MP-8 sequence (pAAH5/MP-8) showed a ferrous-CO spectrum typical of the P-450 proteins . Immunoblotting with anti-P450MP revealed that pAAH5/MP-8 microsomes contained a protein with an Mr similar to that of P-450MP-1 (approximately 48,000) that was not present in microsomes from yeast transformed with pAAH5 alone (1.7 X 10(4) molecules of the expressed P-450 per cell) . Microsomes from pAAH5/MP-8 contained no detectable mephenytoin 4'-hydroxylase activity but were more active in tolbutamide hydroxylation, on a nanomoles of P-450 basis, than human liver microsomes . The pAAH5/MP-8 microsomes also contained hexobarbital 3'-hydroxylase activity, although the enrichment compared to liver microsomes was not great with respect to the tolbutamide hydroxylase activity.(ABSTRACT TRUNCATED AT 250 WORDS)

FEBS Lett, 1989 Jun 5, 249(2), 396 - 400
Binding properties of T4 gene 32 protein fragments carrying partially cleaved terminal domains; Casas-Finet JR; Analysis of fluorimetric equilibrium-binding isotherms of a proteolytic fragment of bacteriophage T4 gene 32 protein (g32P) lacking residues 1-9 shows that this region contains the site responsible for the function of the NH2-terminal 'B' domain (residues 1-21) . The end codon of the frameshift mutant g32P-PR201 has been identified as TAG at nucleotide position 852 . The PR201 gene 32 product ends at Ser283 and carries a truncated COOH-terminal 'A' domain (residues 253-301) . Fluorimetric titrations of g32P-PR201 with double-stranded DNA show that the functional residues of the A domain are located within the region spanning residues 284-301.

J Mol Biol, 1989 Jun 5, 207(3), 555 - 61
Nucleotide sequence and complementation studies of the gene 10 region of bacteriophage T3; Condreay JP et al.; The nucleotide sequence of bacteriophage T3 gene 10 and surrounding regulatory elements has been determined and compared to the analogous region of bacteriophage T7 . T3 genes 9, 10 and 11 have been shown to complement T7 mutants . The DNA sequences of T3 and T7 gene 10A are homologous, as are the amino acid sequences of the respective products . The translational shift to the -1 frame is predicted to occur at the same position in gene 10 of T3 and T7, though different nucleotide sequences are probably responsible . The resulting gp10B products have completely different C termini.

J Mol Biol, 1989 Jun 5, 207(3), 563 - 74
Mutants of bacteriophage T7 that escape F restriction; Molineux IJ et al.; Mutants of bacteriophage T7 that escape F restriction have been isolated . Two mutations in gene 10, which codes for the capsid protein, and one mutation in gene 1.2 are required for these phages to grow on F-containing strains . The products of these two genes are the two targets of the exclusion system; the presence of either wild-type product results in an abortive infection . Phages that grow normally in male hosts still lead to membrane dysfunction and nucleotide efflux from the infected cell . This type of membrane damage and the abortive infection are therefore separable phenomena.

J Mol Biol, 1989 Jun 5, 207(3), 543 - 54
Synthesis of the capsid protein inhibits development of bacteriophage T3 mutants that abortively infect F plasmid-containing cells; Condreay JP et al.; Mutants of bacteriophage T3 that lack gene 1.2 resemble wild-type phage T7 in that they are unable productively to infect F plasmid-containing cells of Escherichia coli . Pseudorevertants of a T3 gene 1.2 deletion mutant that have regained the ability to plate efficiently on male cells have been isolated and characterized . At least two mutations in the gene for the major capsid protein are necessary for these phages to bypass F-mediated restriction . One mutation serves to reduce the rate of synthesis of the capsid protein; a second mutation apparently alters an unknown property that is intrinsic to the free, or unassembled form of the protein . During the abortive infection of an F-containing host, synthesis of the wild-type capsid protein directly inhibits further phage development.

Biophys J, 1989 Jun, 55(6), 1237 - 49
Low angle light scattering studies on whole, half, and quarter molecules of T2 bacteriophage DNA; Harpst JA et al.; Static light scattering measurements have been made at angles as low as 8 degrees on whole, half, and quarter molecules of native, T2 bacteriophage DNA in 0.195 M Na+ . The fragments were obtained by high-speed stirring of the native DNA, and fractionated on methylated-albumin-kieselguhr columns . Accompanying measurements of sedimentation coefficients and intrinsic viscosities were made . Because linear extrapolations of light scattering data above 8 degrees for these samples were suspect, the measurements were analyzed by fitting curves calculated from the theory of wormlike coils to experimental curves at c = 0 . Results showed that the excluded volume parameter, epsilon, must be used in analyzing the scattering curves; a reasonable value of epsilon was 0.08, in agreement with that found for T7 DNA (Harpst, J . A . 1980 . Biophys . Chem . 11:295-302) . The persistence length of all three DNAs in this paper was 50 +/- 5 nm, showed no dependence on molecular weight, but was somewhat below that reported previously for T7 DNA (60 nm) . Theoretical curves calculated with the preceding parameters had a clear upward curvature in scattering envelopes below 8 degrees for quarter and half molecules, but such curvature was minimal for whole T2 DNA, so that linear extrapolations of experimental data above 8 degrees gave a molecular weight and root-mean-square radius which were nearly the same as those from theory . The molecular weight and radius for whole T2, derived from the comparison of theory and experiment, were 115 X 10(6) and 1,224 nm, respectively . The measurements on T2 DNA were clearly at the upper limit of current techniques.

Ann Allergy, 1989 Jun, 62(6), 547 - 52
IgM deficiency: clinical spectrum and immunologic assessment; Guill MF et al.; Selective IgM deficiency has been associated with recurrent infections and enteric protein loss . We have evaluated eight patients with IgM deficiency presenting with recurrent infections and manifestation of atopy . A variety of subtle immunologic aberrations were noted, including depressed IgM and elevated IgG responses to immunization with bacteriophage OX174 . Despite normal quantitative IgG in most patients, IgG antibody responses to diphtheria-tetanus and/or pneumococcal polysaccharide antigens were generally depressed . No correlation could be made between antibody responses (or lack thereof) and the nature of infections in any given patient.

J Gen Virol, 1989 Jun, 70 ( Pt 6), 1321 - 7
Further characterization of a bacteriophage recovered from an avian strain of Chlamydia psittaci; Storey CC et al.; The genome of a 22 nm icosahedral phage which infects some avian Chlamydia psittaci strains recovered from domestic ducks has been characterized as a ss circular DNA molecule of about 4850 bases . The replicative form of this genome was isolated from purified chlamydial organisms . A restriction endonuclease cleavage site map of the genome was constructed from dsDNA synthesized in vitro from ss phage DNA and EcoRI fragments were then cloned into pUC9 . The phage genome was detected only by Southern blot hybridization in C . psittaci which was productively infected with phage; no evidence was found for the integration of phage DNA into the chlamydial chromosome . Three viral polypeptides, of approximate Mr values 75K, 30K and 16.5K were identified when phage was analysed by SDS-PAGE . This virus, which we have designated Chp 1, is either an aberrant member of the Microviridae or the first member of a new bacteriophage family.

J Virol, 1989 Jun, 63(6), 2427 - 36
Analysis of near-neighbor contacts in bacteriophage T4 wedges and hubless baseplates by using a cleavable chemical cross-linker; Watts NR et al.; Although bacteriophage T4 baseplate morphogenesis has been analyzed in some detail, there is little information available on the spatial arrangement and associations of its 150 subunits . We have therefore carried out the first analysis of its near-neighbor interactions by using the cleavable chemical cross-linker ethylene glycolbis(succinimidylsuccinate) . In this report, we describe the cross-linked complexes that have been identified in the one-sixth arms or wedges and also in baseplatelike structures called rings consisting of six wedges but lacking the central hub, both of which are purified from T4 gene 5- -infected cells . Thirty different complexes were identified, of which about half contain multimers of a single species and half contain two different species . In general, the complexes reflect and support the assembly pathway derived by Kikuchi and King (Y . Kikuchi and J . King, J . Mol . Biol . 99:695-716, 1975) but broaden its scope to include such complexes as gp25-gp53, gp25-gp48, and gp48-gp53, which locate the gp48 binding site over the inner edge of the ring but outside the central hub . The data also supports the view that wedges are assembled from the outer edge inward toward the central hub . Wedge-wedge contact in rings was mediated primarily by gp12 and gp9, the absence of which dramatically destabilized the ring----wedge equilibrium in favor of wedges . Although no heterologous complexes containing gp9 were identified, gp12 contacts unique to rings were observed with both gp10 and gp11.

J Bacteriol, 1989 Jun, 171(6), 3579 - 82
Overproduced bacteriophage T4 gene 33 protein binds RNA polymerase; Williams KP et al.; Bacteriophage T4 gene 33 protein (gp33), which is required for viral late transcription, has been overproduced . The purified gp33 binds to RNA polymerase core from uninfected or T4-infected Escherichia coli, but the major E . coli transcription initiation factor, sigma 70, competed effectively for this binding.

J Bacteriol, 1989 Jun, 171(6), 3128 - 32
Streptomyces lipmanii expresses two restriction systems that inhibit plasmid transformation and bacteriophage plaque formation; Matsushima P et al.; Bacteriophage host range studies suggested that several beta-lactam-producing streptomycetes express similar restriction-modification systems . Streptomyces lipmanii LE32 expressed two restriction-modification systems, designated SliI and SliII . A mutant strain, PM87, was defective only in SliI restriction but expressed both SliI and SliII modification . Streptomyces sp . strain A57986, a natural isolate partially deficient in the expression of SliI and SliII restriction, nevertheless modified bacteriophage DNA for both SliI and SliII specificities . Protoplasts of PM87 and A57986 were transformed by several plasmids, and the modified plasmids isolated from these strains transformed wild-type S . lipmanii efficiently.

Proc Natl Acad Sci U S A, 1989 Jun, 86(11), 4076 - 80
Effect of manganese ions on the incorporation of dideoxynucleotides by bacteriophage T7 DNA polymerase and Escherichia coli DNA polymerase I; Tabor S et al.; Incorporation of dideoxynucleotides by T7 DNA polymerase and Escherichia coli DNA polymerase I is more efficient when Mn2+ rather than Mg2+ is used for catalysis . Substituting Mn2+ for Mg2+ reduces the discrimination against dideoxynucleotides approximately 100-fold for DNA polymerase I and 4-fold for T7 DNA polymerase . With T7 DNA polymerase and Mn2+, dideoxynucleotides and deoxynucleotides are incorporated at virtually the same rate . Mn2+ also reduces the discrimination against other analogs with modifications in the furanose moiety, the base, and the phosphate linkage . A metal buffer, isocitrate, expands the MnCl2 concentration range effective in catalyzing DNA synthesis . The lack of discrimination against dideoxynucleoside triphosphates using T7 DNA polymerase and Mn2+ results in uniform terminations of DNA sequencing reactions, with the intensity of adjacent bands on polyacrylamide gels varying in most instances by less than 10%.

Proc Natl Acad Sci U S A, 1989 Jun, 86(11), 4002 - 6
Translational repression in bacteriophage f1: characterization of the gene V protein target on the gene II mRNA; Michel B et al.; Previous studies have shown that the single-stranded DNA binding protein of bacteriophage f1 (gene V protein) represses the translation of the mRNA of the phage-encoded replication protein (gene II protein) . We have characterized phage mutations in the repressor and in its target . Using a gene II-lacZ translational fusion, we have defined a 16-nucleotide-long region in the gene II mRNA sequence that is required in vivo for repression by the gene V protein . We have shown that in vitro the binding affinity of the gene V protein is at least 10-fold higher to an RNA carrying this sequence than to an RNA lacking it . We propose that this sequence constitutes the gene II mRNA operator.

J Bacteriol, 1989 Jun, 171(6), 3331 - 6
Specific localization of the lysis protein of bacteriophage MS2 in membrane adhesion sites of Escherichia coli; Walderich B et al.; Specific localization of the lysis (L) protein of bacteriophage MS2 in the cell wall of Escherichia coli was determined by immunoelectron microscopy . After induction of the cloned lysis gene, the cells were plasmolyzed, fixed, and embedded in either Epon or Lowicryl K4M . Polyclonal L-protein-specific antiserum was purified by preabsorption to membranes from cells harboring a control plasmid . Protein A-gold was used to label the protein-antibody complexes . Between 42.8% (Lowicryl) and 33.8% (Epon) of the label was found in inner and outer membranes, but 30.3% (Lowicryl) and 32.8% (Epon) was present mostly in clusters in the adhesion sites visible after plasmolysis . The remaining label (26.9 and 33.4%, respectively) appeared to be present in the periplasmic space but may also have been part of membrane junctions not visible because of poor contrast of the specimen . In contrast, a quite different distribution of the L protein was found in cells grown under conditions of penicillin tolerance, i.e., at pH 5, a condition that had previously been shown to protect cells from L-protein-induced lysis . At tolerant conditions, only 21.0% of the L protein was in the adhesion sites; most of the protein (68.2%) was found in inner and outer membranes . It is concluded that lysis of the host, E . coli, was a result of the formation of specific L-protein-mediated membrane adhesion sites.

J Bacteriol, 1989 Jun, 171(6), 3002 - 7
Expression of cloned rpoB gene of Escherichia coli: a genetic system for the isolation of dominant negative mutations and overproduction of defective beta subunit of RNA polymerase; Lee JY et al.; The rifampin resistance rifD18 allele of rpoB, carried on the expression plasmid pXT7 beta, is controlled by a strong bacteriophage T7 late promoter and two weak Escherichia coli promoters . Depending on the host strain, pXT7 beta specifies different levels of Rifr beta subunit, providing a system for the isolation, maintenance, and overexpression of dominant lethal alleles of rpoB . In rpoB+ hosts, pXT7 beta confers the Rifr phenotype on the Rifs host . Negative rpoB mutations in the plasmid DNA can thus be scored by screening transformants for Rifs . In an rpoB(Am) supD(Ts) host in which chromosomal rpoB expression is decreased as the temperature goes up, some of the negative plasmid-borne rpoB mutations displayed a dominant phenotype . In a host harboring inducible T7 RNA polymerase, the defective beta subunits could be overexpressed independently of the E . coli transcriptional machinery . With this system, we isolated several negative rpoB mutations induced in vitro by hydroxylamine . Seven of the mutant rpoB alleles, when overexpressed, were found to specify normal-size beta polypeptides . Two of them displayed the dominant lethal phenotype in the rpoB(Am) supD(Ts) background . We also constructed a mutation (rpoB1800) in which 24 carboxy-terminal amino acids were substituted with a random 19-amino-acid sequence . The nonfunctional rpoB1800 beta polypeptide was isolated and assembled in vitro into the core enzyme molecule.

Biotechniques, 1989 Jun, 7(6), 590 - 2
A dedicated database program for cataloging recombinant clones and other laboratory products of molecular biology technology; Jenson HB; A novel computer database program dedicated to storing, cataloging, and accessing information about recombinant clones and libraries has been developed for the IBM (or compatible) personal computer . This program, named CLONES, also stores information about bacterial strains and plasmid and bacteriophage vectors used in molecular biology . The advantages of this method are improved organization of data, fast and easy assimilation of new data, automatic association of new data with existing data, and rapid retrieval of desired records using search criteria specified by the user . Individual records are indexed in the database using B-trees, which automatically index new entries and expedite later access . The use of multiple windows, pull-down menus, scrolling pick-lists, and field-input techniques make the program intuitive to understand and easy to use . Daughter databases can be created to include all records of a particular type, or only those records matching user-specified search criteria . Separate databases can also be merged into a larger database . This computer program provides an easy-to-use and accurate means to organize, maintain, access, and share information about recombinant clones and other laboratory products of molecular biology technology.

Mol Gen Genet, 1989 Jun, 217(2-3), 392 - 400
Regulation of repressor and early gene expression in Mu-like transposable bacteriophage D108; Levin DB et al.; The temperate, transposable bacteriophages D108 and Mu are highly homologous, but differ in their lef-end regulatory regions . We have previously cloned the gene encoding the D108 thermo-sensitive (cts) repressor under the control of the lactUV5 promoter . In this work, we report that crude protein extracts containing highly-expressed D108 repressor protect a 77 bp region of DNA, located between 863 bp and 940 bp from the D108 lef--end, from both exonuclease III and DNase I hydrolysis . Nucleotide sequence analysis of this region reveals that is also contains DNA sequences homologous to the consensus DNA-binding site of the Escherichia coli protein, Integration Host Factor (IHF) . Crude protein extracts containing highly-expressed IHF specifically bind to, and retard the migration of, DNA fragments containing the D108 regulatory region, and the DNA sequence which IHF protects from DNase I cleave lies directly within the D108 repressor binding region . There are two apparent repressor-specific S1 nuclease-resistant RNA suggests that transcription from the early region promoter, Pe may initiate at or about 1000 bp from the left-end of the D108 genome . Thus though, D108 and Mu utilize three analogous proteins (repressor, ner, and IHF) and the same apparent promoters for early gene regulation and the lytic/lysogenic decision, the organization of these regulatory components is apparently different, suggesting different mechanisms of control of gene expression.

Mol Gen Genet, 1989 Jun, 217(2-3), 215 - 22
Genetic deletions between directly repeated sequences in bacteriophage T7; Pierce JC et al.; DNA sequence analysis of genetic deletions in bacteriophage T7 has shown that these chromosomal rearrangements frequently occur between directly repeated DNA sequences . To study this type of spontaneous deletion in more quantitative detail synthetic fragments of DNA, made by hybridizing two complementary oligonucleotides, were introduced into the non-essential T7 gene 1.3 which codes for T7 DNA ligase . This insert blocked synthesis of functional ligase and made the phage that carried an insert unable to form plaques on a host strain deficient in bacterial ligase . The sequence of the insert was designed so that after it is put into the T7 genome the insert is bracketed by direct repeats . Perfect deletion of the insert between the directly repeated sequences results in a wild-type phage . It was found that these deletion events are highly sensitive to the length of the direct repeats at their ends . In the case of 5 bp direct repeats excision from the genome occurred at a frequency of less than 10(-10), while this value for an almost identical insert bracketed by 10 bp direct repeats was approximately 10(-6) . The deletion events were independent of a host recA mutation.

J Virol, 1989 Jun, 63(6), 2829 - 34
Cloning and partial DNA sequencing of two new human papillomavirus types associated with condylomas and low-grade cervical neoplasia; Lorincz AT et al.; Using low-stringency Southern blot analysis and cloning in lambda bacteriophage, two new human papillomavirus types (HPV-43 and HPV-44) were identified and their DNAs were cloned from vulvar tissues . The isolates were characterized by restriction endonuclease mapping and shown to be new HPV types on the basis of their minimal hybridization with all other known HPV types at high stringency . Both HPVs are most closely related to types 6, 11, and 13 . HPV-43 did not exhibit any cross-reactivity with these HPV types at high stringency . HPV-44 showed minimal cross-reactivity to HPV-13, which was in the range of 20 to 25% according to liquid hybridization analysis . The deduced genomic organization of each of the two new HPVs was colinear with HPV-6b . Prevalence studies revealed that HPV-43 and HPV-44 together were found in 6 of 439 normal cervical tissues, in 8 of 195 cervical intraepithelial neoplasms, but in none of 56 cervical cancers tested thus far.

J Virol, 1989 Jun, 63(6), 2820 - 8
Analysis of a large-T-antigen variant expressed in simian virus 40-transformed mouse cell line mKS-A; Dora S et al.; Earlier reports had suggested that the large T antigen expressed in simian virus 40 (SV40)-transformed mKS-A cells may be replication defective . Our experiments support these earlier observations showing that the mKS-A T antigen has a reduced DNA-unwinding activity in vitro . To investigate the molecular basis for this defect, we have isolated from an mKS-A genomic library an EMBL-3 bacteriophage clone carrying in its insert a full-length SV40 DNA element that most likely encodes the expressed T-antigen variant . DNA sequencing revealed only one nonconservative amino acid exchange, Asp to Asn at residue 636 . Surprisingly, when a plasmid clone carrying the mKS-A T-antigen-coding sequence was transfected into monkey cells, we found that it replicated quite efficiently, probably suggesting that a high nuclear concentration of the variant T-antigen form compensates for the partial biochemical defect . However, a high nuclear concentration of T antigen was also found in mKS-A T-antigen-transformed mouse cells, yet a fusion of these cells to permissive monkey cells failed to induce in situ replication and excision of integrated SV40 DNA . We discuss possible reasons for the different behavior of T antigen in monkey cells and in mouse cells and suggest that one possibility for the replication-negative phenotype in transformed cells may be related to the fact that T antigen forms a tight complex with the cellular p53 protein in mouse cells but not in monkey cells.

J Bacteriol, 1989 Jun, 171(6), 3523 - 9
Suppression of recA deficiency in plasmid recombination by bacteriophage lambda beta protein in RecBCD- ExoI- Escherichia coli cells; Berger I et al.; Plasmid recombination, like other homologous recombination in Escherichia coli, requires RecA protein in most conditions . We have found that the plasmid recombination defect in a recA mutant can be efficiently suppressed by the beta protein of bacteriophage lambda . beta protein is required for homologous recombination of lambda chromosomes during lytic phage growth in a recA host and is known to have a strand-annealing activity resembling that of RecA protein . The bioluminescence recombination assay was used for genetic analysis of beta-protein-mediated plasmid recombination . Efficient suppression of the recA mutation by beta protein required the absence of the E . coli nucleases exonuclease I and RecBCD nuclease . These nucleases inhibit a RecA-mediated plasmid recombination pathway that is more efficient than the pathway functioning in wild-type cells . Like RecA-mediated plasmid recombination in RecBCD- ExoI- cells, beta-protein-mediated plasmid recombination depended on concurrent DNA replication and on the activity of the recQ gene . However, unlike RecA-mediated plasmid recombination, beta-protein-mediated recombination in RecBCD- ExoI- cells was independent of recF and recJ activities . We propose that inactivation of exonuclease I and RecBCD nuclease stabilizes a recombination intermediate that is involved in RecA- and beta-protein-catalyzed homologous pairing reactions . We suggest that the intermediate may be linear plasmid DNA with a protruding 3' end, since these nucleases are known to interfere with the synthesis of such linear forms . The different recF and recJ requirements for beta-protein-dependent and RecA-dependent recombinations imply that the mechanisms of formation or processing of the putative intermediate differ in the two cases.

J Bacteriol, 1989 Jun, 171(6), 3427 - 32
Stimulation of IS1 excision by bacteriophage P1 ref function; Lu SD et al.; Lysogenization by a c1ts variant of coliphage P1, P1c1.100, markedly increased the frequency of reversion of a galT::IS1 mutation . The formation of Gal+ colonies presumably occurs by microhomologous recombination between the 9-base-pair repeats in galT (CGCCGCTAC) generated by the transposition of IS1 . The responsible P1 gene, ref, has been cloned and sequenced . ref encodes a 22.8-kilodalton protein and is located near the P1 site-specific recombination function, cre . Expression of ref was repressed by P1 c+ . The absence of a distinctive ribosome-binding site is consistent with a poor translation of ref from an expression vector in vivo . Placement of a ribosome-binding site before ref resulted in the extensive synthesis of the Ref protein . Ref stimulated precise excision in recB or himA cells, but not in recA mutants . Ref was active in lexA3 mutants, suggesting that the recombination activity of RecA was directly involved in the reaction . We have constructed a P1c1.100 ref::Tn10 mutant . The absence of Ref did not appear to restrict dramatically the ability of P1 to grow lytically or to form lysogens . Thus, the role of ref in the physiology of P1 remains to be determined.

J Bacteriol, 1989 Jun, 171(6), 3080 - 4
Transduction and transformation of plasmid DNA in Streptomyces fradiae strains that express different levels of restriction; Matsushima P et al.; We constructed nonrestricting strains of Streptomyces fradiae blocked in different steps in tylosin biosynthesis . Plasmid transformation frequencies were 10(3)- to 10(4)-fold higher and bacteriophage plating efficiencies were 10(4)- to 10(8)-fold higher in the nonrestricting strains than in the restricting strains . The efficiencies of transduction of plasmid pRHB101 in S . fradiae strains varied by over 1,000-fold, depending on growth conditions, and optimum transduction frequencies were obtained when cells were grown to mid-exponential phase at 39 degrees C . Under these conditions, restricting and nonrestricting strains were transduced at frequencies that differed by only two- to fivefold.

EMBO J, 1989 Jun, 8(6), 1833 - 9
Exonucleolytic proofreading increases the accuracy of DNA synthesis by human lymphocyte DNA polymerase alpha-DNA primase; Bialek G et al.; DNA polymerase-primase complex, isolated with an apparently undegraded alpha-subunit, was immunoaffinity-purified to near homogeneity from the human lymphoblast line HSC93 . The undegraded state of the alpha-subunit was monitored by Western-blot analysis of crude cellular extracts and all active fractions obtained during purification . The human polymerase-primase consists of four subunits with molecular weights of 195, 68, 55 and 48 kd . The fidelity of the polymerase-primase in copying bacteriophage phi X174am16 DNA in vitro was determined by measuring the frequency of production of different revertent phages . The overall accuracy was between 4 x 10(-6) and 10 x 10(-6) . This value reflects the spontaneous mutation frequency of phi X174am16 phages in Escherichia coli, and is 10- to 20-fold higher than the accuracy of a conventionally purified enzyme from calf thymus . The frequencies of base pairing mismatches, estimated from pool bias measurements, were 3.5 x 10(-7) (1/2 880,000) for dGMP:Ttemplate mispairs, between 10(-7) and 10(-8) for dCMP:Ttemplate (1/35,000,000), dCMP:Atemplate (1/18,200,000) and dAMP:Gtemplate mispairs (1/16,500,000), and below 10(-8) (1/100,000,000) for dTMP:Ttemplate, dGMP:Atemplate and dGMP:Gtemplate mispairs . In contrast to previous preparations, the intact polymerase-primase possesses a 3'----5' exonuclease activity . This exonuclease removes both matched and mismatched 3'-OH ends, with a preference for mismatched bases . Fidelity was reduced 8-fold by increasing the concentration of the next nucleotide following the incorporated mismatch nucleotide.(ABSTRACT TRUNCATED AT 250 WORDS)

Transplantation, 1989 Jun, 47(6), 1061 - 7
Immune dysfunction associated with graft-versus-host reaction in mice transplanted across minor histocompatibility barriers . I . Depressed antigen-specific antibody responses to bacteriophage phi chi 174; Hamilton BL et al.; The effect of the graft-versus-host reaction in response to minor histocompatibility antigens on the antibody response to a T-dependent antigen was studied in four strains of mice . Lethally irradiated mice were transplanted with bone marrow plus graded numbers of spleen cells from H-2-compatible donors . Recipients of syngeneic bone marrow transplants and recipients of allogeneic bone marrow depleted of T cells made normal antibody responses to bacteriophage phi chi 174 when immunized on day 28 (primary) and day 56 (secondary) after marrow transplantation . Recipients of allogeneic bone marrow plus spleen cells made only small amounts of specific antibody and failed to make IgG antibody after secondary immunization . The pattern of the depressed antibody response suggests that the primary mechanism of immune dysfunction in mice with the minor antigen GVHR is a lack of T helper cell function.

J Bacteriol, 1989 Jun, 171(6), 3440 - 8
Localization and regulation of bacteriophage Mu promoters; Stoddard SF et al.; Mu promoters active during the lytic cycle were located by isolating RNA at various times after induction of Mu prophages, radiolabeling it by capping in vitro, and hybridizing it to Mu DNA fragments on Southern blots . Signals were detected from four new promoters in addition to the previously characterized Pe (early), PcM (repressor), and Pmom (late) promoters . A major signal upstream of C was first observed at 12 min and intensified thereafter with RNA from cts and C amber but not replication-defective prophages; these characteristics indicate that this signal arises from a middle promoter, which we designate Pm . With 20- and 40-min RNA, four additional major signals originated in the C-lys, F-G-I, N-P, and com-mom regions . These signals were missing with RNA from C amber and replication-defective prophages and therefore reflected the activity of late promoters, one of which we presume was Pmom . Uninduced lysogens showed weak signals from five regions, one from the early regulatory region, three between genes B and lys, and one near the late genes K, L, and M . The first of these probably resulted from PcM activity; the others remain to be identified.

Virology, 1989 Jun, 170(2), 442 - 9
Protein-primed replication of bacteriophage PRD1 genome in vitro; Yoo SK et al.; A cell-free system has been developed from cells of an Escherichia coli strain, carrying cloned genes 1 and 8 of bacteriophage PRD1, that catalyzes protein-primed DNA synthesis . DNA synthesis in vitro is entirely dependent upon the addition of PRD1 DNA-protein complex as template, Mg2+, and four deoxyribonucleoside triphosphates . No in vitro DNA synthesis was observed when deproteinized PRD1 DNA was used as template . The origin and direction of PRD1 DNA replication in vitro was determined by restriction enzyme analysis of 32P-labeled PRD1 DNA synthesized in this system . Replication starts at both ends of the linear PRD1 DNA template . Alkaline sucrose gradient centrifugation and agarose gel electrophoresis showed that full-length PRD1 DNA is synthesized in vitro . DNA synthesis in this system is inhibited by the drug aphidicolin . We also observed that dimethyl sulfoxide (DMSO) stimulates in vitro DNA synthesis, although it inhibits bacterial DNA polymerase.

Int J Biol Macromol, 1989 Jun, 11(3), 159 - 64
Dynamics of telescoping Inovirus: a mechanism for assembly at membrane adhesions; Marvin DA; Telescoping of Inovirus (filamentous bacteriophage) into short hollow tubes by organic solvents suggests a molecular mechanism both for infection and for maturation of the virion at adhesions between the inner and outer bacterial membranes . The symmetry of alpha-helix subunit arrangement in the virion is related to the symmetry of leaf arrangement in plants (phyllotaxis) and is conserved in a molecular rearrangement model of the telescope.

Virology, 1989 Jun, 170(2), 392 - 407
Epitope mapping by deletion mutants and chimeras of two vesicular stomatitis virus glycoprotein genes expressed by a vaccinia virus vector; Keil W et al.; Deletion mutants and chimeras of the glycoprotein (G) genes of vesicular stomatitis virus serotypes Indiana (VSV-Ind) and New Jersey (VSV-NJ) were cloned in plasmids and vaccinia virus vectors under control of the bacteriophage T7 polymerase promoter for expression in CV-1 cells co-infected with a T7 polymerase-expressing vaccinia virus recombinant . Truncated and chimeric G proteins expressed by these vectors were tested for their capacity to react with VSV-Ind and VSV-NJ epitope-specific monoclonal antibodies (MAbs) by Western blot analysis for those antigenic determinants not affected by disulfide-bond reducing conditions or by immuno dotblot analysis for those that are . These experiments allowed us to create putative epitope maps for glycoproteins of both serotypes based on binding affinity and cross-reactivity of VSV-Ind and VSV-NJ MAbs for truncated or chimeric G proteins of known amino acid sequences . Seven of the 9 VSV-NJ G epitopes, including all 4 epitopes involved in virus neutralization by MAbs, mapped to the center (amino acid sequence 193-289) of the 517 amino acid VSV-NJ G protein . Four of the 11 VSV-Ind G epitopes, including 2 neutralizable epitopes, mapped to the cysteine-rich amino-terminal domain (amino acid sequence 80-183) of the 511 amino acid VSV-Ind G protein; the remaining 7 VSV-Ind G epitopes, including 2 involved in virus neutralization, were clustered in the cysteine-poor carboxy-terminal domain (amino acid sequence 286-428) . In site-specific mutants of the VSV-Ind G gene defective in one or both glycosylation sites, only the amino-terminal epitopes of the VSV-Ind G protein were affected by deletion of the carbohydrate chain at residue 179; deletion of the carbohydrate chain at residue 336 did not alter reactivity of the G protein with any of the relevant monoclonal antibodies . These results are discussed in relation to earlier attempts to map the antigenic determinants of VSV-NJ and VSV-Ind G proteins by proteolysis of the G protein and by sequencing the G genes of mutant viruses selected for their resistance to neutralization by epitope-specific monoclonal antibodies.

Gene, 1989 May 30, 78(2), 267 - 75
Isolation and structural analysis of the mouse beta-casein gene; Yoshimura M et al.; Three overlapping clones containing the entire beta-casein gene were isolated from a mouse genomic library constructed in bacteriophage lambda EMBL3 . Within the three clones the 6.8-kb casein gene was flanked by about 6-kb (5') and about 10-kb (3') sequences . The complete nucleotide sequence of the gene and its immediate flanking regions was determined . The gene consisted of nine exons ranging from 21 bp to 525 bp separated by introns ranging from 81 bp to 1288 bp . The length of the first exon was 43 bp, as determined by primer extension . The comparison of this sequence with that of the rat beta-casein gene showed that the overall structure of the gene was highly conserved . However, the first intron of the mouse gene was 400 bp shorter than that of the rat gene . Further analysis of homology between this gene and four other casein genes from rat and bovine revealed the presence of four highly conserved sequences . A search for the consensus sequences of transcription-regulatory elements showed that several potential regulatory sequences were present in the gene and its 5' flanking region.

Nucleic Acids Res, 1989 May 25, 17(10), 3909 - 25
Mutational analysis of the lac regulatory region: second-site changes that activate mutant promoters; Rothmel RK et al.; Second-site mutations that restored activity to severe lacP1 down-promoter mutants were isolated . This was accomplished by using a bacteriophage f1 vector containing a fusion of the mutant E . coli lac promoters with the structural gene for chloramphenicol acetyltransferase (CAT), so that a system was provided for selecting phage revertants (or pseudorevertants) that conferred resistance of phage-infected cells to chloramphenicol . Among the second-site changes that relieved defects in mutant lac promoters, the only one that restored lacP1 activity was a T----G substitution at position -14, a weakly conserved site in E . coli promoters . Three other sequence changes, G----A at -2, A----T at +1, and C----A at +10, activated nascent promoters in the lac regulatory region . The nascent promoters conformed to the consensus rule, that activity is gained by sequence changes toward homology with consensus sequences at the -35 and -10 regions of the promoter . However, the relative activities of some promoters cannot be explained solely by consideration of their conserved sequence elements.

Nucleic Acids Res, 1989 May 25, 17(10), 3747 - 55
Flanking DNA-sequences contribute to the specific binding of cI-repressor and OR1; Brenowitz M et al.; The binding of cI-repressor to a series of mutant operators containing OR1 of the right operator of bacteriophage lambda was investigated . Sites OR2 and/or OR3 were inactivated by either point or deletion mutations . The free energy of binding repressor to OR1 in the wildtype operator, delta G1, is -13.7 +/- 0.3 kcal/mol . delta G1 determined for an OR2- operator created by a single point mutation in OR2 is -13.6 +/- 0.2 kcal/mol . In contrast, delta G1 for the binding of repressor to a cloned synthetic OR1 operator containing only 24 bp of lambda sequence is -12.2 +/- 0.1 kcal/mol . When sequence 5' to OR1 is present, the binding affinity increases to -13.0 +/- 0.1 kcal/mol . In addition, the proximity of OR1 to a fragment-end decreases delta G1 from -13.7 to -12.3 +/- 0.1 kcal/mol . These results suggest that the DNA sequence outside the 17 bp OR1 binding-site contributes to the specific binding of cI-repressor.

J Mol Biol, 1989 May 20, 207(2), 311 - 33
Paramyosin gene (unc-15) of Caenorhabditis elegans . Molecular cloning, nucleotide sequence and models for thick filament structure; Kagawa H et al.; Paramyosin is a major structural component of thick filaments isolated from many invertebrate muscles . The Caenorhabditis elegans paramyosin gene (unc-15) was identified by screening with specific antibodies an "exon-expression" library containing lacZ/nematode gene fusions . Short probes recovered from the library were used to identify bacteriophage lambda and cosmid clones that encompass the entire paramyosin (unc-15) gene . From these clones, numerous subclones containing epitopes reacting with anti-paramyosin sera were obtained, providing strong evidence that the initial cloned fragment was, in fact, derived from the structural gene for paramyosin . The complete nucleotide sequence of a 12 x 10(3) base-pair region spanning the gene was obtained . The gene is composed of ten short exons encoding a protein of 866 {corrected} amino acid residues . Paramyosin is highly similar to residues 267 to 1089 of myosin heavy chain rods . For most of its length, paramyosin appears to form an alpha-helical coiled-coil and shows the expected heptad repeat of hydrophobic amino acid residues and the 28-residue repeat of charged amino acids characteristic of myosin heavy chain rods . However, paramyosin differs from myosin in having non-helical extensions at both the N and C termini and an additional "skip" residue that interrupts the 28-residue repeat . The distribution of charges along the length of the paramyosin rod is also significantly different from that of myosin heavy chain rods . Potential charge-mediated interactions between paramyosin rods and between paramyosin and myosin rods were calculated using a model successfully applied previously to the analysis of the myosin rod sequences . Myosin rods aligned in parallel show optimal charge-charge interactions at multiples of 98 residue staggers (i.e . at axial displacements of multiples of 143 A) . Paramyosin rods, in contrast, appear to interact optimally at parallel staggers of 493 residues (i.e . at axial displacements of 720 A) but show only weak interaction peaks at 98 or 296 residues . Similar calculations suggest optimal interactions between paramyosin molecules and myosin rods and in their anti-parallel alignments . The implications of these results for the structure of the bare zone and the assembly of nematode thick filaments are discussed.

Biochemistry, 1989 May 16, 28(10), 4277 - 83
Sequence-dependent termination of bacteriophage T7 transcription in vitro by DNA-binding drugs; White RJ et al.; An in vitro T7 bacteriophage transcription system has been utilized in which the RNA was initiated to a specific length (defined by the absence of the appropriate nucleoside triphosphate) . When the DNA-RNA-RNA polymerase ternary complex was exposed to nonsaturating levels of DNA-binding ligands (i.e., a small fractional occupancy at each site), and the RNA transcript then allowed to elongate in the presence of all four nucleoside triphosphates, there was a synchronous increase of RNA lengths up to sites occupied by ligands . A unique characteristic is that bacteriophage transcription was completely terminated at every ligand site, in contrast to bacterial RNA polymerases where "read-through" past drug sites occurs and results merely in a delay of transcription at each site due primarily to dissociation of drug from the DNA . Similar termination of transcription at each drug site was observed with T3 and SP6 RNA polymerases . The termination at drug sites in the bacteriophage system results in RNA of specific lengths which define the location of ligand sites, and the RNA concentration provides a measure of relative ligand occupancy at that site . Termination of transcription was observed with four drugs with relatively long DNA residence times (half-life greater than or equal to 300 s at 20 degrees C for nogalamycin, actinomycin, mithramycin, and echinomycin) but to a lesser extent with drugs of intermediate residence times {a bis(thiadaunomycin) and an acridine-tripyrrole, with half-lives of 230 and 7 s, respectively, at 20 degrees C}

J Biol Chem, 1989 May 15, 264(14), 7814 - 20
Synergistic action of three recombination gene products of bacteriophage T4, uvsX, uvsY, and gene 32 proteins; Yonesaki T et al.; Previously we proved that uvsX protein catalyzes reactions similar to those thought to occur during T4 recombination (Yonesaki, T., Ryo, Y., Minagawa, T., and Takahashi, H . (1985) Eur . J . Biochem . 148, 127-134; Yonesaki, T., and Minagawa, T . (1985) EMBO J . 4, 3321-3327) . Now we have found that uvsY protein stabilizes binding of uvsX protein and single-stranded DNA and weakly stimulates those reactions catalyzed by uvsX protein, although it has little activity, if any, by itself . Gene 32 protein also stimulates them weakly at low concentrations, but is strongly inhibitory at high concentrations (Yonesaki, T., and Minagawa, T . (1985) EMBO J . 4, 3321-3327; Formosa, T., and Alberts, B . M . (1986) J . Biol . Chem . 261, 6107-6118) . This inhibition is counteracted by uvsY protein . The highest efficiency of uvsX protein is achieved by the co-existence of a small amount of uvsY protein and a large amount of gene 32 protein . We discuss the mechanism and the role of the three proteins in recombination.

Gene, 1989 May 15, 78(1), 85 - 91
Expression vector with two-step control by the cI-pR-Q-p'R-qut-t'R module of coliphage lambda; Petrenko LA et al.; A plasmid expression vector (pCEQ3), using temperature-regulated transcription from the p'R promoter of bacteriophage lambda, has been constructed . The vector is derived from pBR327 in which the EcoRI-ClaI fragment of plasmid DNA is replaced with a 2.2-kb DNA module cI857-pR-Q-p'R-qut-t'R, consisting of two regions of the lambda genome . The first region contains the repressor gene cI857 and promoter pR; the second one contains gene Q and the late promoter p'R . When the repressor protein, product of the cI857 gene, becomes temperature-inactivated, it allows the promoter pR to initiate the transcription of the Q gene . The product of the Q gene, in turn, acts as a positive regulator of transcription from promoter p'R . The promoter activity of pR is fully repressed at a low temperature (30 degrees C) and transcription from p'R is terminated in the absence of Q gene product, but the shift of temperature up to 37 degrees C is sufficient to make the transcription from the p'R promoter highly active . Foreign genes can be inserted into the single EcoRI site downstream from the p'R promoter . The resultant constructions express extremely high levels of the cloned gene product in Escherichia coli.

Ultramicroscopy, 1989 May 11, 27(4), 367 - 73
Imaging of metal-coated biological samples by scanning tunneling microscopy; Garcia R et al.; A method for imaging biological samples by scanning tunneling microscopy (STM) is presented . There are two main difficulties in imaging biological samples by STM: (1) the low conductivity of biological material and (2) finding a method of reliably depositing the sample on a flat conducting surface . The first of these difficulties was solved by coating the samples with a thin film of platinum-carbon . The deposition problem was solved by a method similar to a procedure used to deposit biological molecules onto field ion microscope (FIM) tips . STM images of bacteriophage T7 and filamentous phage fd are shown . The substrate on which the samples were absorbed was atomically flat gold . The images do not show molecular detail due to the metal coating, but the gross dimensions and morphology are correct for each type of virus . Also, the surface density of virus particles increases and decreases in the way expected when the conditions of deposition are changed . These methods allow reliable and reproducible STM imaging of biological samples.

Nucleic Acids Res, 1989 May 11, 17(9), 3459 - 68
Prohead RNA of bacteriophage phi 29: size, stoichiometry and biological activity; Wichitwechkarn J et al.; We previously demonstrated (Guo et al., 1987 . Nucl . Acids Res . 15, 7081-7090) that purified proheads of bacteriophage phi 29 contain an RNA of 120 bases which is essential for DNA packaging . Here we report that this RNA exists primarily as a polymer of ca . 174 residues in phage-infected cells and that ca . 54 bases are cleaved from its 3'-terminus by adventitious nucleases during the purification of proheads . The long and short forms of the RNA had similar activity in in vitro DNA packaging and phage assembly . We report the sequence of the long form of the RNA and show that similar long and short forms can be isolated from the proheads of the phi 29 relatives phi 21, phi 15 and SF5 . The concentration dependence in the reconstitution of RNA-free proheads suggests that one copy of the RNA is sufficient to restore DNA packaging activity to RNA-free proheads . However, quantitative measurements indicate that 5 to 6 copies of the RNA are present on proheads isolated from phage-infected cells.

J Mol Biol, 1989 May 5, 207(1), 99 - 109
Genetic analysis of the lytic replicon of bacteriophage P1 . I . Isolation and partial characterization; Cohen G et al.; Despite the extensive genetic analysis of bacteriophage P1, the region of the viral genome that is responsible for its lytic (vegetative) replication has not been identified . In this paper we describe the identification of various fragments of P1 DNA that can replicate an otherwise replication-defective lambda vector when they are cloned into that vector . The fragments share a 2800 base-pair segment of the P1 genome that is located adjacent to the immI region of the phage . Replication mediated by the cloned P1 fragments is abolished by the product of the P1 c1 gene, the repressor of phage lytic functions . Since these properties resemble those of the P1 lytic replicon, we suggest that the 2800 base-pair segment identified here contains that replicon.

J Mol Biol, 1989 May 5, 207(1), 1 - 13
Genetic structure of the bacteriophage P22 PL operon; Semerjian AV et al.; The sequence of 1416 base-pairs of the P22 PL operon was determined, linking a continuous sequence from PL through abc2 . P22 mutants bearing deletions in the sequenced region were constructed and tested for their phenotypes . Plasmids were constructed to express PL operon genes singly and in combination from Plac UV5 . Two previously known genes, 17 and c3, are located within this sequence . In addition, three new genes have been identified: ral, kil and arf . Genes ral and c3 are homologous, as well as functionally analogous, to lambda ral and cIII, respectively . P22 kil, like lambda kil, kills the host cell when it is expressed . The two kil genes, although analogous in cell killing and map location, have no apparent sequence homology . The functions of the P22 and lambda kil genes are unknown; however, P22 kil is essential for lytic growth in the absence of abc . Gene arf (accessory recombination function) is located just upstream from erf; it is essential for P22 growth in the absence of kil or other genes upstream in PL . The growth defect of P22 bearing a deletion that removes arf is complemented by expression of either arf or the lambda red genes from plasmids . Sequences that include the stop codon for gene 17 may form a small stem-loop structure and are nearly identical to lambda sequences that contain the stop codon for ssb, which is near lambda tL 2b . Plasmids that include the P22 structure negatively regulate kil gene expression in cis.

Cell, 1989 May 5, 57(3), 423 - 31
Replication of RNA by the DNA-dependent RNA polymerase of phage T7; Konarska MM et al.; The DNA-dependent RNA polymerase of bacteriophage T7 utilizes a specific RNA as a template and replicates it efficiently and accurately . The RNA product (X RNA), approximately 70 nucleotides long, is initiated with either pppC or pppG and contains an AU-tich sequence . Replication of X RNA involves synthesis of complementary strands . Both strands are also significantly self-complementary, producing RNA with an extensive hairpin secondary structure . Replication of X RNA by T7 RNA polymerase is both template and enzyme specific . No other RNA serves as template for replication; neither do other polymerases, including the closely related T3 RNA polymerase, replicate X RNA . The T7 RNA polymerase-X RNA system provides an interesting model for studying replication of RNA by DNA-dependent RNA polymerases . Such a mechanism has been proposed to propagate viroids and hepatitis delta, pathogenic RNAs whose replication seems to depend on cellular RNA polymerases.

J Mol Biol, 1989 May 5, 207(1), 135 - 49
Structure and regulation of the lytic replicon of phage P1; Hansen EB; Three replicons, R, L and P1dR, have been previously identified in bacteriophage P1, but only the R (or plasmid) replicon has been functionally and structurally characterized . Evidence is provided here that the L-replicon is the principal replicon used for DNA replication during the lytic cycle . The L-replicon (exclusive of its promoter) is shown to be contained within a 1093-base-pair DNA segment that includes a 281-codon open reading frame, designated repL . L-replicon function requires transcription in the direction that should generate translatable repL message . This transcription is normally under the control of the phage c1 repressor, but a deletion that places the functional L-replicon under alternative control can be constructed . The DNA sequence of the replicon and surrounding regions was established . The sequenced region contains the c4 and ant genes and a hitherto unidentified gene, kilA, which is immediately upstream of repL and is controlled by the c1-regulated promoter . The kilA gene was shown to be non-essential for both replication and lytic development whereas the repL gene probably encodes an essential replication protein.

J Mol Biol, 1989 May 5, 207(1), 111 - 33
Genetic analysis of the lytic replicon of bacteriophage P1 . II . Organization of replicon elements; Sternberg N et al.; The region of bacteriophage P1 DNA containing a lytic (vegetative) replicon has been identified by cloning P1 fragments into a phage lambda vector . We present the sequence of that replicon . Using a novel fusion vector containing two P1 loxP recombination sites, we have developed a transformation assay for replicon function and have used that assay to identify some of the components of the P1 lytic replicon . Among those components is a transcription promoter, P53, whose activity is essential for replicon function . When that promoter is inactivated by the binding of P1 repressor to an operator site, Op53, whose sequence overlaps the promoter, replicon function is blocked . The P53 promoter can be replaced for replicon function by other promoters and, when the lacZ promoter was used, the extent of replication was shown to be proportional to promoter activity . Two open reading frames are located downstream from P53 . The promoter-proximal reading frame is 266 amino acid residues long and is not essential for replicon function . In fact, expression of that open reading frame either interferes with plasmid establishment after transformation or is lethal to cells . The promoter-distal reading frame, designated the repL open reading frame, is either 269 or 281 amino acid residues long and is essential for replicon function . Insertion of a Tn5 transposon into the 266 amino acid residue open reading frame inactivates the cloned lytic replicon probably by interfering with the transcription of the repL open reading frame from P53 . In P1, this Tn5 insertion mutation completely blocks lytic replication, indicating that the replicon identified here is either the only P1 lytic replicon or, if not, is at least necessary for the function of any other lytic replicon . A four base insertion in the repL open reading frame has largely the same inhibitory effect on phage lytic replication as the Tn5 insertion.

J Biol Chem, 1989 May 5, 264(13), 7596 - 602
Identification of C-terminal extensions that protect proteins from intracellular proteolysis; Bowie JU et al.; Revertants of defective mutants in the Arc repressor of bacteriophage P22 were isolated . Five of the six reverting mutations were frameshifts near the end of the coding sequence which resulted in proteins with C-terminal extensions . Each of the reverting mutations prolong the half-lives in vivo of the proteins in which they reside, yet they do not alter the thermodynamic stability, structure, oligomeric form, or DNA-binding properties of these proteins . Fusion of one of these tails to the C-terminal end of a mutant form of the N-terminal domain of lambda repressor also prevented proteolysis of this protein . These C-terminal sequences may prevent degradation by blocking the recognition of unstable proteins by the proteolytic machinery in the cell.

J Mol Biol, 1989 May 5, 207(1), 85 - 98
Genetic analysis of attTn7, the transposon Tn7 attachment site in Escherichia coli, using a novel M13-based transduction assay; Qadri MI et al.; The large (14 kb; kb = 10(3) bases) bacterial transposon, Tn7 (encoding resistance to trimethoprim and streptomycin/spectinomycin), has unusual properties . Like other elements, Tn7 transposes with low efficiency and low target-site specificity, but Tn7 also transposes, with high frequency in a unique orientation, to a preferred "attachment" site, called attTn7, in the Escherichia coli chromosome and similarly into plasmids containing attTn7 . We developed a novel bacteriophage M13-based assay system to measure the transposition frequency of Tn7 to M13mp phage vectors containing attTn7 on a cloned 1 kb fragment of chromosomal DNA . Phage harvested from a Tn7 donor strain were used to infect recipient bacteria with selection for trimethoprim resistance . Transposition frequency, expressed as the number of trimethoprim-resistant colonies per plaque-forming unit, was found to be approximately 10(-4) to M13mp::attTn7, in contrast to 10(-10) to M13mp recombinants with approximately 1 kb insertions of other, "generic brand", DNA . By deletion analysis of M13mp::attTn7, we show that attTn7 is contained within a 64 base-pair region; sequences adjacent to the actual insertion site and encoding the carboxy terminus of the glmS gene are required . This assay also provided evidence for transposition immunity conferred by the right end of Tn7.

Biochemistry, 1989 May 2, 28(9), 3793 - 7
High-resolution structure of the temperature-sensitive mutant of phage lysozyme, Arg 96----His; Weaver LH et al.; The structure of the temperature-sensitive mutant lysozyme of bacteriophage T4 in which arginine 96 is replaced by histidine has been determined crystallographically and refined to a residual of 17.6% at 1.9-A resolution . Overall, the three-dimensional structure of the mutant protein is extremely similar to that of wild type . There are local distortions in the mutant structure suggesting that the substituted His 96 residue is under strain . This appears to be one of the major reasons for the decreased thermostability . In wild-type lysozyme the guanidinium of Arg 96 is located at the carboxy terminus of alpha-helix 82-90 and makes a pair of hydrogen bonds to two of the carbonyl groups in the last turn of the helix . The loss of this "helix dipole" interaction also appears to contribute to the destabilization . The pKa* of His 96 in the mutant lysozyme has been determined by nuclear magnetic resonance and found to be 6.8 at 10 degrees C . This relatively normal value of the histidine pKa* suggests that the protonated and unprotonated forms of the imidazole ring are perturbed equally by the protein environment or, what is equivalent, the mutant lysozyme is equally stable with either histidine species.

Biochemistry, 1989 May 2, 28(9), 3788 - 92
A scanning calorimetric study of the thermal denaturation of the lysozyme of phage T4 and the Arg 96----His mutant form thereof; Kitamura S et al.; High-sensitivity scanning calorimetry has been employed to study the reversible thermal unfolding of the lysozyme of T4 bacteriophage and of its mutant form Arg 96----His in the pH range 1.80-2.84 . The values for t1/2, the temperature of half-denaturation, in degrees Celsius and for the enthalpy of unfolding in kilocalories per mole are given by (standard deviations in parentheses) wild type t1/2 = 9.63 + 14.41 pH (+/- 0.58) delta Hcal = 5.97 + 2.33t (+/- 4.20) mutant form t1/2 = -19.84 + 21.31 pH (+/- 0.51) delta Hcal = -8.58 + 2.66t (+/- 4.48) At any temperature within the range -20 to 60 degrees C, the free energy of unfolding of the mutant form is more negative than that of the wild type by 3-5 kcal mol-1, indicating an apparent destabilization resulting from the arginine to histidine replacement . The ratio of the van't Hoff enthalpy to the calorimetric enthalpy deviates from unity, the value expected for a simple two-state process, by +/- 0.2 depending on the pH . It thus appears that the nature of the unfolding of T4 lysozyme varies with pH in unknown manner . This complication does not invalidate the values reported here for the temperature of half-completion of unfolding, the calorimetric enthalpy, the heat capacity change, or the free energy of unfolding.

Mol Gen Genet, 1989 May, 217(1), 132 - 40
The linear mitochondrial plasmid pClK1 of the phytopathogenic fungus Claviceps purpurea may code for a DNA polymerase and an RNA polymerase; Oeser B et al.; Plasmid pClK1, a linear mitochondrial plasmid of Claviceps purpurea, was completely sequenced . The sequence contains two long open reading frames (ORF1, 3291 bp; ORF2, 2910 bp), and at least four smaller ORFs . The potential polypeptide derived from ORF1 shows homology to the family B type DNA polymerases . The product of ORF2 has significant homology to the mitochondrial RNA polymerase of yeast and RNA polymerases from bacteriophages . ORF1 and ORF2 show homology to URF3 and URF1 of the maize plasmids S1 and S2, respectively . No homology to any published protein sequence was found for the smaller ORFs . The origin of the terminal protein attached to the 5' ends of pClK1 remains open; several alternatives for its origin are discussed . The sequence data as a whole confirm the virus-like character of pClK1 already postulated from structural properties . Thus pClK1 together with S plasmids of maize and several other linear plasmids make up a distinct class of DNA species of plants and fungi probably derived from a common virus-like ancestor.

Biofizika, 1989 May-Jun, 34(3), 396 - 9
{Kinetic features of aberration occurrence in thermally-induced reorganization of T4 phage}; Khusainov AA et al.; The reorganization process of bacteriophage T4B in the course of heating at various rates was studied . Reduction of the heating rate from 1 to 8.10(-4) degree per min showed that the content of normally reorganized particles was increased.

Appl Environ Microbiol, 1989 May, 55(5), 1230 - 3
Molecular cloning of genes from Ruminococcus flavefaciens encoding xylanase and beta(1-3,1-4)glucanase activities; Flint HJ et al.; Clones expressing activity against xylan or beta(1-3,1-4)glucan (lichenan) were isolated from a library of Ruminococcus flavefaciens 17 DNA made in bacteriophage lambda EMBL3 . Hybridization analyses indicated the recovery of four separate genes encoding xylanases that showed no detectable associated carboxylmethylcellulase activity . One of these genes was associated with clones that also expressed beta(1-3,1-4)glucanase and beta-xylosidase activities.

Photochem Photobiol, 1989 May, 49(5), 599 - 605
Formation of purine photoproducts in a defined human DNA sequence; Gallagher PE et al.; The formation of DNA base damages by broad spectrum ultraviolet irradiation (250-400 nm) was investigated using a defined sequence of human DNA . The irradiated, 92 base pair, 3'-end of the human alphoid segment was incubated with an enzyme fraction purified from bacteriophage T4-infected E . coli . As previously reported, analysis of reaction products by sequencing gels showed enzymic incision of purine-containing photoproducts as well as pyrimidine cyclobutane photodimers . The purine-incising activity does not require metal ions and was unaffected by beta-mercaptoethanol or dithiothreitol . The formation of the purine photoproducts is independent of buffer; these lesions are produced by irradiation of DNA in Tris, Hepes or phosphate buffers . They are produced at biologically significant wavelengths between 260 to 300 nm . Only low levels were detected above or below this range . The formation of purine photoproducts is dose dependent with similar yields at some specific loci to pyrimidine dimers . These results suggest that purine-containing photoproducts could be of consequence in ultraviolet carcinogenesis.

Proc Natl Acad Sci U S A, 1989 May, 86(10), 3509 - 13
Infectious in vitro transcripts from cloned cDNA of a potyvirus, tobacco vein mottling virus; Domier LL et al.; Full-length cDNA copies of tobacco vein mottling virus (TVMV) RNA were constructed downstream from bacteriophage T7 or T3 RNA polymerase promoters . The plasmids were designed to produce in vitro transcripts containing, respectively, one or two guanosine residues at the 5' terminus not derived from the TVMV sequence and a single cytidine residue at the 3' terminus following the poly(A) tail . Introduction of transcripts from either plasmid into tobacco mesophyll protoplasts resulted in the accumulation of TVMV coat protein and RNA . Neither coat protein nor viral RNA accumulated in protoplasts inoculated with linearized cDNA or with in vitro transcripts synthesized in the absence of 7-methylguanosine(5')triphospho(5')guanosine (m7GpppG) . Tobacco seedlings inoculated with native TVMV RNA developed symptoms a few days before those inoculated with in vitro transcripts; however, 3 weeks after inoculation, the symptoms produced by the two inocula were indistinguishable.

Hum Genet, 1989 May, 82(2), 197 - 8
Demonstration of spontaneous XX/XY chimerism by DNA fingerprinting; Farber CM et al.; The M13 bacteriophage probe, which makes possible the establishment of DNA fingerprints, was used to study a phenotypically normal woman with a 46XY karyotype and her twin brother . Identical fingerprints and positive hybridization with a series of Y-specific probes were obtained on blood samples from the siblings . DNA from a skin biopsy of the woman yielded a clearly different pattern and displayed no Y-specific hybridization, indicating that she is a spontaneous chimera . This study illustrates the use of DNA fingerprinting as a powerful and simple aid to the diagnosis of natural chimerism.

Virology, 1989 May, 170(1), 238 - 42
Nucleotide sequence from the ssRNA bacteriophage JP34 resolves the discrepancy between serological and biophysical classification; Adhin MR et al.; The nucleotide sequence of the coat and lysis genes of the single-stranded RNA bacteriophage JP34 is presented . Serological inactivation studies classified this phage as an intermediate between groups I and II . We show that the nucleotide similarity with group I is less than 45% but more than 95% for group II, classifying JP34 as a member of group II . The altered serotype of JP34 is most likely due to the change of three critical amino acids of the coat protein to residues present in group I phage MS2 at the homologous positions . Serological characterization of RNA bacteriophages is thus not unambiguous . Phylogenetic sequence comparison between JP34, GA, and MS2 confirms the existence of a conserved helix in the coat gene of group I and group II phages . We also show that the JP34 coat and lysis genes can be expressed in cDNA clones and that the translation of the lysis gene is coupled to coat gene translation analogous to the regulation found in the group I phages.

J Bacteriol, 1989 May, 171(5), 2347 - 52
Characterization of mutations of the bacteriophage P1 mod gene encoding the recognition subunit of the EcoP1 restriction and modification system; Rao DN et al.; This study characterized several mutations of the bacteriophage P1 mod gene . This gene codes for the subunit of the EcoP1 restriction enzyme that is responsible for DNA sequence recognition and for modification methylation . We cloned the mutant mod genes into expression vectors and purified the mutant proteins to near homogeneity . Two of the mutant mod genes studied were the c2 clear-plaque mutants described by Scott (Virology 41:66-71, 1970) . These mutant proteins can recognize EcoP1 sites in DNA and direct restriction but are unable to modify DNA . Methylation assays as well as S-adenosylmethionine (SAM) binding studies showed that the c2 mutants are methylation deficient because they do not bind SAM, and we conclude that the mutations destroy the SAM-binding site . Both of the c2 mutations lie within a region of the EcoP1 mod gene that is not conserved when compared with the mod gene of the related EcoP15 system . EcoP15 and EcoP1 recognize different DNA sequences, and we believe that this region of the protein may code for the DNA-binding site of the enzyme . The other mutants characterized were made by site-directed mutagenesis at codon 240 . Evidence is presented that one of them, Ser-240----Pro, simultaneously lost the capacity to bind SAM and may also have changed its DNA sequence specificity.

J Cell Sci, 1989 May, 93 ( Pt 1), 71 - 83
Expression in Escherichia coli of fragments of glial fibrillary acidic protein: characterization, assembly properties and paracrystal formation; Quinlan RA et al.; We have expressed in Escherichia coli a 1258 bp cDNA fragment corresponding to 97% of mouse glial fibrillary acidic protein (GFAP), the principal intermediate filament protein of astrocytes . High levels of expression were obtained, as a fusion protein with 32 residues of the bacteriophage lambda cII protein, using the pLcII expression vector system of K . Nagai and H.-C . Thogersen . Although removal of the cII protein fragment by proteolysis using factor X proved difficult, a protein corresponding to most of the cDNA fragment was obtained by cleaving at the endogenous thrombin site near the middle of the N-terminal non-helical domain of GFAP . A shorter 1047 bp fragment, in which the C-terminal non-helical domain of GFAP was deleted, was also produced using oligonucleotide-directed site-specific mutagenesis of the original cDNA clone . After proteolysis with thrombin, this material gave a fragment that corresponded to the alpha-helical coiled-coil rod region of the GFAP molecule, together with a portion of the non-helical N-terminal domain . The fragments produced were characterized both biochemically and ultrastructurally, and appeared to retain the conformation of native GFAP . Crosslinking showed that all fragments formed molecules containing two chains ('dimers') that associated to form four-chain molecular dimers ('tetramers') analogous to those formed by intact intermediate filament proteins . Shadowed preparations showed the presence of rod-like particles that closely resembled those observed for other intermediate filament proteins and proteolytically prepared rod domains . Remarkably, the fusion protein produced from the entire 1258 bp cDNA fragment and the cII peptide was able to form filaments that closely resembled those produced by native GFAP . However, fragments in which either the cII peptide or the C-terminal non-helical domain were removed, or in which both were removed, failed to form filaments under standard assembly conditions . Although preliminary in nature, these results suggest that both N- and C-terminal non-helical domains may have a role in intermediate filament formation . Moreover, the fragment corresponding approximately to the GFAP rod formed paracrystals similar to those observed with other coiled-coil proteins . The molecules in these paracrystals were arranged antiparallel with the two molecules in the unit cell, which may correspond to the four-chain molecular dimer (tetramer), overlapping by approximately two-thirds of their length.

Mol Biol (Mosk), 1989 May-Jun, 23(3), 822 - 9
{Kinetics of inhibition by 8-oxy-GTP and 8-bromo-GTP of Escherichia coli RNA-polymerase synthesis of pppApU dinucleotide on the promotor a1 of phage T7deltaD111 DNA in a limited set of substrates}; Kuriavyi VV et al.; Detailed analysis of the kinetics of inhibition of E . coli RNA-polymerase-catalyzed synthesis of dinucleotide pppApU by 8-oxy-GTP and 8-Br-GTP on promoter A1 of the bacteriophage T7 delta D111 with an incomplete set of substrates was carried out . In accordance with the mathematical models obtained, we calculated quantitative parameters of binding of these nucleotide analogs to the centers whose geometry is suitable for incorporation of ATP and UTP . 8-oxy-GTP and 8-Br-GTP compete with ATP for the binding center (their steady-state dissociation constant ratios are 2.1 and 2.4, respectively, whereas the constant for ATP is 0.3 mM) but, unlike ATP, they are not incorporated into the product . 8-oxy-GTP competes also with UTP (its steady-state dissociation constant ratio is 21.6, the constant for UTP is 0.03 mM) . 8-Br-GTP does not interact with the binding center of UTP.

Mol Biol (Mosk), 1989 May-Jun, 23(3), 739 - 49
{A new early gene in front of the middle gene 31 of bacteriophage T4: cloning and expression}; Nivinskas RG et al.; We have identified a new gene, which we designated gene 31.1, as the nearest upstream neighbour of gene 31 . We cloned the 1.03 k.b . EcoRI/BglII fragment of T4 DNA, and expressed both genes using the T7 RNA polymerase/promoter two plasmid system . Gene 31.1 encodes a protein with molecular weight of about 10 kDa, and the C-terminal portion of this gene is the incomplete open reading frame ORF 31.1 determined earlier . Using primer extension sequencing on RNA templates isolated from T4 mot A+ and mot A- infected cells, we have shown that gene 31 has a middle mode mot A-dependent transcript starting at two points and which is initiated from gene's 31 own promoter . Gene 31 is also transcribed from an early upstream promoter into a polycistronic mRNA which covers an early gene 31.1 as well.

DNA, 1989 May, 8(4), 261 - 70
Tilapia prolactin: molecular cloning of two cDNAs and expression in Escherichia coli; Rentier-Delrue F et al.; We have isolated cDNA clones encoding tilapia prolactin (tiPRL) from a cDNA library prepared from tilapia (Oreochromis niloticus) anterior pituitary glands . A trout PRL cDNA fragment was used as hybridization probe to select the recombinant plasmids carrying the tiPRL coding sequence . Two types of PRL cDNA were isolated and their complete nucleotide sequence determined . The larger cDNA (tiPRL-I) codes for a polypeptide of 212 amino acids, including a putative signal sequence of 24 amino acids, and contains a 3' untranslated region of 787 bp . The second prolactin cDNA (tiPRL-II) encodes a polypeptide of 200 amino acids, including a presumptive signal peptide of 23 amino acids, and contains a noncoding region of 512 bp . tiPRL-I and tiPRL-II cDNA sequences are 81% similar, whereas the encoded proteins share 69% amino acid identity at optimal alignment . Mature tiPRL-I was efficiently expressed in Escherichia coli carrying a plasmid in which the tiPRL-I cDNA was under the control of the phi 10 promoter of T7 bacteriophage . The new recombinant protein representing about 45% of the total cellular proteins was found in inclusion bodies and cross-reacted with salmon PRL antiserum.

Trends Genet, 1989 May, 5(5), 149 - 54
Mitochondrial RNA polymerase: dual role in transcription and replication; Schinkel AH et al.; Mitochondrial RNA polymerases from humans, Xenopus laevis and Saccharomyces cerevisiae are very similar in protein composition and function . They consist of a nonspecific core RNA polymerase and a protein factor that confers promoter selectivity on the core component, and they participate in transcription as well as in DNA replication . Amino acid sequence comparisons indicate that the yeast mitochondrial core component is related to bacteriophage T3 and T7 RNA polymerases; mitochondrial and phage polymerases may therefore belong to a family of related polymerases.

J Bacteriol, 1989 May, 171(5), 2748 - 55
The heat-shock-regulated grpE gene of Escherichia coli is required for bacterial growth at all temperatures but is dispensable in certain mutant backgrounds; Ang D et al.; Previous work has established that the grpE+ gene product is a heat shock protein that is essential for bacteriophage lambda growth at all temperatures and for Escherichia coli growth at temperatures above 43 degrees C . Here it is shown that the grpE+ gene product is essential for bacterial viability at all temperatures . The strategy required constructing a grpE deletion derivative carrying a selectable chloramphenicol drug resistance marker provided by an omega insertion and showing that this deletion construct can be crossed into the bacterial chromosome if and only if a functional grpE+ gene is present elsewhere in the same cell . As a control, the same omega insertion could be placed immediately downstream of the grpE+ coding sequence without any observable effects on host growth . This result demonstrates that the inability to construct a grpE-deleted E . coli strain is not simply due to a lethal polar effect on neighboring gene expression . Unexpectedly, it was found that the grpE deletion derivative could be crossed into the bacterial chromosome in a strain that was defective in DnaK function . Further analysis showed that it was not the lack of DnaK function per se that allowed E . coli to tolerate a deletion in the grpE+ gene . Rather, it was the presence of unknown extragenic suppressors of a dnaK mutation that somehow compensated for the deficiency in both DnaK and GrpE function.

J Bacteriol, 1989 May, 171(5), 2639 - 45
Translational coupling of the two proximal genes in the S10 ribosomal protein operon of Escherichia coli; Lindahl L et al.; We have examined the translational coupling between the first two genes in the S10 ribosomal protein operon . We isolated mutations blocking the translation of the first gene of the operon, coding for S10, and monitored their effects on translation of the downstream gene, coding for L3 . All of the mutations inhibiting S10 synthesis also affected the synthesis of L3 . However, these experiments were complicated by decreased mRNA synthesis resulting from transcription polarity, which we could only partially eliminate by using a rho-100 strain . To completely eliminate the problem of transcription polarity and obtain a more accurate measurement of the coupling, we replaced the natural S10 promoter with a promoter used by the bacteriophage T7 RNA polymerase . As expected, the T7 RNA polymerase was not subject to transcription polarity . Using this system, we were able to show that a complete abolishment of S10 translation resulted in an 80% inhibition of L3 synthesis . Other experiments show that the synthesis of L3 goes up as a function of increasing S10 synthesis, but the translational coupling does not assure strictly proportional output from the two genes.

J Bacteriol, 1989 May, 171(5), 2528 - 32
Hypervariable DNA fingerprinting in Escherichia coli: minisatellite probe from bacteriophage M13; Huey B et al.; Extensive restriction-fragment-length polymorphism was revealed in Escherichia coli strains by using a region of the bacteriophage M13 genome as a DNA hybridization probe . This variation was observed across natural strains, in clinical samples, and to a lesser extent in laboratory strains . The sequence in M13 which revealed this fingerprint pattern was a region of the gene III coat protein, which contains two clusters of a 15-base-pair repeat . Oligonucleotides made to a consensus of these repeats also revealed the fingerprint profile . While this consensus sequence has significant homology to the lambda chi site sequence, an oligonucleotide made of the chi sequence did not reveal polymorphic fingerprint patterns in E . coli . The strain variation revealed by the M13 and M13-derived oligonucleotide probes will be useful for bacterial characterization and should find use in studies of bacterial evolution and population dynamics . The findings raise questions about what these repeated sequences are and why they are so variable.

Am J Hum Genet, 1989 May, 44(5), 671 - 8
Identification and characterization of 23 RFLP loci by screening random cosmid genomic clones; Bowden DW et al.; As part of our search for polymorphic DNA probes, we have screened cosmids from a human genomic DNA library for their ability to reveal RFLPs . A total of 101 randomly isolated cosmid clones were tested in Southern hybridizations for polymorphic band patterns . Fifty-four of these clones revealed RFLPs with one or more of nine restriction enzymes . Twenty-three of these clones have been further characterized and assigned to 10 different chromosomes by linkage analysis or by hybridization to panels of human-hamster hybrid cell lines . Fifteen of the probes have heterozygosities greater than or equal to .5 . The relative efficiency of RsaI and PstI restriction enzymes in detecting polymorphism was different from results obtained with libraries constructed in bacteriophage vectors . Screening randomly selected cosmid probes is an efficient method for detecting RFLPs.

DNA, 1989 May, 8(4), 287 - 95
Using transposon Tn5 insertions to sequence bacteriophage T4 gene 11; Barrett BK et al.; A simple and rapid method of creating an overlapping set of deletions in cloned DNA in preparation for sequencing has been developed . The method is based on a positive selection for Tn5 transposition into the cloned DNA fragment on a high-copy-number filamid, resolution of potential filamid dimers by filamentous phage infection, and the use of Tn5 both as a "portable" restriction enzyme site for in vitro DNA deletion and a "portable" sequencing primer binding site to initiate DNA sequencing reactions using a custom primer complementary to the outside ends of IS50 . This new method has been utilized to sequence bacteriophage T4 gene 11, encoding the T4 baseplate protein gp11 . The coding sequence of gene 11 is 657 bp in length, and predicts a primary structure of 219 amino acids that agrees well with the biochemical data previously obtained . DNA sequence around gene 11 suggests that the expression of genes 10, 11, and 12 of phage T4 are translationally coupled . Plasmids carrying deletions generated using this method have been used to map genetically five amber alleles of gene 11 . These amber alleles were sequenced to confirm the proposed reading frame . The five amber alleles actually represent two different mutational changes at either codon 206 or 207, changing these adjacent glutamine codons to amber . The position of these amber alleles lends support to earlier studies identifying the carboxyl terminus of gp11 as essential in the interaction of P11 with baseplate protein P10 (Plishker and Berget, 1984).

Genetika, 1989 May, 25(5), 829 - 37
{Characteristics of Escherichia coli K-12 mutants with altered effectiveness of excision of Tn5 and Tn9 transposons}; Mukhamedshin EK et al.; The properties of Escherichia coli K-12 mutans HFETn5, HFETn9 and LFETn9 have been studied . The majority of mutations were shown to have pleiotropic effect . Some of them increase cell sensitivity to UV light and mitomycin C and affect efficiency of homologous recombination in transduction and conjugation . The level of spontaneous mutagenesis is increased in a number of mutants . None of the mutations isolated affect frequency of transposition of Tn5 from bacteriophage lambda::Tn5 into the chromosome . Based on analysis of properties of hfeTn5-09 and hfeTn9 mutations and on the date of preliminary mapping of hfeTn5-09 mutation, these mutations were considered to be novel . It is shown that the processes of precise excision of Tn5 and Tn9 transposons may be accomplished by at least two pathways, one of them being dependent on recA gene functions.

J Bacteriol, 1989 May, 171(5), 2581 - 90
Genetic analysis of the rnc operon of Escherichia coli; Takiff HE et al.; RNase III, an Escherichia coli double-stranded endoribonuclease, is known to be involved in maturation of rRNA and regulation of several bacteriophage and Escherichia coli genes . Clones of the region of the E . coli chromosome containing the gene for RNase III (rnc) were obtained by screening genomic libraries in lambda with DNA known to map near rnc . A phage clone with the rnc region was randomly mutagenized with a delta Tn10 element, and the insertions were recombined onto the chromosome, generating a series of strains with delta Tn10 insertions in the rnc region . Two insertions that had Rnc- phenotypes were located . One of them lay in the rnc gene, and one was in the rnc leader sequence . Polarity studies showed that rnc is in an operon with two other genes, era and recO . The sequence of the recO gene beyond era indicated it could encode a protein of approximately 26 kilodaltons and, like rnc and era, had codon usage consistent with a low level of expression . Experiments using antibiotic cassettes to disrupt the genes rnc, era, and recO showed that era is essential for E . coli growth but that rnc and recO are dispensable.

EMBO J, 1989 May, 8(5), 1601 - 8
Initiation of lambda DNA replication with purified host- and bacteriophage-encoded proteins: the role of the dnaK, dnaJ and grpE heat shock proteins; Zylicz M et al.; Based on previous in vivo genetic analysis of bacteriophage lambda growth, we have developed two in vitro lambda DNA replication systems composed entirely of purified proteins . One is termed 'grpE-independent' and consists of supercoiled lambda dv plasmid DNA, the lambda O and lambda P proteins, as well as the Escherichia coli dnaK, dnaJ, dnaB, dnaG, ssb, DNA gyrase and DNA polymerase III holoenzyme proteins . The second system includes the E.coli grpE protein and is termed 'grpE-dependent' . Both systems are specific for plasmid molecules carrying the ori lambda DNA initiation site . The major difference in the two systems is that the 'grpE-independent' system requires at least a 10-fold higher level of dnaK protein compared with the grpE-dependent one . The lambda DNA replication process may be divided into several discernible steps, some of which are defined by the isolation of stable intermediates . The first is the formation of a stable ori lambda-lambda O structure . The second is the assembly of a stable ori lambda-lambda O-lambda P-dnaB complex . The addition of dnaJ to this complex also results in an isolatable intermediate . The dnaK, dnaJ and grpE proteins destabilize the lambda P-dnaB interaction, thus liberating dnaB's helicase activity, resulting in unwinding of the DNA template . At this stage, a stable DNA replication intermediate can be isolated, provided that the grpE protein has acted and/or is present . Following this, the dnaG primase enzyme recognizes the single-stranded DNA-dnaB complex and synthesizes RNA primers . Subsequently, the RNA primers are extended into DNA by DNA polymerase III holoenzyme . The proposed model of the molecular series of events taking place at ori lambda is substantiated by the many demonstrable protein-protein interactions among the various participants.

EMBO J, 1989 May, 8(5), 1591 - 9
Mutations of the phage lambda attachment site alter the directionality of resolution of Holliday structures; de Massy B et al.; Integrative recombination of bacteriophage lambda occurs by two sequential, reciprocal strand exchanges at specific positions within the attachment sites . Both exchanges are promoted by the lambda Int protein; the first forms a Holliday structure, and the second resolves it to recombinant products . Recombination requires sequence homology within the 7 bp 'overlap' region that separates the two points of strand exchange . To see if homology promotes the second strand exchange, we constructed attachment site Holliday structures by annealing DNA strands and then assayed Int-promoted resolution . Holliday structures corresponding to strand exchange between sites with homologous overlap regions were efficiently resolved to give mixtures of recombinants and parents . Holliday structures corresponding to exchanges between heterologous sites fell into two classes . Members of the first class, in which heterology limited but did not completely prevent migration of the branchpoint within the overlap region, were resolved efficiently and preferentially to parental molecules . We propose that resolution to recombinants occurs only if homology allows branch migration from the first to the second exchange site . Members of the second class, in which heterology constrained the branchpoint within an Int binding site, were resolved poorly . We suggest that Holliday structures that have a branchpoint within an Int binding site are poor substrates for Int.

J Bacteriol, 1989 May, 171(5), 2563 - 72
Genetic analysis of bacteriophage lambda cIII gene: mRNA structural requirements for translation initiation; Kornitzer D et al.; The bacteriophage lambda cIII gene product regulates the lysogenic pathway . The cIII gene is located in the leftward operon, which is transcribed from the pL promoter . We have previously shown (S . Altuvia and A . B . Oppenheim, J . Bacteriol . 167:415-419, 1986) that mutations that show elevated expression lie within the cIII coding sequence . We isolated mutants that show decreased CIII activity . All the mutations were found to cause a drastic reduction in the rate of initiation of cIII translation . Several mutations were found to be scattered within the first 40 nucleotides of the cIII coding region . Additional mutations affected the AUG initiation codon, the Shine-Dalgarno sequence, and the upstream RNaseIII processing site . Computer folding of the cIII mRNA suggested the presence of two alternative RNA structures . All the mutations within the coding region that reduce expression reduce the stability of one specific mRNA structure (structure B) . Mutations that increase expression lie in the loops of this structure and may in fact stabilize it by interfering with the formation of the alternative structure (structure A) . Thus, it appears that a specific mRNA secondary structure at the beginning of the cIII coding region is essential for efficient translation, suggesting that changes in mRNA structure regulate cIII expression.

Clin Immunol Immunopathol, 1989 May, 51(2), 252 - 63
Human antibody responses to bacteriophage phi X 174: sequential induction of IgM and IgG subclass antibody; Pyun KH et al.; We studied the appearance of antigen-specific immunoglobulin classes and IgG subclasses in normal adult human subjects in response to primary, secondary, and tertiary immunization with the T-cell-dependent neo-antigen bacteriophage phi X 174 . To complete the study we developed a sensitive, specific, and reproducible ELISA assay which was closely comparable to the widely used neutralization assay for total antibody (r = +0.97) and for IgG antibody (r = +0.93), and reasonably comparable for IgM antibody (r = +0.76) . We confirmed that the initial response to primary immunization was predominantly, but not exclusively, IgM antibody . The secondary and tertiary responses demonstrated memory, amplification, and switch from IgM to IgG antibody . There was an orderly appearance of phage-specific IgG subclasses . IgG3 and IgG1 antibodies appeared 2 to 6 weeks after primary immunization . In all subjects there was a marked increase in IgG1 and IgG3 antibody after secondary immunization, and IgG2 antibody followed closely; IgG4 antibody appeared in some subjects . IgM antibody persisted in significant amounts (approx 50%) throughout the secondary response period . Following tertiary immunization, IgG1, IgG2, and IgG3 antibody consistently increased, and IgG4 antibody appeared in all subjects; IgG1 antibody predominated . Low levels of IgM antibody (approx 1% of total) persisted during the tertiary response . The persisting antibody on long-term follow-up (median 4 years after immunization) was virtually all (greater than 90%) IgG1.

Biochimie, 1989 May, 71(5), 681 - 5
Expression of rubella virus cDNA encoding the E1 structural protein; de Mazancourt A et al.; cDNAs synthesized from the 40S RNA genome of rubella virus were cloned into the lambda gt10 bacteriophage . A clone (pRV # 1475) which contains a truncated version of the E1 envelope glycoprotein (amino acid residues 17-481) and the 3' non-coding region was constructed from two overlapping cDNAs . pRV # 1475 was inserted into a eukaryotic expression vector under the control of the cytomegalovirus immediate early promoter . After transfection of 293 cells, a protein with intact antigenic domains could be detected.

Mol Biochem Parasitol, 1989 May 1, 34(2), 135 - 46
Two variant surface glycoprotein genes distinguish between different substrains of Trypanosoma brucei gambiense; Barnes DA et al.; Trypanosoma brucei gambiense differs from other T . brucei subspecies in the stability and conservation of its bloodstream form antigenic repertoire . Two variant surface glycoprotein (VSG) cDNA clones corresponding to the antigens U1 and L2 were isolated from T . b . gambiense bacteriophage lambda gt11 expression libraries and characterized . A third VSG cDNA clone, P1, was also examined . The L2 and U1 VSG genes are present in a large number of T . b . gambiense stocks isolated over a thirty-year period from different geographical areas of Africa, suggesting that they are stably maintained in the T . b . gambiense genome . These probes may be useful in epidemiological studies of Gambian sleeping sickness to differentiate between T . b . gambiense isolates.

J Bacteriol, 1989 May, 171(5), 2542 - 6
Role of exonuclease III and endonuclease IV in repair of pyrimidine dimers initiated by bacteriophage T4 pyrimidine dimer-DNA glycosylase; Saporito SM et al.; The role of exonuclease III and endonuclease IV in the repair of pyrimidine dimers in bacteriophage T4-infected Escherichia coli was examined . UV-irradiated T4 showed reduced survival when plated on an xth nfo double mutant but showed wild-type survival on either single mutant . T4 denV phage were equally sensitive when plated on wild-type E . coli or an xth nfo double mutant, suggesting that these endonucleases function in the same repair pathway as T4 pyrimidine dimer-DNA glycosylase . A uvrA mutant of E . coli in which the repair of pyrimidine dimers was dependent on the T4 denV gene carried on a plasmid was constructed . Neither an xth nor an nfo derivative of this strain was more sensitive than the parental strain to UV irradiation . We were unable to construct a uvrA xth nfo triple mutant . In addition, T4, which turns off the host UvrABC excision nuclease, showed reduced plating efficiency on an xth nfo double mutant.

J Biol Chem, 1989 Apr 25, 264(12), 6748 - 54
Benzo{a}pyrene-DNA adducts inhibit translocation by the gene 4 protein of bacteriophage T7; Brown WC et al.; Bacteriophage T7 gene 4 protein is an essential component of the T7 DNA replication system, acting as both a primase and a helicase . The gene 4 protein has been shown to translocate along single-stranded DNA in the 5'----3' direction, using an energy source for this movement the hydrolysis of nucleoside 5'-triphosphates, preferably dTTP . Thus, measuring the rate and extent of dTTP hydrolysis provides a means to directly measure translocation . We have determined that the hydrolysis of dTTP by the gene 4 protein is strongly inhibited by the presence of benzo{a}pyrene (B{a}P) adducts on the DNA . Time course experiments on adduct-containing DNA show that after an initial burst of hydrolysis, which parallels what is observed on unmodified DNA, further hydrolysis abruptly ceases . Addition of excess unmodified DNA does not restore the hydrolysis activity . These data suggest that the gene 4 protein is blocked and sequestered on the DNA at the site of the adduct . This was confirmed by experiments in which gene 4 protein preferentially protected the radiolabeled adduct-containing DNA but not randomly labeled M13 DNA . The gene 4 protein bound to the B{a}P-modified DNA was isolated, and the complex was found only to contain dTTP . These results have been used to formulate a model for gene 4 protein translocation in which we speculate that the power stroke for unidirectional movement along the single-stranded DNA is the displacement of dTDP by dTTP . Finally, we observe a constant ratio of DNA synthesis to dTTP hydrolysis regardless of the number of B{a}P adducts in the template suggesting that a significant portion of the inhibition of DNA synthesis is a direct consequence of the inhibition of gene 4 translocation.

Nucleic Acids Res, 1989 Apr 25, 17(8), 3023 - 36
Unusual promoter-independent transcription reactions with bacteriophage RNA polymerases; Krupp G; Efficient transcription reactions of DNA-dependent RNA polymerases require the presence of a specific promoter sequence . This report shows that in the absence of their cognate promoter, two bacteriophage RNA polymerases are capable of performing unusual transcription reactions: (i) the DNA template serves also as a primer for RNA synthesis and this leads to hybrid DNA/RNA molecules, (ii) if the DNA template forms a hairpin structure, the linear DNA can be transcribed via the 'rolling circle' mechanism.

Nature, 1989 Apr 20, 338(6217), 656 - 8
Interaction of distinct domains in Mu transposase with Mu DNA ends and an internal transpositional enhancer; Leung PC et al.; Bacteriophage Mu is the largest and most efficient transposable element known . The Mu transposase (A protein) of relative molecular mass 75,000 is a central component of the transposition machinery . We report here that the N-terminal region of Mu transposase contains two distinct DNA-binding domains, one which binds the two Mu DNA ends, and another which binds an internal operator region . This internal operator is required for the transposase-mediated synapsis and nicking of Mu ends in vitro, and stimulates transposition more than 100-fold in vivo . The orientation of the operator with respect to the ends is critical to its function, whereas its distance from the ends seems to be relatively unimportant . We propose that the operator enhances transposition by transiently interacting with the transposase and Mu DNA end(s) to form a complex in which synapsis of the ends occurs.

Biochemistry, 1989 Apr 18, 28(8), 3510 - 7
Thermodynamic and enzymological characterization of the interaction between transcription termination factor rho and lambda cro mRNA; Faus I et al.; Termination of transcription at tR1, the rho-dependent terminator between genes cro and cII of bacteriophage lambda, is mediated by interactions between rho protein and an RNA sequence element called rut . We show, using a filter retention assay technique, that rho protein binds with about 10-fold lower affinity to variants of cro RNA lacking both parts of rut or to normal cro RNA having one or the other part of rut bound to a complementary DNA oligonucleotide than it binds to unmodified cro RNA . These same variant and modified forms are nearly devoid of the strong rho ATPase cofactor activity of cro RNA . Estimates of binding energies of the rho-cro RNA interaction under different conditions reveal that termination function correlates with about 12.6 kcal of binding energy, of which two-thirds is due to nonelectrostatic interactions . The rut segment is shown to contribute about 1 kcal, nearly all to nonelectrostatic interactions . KCl is found to be more effective than potassium glutamate as a competitive counterion, and a decrease in 1.4 kcal of binding energy due to counterion competition correlates with a loss of termination and ATPase activities . In sum, the results indicate that the rut sequence contributes substantially to the overall binding affinity, that ionic interactions are also important, and that mere binding of rho to RNA is not sufficient for rho ATPase activation.

Gene, 1989 Apr 15, 77(1), 69 - 78
Expression vector pT7:TKII for the synthesis of authentic biologically active RNA encoding vaccinia virus thymidine kinase; Wilson EM et al.; A transcription vector, pT7: TKII, was constructed by a novel application of the polymerase chain reaction . Chimeric oligodeoxynucleotides were used to direct the synthesis of a DNA fragment which consisted of a truncated bacteriophage T7 promoter element fused to the vaccinia virus (VV) thymidine kinase gene (tk) . This fragment was cloned into a pUC118 plasmid and sequenced to ensure no mutations had occurred during its synthesis . When linearized at the 3' end of the VV tk gene at the BamHI site located in the polylinker region of the vector, pT7:TKII was efficiently transcribed by T7 RNA polymerase into a 595 nucleotide transcript whose 5' end was identical to that found on authentic nascent VV tk mRNA . When translated in a rabbit reticulocyte lysate system, the synthetic VV tk RNA was shown to be biologically active in that it directed the synthesis of a 20-kDa protein which assembled into an enzymatically active 80-kDa tetrameric complex which was indistinguishable from VV thymidine kinase (TK) enzyme isolated from VV-infected cells . The pT7:TKII vector provides a powerful approach with which: (i) to investigate the translational and posttranslational regulation of the VV tk gene; (ii) to use directed genetics to identify potential cis-acting regulatory sequences or structures present within the VV tk RNA; and (iii) to apply protein engineering procedures to identify the catalytic, allosteric and subunit interactive domains of the VV TK enzyme . As an example, the translational effects of adding a m7G cap structure to the pT7:TKII-derived VV tk RNA are presented.

Biochem J, 1989 Apr 15, 259(2), 549 - 53
Entrapment of high-molecular-mass DNA molecules in liposomes for the genetic transformation of animal cells; Szelei J et al.; We modified the Ca/EDTA procedure for the production of liposomes {Papahadjopoulos, Vail, Jacobson & Poste (1975) Biochim . Biophys . Acta 394, 483-491} to entrap intact DNA molecules of very high molecular mass into large unilamellar phospholipid vesicles . The use of DNA-protein complexes and phage particles instead of naked linear DNA increases the efficiency of entrapment and protects the integrity of DNA molecules . We investigated the interaction of mammalian cells with liposome-encapsulated recombinant lambda bacteriophages carrying marker genes . The liposomes bind surprisingly fast to the cellular surface and are taken up by the cells . A significant proportion of the encapsulated DNA is transported to and soon located in or around the nuclei . Experiments prove that these liposomes can be used for the genetic transformation of mammalian cells.

J Biol Chem, 1989 Apr 15, 264(11), 6447 - 58
Selective inactivation of the exonuclease activity of bacteriophage T7 DNA polymerase by in vitro mutagenesis; Tabor S et al.; The 3' to 5' exonuclease activity of bacteriophage T7 DNA polymerase (gene 5 protein) can be inactivated selectively by reactive oxygen species . Differences in the enzymatic properties between the two forms are exploited to show by a chemical screen that modification of a histidine residue reduces selectively the exonuclease activity . In vitro mutagenesis of the histidine at residue 123, and of the neighboring residues, results in varying reduction of the exonuclease activity, including mutant enzymes that have no detectable exonuclease activity; as a consequence their polymerase activity is increased up to 9-fold . T7 phage containing the mutant genes have a greatly reduced burst size and demonstrate up to a 14-fold increase in the spontaneous mutation rate.

Biochem Biophys Res Commun, 1989 Apr 14, 160(1), 121 - 5
Footprint of the sigma protein; Ramesh U et al.; Escherichia coli RNA Polymerase is a multi-subunit enzyme that catalyzes RNA synthesis, using DNA as a template . The sigma subunit of this enzyme plays an important role in the recognition of promoter sites on DNA . Using DNase I footprinting, we have found that in the absence of the other subunits, sigma binds specifically to the bacteriophage lambda PR promoter DNA sequence . In the presence of the sigma subunit alone, a protective footprint encompassing the region between residue positions -41 and +17 was observed (where +1 is the transcription start site) . The holoenzyme gave a footprint covering the same region . Thus not only does the sigma subunit interact with the DNA promoter site in the absence of the other components of RNA polymerase, but also the footprint of sigma is indistinguishable from that of the holoenzyme.

Biochim Biophys Acta, 1989 Apr 12, 1007(3), 359 - 62
Mutational analysis of the bacteriophage alpha 3 origin of complementary DNA synthesis: in vivo properties of mutants; Kodaira K et al.; Bacteriophage alpha 3 origin of complementary strand DNA synthesis contains two potential secondary loop structures, I and II, which have been implicated in direct recognition sites for host Escherichia coli dnaG protein . To elucidate the function of the hairpin loops, we have introduced point mutations within the stem of the hairpin II so as to disturb its base-pairings . A mutant, oriAA, which had two point mutations in the region, formed minute plaques on E . coli host cells and its mean burst size at 37 degrees C was about 50, whereas that of wild-type was 250 . In addition, the growth of oriAA at 42 degrees C was thermosensitive and the burst size was reduced to 5 . From the oriAA, a revertant-like phage oriGA occurred spontaneously with a high-frequency of about 2.10(-2) . It retained one point mutation and the plaque size and phage yield were nearly same as those of wild-type . These results are discussed with respect to the role of secondary structure as well as specific nucleotide sequence in the recognition site for the dnaG protein.

Nucleic Acids Res, 1989 Apr 11, 17(7), 2463 - 76
Modulations of the in vitro translational efficiencies of Yellow Fever virus mRNAs: interactions between coding and noncoding regions; Ruiz-Linares A et al.; As an approach to define the structural features within the 5' noncoding region of Yellow Fever virus (YFV) that modulate mRNA translational efficiency, we have studied how minor changes in this region affect the translational capacity in vitro of the corresponding mRNAs . A cDNA sequence coding for part of the YFV structural proteins was inserted into the vector pGEM3 containing the bacteriophage T7 promoter . This vector was engineered by site-directed mutagenesis to permit in vitro synthesis of transcripts containing only 5 vector nucleotides at their 5' end . The sequence of the YFV 5' untranslated region was further modified in order to alter the secondary structure of resulting T7 transcripts . The efficiency of these messengers in programming cell-free translation systems varied from 1- to 15-fold, correlating inversely with the potential of the 5' untranslated sequences to form stable secondary structures . A chimaeric messenger containing the YFV 5' noncoding (5' NC) region linked to a heterologous mRNA derived from Germiston virus, was tested for its in vitro translatability . We found a translational efficiency about 2-fold higher than that obtained with homologous transcripts, suggesting that YFV 5' NC region can function as a potential enhancer for gene expression . Data obtained with a series of plasmids constructed by linking the native YFV 5'NC region to various coding regions of the YFV genome indicated that interactions between the untranslated sequence and protein coding regions influence mRNAs translational efficiency.

J Biol Chem, 1989 Apr 5, 264(10), 5495 - 502
Structure of the gene for human plasminogen activator inhibitor-2 . The nearest mammalian homologue of chicken ovalbumin; Ye RD et al.; Plasminogen activator inhibitor-2 (PAI-2) can regulate the formation of plasmin by inhibiting urokinase and tissue plasminogen activator . PAI-2 is induced in monocytes and endothelium by inflammatory mediators, and it is made in the placenta during pregnancy . PAI-2 is a member of the serine protease inhibitor gene family, and it is particularly similar to chicken ovalbumin . Like ovalbumin, PAI-2 is secreted without cleavage of a signal peptide . To determine the structure of the PAI-2 gene, two bacteriophage lambda human genomic DNA libraries were screened with PAI-2 cDNA probes . Characterization of three positive clones shows that the human PAI-2 gene spans 16.5 kilobases and has eight exons . The 5'-untranslated sequence of the PAI-2 mRNA is 77 base pairs in length as suggested by primer extension and S1 nuclease mapping . The eukaryotic consensus sequence TATAAAA is found 22 base pairs 5' of the proposed cap site . The PAI-2 gene is on chromosome 18q21-23 as determined by hybridization to flow-sorted chromosomes and by in situ hybridization . There appear to be two common PAI-2 alleles that differ by six nucleotides in exons 1, 4, and 8 . The structure of the PAI-2 gene is quite different from that of PAI-1 although these two inhibitors have common target protease specificity . In contrast, the structure of the PAI-2 gene is very similar to that of the chicken ovalbumin gene . When protein sequences are aligned to obtain maximal identity, six of the seven intron positions in the PAI-2 gene are identical to those in the chicken ovalbumin gene . We conclude that PAI-2 is the closest mammalian homologue of avian ovalbumin.

Biochemistry, 1989 Apr 4, 28(7), 2849 - 55
Determination of RNA-protein contacts using thiophosphate substitutions; Milligan JF et al.; The binding of the bacteriophage R17 coat protein to its RNA binding site is an example of a specific RNA-protein interaction . Extensive analysis has revealed that the binding is dependent upon a unique hairpin structure that contains four essential single-stranded nucleotides . Additional specificity is thought to be due to four or five ionic contacts between the protein and phosphates on the RNA . Transcription of synthetic DNA with T7 RNA polymerase, using one of the nucleoside 5'-O-(1-thiotriphosphates) {NTP(alpha S)s}, allows the synthesis of RNAs specifically substituted with thiophosphates . Eleven sequence variants of the R17 coat protein binding site were synthesized with different NTP(alpha S)s and tested for coat protein binding to deduce positions of thiophosphates that alter the binding affinity . Of the twenty-one phosphate positions in the molecule, two were found to decrease the Ka 3-fold when substituted with a thiophosphate, one position decreased the Ka 10-fold, and one position increased the Ka 10-fold . Substitution of any of the other 17 positions with thiophosphates does not alter the Ka . The four positions that alter the Ka are located in a uniquely structured region of the RNA, and it is postulated that these thiophosphates affect binding because they contact coat protein directly.

AIDS Res Hum Retroviruses, 1989 Apr, 5(2), 159 - 71
Large-scale production and purification of a vaccinia recombinant-derived HIV-1 gp160 and analysis of its immunogenicity; Barrett N et al.; The human immunodeficiency virus (HIV-1) envelope gene was expressed in large-scale microcarrier cultures of Vero cells using a system involving coinfection with two recombinant vaccinia viruses . One recombinant contained the bacteriophage T7 RNA polymerase gene under control of a vaccinia virus promoter . The second contained the HIV-1 gp160 gene flanked by T7 promoter and termination sequences . The protein was expressed on the surface of infected cells, and it was shown to have a molecular weight of 160 kD and to react with gp41 and gp120 specific monoclonal antibodies . After purification by successive affinity and ion-exchange chromatography, the protein was demonstrated to be present in a particulate form with a diameter in the range of 15-30 nm . When injected into goats a high-titer gp160 specific antibody response was elicited and group-specific neutralizing activity could be demonstrated in vitro . The immunogenicity of the protein was also studied in conjunction with a number of adjuvant formulations, and the highest potency in mice was obtained using a preparation with 0.2% Al(OH)3 and 0.25% deoxycholate.

Hum Genet, 1989 Apr, 82(1), 40 - 4
Mapping of the gene encoding the multifunctional protein carrying out the first three steps of pyrimidine biosynthesis to human chromosome 2; Chen KC et al.; The CAD gene encodes a trifunctional protein that carries the activities of the first three enzymes (carbamyl phosphate synthetase II, aspartate transcarbamylase, and dihydroorotase) of de novo pyrimidine biosynthesis . Genomic fragments of the human CAD gene have been obtained by screening a human genomic library in bacteriophage lambda using a Syrian hamster cDNA clone as a probe . These human genomic clones have been used to assign the CAD gene to human chromosome 2 using in situ hybridization to human metaphase chromosomes and Southern blot hybridization analysis of DNA isolated from a panel of Chinese hamster/human hybrid cells . In situ hybridization analysis has allowed further localization of this gene to the chromosomal region 2p21-p22.

Proc Natl Acad Sci U S A, 1989 Apr, 86(8), 2587 - 91
Characterization of the human 5-lipoxygenase gene; Funk CD et al.; The gene for human 5-lipoxygenase has been isolated from three different bacteriophage genomic libraries and a genomic cosmid library . The gene spans greater than 82 kilobases and consists of 14 exons . The size range for the exons is 82-613 base pairs, whereas that for the introns is approximately 200 bp to greater than 26 kb . A major site of transcription initiation in leukocytes was mapped to a thymidine residue 65 base pairs upstream of the ATG initiation codon by nuclease S1 protection and primer extension experiments . Other potential minor initiation sites were found . The putative promoter region contains no TATA and CCAAT sequences in the expected positions upstream of the major transcription initiation site but contains multiple GC boxes within a (G + C)-rich region, as does the immediate 5' region of the first intron . Characteristics common to the 5' end of the human 5-lipoxygenase gene and the promoter regions of the housekeeping genes raise important questions concerning the regulation of 5-lipoxygenase gene expression.

J Virol, 1989 Apr, 63(4), 1661 - 8
The rhesus rotavirus outer capsid protein VP4 functions as a hemagglutinin and is antigenically conserved when expressed by a baculovirus recombinant; Mackow ER et al.; Rhesus rotavirus (RRV) gene 4 was cloned into lambda bacteriophage, inserted into a polyhedrin promoter shuttle plasmid, and expressed in Sf9 cells by a recombinant baculovirus . The baculovirus-expressed VP4 protein made up approximately 5% of the Spodoptera frugiperda-infected cell protein . Monoclonal antibodies that neutralize the virus bound to the expressed VP4 polypeptide, indicating that the expressed VP4 protein was antigenically indistinguishable from viral VP4 . In addition, we have determined that the baculovirus-expressed VP4 protein bound to erythrocytes and functions as the RRV hemagglutinin . The endogenous hemagglutinating activity of the VP4 protein, like the virus, was inhibited by guinea pig antirotavirus hyperimmune serum and by VP4-specific neutralizing monoclonal antibodies . The human erythrocyte protein, glycophorin, also inhibited hemagglutination by RRV or the expressed VP4 protein and appears to be the rotavirus erythrocyte receptor . The baculovirus-expressed VP4 protein was conserved functionally and antigenically in the absence of other outer or inner capsid rotavirus components and represents a logical candidate for future immunological studies.

J Biomol Struct Dyn, 1989 Apr, 6(5), 1027 - 38
Linguistics of nucleotide sequences . II: Stationary words in genetic texts and the zonal structure of DNA; Pevzner PA et al.; Words are irregularly distributed in genetic texts . The analysis of this irregularity leads to the notion of stationary and non-stationary words . The polyW and polyS tracts are shown to be the most non-stationary words in genetic texts (here W-{A,T}, S-{G,C}, a polyW tract is a sequence of A,T nucleotides and a polyS tract is a sequence of G,C nucleotides . The distribution of stationary words suggests a method for partitioning DNA into zones . The zones obtained in the case of the phage are interpreted in the light of the Dowe hypothesis of the modular structure of bacteriophage genomes.

Mol Gen Genet, 1989 Apr, 216(2-3), 364 - 71
mRNA stabilizing signals encoded in the genome of the bacteriophage phi x174; Hayashi MN et al.; In Escherichia coli cells infected with bacteriophage phi x174, mRNAs initiated by promoters PB and PD terminate after genes J, F, G, or H (TJ, TF, TG, or TH) . These RNAs are relatively stable and contain mRNA-stabilizing signals at their 3' ends . These signals were cloned after gene D of phi x174 in an expression vector plasmid . The cloned signals stabilize mRNA of the upstream gene D and the stabilized mRNA is translationally functional . When these signals are inserted in reverse, no stabilizing effect on mRNA is observed indicating that the correct sequences at the 3' ends of transcripts determine their stability . When a stabilizing signal (+) and a mutated stabilizing signal (-) which has reduced stabilizing activity are tandemly inserted after gene D, two sets of 3' termini of the transcript are observed indicating that both signals also function as terminators . The amount of gpD synthesized from these constructs varies depending upon the relative positions of the (+) or (-) signals after gene D . The stabilizing function seems to act by preventing mRNA degradation from the 3' to 5' direction . Several common features of these stabilizers are described.

J Bacteriol, 1989 Apr, 171(4), 2019 - 27
Role of bacteriophage Mu C protein in activation of the mom gene promoter; Bolker M et al.; The phage Mu C gene product is a specific activator of Mu late gene transcription, including activation of the mom operon . Fusion of the C gene to the efficient translation initiation region of the Escherichia coli atpE gene allowed significant overproduction of C protein, which was subsequently purified and assayed for DNA binding by gel retardation and nuclease footprinting techniques . C protein binds to a site immediately upstream of the -35 region both of the mom promoter and the related phage D108 mod promoter . The location of the mom promoter has been determined by primer extension . Upstream deletions extending more than 3 base pairs into the C-binding site abolished activation of the mom promoter in vivo . In vitro binding of C was not significantly affected by DNA methylation . A second, C-dependent promoter was identified just downstream of the C coding region; comparison with the mom promoter revealed common structural elements.

J Bacteriol, 1989 Apr, 171(4), 2003 - 18
Bacteriophage Mu late promoters: four late transcripts initiate near a conserved sequence; Margolin W et al.; Late transcription of bacteriophage Mu, which results in the expression of phage morphogenetic functions, is dependent on Mu C protein . Earlier experiments indicated that Mu late RNAs originate from four promoters, including the previously characterized mom promoter . S1 nuclease protection experiments were used to map RNA 5' ends in the three new regions . Transcripts were initiated at these points only in the presence of C and were synthesized in a rightward direction on the Mu genome . Amber mutant marker rescue analysis of plasmid clones and limited DNA sequencing demonstrated that these new promoters are located between C and lys, upstream of I, and upstream of P within the N gene . A comparison of the promoter sequences upstream from the four RNA 5' ends yielded two conserved sequences: the first (tA . . cT, where capital and lowercase letters indicate 100 and 75% base conservation, respectively), at approximately -10, shares some similarity with the consensus Escherichia coli sigma 70 -10 region, while the second (ccATAAc CcCPuG/Cac, where Pu indicates a purine), in the -35 region, bears no resemblance to the E . coli -35 consensus . We propose that these conserved Mu late promoter consensus sequences are important for C-dependent promoter activity . Plasmids containing transcription fusions of these late promoters to lacZ exhibited C-dependent beta-galactosidase synthesis in vivo, and C was the only Mu product needed for this transactivation . As expected, the late promoter-lacZ fusions were activated only at late times after induction of a Mu prophage . The C-dependent activation of lacZ fusions containing only a few bases of the 5' end of Mu late RNA and the presence of altered promoter sequences imply that C acts at the level of transcription initiation.

Proc Natl Acad Sci U S A, 1989 Apr, 86(8), 2799 - 803
Structural homology of complement protein C6 with other channel-forming proteins of complement; Chakravarti DN et al.; The amino acid sequence of the amino-terminal half of the complement protein C6 has been found to show overall structural homology with the homologous regions of the channel-forming proteins C7, C8 alpha, C8 beta, and C9 . In addition, two specific cysteine-rich segments common to the amino-terminal regions of C7, C8 alpha, C8 beta, and C9 also occur in their expected positions in C6, suggesting functional significance . Two cDNA clones encoding C6 were isolated from a human liver library in the bacteriophage vector lambda gt11 . The predicted protein sequence contains an apparent initiation methionine and a putative signal peptide of 21 residues, as well as a site for N-glycosylation at residue 303 . The sequence of the C6 protein reported here has 47-52% similarity with C7, C8 alpha, C8 beta, and C9, as well as 31-38% similarity with thrombospondin, thrombomodulin, and low density lipoprotein receptor . The sequence data have been interpreted by using computer algorithms for estimation of average hydrophobicity and secondary structure.

J Biol Chem, 1989 Mar 25, 264(9), 5031 - 5
Translation initiation at non-AUG triplets in mammalian cells; Peabody DS; In a previous report it was shown that mammalian ribosomes were capable of initiating translation at a non-AUG triplet when the initiation codon of mouse dihydrofolate reductase (dhfr) was mutated to ACG (Peabody, D.S . (1987) J . Biol . Chem . 262, 11847-11851) . In order to assess the capacity of the mammalian translation apparatus to initiate at other non-AUG triplets, the initiator AUG of dihydrofolate reductase was converted to GUG, UUG, CUG, AGG, AAG, AUA, AUC, and AUU . These represent (with ACG) all the possible triplets that differ from AUG by only one nucleotide . The ability of each mutant to produce dihydrofolate reductase was assessed by in vitro transcription/translation of the mutant dhfr sequences under control of the bacteriophage SP6 promoter . Each of the triplets (with the exceptions of AGG and AAG) was able to direct the synthesis of apparently normal dihydrofolate reductase . Incorporation of {35S}tRNAifMet into the products of in vitro translation indicates that in each case the non-AUG triplet is able to direct initiation of the polypeptide chain with methionine . The mutant dhfr sequences were also inserted into the mammalian expression vector SVGT5 for expression in cultured monkey cells . The hierarchy of relative translation efficiencies was similar in vivo and in vitro.

Biochemistry, 1989 Mar 21, 28(6), 2410 - 8
NMR spectroscopy of 113Cd(II)-substituted gene 32 protein; Giedroc DP et al.; Gene 32 protein (g32P), the single-stranded DNA binding protein from bacteriophage T4, contains 1 mol of Zn(II)/mol of protein . This intrinsic zinc is retained within the DNA-binding core fragment, g32P-(A+B) (residues 22-253), obtained by limited proteolysis of the intact protein . Ultraviolet circular dichroism provides evidence that Zn(II) binding causes significant changes in the conformation of the peptide chain coupled with alterations in the microenvironments of tryptophan and tyrosine side chains . NMR spectroscopy of the 113Cd(II) derivative of g32P-(A+B) at both 44.4 and 110.9 MHz shows a single 113Cd resonance, delta 637, a chemical shift consistent with coordination to three of the four sulfhydryl groups in the protein . In vitro mutagenesis of Cys166 to Ser166 creates a mutant g32P that still contains 1 Zn(II)/molecule . This mutant protein when substituted with 113Cd(II) shows a 113Cd signal with a delta and a line width the same as those observed for the wild-type protein . Thus, the S-ligands to the metal ion appear to be contributed by Cys77, Cys87, and Cys90 . Relaxation data suggest that chemical shift anisotropy is the dominant, but not exclusive, mechanism of relaxation of the 113Cd nucleus in g32P, since a dipolar modulation from ligand protons is observed at 44.4 MHz but not at 110.9 MHz . Complexation of core 113Cd g32P with d(pA)6 or Co(II) g32P with poly(dT) shows only minor perturbation of the NMR signal or d-d electronic transitions, respectively, suggesting that the metal ion in g32P does not add a ligand from the bound DNA.(ABSTRACT TRUNCATED AT 250 WORDS)

J Mol Biol, 1989 Mar 20, 206(2), 333 - 48
Structure and stability of mRNA synthesized by vaccinia virus-encoded bacteriophage T7 RNA polymerase in mammalian cells . Importance of the 5' untranslated leader; Fuerst TR et al.; We have analyzed the structure and stability of RNA synthesized by bacteriophage T7 RNA polymerase in mammalian cells . The T7 polymerase, expressed by a recombinant vaccinia virus, transcribed the Escherichia coli lacZ gene flanked by T7 promoter and terminator signals . The lacZ gene cassette was introduced into infected cells within either a transfected plasmid or a second recombinant vaccinia virus . The T7-lacZ transcripts, which had a half-life of approximately 75 minutes, represented approximately 30% of total cytoplasmic RNA after a 24 hour period . The latter estimation indicated a disparity between the levels of lacZ RNA and beta-galactosidase synthesis . Analysis of the T7 transcripts indicated that they were initiated correctly but that only 5 to 10% contained terminal cap structures, providing an explanation for the low translatability of the RNA . Since the 5' end of the T7 transcripts can form a stem-loop structure that might interfere with capping by vaccinia virus RNA guanylyltransferase, as well as ribosome binding and scanning, a similar vector lacking such sequences was constructed . In vitro experiments demonstrated that T7 RNA polymerase transcribed both templates with similar efficiency and that the RNA lacking the potential to form the stem-loop was capped more rapidly by the purified vaccinia virus enzyme . Nevertheless, when the stem-loop was removed, beta-galactosidase was not expressed in infected cells; moreover, no T7 transcripts could be detected, suggesting that the RNA was not made or more likely was degraded during or shortly after synthesis . There is previous evidence that vaccinia virus RNA guanylyltransferase is associated with the viral transcription complex, thereby allowing RNA synthesis and capping to occur concurrently . We suggest that a lack of coupling between the vaccinia viral RNA guanylyltransferase and bacteriophage T7 RNA polymerase delays capping of T7 transcripts and that, under these conditions, the 5'-terminal double-stranded stem is required to stabilize the nascent RNA against degradation . Although deletion of the 3' palindromic sequence specifying T7 transcriptional termination from the expression cassette resulted in RNA of more heterogeneous lengths, neither the apparent turnover rate nor translation of the RNAs was diminished appreciably.

Eur J Biochem, 1989 Mar 15, 180(2), 301 - 8
Molecular cloning, primary structure and disruption of the structural gene of aldolase from Saccharomyces cerevisiae; Schwelberger HG et al.; A yeast cDNA genetic library in a bacteriophage expression vector was screened using an antiserum reacting with fructose 1,6-bisphosphate aldolase from Saccharomyces cerevisiae . Radio-labelled probes of selected immunopositive clones were used for screening of a yeast genomic library . From the genomic clones a yeast/Escherichia coli shuttle plasmid was constructed containing on a 1990-base-pair fragment the entire structural gene FBA1 coding for yeast aldolase . The primary structure of the FBA1 gene was determined . An open reading frame comprises 1077 base pairs coding for a protein of 359 amino acids with a predicted molecular mass of 39,608 Da . As observed for other strongly expressed yeast genes, codon usage is extremely biased . The 810 base pairs at the 5' end and the 90 base pairs at the 3' end of the coding region of the cloned FBA1 gene are sufficient for normal expression and show characteristic elements present in the noncoding sequences of other yeast genes . Aldolase is the major protein in yeast cells transformed with a high-copy-number plasmid containing the FBA1 gene . The aldolase gene was disrupted by insertion of the yeast URA3 gene into the coding region of one FBA1 allele in a homozygous diploid ura3 strain . The haploid offsprings with the defective aldolase allele fba1::URA3 lack aldolase enzymatic activity and fail to grow in media containing as a carbon source metabolites of only one side of the aldolase reaction.

J Biol Chem, 1989 Mar 15, 264(8), 4725 - 31
Characterization of the bacteriophage T4 gene 41 DNA helicase; Richardson RW et al.; The T4 gene 41 protein and the gene 61 protein function together as a primase-helicase within the seven protein bacteriophage T4 multienzyme complex that replicates duplex DNA in vitro . We have previously shown that the 41 protein is a 5' to 3' helicase that requires a single-stranded region on the 5' side of the duplex to be unwound and is stimulated by the 61 protein (Venkatesan, M., Silver L . L., and Nossal, N . G . (1982) J . biol . Chem . 257, 12426-12434) . The 41 protein, in turn, is required for pentamer primer synthesis by the 61 protein . We now show that the 41 protein helicase unwinds a partially duplex DNA molecule containing a performed fork more efficiently than a DNA molecule without a fork . Optimal helicase activity requires greater than 29 nucleotides of single-stranded DNA on the 3' side of the duplex (analogous to the leading strand template) . This result suggests the 41 protein helicase interacts with the leading strand template as well as the lagging strand template as it unwinds the duplex region at the replication fork . As the single-stranded DNA on the 3' side of a short duplex (51 base pairs) is lengthened, the stimulation of the 41 protein helicase by the 61 protein is diminished . However, both the 61 protein and a preformed fork are essential for efficient unwinding of longer duplex regions (650 base pairs) . These findings suggest that the 61 protein promotes both the initial unwinding of the duplex to form a fork and subsequent unwinding of longer duplexes by the 41 protein . A stable protein-DNA complex, detected by a gel mobility shift of phi X174 single-stranded DNA, requires both the 41 and 61 proteins and a rNTP (preferably rATP or rGTP, the nucleotides with the greatest effect on the helicase activity) . In the accompanying paper, we report the altered properties of a proteolytic fragment of the 41 protein helicase and its effect on in vitro DNA synthesis in the T4 multienzyme replication system.

J Biol Chem, 1989 Mar 15, 264(8), 4732 - 9
Trypsin cleavage in the COOH terminus of the bacteriophage T4 gene 41 DNA helicase alters the primase-helicase activities of the T4 replication complex in vitro; Richardson RW et al.; The bacteriophage T4 gene 41 protein is a 5' to 3' DNA helicase which unwinds DNA ahead of the growing replication fork and, together with the T4 gene 61 protein, also functions as a primase to initiate DNA synthesis on the lagging strand . Proteolytic cleavage by trypsin approximately 20 amino acids from the COOH terminus of the 41 protein produces 41T, a 51,500-dalton fragment (possibly still associated with small COOH-terminal fragments) which still retains the ssDNA-stimulated GTPase (ATPase) activity, the 61 protein-stimulated DNA helicase activity, and the ability to act with 61 protein to synthesize pentaribonucleotide primers . In the absence of the T4 gene 32 ssDNA binding protein, the primase-helicase composed of the tryptic fragment (41T) and 61 proteins efficiently primes DNA synthesis on circular ssDNA templates by the T4 DNA polymerase and the three T4 polymerase accessory proteins . In contrast, the 41T protein is defective as a helicase or a primase component on 32 protein-covered DNA . Thus, unlike the intact protein, 41T does not support RNA-dependent DNA synthesis on 32 protein-covered ssDNA and does not stimulate strand displacement DNA synthesis on a nicked duplex DNA template . High concentrations of 32 protein strongly inhibit RNA primer synthesis with either 41 T or intact 41 protein . The 44/62 and 45 polymerase accessory proteins (and even the 44/62 proteins to some extent) substantially reverse the 32 protein inhibition of RNA primer synthesis with intact 41 protein but not with 41T protein . We propose that the COOH-terminal region of the 41 protein is required for its interaction with the T4 polymerase accessory proteins, permitting the synthesis and utilization of RNA primers and helicase function within the T4 replication complex . When this region is altered, as in 41T protein, the protein is unable to assemble a functional primase-helicase in the replication complex . An easy and rapid purification of T4 41 protein produced by a plasmid encoding this gene (Hinton, D . M., Silver, L . L., and Nossal, N . G . (1985) J . Biol . Chem . 260, 12851-12857) is also described.

J Biol Chem, 1989 Mar 15, 264(8), 4552 - 8
Evidence for membrane-bound oligomerization of bacteriophage phi X174 lysis protein-E; Blasi U et al.; The expression of cloned bacteriophage phi X174 lysis gene E was analyzed in minicells of Escherichia coli using two-dimensional gel electrophoresis . Beside the 10-11-kDa protein-E, at least two additional protein bands were detected, associated with the inner membrane, which showed the same isoelectric point as E . To clarify whether these proteins were E-specific, two different antibodies directed against a beta-galactosidase-E' hybrid protein and a synthetic oligopeptide corresponding to the C-terminal end of protein-E were raised . Immunoadsorption studies with anti-peptide-specific antibodies resulted in the detection of protein-E as well as in the detection of proteins of higher molecular weight . Two of these protein bands were positively recognized by anti beta-galactosidase-E' antibodies . The latter protein bands had the same molecular weight as the putative protein-E bands detected by two-dimensional gel electrophoresis indicating that these bands represent protein-E-specific oligomers . These data support the idea that an E-specific oligomeric structure penetrating the inner and outer membrane of E . coli is formed during the lytic action of protein-E.

Nucleic Acids Res, 1989 Mar 11, 17(5), 1839 - 43
Codon usage and secondary structure of MS2 phage RNA; Bulmer M; MS2 is an RNA bacteriophage (3569 bases) . The secondary structure of the RNA has been determined, and is known to play an important role in regulating translation . Paired regions of the genome have a higher G+C content than unpaired regions . It has been suggested that this reflects selection for high G+C content to encourage pairing, but a re-analysis of the data together with computer simulation suggest that it is an automatic consequence in any RNA sequence of the way it folds up to minimise its free energy . It has also been suggested that the three registers in which pairing can occur in a coding region are used differentially to optimise the use of the redundancy of the genetic code, but re-analysis of the data shows only weak statistical support for this hypothesis.

Biochemistry, 1989 Mar 7, 28(5), 2245 - 51
Perturbation of tryptophan residues by point mutations in bacteriophage T4 lysozyme studied by optical detection of triplet-state magnetic resonance spectroscopy; Zang LH et al.; We have investigated perturbations of the triplet-state properties of Trp residues in bacteriophage T4 lysozyme caused by point mutations using low-temperature phosphorescence and optical detection of triplet-state magnetic resonance (ODMR) spectroscopy . Five temperature-sensitive mutants have been studied in detail . These include lysozymes with the point mutations Gln-105----Ala, Gln-105----Gly, Gln-105----Glu, Ala-146----Thr, and Trp-126----Gln . Changes in phosphorescence 0,0 band wavelength, intensity, the triplet-state zero-field splitting (ZFS), and the wavelength dependence of the ZFS were detected only from Trp-138 in each mutant . In the case of the Q105A mutation, the perturbations on Trp-138 have been ascribed to the combination of an increase in the polarizability of the environment and to the loss of hydrogen bonding of the enamine nitrogen of indole . For the Q105G mutation, we believe that Q is replaced by a solvent molecule in H bonding, leading to relatively small changes . In the Q105E mutation, the perturbation results largely from the introduction of a charged residue . In the case of the mutation A146T, the perturbation is associated with a local conformational change in which Trp-138 is shifted to a more solvent-exposed location . On the other hand, no significant spectroscopic changes in Trp-126 and Trp-158 were found in any of the mutants, suggesting that the perturbations are probably localized near Trp-138 for the mutations of positions 105 and 146 . However, in the mutation W126Q, which occurs approximately 16 A away from Trp-138, significant changes of Trp-138 are detected, suggesting that the effects of this mutation are propagated over large distances.

J Mol Biol, 1989 Mar 5, 206(1), 133 - 52
Two-dimensional 1H nuclear magnetic resonance studies on the gene V-encoded single-stranded DNA-binding protein of the filamentous bacteriophage IKe . II . Characterization of the DNA-binding wing with the aid of spin-labelled oligonucleotides; de Jong EA et al.; The DNA-binding domain of the single-stranded DNA-binding protein IKe GVP was studied by means of 1H nuclear magnetic resonance, through use of oligonucleotides of two and three adenyl residues in length, that were spin-labelled at their 3' and/or 5' termini . These spin-labelled ligands were found to cause line broadening of specific protein resonances when bound to the protein, although they were present in small quantities, i.e . of the order of 0.04 molar equivalent and less . The line broadening of protein resonances was made manifest by means of difference one and two-dimensional spectroscopy . Difference one-dimensional experiments revealed line broadening of the same protein resonances upon binding of either 3' or 5' spin-labelled oligonucleotides . Evidence in favour of the existence of a fixed 5' to 3' orientation in the binding of oligonucleotides to the protein surface was therefore not obtained from the spin-labelled oligonucleotide binding studies . Residue-specific assignments of broadened resonances could not, or could only sparsely, be derived from the difference one-dimensional spectra, because of the tremendous overlap in the aliphatic region of the spectrum . In contrast, such assignments were easily obtained from the difference two-dimensional spectra, which were recorded by means of both total correlated spectroscopy and nuclear Overhauser effect spectroscopy . Difference signals were detected for 15 spin systems; ten out of these were assigned to the residues I29, Y27, S20, G18, R16, T28, K22, Q21, V19 and S17 in the amino acid sequence of IKe GVP; the other five spin systems could be assigned to a phenylalanyl residue, an arginyl or lysyl residue, an aspartic acid or asparagyl residue, a glycyl residue and a glutamic acid or glutamyl residue . From the evaluation of the relative difference signals, it was concluded that the direct surroundings of the spin-label group of the labelled oligonucleotide in the bound state is composed of the first five residues in the former group of residues and the five residues in the latter group.(ABSTRACT TRUNCATED AT 400 WORDS)

J Mol Biol, 1989 Mar 5, 206(1), 119 - 32
Two-dimensional 1H nuclear magnetic resonance studies on the gene V-encoded single-stranded DNA-binding protein of the filamentous bacteriophage IKe . I . Structure elucidation of the DNA-binding wing; de Jong EA et al.; Two-dimensional nuclear magnetic resonance techniques were used to obtain residue- and sequence-specific assignments in the 1H spectrum of the single-stranded DNA-binding protein encoded by gene V of the filamentous phage IKe (IKe GVP) . The residue-specific assignments are based on the analysis of J-correlated spectra, i.e . correlated spectroscopy and homonuclear-Hartmann-Hahn total correlated spectroscopy . Complete assignments of side-chain spin systems, e.g . long side-chains, were, to a major part, derived from two-dimensional spectra obtained by means of the latter technique . Sequence-specific residue assignments were obtained for the two neighbouring residues V41 and Y42, and the amino acid sequence segment encompassing residues S17 through I29 . The structure of this segment, a beta-loop, was deduced from the interresidue nuclear Overhauser effect pattern . Residues S17 through V19 and P26 through I29 form an anti-parallel beta-ladder segment, whereas residues Q21 to K25 constitute the loop region . The beta-loop is expected to project into the solution and is intimately involved in binding to single-stranded DNA; it is therefore designated the "DNA-binding wing" . By analogy with the structure of the DNA-binding wing deduced from IKe GVP, a similar structure is proposed for the corresponding domain of the gene V protein encoded by the filamentous phage Ff for which, from X-ray diffraction studies, a three-dimensional structure has been deduced . Essential differences appear to exist between the DNA-binding domain in the X-ray structure and that proposed in this paper . Possible reasons for these differences are discussed.

J Mol Biol, 1989 Mar 5, 206(1), 29 - 39
Lambda repressor mutants that are better substrates for RecA-mediated cleavage; Gimble FS et al.; RecA-mediated cleavage of the bacteriophage lambda repressor results in inactivation of the protein and leads to induction of the lambda prophage . Here, we report the identification of three mutations in lambda repressor that significantly increase the rate of RecA-mediated cleavage . These mutations were isolated as intragenic second-site suppressors of a mutation (ind-) which prevents cleavage . Purified repressor proteins that contain both the ind- mutation and one of the second-site mutations undergo cleavage at near wild-type rates . Purified repressors that contain the second-site mutations in otherwise wild-type backgrounds undergo RecA-mediated cleavage at significantly faster rates than wild-type, and form dimers more poorly than the wild-type protein . In related experiments, we found that other repressor mutants that dimerize poorly are also better substrates for RecA-mediated cleavage . Conversely, we show that a covalent disulfide-bonded repressor dimer is resistant to cleavage . These results support a model in which repressor monomers are the only substrate in the cleavage reaction.

J Mol Biol, 1989 Mar 5, 206(1), 19 - 27
UvsY protein of bacteriophage T4 is an accessory protein for in vitro catalysis of strand exchange; Harris LD et al.; The uvsX and uvsY genes are essential to genetic recombination, recombination-dependent DNA synthesis and to the repair of DNA damage in bacteriophage T4 . Purified UvsX protein has been shown to catalyze strand exchange and D-loop formation in vitro, but the role of UvsY protein has been unclear . We report that UvsY protein enhances strand exchange by UvsX protein by interacting specifically with UvsX protein: gene 32 protein (gp32) is not necessary for this effect and UvsY protein has no similar effect on the RecA protein of E . coli . UvsY protein, like UvsX protein, protects single-stranded DNA from digestion by nucleases, but, unlike UvsX protein, shows no ability to protect double-stranded DNA . UvsY protein enhances the rate of single-stranded-DNA-dependent ATP hydrolysis by UvsX protein, particularly in the presence of gp32 or high concentrations of salt, factors that otherwise reduce the ATPase activity of UvsX protein . The enhancement of ATP hydrolysis by UvsY protein is shown to result from the ability of UvsY protein to increase the affinity of UvsX protein for single-stranded DNA.

Appl Environ Microbiol, 1989 Mar, 55(3), 661 - 5
Use of a biotinylated DNA probe to detect bacteria transduced by bacteriophage P1 in soil; Zeph LR et al.; Presumptive bacteriophage P1 transductants of Escherichia coli, isolated from soil inoculated with lysates of transducing phage P1 and E . coli, were confirmed to be lysogenic for phage P1 by hybridization with a biotinylated DNA probe prepared from the 1.2-kilobase-pair HindIII 3 fragment of bacteriophage P1 . No P1 lysogens of indigenous soil bacteria were detected with the DNA probe . The sensitivity and specificity of the DNA probe were assessed with purified and dot blot DNA, respectively . In addition, two techniques for the lysis and deproteinization of bacteria and bacteriophages on nitrocellulose filters were compared . These studies indicated that biotinylated DNA probes may be an effective alternative to conventional radiolabeled DNA probes for detecting specific gene sequences in bacteria indigenous to or introduced into soil.

Biochim Biophys Acta, 1989 Mar 1, 1007(2), 151 - 7
Singlet molecular oxygen causes loss of biological activity in plasmid and bacteriophage DNA and induces single-strand breaks; Di Mascio P et al.; Damage of plasmid and bacteriophage DNA inflicted by singlet molecular oxygen (1O2) includes loss of the biological activity measured as transforming capacity in E . coli and single-strand break formation . Three different sources of 1O2 were employed: (i) photosensitization with Rose bengal immobilized on a glass plate physically separated from the solution; (ii) thermal decomposition of the water-soluble endoperoxide 3,3'-(1,4-naphthylidene) dipropionate (NDPO2); and (iii) microwave discharge . Loss of transforming activity was documented after exposing bacteriophage M13 DNA to 1O2 generated by photosensitization employing immobilized Rose bengal, and with bacteriophage luminal diameter X174 DNA, using the thermodissociable endoperoxide (NDPO2) as a source of 1O2 . These findings are in agreement with experiments in which plasmid DNA pBR322 was exposed to a gas stream of 1O2 generated by microwave discharge . The effects of 1O2 quenchers and of 2H2O indicate 1O2 to be the species responsible . Strand-break formation in pBR322 and luminal diameter X174, measured as an increase of the open circular form at the expense of the closed circular supercoiled form, was observed without alkaline treatment after exposing the DNA to 1O2, using either agarose gel electrophoresis or sucrose gradient separation . The effect of quenchers and 2H2O indicate the involvement of 1O2 in DNA damage . We conclude that singlet oxygen can cause loss of biological activity and DNA strand breakage.

EMBO J, 1989 Mar, 8(3), 973 - 80
The integrated conjugative plasmid pSAM2 of Streptomyces ambofaciens is related to temperate bacteriophages; Boccard F et al.; Streptomyces ambofaciens ATCC23877 and derivatives contain the 11-kb element pSAM2 present in an integrated state or as a free and integrated plasmid . This element, able to integrate site-specifically in the genome of different Streptomyces species, is conjugative and mobilizes chromosomal markers . Besides these plasmid functions, we have shown that the site-specific recombination system of pSAM2 presents strong similarities with that of several temperate phages . The integration event is promoted by a site-specific recombinase of the integrase family . The int gene encoding this integrase is closely linked to the plasmid attachment site (attP) . A small open reading frame (ORF) overlaps the int gene and the predicted protein exhibits similarities with Xis proteins involved in phages excision . The integrated copy of pSAM2 in strain ATCC23877 is flanked by att sequences (attL and attR) . Another att sequence (attX) is present in this strain and attX and attL are the boundaries of a 42-kb fragment (xSAM1) absent, as well as pSAM2, from S.ambofaciens DSM40697 . Sequences partially similar to pSAM2 int gene are found near the chromosomal integration zone in both S.ambofaciens strains . The possible origin of pSAM2, an element carrying plasmid as well as phage features, is discussed.

Biofizika, 1989 Mar-Apr, 34(2), 225 - 9
{Study of the binding of RNA polymerase by a recombinant plasmid using electron microscopy}; Beritashvili DR et al.; RNA-polymerase of E . coli was bound in vitro under physiological conditions to a recombinant plasmid pBR322 carrying two identical segments of bacteriophage T4 DNA, each containing a complete gene coding for T4 DNA ligase . After fixation of the complex with formaldehyde it was analysed by electron microscopy . A map of binding sites of the enzyme to DNA was obtained after a statistical assessment of micrographs . The inserted repeat revealed itself in the map as two regions of identical binding patterns, thus proving the adequacy of the preparation procedure . In pBR322 the strong binding sites correlate with the position of promoters . Also, apart from this, there are other strong binding sites within the T4 sequences which correlate strongly with the regions of abnormally high AT content . That means that under physiological conditions the RNA polymerase forms strong binding complexes with any AT rich DNA regions, as well as with real promoters.

Mol Gen Mikrobiol Virusol, 1989 Mar, (3), 35 - 9
{Mutagenesis directed by phosphotriester analogs of oligonucleotides: a way to site-specific mutagenesis in vivo}; Petrenko VA et al.; A new approach is proposed to obtain the directed mutations in the gene under study . The technique is based on using alkylphosphotriester analogues of oligodeoxyribonucleotides as site-specific mutagens . The deletion C in lacZ' gene of bacteriophage M13mpB was obtained by cotransfection of Escherichia coli cells with a mix of DNA and phosphotriester analogues of oligonucleotides.

Radiobiologiia, 1989 Mar-Apr, 29(2), 278 - 80
{Effect of He-Ne laser radiation on the bacteriophage T4-Escherichia coli system}; Tiflova OA et al.; Exposure of T4 bacteriophage, having no red light chromophores, to He-Ne laser (lambda = 632.8 nm) of 10(3)-6 X 10(4) J/m2 does not influence its lytic properties . Irradiation of E . coli WP2 bacteria with doses of 4-6 X 10(3) J/m2 causes a 1.25-1.35-fold increase in their ability to keep on the development of nonirradiated bacteriophage T4.

Rev Infect Dis, 1989 Mar-Apr, 11 Suppl 2, S404 - 10
Mycobacteriophage vector systems; Jacobs WR Jr et al.; Successful application of molecular genetic approaches to the study of mycobacteria necessitates the introduction of recombinant DNA molecules into mycobacterial cells . Efficient methods of introducing DNA into Mycobacterium smegmatis protoplasts have been developed, and the construction of mycobacteriophage recombinant DNA vectors has been initiated . Novel Escherichia coli-Mycobacterium shuttle vectors, termed shuttle phasmids, have been constructed . These vectors were constructed by inserting E . coli cosmids into nonessential regions of mycobacteriophage DNAs . Shuttle phasmids are multifunctional vectors that replicate in E . coli as plasmids and replicate in mycobacteria as phage . The presence of the bacteriophage lambda cos sequences permits the use of the lambda in vitro packaging system for efficient cloning of additional genes into these vectors . Temperate shuttle phasmids have been constructed that can infect and lyse mycobacterial cells or lysogenize mycobacterial cells to stably integrate and express cloned DNA into mycobacterial genomes . Shuttle phasmids can be transduced into a wide variety of mycobacterial species and thus should permit the development of molecular genetic systems for the mycobacteria.

Eur J Biochem, 1989 Mar 1, 180(1), 149 - 52
Comparison of the amino acid sequence of the lytic enzyme from broad-host-range bacteriophage PRD1 with sequences of other cell-wall-peptidoglycan lytic enzymes; Pakula TM et al.; The gene for the lytic enzyme of the lipid-containing, broad-host-range bacteriophage PRD1 codes for a protein of 149 amino acids (17271 Da) . The sequence of the protein is unique when compared to other lytic enzymes sequenced . However, three regions of weak similarity with other phage lytic enzymes were observed . The C-terminal region shared seven amino acids in common with phage P22 lysozyme at a site which is conserved in phage-type lysozymes.

J Bacteriol, 1989 Mar, 171(3), 1379 - 85
The groES and groEL heat shock gene products of Escherichia coli are essential for bacterial growth at all temperatures; Fayet O et al.; The products of the groES and groEL genes of Escherichia coli, constituting the groE operon, are known to be required for growth at high temperature (42 degrees C) and are members of the heat shock regulon . Using a genetic approach, we examined the requirement for these gene products for bacterial growth at low temperature (17 to 30 degrees C) . To do this, we constructed various groES groEL heterodiploid derivative strains . By inactivating one of the groE operons by a polar insertion, it was shown by bacteriophage P1 transduction that at least one of the groE genes was essential for growth at low temperature . Further P1 transduction experiments with strains that were heterodiploid for only one of the groE genes demonstrated that both groE gene products were required for growth at low temperature, which suggested a fundamental role for the groE proteins in E . coli growth and physiology.

Genetika, 1989 Mar, 25(3), 396 - 405
{Deletion-insertion mapping of the region non-essential for functioning of the beta-subunit of Escherichia coli RNA polymerase}; Kashlev MV et al.; A plasmid has been constructed containing the gene of beta-subunit of RNA polymerase of Escherichia coli under control of the PR promoter of bacteriophage lambda . PR promoter may be induced by heating up to 42 degrees C . In frame insertions of different sequences between 989 and 990 or 1010 and 1011 codons of the rpoB gene do not inactivate the beta-subunit function . Deletions in the region of 1011-1027 codons result in inactivation of beta-subunit . We localized antigene determinant of monoclonal anti-beta-antibodies which do not inactivate RNA polymerase in vitro . The borders of non-essential region of beta-subunit were accurately determined.

Mol Gen Genet, 1989 Mar, 216(1), 31 - 6
Suppression of the thermosensitive DNA ligase mutations in Escherichia coli K12 through modulation of gene expression induced by phage Mu; Ghelardini P et al.; We have previously shown that Mu can sustain the growth at non-permissive temperature of an Escherichia coli strain harbouring a thermosensitive mutation in the DNA ligase structural gene . This "complementation" reaches a maximal level with the Mu lig3 mutant which restores the viability of a ligts7 strain to the level of the wild type (Ghelardini et al . 1980; Paolozzi et al . 1980) . In this study we analysed the characteristics of this phenotypic suppression in order to clarify its molecular mechanism . We found that an E . coli ligts7 strain lysogenic for the Mu lig3 mutant shows: (i) an increment in the host DNA ligase activity; (ii) an increase in the specific mRNA of the host lig gene; (iii) an increase (towards the relaxed state) in the average linking number of a resident plasmid; and (iv) a reduction in DNA gyrase activity . These results are compatible with the hypothesis that the Mu lig gene product by interfering with the host enzymatic apparatus controlling DNA topology leads to a reduction in chromosomal supercoiling . The relaxation of the chromosome could affect the transcription of the DNA ligase gene, amongst others . Thus, through this mechanism, the Mu lig gene product is able to modulate gene expression and hence suppress the effects of the E . coli ligts7 mutation . On the basis of the identification of this mechanism of action, we propose to change the name of the Mu lig gene (thought originally to be the structural gene for a bacteriophage ligase) to gem (gene expression modulation).

Mol Gen Genet, 1989 Mar, 216(1), 106 - 12
Modulation of the SOS response by truncated RecA proteins; Larminat F et al.; RecA protein plays several key roles in the SOS response . We have constructed truncated proteins and examined their capacity to accomplish Weigle reactivation and mutagenesis of bacteriophage lambda and recombination in Escherichia coli . Our data indicate that the 17 carboxyl terminal amino acids are not essential to RecA function . However in the presence of wild-type RecA protein, the truncated protein reduces the efficiency of recombination without affecting either mutagenesis or induction of an SOS gene or Weigle reactivation . The data presented here suggest that activation of RecA protein does not involve mixed multimers or is not affected by their presence.

Genetics, 1989 Mar, 121(3), 401 - 9
Phage genetic sites involved in lambda growth inhibition by the Escherichia coli rap mutant; Guzman P et al.; The rap mutation of Escherichia coli prevents the growth of bacteriophage lambda . We have isolated phage mutants that compensate for the host deficiency . The mutations, named bar, were genetically located to three different loci of the lambda genome: barI in the attP site, barII in the cIII ea10 region, and barIII within or very near the imm434 region . The level of lambda leftward transcription correlates with rap exclusion . Phage lambda mutants partially defective in the pL promoter or in pL-transcript antitermination showed a Bar- phenotype . Conversely, mutants constitutive for transcription from the pI or pL promoters were excluded more stringently by rap bacteria . We conclude that rap exclusion depends on the magnitude of transcription through the wild type bar loci in the phage genome.

Mutat Res, 1989 Mar-May, 220(2-3), 263 - 8
Lambda phage shuttle vectors for analysis of mutations in mammalian cells in culture and in transgenic mice; Summers WC et al.; Foreign DNA sequences contained in lambda bacteriophage genomes integrated in mammalian DNA can be efficiently rescued into infectious phage particles by treatment of the mammalian DNA with lambda-packaging extracts prepared in E . coli . This system provides for rapid, non-selective recovery of stably integrated, chromosomal sequences into lambda phage for subsequent analysis in bacterial systems . Since rescue is prior to selection, mutations can be recovered from intact animals made transgenic for the phage-target gene sequences . Such approaches allow study of physiologically relevant aspects of mammalian mutagenesis at the molecular level.

J Bacteriol, 1989 Mar, 171(3), 1590 - 6
Escherichia coli DnaK and GrpE heat shock proteins interact both in vivo and in vitro; Johnson C et al.; Previous studies have demonstrated that the Escherichia coli dnaK and grpE genes code for heat shock proteins . Both the Dnak and GrpE proteins are necessary for bacteriophage lambda DNA replication and for E . coli growth at all temperatures . Through a series of genetic and biochemical experiments, we have shown that these heat shock proteins functionally interact both in vivo and in vitro . The genetic evidence is based on the isolation of mutations in the dnaK gene, such as dnaK9 and dnaK90, which suppress the Tr- phenotype of bacteria carrying the grpE280 mutation . Coimmunoprecipitation of DnaK+ and GrpE+ proteins from cell lysates with anti-DnaK antibodies demonstrated their interaction in vitro . In addition, the DnaK756 and GrpE280 mutant proteins did not coimmunoprecipitate efficiently with the GrpE+ and DnaK+ proteins, respectively, suggesting that interaction between the DnaK and GrpE proteins is necessary for E . coli growth, at least at temperatures above 43 degrees C . Using this assay, we found that one of the dnaK suppressor mutations, dnaK9, reinstated a protein-protein interaction between the suppressor DnaK9 and GrpE280 proteins.

J Bacteriol, 1989 Mar, 171(3), 1235 - 44
Structural analysis of the carboxy terminus of bacteriophage lambda repressor determined by antipeptide antibodies; Sussman R et al.; To analyze lambda repressor function and structure, antibodies were generated with synthetic peptides corresponding to sequences believed to be involved in prophage induction . These site-directed antibodies seemed to recognize preferentially the primary sequence of repressor because they reacted better in competition experiments with the oligopeptide and with the partially denatured forms of repressor than with the native molecules . This information, together with the characteristic ability of the antibodies to immunoprecipitate or react with repressor in immunoblots, allowed us to infer some conformational properties of the specific regions that the antibodies recognized . The antibodies reacted less with some mutant repressors that had a single amino acid substitution within the cognitive sequences . RecA-catalyzed cleavage of repressor was inhibited to different extents in relation to the proportion of repressor that each antipeptide immunoglobulin G (IgG) was able to immunoprecipitate . The antipeptide IgGs did not affect specific binding of repressor to operator DNA, whereas the antirepressor IgG was inhibitory . The three different IgGs competed for binding to repressor in an enzyme-linked immunosorbent assay additivity test, which suggested that the three regions of conserved amino acids are probably located on the same side of the carboxyl domain of repressor and possibly close together in the tertiary structure.

FEBS Lett, 1989 Feb 27, 244(2), 369 - 75
Purification and characterization of the Ner repressor of bacteriophage Mu; Kukolj G et al.; The Ner protein of bacteriophage Mu acts as a lambda cro-like negative regulator of the phage's early (transposase) operon . Using the band retardation assay to monitor ner-operator-specific DNA-binding activity, the 8 kDa Ner protein was purified to homogeneity . DNase I footprinting revealed that the purified protein bound and protected a specific DNA operator that contains two 12 bp sites with the consensus sequence 5'-ANPyTAPuCTAAGT-3', separated by a 6 bp spacer region . Moreover, regions corresponding to a turn of the DNA helix flanking these 12 bp repeats are also protected by Ner . Unlike the functionally similar lambda cro protein, gel filtration experiments show that the native molecular mass of Mu Ner to be approx . 8 kDa . These results, plus the pattern of DNase I protection, suggest that the protein may bind as a monomer to each of its specific DNA substrates.

Nucleic Acids Res, 1989 Feb 25, 17(4), 1605 - 18
Abortive initiation by bacteriophage T3 and T7 RNA polymerases under conditions of limiting substrate; Ling ML et al.; Initiation of RNA synthesis by the phage polymerases is abortive if the concentration of pyrimidine triphosphates is limiting . Under abortive initiation conditions the polymerases repeatedly initiate transcription but produce ribooligonucleotides that terminate just prior to the first occurrence of the limiting substrate . Abortive initiation is most severe if the limiting substrate occurs within the first 8-12 nucleotides of the nascent RNA chain and is particularly evident when UMP is limiting . The formation of stable elongation complexes (as determined by gel retardation experiments) occurs after the synthesis of an RNA product 8-12 nucleotides in length.

J Biol Chem, 1989 Feb 25, 264(6), 3611 - 7
Three additional operators, Op21, Op68, and Op88, of bacteriophage P1 . Evidence for control of the P1 dam methylase by Op68; Citron M et al.; The repressor of bacteriophage P1, encoded by the c1 gene, is responsible for maintaining the P1 prophage in the lysogenic state . Previously, 11 c1 repressor binding sites or operators scattered over the whole genome of P1 have been found . From sequence analysis an asymmetric, 17-base pair consensus sequence, ATTGCTCTAATAAATTT, was derived . Using a synthetic 15-base-long oligodeoxyribonucleotide as operator probe, we have identified three additional operators . We have mapped the operators at the positions 21,68, and 88 of the P1 genome and determined their sequence . These operators are controlled by c1 because corresponding P1 DNA fragments (i) require c1 repressor in vivo in order to be clonable in multicopy plasmids, (ii) exhibit a c1-repressible promoter activity, (iii) are retarded by c1 repressor protein during electrophoresis, and (iv) contain the 17-base pair consensus sequence with one mismatch base each . Furthermore, we suggest that expression of the DNA adenine methylase (dam) encoded by P1 is controlled via Op68.

Biochemistry, 1989 Feb 21, 28(4), 1471 - 7
Solid-phase synthesis and side reactions of oligonucleotides containing O-alkylthymine residues; Borowy-Borowski H et al.; As part of our studies on the molecular mechanism of mutation {Chambers, R . W . (1982) in Molecular and Cellular Mechanisms of Mutagenesis (Lemontt, J . F., & Generoso, W . M., Eds.) pp 121-145, Plenum, New York and London}, we wanted to prepare specific oligonucleotides carrying O2- or O4-alkylthymidine residues . Since O-alkylthymine moieties are known to be alkali labile, side reactions were expected during the deprotection procedures used for synthesis of oligonucleotides on a solid support by the classical phosphoramidite method . We have studied these side reactions in detail . Kinetic data show the deprotection procedures displace most O-alkyl groups at rates that make these procedures inappropriate for synthesis of most oligonucleotides carrying O-alkylthymine moieties . We describe alternative deprotection procedures, using readily accessible reagents, that we have used successfully to synthesize a series of oligonucleotides carrying several different O-alkylthymine moieties . The oligonucleotides synthesized are d(A-A-A-A-G-T-alkT-T-A-A-A-A-C-A-T), where alk = O2-methyl, O2-isopropyl, O4-methyl, O4-isopropyl, and O4-n-butyl . This work extends the previously described procedure for the chemical synthesis of oligonucleotides carrying an O4-methylthymine moiety {Li, B . F., Reese, C . B., & Swann, P . F . (1987) Biochemistry 26, 1086-1093} and reports the first chemical synthesis of an oligonucleotide carrying an O2-alkylthymine . The oligonucleotides synthesized have a sequence corresponding to the minus strand that is complementary to the viral strand at the start of gene G in bacteriophage phi X174 replicative form DNA where the normal third codon has been replaced with the ocher codon, TAA.

Gene, 1989 Feb 20, 75(2), 323 - 7
New plasmid vectors for high level synthesis of eukaryotic fusion proteins in Escherichia coli; Rimm DL et al.; Production of eukaryotic proteins in Escherichia coli has become rather simple since commercially available bacteriophage and plasmid vector systems allow investigators to select the optimal system for their particular problem . A common question is which system to use to produce the largest quantity of soluble recombinant protein with minimal, if any, bacterial protein fused to it . We have constructed a new set of plasmid vectors that produce large amounts of a fusion proteins that contain less than 25 amino acids of bacterial protein . We started with pATH-1, a plasmid expression vector comprised of the trpEp promoter and 37 kDa of the TrpE protein followed by a M13mp13 multiple cloning site for insertion of sequences to be expressed . We deleted the majority of the eukaryotic trpE sequence to produce a multiple frame, multiple enzyme cloning site, plasmid expression vector set called pRX . Transformation of E . coli CAG-456 (Baker et al., 1984) with this vector with an Acanthamoeba myosin tail sequence inserted in the correct frame yields a fusion protein that represents 45% of the total soluble protein . We have produced and purified 100 mg of this Acanthamoeba myosin-II fusion protein per liter of cell suspension.

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