Bacteriophage

Proc Natl Acad Sci U S A, 1985 Jun, 82(11), 3702 - 6
Isolation of recombinant cDNAs encoding chicken erythroid delta-aminolevulinate synthase; Yamamoto M et al.; We report the isolation of cDNA clones encoding delta-aminolevulinate synthase (ALA synthase; EC 2.3.1.37), the first enzyme in the heme biosynthetic pathway in animal cells . The gene was isolated from a chicken erythroid cDNA library prepared in the bacteriophage lambda fusion/expression vector gt11, using rabbit antibody raised against the relatively abundant chicken liver enzyme . The chicken liver and red cell ALA synthase isozymes share substantial crossreactivity to the antibody, thereby allowing isolation of the erythroid-specific gene by using the heterologous antibody in immune screening of the red cell cDNA library . Preliminary analysis documenting the tissue specificity of transcription indicates that the enzyme is encoded by a highly homologous set of messages, which appear to differ in size in various avian tissues . From analysis using strand-specific RNA probes, it appears that the different ALA synthase mRNAs detected may be transcribed from a family of genes that are closely related in nucleotide sequence and are each regulated in a developmentally specific manner.

Mol Gen Mikrobiol Virusol, 1985 Jun, (6), 15 - 20
{Development of bacteriophage Mu in E . coli gyrBts mutant strain}; Velikodvorskaia GA et al.; The study deals with Mu growth in cells carrying a temperature-sensitive mutation in the gene of DNA gyrase B subunit . At a nonpermissive temperature the Mu growth is shown to be blocked in the host gyrB ts mutant both on infection and on prophage induction . Mu DNA does not get integrated in the host chromosome upon the infection of mutant cells, as demonstrated by DNA-DNA hybridization experiments . In the case of prophage induction in mutant cells, as opposed to the wild type cells early mRNA synthesis is practically fully inhibited while the total RNA synthesis is three times reduced after 20 min of induction . The transcription of phage DNA associated with the changed superhelicity of DNA in the cell.

Genetika, 1985 Jun, 21(6), 927 - 35
{Transposition immunity in bacteriophage Mu . The effect of a mutation at the kil gene on the establishment of immunity}; Mogutov MA et al.; Bacteriophage Mu is characterized by a phenomenon similar to the transposition immunity of TnA: the frequency of transposition of Mu or mini-Mu into plasmids containing certain phage sequences is reduced by two orders of magnitude . In order to lend transposition immunity to Mu, the recipient replicon must contain a sequence of phage DNA including a 5.1 kb early region from the c-end of Mu . The product of the kil (or cim) gene takes part in establishing the immunity . The transposition immunity of Mu is connected with the disturbance of cointegrate formation.

Proc Natl Acad Sci U S A, 1985 Jun, 82(12), 4070 - 4
Evidence that ribosomal protein S10 itself is a cellular component necessary for transcription antitermination by phage lambda N protein; Das A et al.; Bacteriophage lambda N gene product acts to modify host RNA polymerase allowing the formation of a termination-resistant transcription apparatus . Previous studies have demonstrated that the nusE71 mutation that has altered the ribosomal protein S10 prevents N action in vivo . Using a coupled transcription-translation system, we demonstrate here that purified S10 protein as well as the 30S ribosomal subunit is sufficient to restore N activity in the nusE mutant extract, allowing antitermination of Rho-dependent and Rho-independent terminators . This provides direct biochemical evidence that the S10 protein itself is one of the cellular components necessary for the formation of an antitermination apparatus.

Proc Natl Acad Sci U S A, 1985 Jun, 82(11), 3889 - 93
Reduced in vivo mutagenesis by mutant herpes simplex DNA polymerase involves improved nucleotide selection; Hall JD et al.; We present evidence that mutation frequencies in a mammalian system can vary according to the replication fidelity of the DNA polymerase . We demonstrated previously that several derivatives of herpes simplex virus type 1 that encode polymerases resistant to various antiviral drugs (e.g., nucleotide analogues) also produce reduced numbers of spontaneous mutants . Here we show that the DNA polymerase from one antimutator virus exhibits enhanced replication fidelity . First, the antimutator virus showed a reduced response to known mutagens that promote base mispairing during DNA replication (N-methyl-N'-nitro-N-nitrosoguanidine, 5-bromo-deoxyuridine) . Second, purified DNA polymerase from the antimutator produced fewer replication errors in vitro, based on incorporation of mispaired nucleotides or analogues with abnormal sugar rings . We have investigated possible mechanisms for the enhanced fidelity of the antimutator polymerase . We show that the mutant enzyme has altered interactions with nucleoside triphosphates, as indicated by its resistance to nucleotide analogues and elevated Km values for normal nucleoside triphosphates . We present evidence against increased proofreading by an associated 3',5' exonuclease (as seen for T4 bacteriophage antimutator polymerases), based on nuclease levels in the mutant polymerase . We propose that reduced affinity of the polymerase for nucleoside triphosphates accounts for the antimutator phenotype by accentuating differences in base-pair stability, thus facilitating selection of correct nucleotides.

J Bacteriol, 1985 Jun, 162(3), 1092 - 9
Transposable lambda placMu bacteriophages for creating lacZ operon fusions and kanamycin resistance insertions in Escherichia coli; Bremer E et al.; We have constructed several derivatives of bacteriophage lambda that translocate by using the transposition machinery of phage Mu (lambda placMu phages) . Each phage carries the c end of Mu, containing the Mu cIts62, ner (cII), and A genes, and the terminal sequences from the Mu S end (beta end) . These sequences contain the Mu attachment sites, and their orientation allows the lambda genome to be inserted into other chromosomes, resulting in a lambda prophage flanked by the Mu c and S sequences . These phages provide a means to isolate cells containing fusions of the lac operon to other genes in vivo in a single step . In lambda placMu50, the lacZ and lacY genes, lacking a promoter, were located adjacent to the Mu S sequence . Insertion of lambda placMu50 into a gene in the proper orientation created an operon fusion in which lacZ and lacY were expressed from the promoter of the target gene . We also introduced a gene, kan, which confers kanamycin resistance, into lambda placMu50 and lambda placMu1, an analogous phage for constructing lacZ protein fusions (Bremer et al., J . Bacteriol . 158:1084-1093, 1984) . The kan gene, located between the cIII and ssb genes of lambda, permitted cells containing insertions of these phages to be selected independently of their Lac phenotype.

Arch Pathol Lab Med, 1985 Jun, 109(6), 546 - 50
Necrotizing lymphoid vasculitis in X-linked lymphoproliferative syndrome; Loeffel S et al.; An 8-year-old maternally related relative of three boys who had developed agammaglobulinemia associated with Epstein-Barr virus (EBV)-induced infectious mononucleosis was studied for X-linked lymphoproliferative syndrome (XLP) in 1979 . At that time, he demonstrated no striking immunologic aberrations and was seronegative for EBV . Subsequently, immunologic abnormalities including failure to switch from IgM to IgG antibody synthesis after secondary immunization with bacteriophage phi X174 were detected . In 1983, he experienced episodic intracerebral hemorrhages, with the second being fatal . At autopsy, necrotizing vasculitis and aneurysms involving arteries of the central nervous system were observed . Studies of blood obtained immediately before and after death failed to show antibodies to EBV . However, EBV genome was demonstrated in tissues obtained at autopsy by DNA hybridization studies . Fatal lymphoid vasculitis in this patient is unique among boys with XLP in the registry . These findings probably extend the phenotypic expressions of XLP.

Proc Natl Acad Sci U S A, 1985 Jun, 82(12), 3988 - 92
Initiation of DNA replication on single-stranded DNA templates catalyzed by purified replication proteins of bacteriophage lambda and Escherichia coli; LeBowitz JH et al.; Initiation of bacteriophage lambda DNA replication at the chromosomal origin depends on the lambda O and P replication proteins . These two viral initiators, together with an Escherichia coli protein fraction, promote the replication in vitro of single-stranded circular DNA chromosomes such as that of bacteriophage M13 . This nonspecific strand initiation reaction, which we have termed the "lambda single-strand replication reaction," has now been established with eight purified proteins, each of which is also required for replication of the phage lambda chromosome in vivo . An early rate-limiting step in the overall reaction is the ATP-dependent assembly of an activated nucleoprotein prepriming complex . In this step the lambda O and P initiators cooperate with the E . coli dnaJ and dnaK proteins to transfer the bacterial dnaB protein onto M13 DNA that is coated with the single-stranded DNA-binding protein . Multiple RNA primers are synthesized on each DNA circle when isolated prepriming complex is incubated with primase and rNTPs . In the complete system, DNA polymerase III holoenzyme extends the first primer synthesized into full-length complementary strands . Because the properties of this system are closely analogous to those found for the replication of phi X174 viral DNA by E . coli proteins, we infer that a mobile prepriming or priming complex (primosome) operates in the lambda single-strand replication reaction.

Biochim Biophys Acta, 1985 May 28, 815(3), 369 - 79
Reconstitution of M13 bacteriophage coat protein . A new strategy to analyze configuration of the protein in the membrane; Bayer R et al.; The configuration of M13 bacteriophage coat protein in a model membrane was analyzed using protease digestion followed by gel permeation chromatography on Fractogel TSK in formic acid/ethanol . Important information is contained in the chromatographic patterns of the membrane-bound fragments, as well as of the fragments released by the digestion . A new reconstitution was thereby developed which involves adding a small volume of a concentrated solution of cholate-solubilized coat protein to preformed vesicles (with the amount of detergent added being less than that required to solubilize the vesicles), freezing in liquid nitrogen, thawing, followed by dialysis to remove excess detergent . The coat protein is incorporated with high efficiency (95 percent) making subsequent fractionation unnecessary . In addition, the incorporated protein is not aggregated, and is incorporated with most molecules spanning the membrane, oriented in the same manner as in vivo (N-terminus outwards) . Two previously described reconstitutions, using sonication or cholate dialysis, are analyzed and found to be less satisfactory.

J Mol Biol, 1985 May 25, 183(2), 129 - 40
An Escherichia coli mutant unable to support site-specific recombination of bacteriophage lambda; Kikuchi A et al.; We report the isolation of mutations in, and the characterization of, an Escherichia coli gene, hip, that is required for site-specific recombination of phage lambda . hip mutants are recessive and are located near minute 20 on the linkage map . The gene product is not vital to bacterial growth, since deletion mutants are viable . The absence of hip product reduces lambda integration to barely detectable levels and also reduces prophage excision, but less drastically . Certain mutations in the lambda int gene partially restore integration and excision in hip- hosts . Homologous recombination promoted by recA does not require hip function . In addition to their defect in site-specific recombination, hip mutants are unable to support lytic growth of phage Mu or of certain lambda mutants . Their pleiotropic phenotype closely resembles that of himA mutants, but complementation, mapping and DNA sequencing show that hip and himA are different genes.

J Mol Biol, 1985 May 25, 183(2), 225 - 38
The terminase of bacteriophage lambda . Functional domains for cosB binding and multimer assembly; Frackman S et al.; Terminase is a protein complex involved in lambda DNA packaging . The subunits of terminase, gpNul and gpA, are the products of genes Nul and A . The actions of terminase include DNA binding, prohead binding and DNA nicking . Phage 21 is a lambdoid phage that also makes a terminase, encoded by genes 1 and 2 . The terminases of 21 and lambda are not interchangeable . This specificity involves two actions of terminase; DNA binding and prohead binding . In addition, the subunits of lambda terminase will not form functional multimers with the subunits of 21 terminase . lambda-21 hybrid phages can be produced as a result of recombination . We describe here lambda-21 hybrid phages that have hybrid terminase genes . The packaging specificities of the hybrids and the structure of their genes were compared in order to identify functional domains of terminase . The packaging specificities were determined in vivo by complementation tests and helper packaging experiments . Restriction enzyme site mapping and sequencing located the sites at which recombination occurred to produce the hybrid phages . lambda-21 hybrid 51 carries the lambda A gene, and a hybrid 1/Nul gene . The crossover that produced this phage occurred near the middle of the 1 and Nul genes . The amino-terminal portion of the hybrid protein is homologous to gp1 and the carboxy-terminal portion is homologous to gpNul . It binds to 21 DNA and forms functional multimers with gpA, providing evidence that the amino-terminal portion of gpNul is involved in DNA binding and the carboxy-terminal portion of gpNul is involved in the interaction with gpA . lambda-21 hybrid 54 has a hybrid 2/A gene . The amino terminus of the hybrid protein of lambda-21 hybrid 54 is homologous with gp2 . This protein forms functional multimers only with gp1, providing evidence that the amino terminus of gpA is involved in the interaction with gpNul . These studies identify three functional domains of terminase.

Nucleic Acids Res, 1985 May 24, 13(10), 3739 - 54
RNA sequence and secondary structure requirements for rho-dependent transcription termination; Morgan WD et al.; The interaction of E . coli termination factor rho with the nascent RNA transcript appears to be a central feature of the rho-dependent transcription termination process . Based on in vitro studies of the rho-dependent termination of the transcript initiated at the PR promoter of bacteriophage lambda, and on earlier studies, Morgan, Bear and von Hippel (J . Biol . Chem . 258, 9565-9574, 1983) proposed a model defining the features of a potential binding site for rho protein on transcripts subject to rho-dependent termination . This model suggested that an effective rho binding site on a nascent RNA transcript should be: (i) greater than 70-80 nucleotide residues in length; (ii) essentially unencumbered with stable secondary structure; (iii) relatively sequence non-specific; and (iv) located within a few hundred nucleotide residues upstream of the potential rho-dependent terminus . In this paper we examine the sequences and secondary structures of several transcripts that exhibit rho-dependent termination to test this hypothesis further . Unstructured regions of approximately the expected size and location were found on all the transcripts examined . Though several short specific sequence elements were found to occur in a very similar arrangement on the lambda PR- and lambda PL-initiated transcripts of lambda phage, no such elements of sequence regularity were found on any of the other rho-dependent transcripts . The results of the sequence comparisons reported here strongly support the generality of the "unstructured binding site" hypothesis for rho-dependent termination.

Eur J Biochem, 1985 May 15, 149(1), 85 - 93
Time-resolved fluorescence of bacteriophage Pf1 DNA-binding protein . Determination of oligonucleotide and polynucleotide binding parameters; Kneale GG et al.; The binding of oligonucleotides and polynucleotides to the Pf1 DNA-binding protein was followed by fluorescence spectral shift and lifetime measurements, which gave an anomalous value for the stoichiometry of binding . The anomaly was investigated in detail using fluorescence depolarisation to measure the aggregation during the titration and showed that all the fluorescence parameters are related to the specific aggregation of dimers on ligand binding . At saturation, complexes of the protein with the octanucleotide d(GCGTTGCG) and the hexadecanucleotide (dT)16 have rotational correlation times, phi, of 50 ns and 85 ns, corresponding to protein tetramers and octamers, respectively . In the presence of the tetranucleotide d(CGCA) the protein remains as the native dimer (phi = 19 ns) . The titration curves could be analysed in terms of two non-equivalent binding sites, with binding constants K1 and K2 . Comparison of K1 values for oligonucleotide binding leads to an estimated (single-site) intrinsic binding constant Kint approximately equal to 3 X 10(4) M-1 and a cooperativity parameter omega approximately equal to 100, in agreement with the apparent binding constant Kapp approximately equal to 3 X 10(6) M-1 for polynucleotides . Binding to the second site on the protein dimer is greatly reduced and cannot be determined accurately . The results suggest that the protein dimers bind cooperatively by lateral association along the DNA and that occupation of only one of the two DNA-binding sites of the protein dimers is sufficient to stabilize the nucleoprotein complexes.

Anal Biochem, 1985 May 15, 147(1), 63 - 74
Comparison of controlled pore glass and Kieselguhr-polydimethylacrylamide composite as supports for solid-phase synthesis of 23-residue oligodeoxyribonucleotides in milligram amounts; Minganti C et al.; Two 23-residue oligodeoxyribonucleotides, corresponding to both strands of a DNA duplex at the OR3 site of bacteriophage lambda, have been synthesized in good yields and in milligram quantities by a solid-phase phosphotriester method using two different supports, Kieselguhr-polydimethylacrylamide composite and controlled pore glass . Rapid purification was possible using high-performance liquid chromatography on radial compression ion-exchange columns . The results and utility of the two supports are compared.

Arch Biochem Biophys, 1985 May 15, 239(1), 137 - 46
3-Methylcholanthrene-induced expression of the cytochrome P-450c gene; Foldes RL et al.; Transcriptional control of 3-methylcholanthrene-dependent cytochrome P-450c nuclear RNA induction was directly observed in an in vitro rat liver nuclear transcription system . Mercurated and radiolabeled ribonucleotides were incorporated into nuclear RNA transcribed in vitro, which was then isolated using thiopropyl-Sepharose 6B affinity chromatography . Dot hybridization experiments were carried out using bacteriophage M13 subclones of pRSA57 (a cDNA clone for rat serum albumin), pEB339 (a cDNA clone for rat cytochrome P-450c), and clone 46 (a cDNA clone for mouse cytochrome P1-450) . The results of these studies demonstrate that 3-methylcholanthrene does not significantly influence the transcription of the rat serum albumin gene, but does increase the transcription of the cytochrome P-450c gene . Nuclear RNA precursors to the cytochrome P-450c mRNA were characterized by Northern blot analysis . Clone 46 hybridized to nuclear RNA species of 6.7 and 4.0 kb, in addition to the 3.0-kb cytochrome P-450c mRNA . pA8 (a genomic clone for rat cytochrome P-450c), hybridized to the same nuclear RNA species in addition to nuclear RNA species of 4.3, 3.4, and 2.2 kb . M13pd15 (a genomic clone containing information for the first intron of the cytochrome P-450c gene) hybridized to nuclear RNA species of 6.7 and 4.3 kb . All of these nuclear RNA species are polyadenylated . The mRNA coding for cytochrome P-450c was induced maximally in hepatic nuclei at 3 h following 3-methylcholanthrene administration . Maximal accumulation of cytochrome P-450c mRNA in hepatic cytosol has been previously shown to occur at approximately 15 h following 3-methylcholanthrene administration (Bresnick, E., Brosseau, M., Levin, W., Reik, L., Ryan, D . E., and Thomas, P . E . (1981) Proc . Natl . Acad . Sci . USA 78, 4083-4087) . These data implicate a possible role of nuclear RNA transport in the regulation of induction of cytochrome P-450c, although further investigations are indicated.

Biochem J, 1985 May 15, 228(1), 193 - 9
Identification of lysine residues at the binding site of bacteriophage-Pf1 DNA-binding protein; Tsugita A et al.; The accessibility of NH2 groups in the DNA-binding protein of Pf1 bacteriophage has been investigated by differential chemical modification with the reagent ethyl acetimidate . The DNA-binding surface was mapped by identification of NH2 groups protected from modification when the protein is bound to bacteriophage-Pf1 DNA in the native nucleoprotein complex and when bound to the synthetic oligonucleotide d(GCGTTGCG) . The ability of the modified protein to bind to DNA was monitored by fluorescence spectroscopy . Modification of the NH2 groups in the native nucleoprotein complex showed that seven out of the eight lysine residues present, and the N-terminus, were accessible to the reagent, and were not protected by DNA or by adjacent protein subunits . Modification of these residues did not inhibit the ability of the protein to bind DNA . Lysine-25 was identified by peptide mapping as being the major protected residue . Modification of this residue does abolish DNA-binding activity . Chemical modification of the accessible NH2 groups in the complex formed with the octanucleotide effectively abolishes binding to DNA . Peptide mapping established that, in this case, lysine-17 was the major protected residue . The differences observed in protection from acetimidation, and in the ability of the modified protein to bind DNA, indicate that the oligonucleotide mode of binding is not identical with that found in the native nucleoprotein complex with bacteriophage-Pf1 DNA.

J Biol Chem, 1985 May 10, 260(9), 5826 - 31
Transcription termination factor rho mediates simultaneous release of RNA transcripts and DNA template from complexes with Escherichia coli RNA polymerase; Andrews C et al.; Dissociation of RNA and DNA from Escherichia coli RNA polymerase in transcription complexes prepared with enzyme molecules located within and near a rho-dependent transcription termination region on bacteriophage T7 D111 DNA has been studied using a membrane filter-binding assay . Rho protein with ATP present mediated rapid (half-time approximately 27 s) simultaneous dissociation of about 50% of both RNA and DNA . RNA molecules were preferentially released from enzyme molecules located within the termination region . Rapid release of RNA and DNA depended on a nucleoside triphosphate but did not depend on sigma factor . Pretreatment of complexes with ribonuclease prevented dissociation of DNA . Nearly simultaneous dissociation of both RNA and DNA was also detected after a lag of 3 min when the isolated transcription complexes were incubated with all four ribonucleoside triphosphates in the absence of rho factor . In this case, release presumably occurred at the rho-independent termination site that is 5990 nucleotides downstream from the A1 promoter . Thus, the dissociation of DNA from RNA polymerase at rho-dependent and rho-independent transcription termination sites is coupled with or occurs spontaneously soon after the release of transcripts at both sites.

Nucleic Acids Res, 1985 May 10, 13(9), 3371 - 88
Effect of NusA protein on expression of the nusA,infB operon in E . coli; Plumbridge JA et al.; Protein and operon fusions between lacZ and various genes of the nusA,infB operon have been constructed on lambda bacteriophages and used to show that the operon is negatively regulated by the level of NusA protein . Overproducing NusA (but not IF2) from a multicopy plasmid reduces the level of beta-galactosidase from the fusions indicating repression of the operon . Introducing the lambda carrying the fusions into nusA mutant strains produces a higher level of beta-galactosidase-indicative of derepression of the operon . In particular, a larger form of the NusA protein which does not affect bacterial growth per se causes a derepression of the operon . As both protein and operon fusions respond equivalently, we conclude that the nusA protein is acting at the transcriptional level to regulate expression of the nusA, infB operon.

J Biol Chem, 1985 May 10, 260(9), 5804 - 7
Cleavage of stem-and-loop structure DNA by bleomycin . Reaction on the bacteriophage G4 origin of complementary strand synthesis; Ueda K et al.; The cleavage by bleomycin-Fe(II) complex in the presence of dithiothreitol of 3'-or 5'-end-labeled DNA from the region of the bacteriophage G4 origin of complementary strand synthesis was investigated by using the DNA-sequencing technique . Bleomycin cleaved a single-stranded DNA substrate preferentially at inverted repeat sequences, which potentially form stem-and-loop structures, while it cleaved double-stranded DNA substrates with different specificity . The results support the formation of three adjoining stem-and-loop structures in the region of the phage G4 origin of complementary strand synthesis under the low-salt conditions used and suggest a difference in the form of the double helix between the stem and the double-stranded DNA fragment . Bleomycin appears to be a useful reagent for searching stem-and-loop structures . The results may also contribute to the understanding of the mode of action of bleomycin as an antitumor antibiotic.

J Mol Biol, 1985 May 5, 183(1), 79 - 88
Three-dimensional reconstruction of bacteriophage phi 29 neck particles at 2 X 2 nm resolution; Carazo JM et al.; The three-dimensional structure of the head-to-tail connecting region of bacteriophage phi 29 has been studied by analysing two-dimensional, hexagonal ordered aggregates of negatively stained viral necks to a resolution of 2 X 2 nm . These necks are composed of two proteins, p10 and p11; p10 being the connector protein . A 12-folded and a 6-folded axially symmetric domain are present in the specimen . The 12-folded domain is the larger part of the structure; it consists of 12 subunits associated in pairs . These subunits appear to be more closely paired towards the centre, where only six subunits are resolved forming the 6-folded domain . The pairs of subunits present an important twist between the 12-folded and the 6-folded areas . A conical hole is formed at the centre of the structure . This hole is more open at the 12-folded domain than at the level of the possible zone of interaction between p10 and p11, where it is almost closed . Protein p11 is very poorly represented in the reconstruction, probably due to lack of staining . The structure described for the phi 29 neck has many of the attributes expected for an active device involved in bacteriophage DNA encapsidation.

Mol Biochem Parasitol, 1985 May, 15(2), 215 - 30
Organization of the ribosomal RNA genes in Schistosoma mansoni; van Keulen H et al.; The organization of the rRNA genes of Schistosoma mansoni has been determined by Southern blot analysis of genomic DNA digested with restriction enzymes, by isolation of the entire repeat on a single fragment of about 11 kilobase pairs from a genomic DNA library constructed in bacteriophage lambda and by characterization of three cloned EcoRI fragments which span the entire repeat . The segments encoding both the large and small rRNA subunits have been identified using specific cloned yeast rDNA fragments as probes and EcoRI, HindIII and BamHI restriction enzyme maps of the rRNA genes were constructed . The ends of the RNAs have been precisely mapped on the genomic DNA by S1 protection experiments . Our data indicate that the rRNA genes are present as a tight cluster . The total length of the rDNA repeat is about 10 kilobase pairs . There appears to be no variation in the size of transcribed and non-transcribed spacer DNA . At the RNA level we have characterized and mapped a small gap in the 28S RNA molecule . The interruption causes the RNA to dissociate into two equal sized fragments when analyzed under denaturing conditions.

Proc Natl Acad Sci U S A, 1985 May, 82(10), 3345 - 9
A defective phage system reveals bacteriophage T4 replication origins that coincide with recombination hot spots; Kreuzer KN et al.; Plasmid transduction mediated by bacteriophage T4 has been used to study putative T4 DNA replication origins cloned as inserts in the Escherichia coli plasmid pBR322 . Two particular inserts from the T4 genome allow high-frequency plasmid transduction, suggesting that each insert might contain a T4 replication origin . T4 infection of these plasmid-containing cells produces large numbers of defective phage particles that contain long linear concatamers of the plasmid DNA . During a second cycle of infection, these defective phage genomes can be replicated better than normal phage chromosomes present in the same infected cell; consequently, the T4 DNA inserts must be functioning as replication origins . Both of these origins appear to utilize a previously unrecognized mode of T4 replication initiation . Moreover, each origin coincides with a major recombination hot spot in the phage genome, and therefore this mode of replication initiation seems to involve a local stimulation of homologous genetic recombination . From a purely practical standpoint, additional DNA fragments can be cloned in an origin-containing plasmid, allowing isolation of large amounts of any DNA sequence with the glucosylated hydroxymethylcytosine modifications of T4 DNA.

Arch Biochem Biophys, 1985 May 1, 238(2), 636 - 41
Two modes of amber codon read-through in vitro; Ryoji M et al.; Read-through translation of bacteriophage R17 amB2 coat cistron carrying an amber mutation at the seventh codon was studied in vitro using the crude cell extract (S30) derived from an Escherichia coli nonsuppressor strain . Despite the presence of termination factors as well as ribosome-releasing factor (RRF) which prevent the read-through translation {M . Ryoji, J . W . Karpen, and A . Kaji (1981) J . Biol . Chem . 256, 5798-5801}, synthesis of coat-like protein still persists at a low level in this system . Characterization of this protein by peptide fingerprinting and amino acid sequencing was performed to reexamine the generally accepted notion that it is produced by amino acid misinsertion to the amber mutation codon . The results indicated, however, that the major population of this coat-like protein is produced as a result of reinitiation of translation from the eighth codon . Read-through by amino acid misinsertion in this system becomes predominant only when the Mg2+ concentration is higher than 16 mM.

J Bacteriol, 1985 May, 162(2), 621 - 5
Irreversible binding to the receptor of bacteriophages T5 and BF23 does not occur with the tip of the tail; Heller KJ et al.; Treatment of purified tails of bacteriophage T5 with 0.05% sodium dodecyl sulfate specifically removed pb2, a protein of 108,000 molecular weight (108K), from the tail . Although these tails were devoid of the single straight tail fiber, they still inhibited adsorption of T5 to Escherichia coli cells . Reconstitution of these tails with pb2 increased the efficiency of inhibition of T5 adsorption . Treatment of tails with 0.1% sodium dodecyl sulfate removed, in addition to pb2, a protein of 67K from phage T5 and one of 60K from phage BF23 . These tails failed to inhibit phage adsorption, and no reconstitution was achieved . Reconstitution of T5 tails with pb2 from BF23, and of BF23 tails with pb2 from T5, did not alter the host receptor specificity of the tails . Binding of untreated T5 tails to small FhuA receptor particles revealed that binding occurred with the conical part of the tail and that pb2 was most likely released from the tail upon binding . From these results and from recent observations with T5-BF23 hybrid phages (K.J . Heller, Virology 139:11-21, 1984), we conclude that the receptor-binding proteins of T5 and BF23 are the 67K and 60K proteins, respectively, and that they are not located at the tip of the tail but rather at or near the site where the straight tail fiber is attached to the conical part of the tail.

Proc Natl Acad Sci U S A, 1985 May, 82(9), 2588 - 92
P1 plasmid replication: multiple functions of RepA protein at the origin; Chattoraj DK et al.; Replication functions of a bacteriophage P1 miniplasmid are carried on a 1.2-kilobase pair (kb) segment that can be subdivided into a 245-base pair (bp) replication origin and a 959-bp region that encodes a protein required for replication (RepA) . The origin region contains five 19-bp direct repeats . By using primer extension and gene-fusion assays, we mapped the promoter of the repA gene within the repeated sequences and showed that the promoter is repressed by RepA . Regulation of RepA synthesis is apparently achieved by the binding of RepA to the repeat sequences . This regulation might be a key step in the replication-control circuit, as we found that overproduction of RepA (from a foreign promoter) inhibits replication . Thus, in addition to being an autoregulated activator of replication, the protein also can have a negative regulatory role.

J Gen Microbiol, 1985 May, 131 ( Pt 5), 1107 - 14
Lysis of Escherichia coli by cloned phi X174 gene E depends on its expression; Blasi U et al.; The lysis gene E of bacteriophage phi X174 was cloned under transcriptional control of the lefthanded lambda promoter, giving rise to plasmid pSB12 . Plasmid pSB22, identical to pSB12 except for an amber mutation in gene E, was constructed in the same way . Induction of the cloned wild-type gene by heat inactivation of the thermosensitive lambda cI857 repressor resulted in lysis of the host bacteria . With plasmid pSB22 only amber suppressor strains of Escherichia coli lysed after heat inactivation of lambda cI857 . Lysis of E . coli was shown to depend on the rate of gene E translation and on the growth phase of the bacteria . Stationary cells could not be lysed by the gene E product (gpE), even if present in sufficient amounts to lyse growing cells . By isotopic labelling gpE could be detected among the proteins synthesized in normal E . coli as well as in minicells . Determination of gene E expression suggested that gpE synthesis is translationally regulated.

Plasmid, 1985 May, 13(3), 173 - 81
Preferential binding of bacteriophage Mu repressor to supercoiled Mu DNA; Roulet E et al.; It was shown, using a relatively simple assay, that Mu repressor, cI, binds specifically to a region which spans the leftmost HindIII cleavage site on the phage genome . This extends the observations of Kwoh and Zipser {Nature (London) 277, 489-491 (1979)}, who were able to define a binding region to the left of this site . These results provide support for the idea that the eight blocks of repeated DNA sequences, which also span the HindIII cleavage site, are involved in repressor binding . These results also indicate that cI repressor has a marked preference for supercoiled DNA.

Proc Natl Acad Sci U S A, 1985 May, 82(10), 3134 - 8
A cII-dependent promoter is located within the Q gene of bacteriophage lambda; Hoopes BC et al.; We have found a cII-dependent promoter, PaQ, within the Q gene of bacteriophage lambda . Transcription experiments and abortive initiation assays performed in vitro showed that the promoter strength and the cII affinity of PaQ were comparable to the other cII-dependent lambda promoters, PE and PI . The location and leftward direction of PaQ suggests a possible role in the delay of lambda late-gene expression by cII protein, a phenomenon that has been called cII-dependent inhibition . We have constructed a promoter down mutation, paq-1, by changing a single base pair in the putative cII binding site of the promoter by oligonucleotide site-directed mutagenesis . The paq-1 mutant promoter required about 4-fold higher cII concentrations for maximal activation compared to the wild-type PaQ . We tested the hypothesis that PaQ is responsible in part for the delay of lambda late-gene expression by recombining the paq-1 mutation into a phage showing severe cII-dependent inhibition . We found that the paq-1 mutation relieved the cII-dependent growth defect of this phage . The paq-1 mutation (in combination with lambda cI857) resulted in a clear-plaque phenotype at the permissive temperature of 32 degrees C . The role of the PaQ-initiated antisense transcript in the control of lambda development is discussed.

Proc Natl Acad Sci U S A, 1985 May, 82(10), 3124 - 8
Genetic rearrangement of DNA induces knots with a unique topology: implications for the mechanism of synapsis and crossing-over; Griffith JD et al.; We have determined the topological sign of the knots produced by a cycle of phage lambda integrative recombination . To insure that these knots reflect intrinsic features of the reaction mechanism, the substrate was constructed so that random interwrapping of segments of DNA played a minimal role in the topological outcome . The knotted DNA was coated with the bacteriophage T4 uvsX gene product and examined in the electron microscope to determine the nature of each crossing point or node . All of the knots were identical; they were trefoils with three nodes of positive sign . We interpret this result to mean that one recombination site, which previous work had indicated is organized into a nucleosome-like structure, is wrapped with a handedness identical to that found in nucleosomes . Therefore, this wrapping may explain the dependence of recombination on supercoiling of the substrate DNA . Moreover, we show that the topological result sharply limits acceptable mechanisms for the details of strand exchange.

Mol Cell Biol, 1985 May, 5(5), 1136 - 42
Bacteriophage lambda vector for transducing a cDNA clone library into mammalian cells; Okayama H et al.; We have developed a bacteriophage lambda vector (lambda NMT) that permits efficient transduction of mammalian cells with a cDNA clone library constructed with the pcD expression vector (H . Okayama and P . Berg, Mol . Cell . Biol . 3:280-289, 1983) . The phage vector contains a bacterial gene (neo) fused to the simian virus 40 early-region promoter and RNA processing signals, providing a dominant-acting selectable marker for mammalian transformation . The phage DNA can accommodate pcD-cDNA recombinants with cDNA of up to about 9 kilobases without impairing the ability of the phage DNA to be packaged in vitro and propagated in vivo . Transfecting cells with the lambda NMT-pcD-cDNA recombinant phage yielded G418-resistant clones at high frequency (approximately 10(-2} . Cells that also acquired a particular cDNA segment could be detected among the G418-resistant transformants by a second selection or by a variety of screening protocols . Reconstitution experiments indicated that the vector could transduce 1 in 10(6) cells for a particular phenotype if the corresponding cDNA was present as 1 functional cDNA clone per 10(5) clones in the cDNA library . This expectation was confirmed by obtaining two hypoxanthine-guanine phosphoribosyltransferase (HPRT)-positive transductants after transfecting 10(7) HPRT-deficient mouse L cells with a simian virus 40-transformed human fibroblast cDNA library incorporated into the lambda NMT phage vector . These transductants contained the human HPRT cDNA sequences and expressed active human HPRT.

Proc Natl Acad Sci U S A, 1985 May, 82(9), 2880 - 4
Propagation of some human DNA sequences in bacteriophage lambda vectors requires mutant Escherichia coli hosts; Wyman AR et al.; The growth of clones of human genomic DNA fragments in a bacteriophage lambda vector has been examined in a number of different Escherichia coli hosts . A large proportion (8.9%) of the phages carrying different fragments of the human genome fail to grow on standard rec+ hosts but will grow on hosts carrying mutations in the recB, recC, and sbcB genes . Heteroduplex analysis in the electron microscope of DNA from four of these phages revealed substantial secondary structure, including snap-back regions 200-500 base pairs in length . Such structures were not found in phages from the same DNA library that grow in rec+ hosts . These results are interpreted in the light of prior observations {Leach, D.R.F . & Stahl, F . (1983) Nature (London) 305, 448-451} showing that inverted repetitions cloned in phage lambda can be propagated in recB recC sbcB hosts but not in rec+ hosts.

J Virol, 1985 May, 54(2), 460 - 6
Isolation and characterization of the bacteriophage T4 tail-associated lysozyme; Nakagawa H et al.; Direct evidence has been obtained that the tail-associated lysozyme of bacteriophage T4 (tail-lysozyme) is gp5, which is a protein component of the hub of the baseplate . Tails were treated with 3 M guanidine hydrochloride containing 1% Triton X-100, and the tail-lysozyme was separated from other tail components by preparative isoelectric focusing electrophoresis as a peak with a pI of 8.4 . The molecular weight as determined from sodium dodecyl sulfate electrophoresis was 42,000 . The tail-lysozyme was unambiguously identified as gp5 when the position of the lysozyme was compared with that of gp5 of tube-baseplates from 5ts1/23amH11/eL1ainfected Escherichia coli cells by two-dimensional gel electrophoresis . The tail-lysozyme has N-acetylmuramidase activity and the same substrate specificity as gene e lysozyme; the optimum pH is around 5.8, about 1 pH unit lower than for the e lysozyme . We assume that the tail-lysozyme plays an essential role in locally digesting the peptidoglycan layer to let the tube penetrate into the periplasmic space . The tail-lysozyme is presumably also responsible for "lysis from without."

J Virol, 1985 May, 54(2), 345 - 50
The J gene of bacteriophage phi X174: in vitro analysis of J protein function; Hamatake RK et al.; The J protein of phi X174 is a small, highly basic protein and is a component of the phage capsid . We have investigated the role of J protein during single-stranded viral DNA synthesis and phage morphogenesis by using an in vitro system composed of purified viral and host components (Aoyama et al., Proc . Natl . Acad . Sci . U.S.A . 80:4195-4199, 1983) . The characterization of the products made in the presence and absence of J protein shows that J protein is not required for viral DNA synthesis, but is required for the packaging of DNA into infectious phage . The ability of J protein to bind to double-stranded DNA as well as single-stranded DNA and other interactions with DNA suggest a model in which J protein binds to double-stranded, replicative form DNA and enters the phage prohead by remaining bound to viral DNA as it is encapsidated.

Virology, 1985 May, 143(1), 347 - 51
Crossover sites cix for inversion of the invertible DNA segment C on the bacteriophage P7 genome; Iida S et al.; The bacteriophage P7 genome contains an invertible DNA segment called C which determines its host range . P7 C(+) phages produce plaques on Escherichia coli K12 . The C segment consists of a 3-kb unique sequence and 0.62-kb inverted repeats of which one carries an internal 0.2-kb deletion . This deletion has been mapped within the right inverted repeat in the C(+) orientation . The crossover sites cix for inversion of the C segment do not map at the inside boundaries of the inverted repeats, as had been proposed . They are localized at the external ends of these repeats . Thus organization of the C segment in phage P7 is analogous to that in the related phage P1.

J Bacteriol, 1985 May, 162(2), 777 - 83
Phasmid vectors for identification of genes by complementation of Escherichia coli mutants; Elledge SJ et al.; A bacteriophage lambda cloning vector was designed to facilitate the isolation of genes from procaryotic organisms by complementation of Escherichia coli mutants . This vector, lambda SE4, was constructed by attaching a very-low-copy-number replication system (from the plasmid NR1) and a spectinomycin resistance gene to the left arm of lambda 1059 (Karn et al., Proc . Natl . Acad . Sci . U.S.A . 77:5172-5176, 1980) . This phasmid cloning vector is capable of growing lytically as a phage in a nonimmune host or lysogenically as a phasmid in an immune host . This phasmid utilizes the Spi- selection for insertions of DNA into the vector and has the ability to accept 2- to 19-kilobase Sau3A1, BamHI, BglII, BclI, or XhoII fragments; recombinants lysogenize immune hosts as single-copy-number selectable plasmids at 100% frequency . An E . coli library was constructed by using the initial vector lambda SE4, and clones of a number of representative genes were identified . A typical clone, lambda ant+, was shown to be readily mutagenized by a mini-Tn10 transposon . A general method for transferring cloned DNA segments onto bacteriophage lambda was developed . The method involves the use of in vivo recombination with a selection and was used to construct two derivatives of lambda SE4 . Possible uses of these vectors and of the method for transferring cloned DNA onto phage lambda are discussed.

J Bacteriol, 1985 May, 162(2), 598 - 606
Physical analysis of deletion mutations in the ilvGEDA operon of Escherichia coli K-12; Driver RP et al.; DNA-DNA hybridization of cloned segments of the Escherichia coli K-12 ilvGEDA operon to genomic blots was used to determine the physical dimensions of a series of deletion mutations of the ilvGEDA operon . The smallest mutation resulted from the deletion of approximately 200 base pairs from within ilvD, whereas the largest mutation resulted from the deletion of 17 kilobases including the rep gene . The structure of three of these mutants indicates that formation of the deletions was mediated by Tn5 (or Tn5-131) that is retained in the chromosome . This is the first observation of this type of Tn5-mediated event . Our analysis of the total acetohydroxy acid synthase activity of strains containing deletions of ilvG indicates that the truncated ilvG polypeptide of wild-type E . coli K-12 lacks enzyme activity . The small 200-base-pair deletion of ilvD confirms the presence of a strong polar site 5' to ilvA . The detailed structure of these deletions should prove useful for the investigation of other genes in this region . This genomic analysis demonstrates that the ilv restriction site map that was established previously by the analysis of recombinant bacteriophage and plasmids is identical to that on the genome.

Mol Gen Mikrobiol Virusol, 1985 May, (5), 23 - 8
{Mutagenic effect of o-methylhydroxylamine on the prophage and extracellular phage lambda}; Kalinin VL et al.; Induction of c-mutations in extracellular bacteriophage and prophage lambda cI857 ind-treated with 1 M O-methylhydroxylamine (OMHA) at 32 degrees and pH 5.6 has been studied . The frequency of c-mutations increases proportionally to the time of treatment of extracellular phage and does not depend on cellular recA+ or polA+ functions and on induction of SOS-repair system caused by UV-irradiation of host cells . Prophage is inactivated and mutagenized approximately 10-fold faster than extracellular phage immediately after treatment of lysogenic cells during prophage induction . Thus, prophage survival does not depend on repair functions of the host cells, and the frequency of c-mutations in recA and, especially, in polA lysogens is significantly lower, than in the wild-type cells.Delayed thermoinduction (90 min) of prophage causes significant enhancement of survival and decreases the frequency of c-mutations in all strains studied . Preliminary treatment of non-lysogens with OMHA does not increase the frequency of c-mutations in undamaged phage or in phage treated with OMHA in vitro.

J Biol Chem, 1985 Apr 25, 260(8), 5055 - 60
The human alpha-fetoprotein gene . Sequence organization and the 5' flanking region; Sakai M et al.; The human alpha-fetoprotein (AFP) gene was isolated into three overlapping clones in bacteriophage lambda vectors and its sequence organization analyzed by restriction endonuclease mapping and nucleotide sequencing . The human AFP gene is about 20 kilobase pairs long and contains 15 exons and 14 introns . The overall organization of the human AFP gene is similar to that of the mouse AFP gene, with all but two exons showing identical sizes . Nucleotide sequences at all exon/intron junctions display similarity to the consensus boundary sequence (Breathnach, R., and Chambon, P . (1981) Annu . Rev . Biochem . 50, 349-383), with the GT-AG rule applied to the splicing point . The cap site maps 44 nucleotides upstream from the translation initiation site . The "TATA box" is located 27 nucleotides upstream from the putative cap site and is flanked by sequences with dyad symmetry . The TATA box can thus be placed in the loop portion of a possible stem-loop structure formed by intrastrand base-pairing . Other characteristic nucleotide sequences in the 5' flanking region include a CCAAC pentamer, a 14-base pair (bp) enhancer-like sequence, and a 9-bp sequence homologous to the glucocorticoid responsive element . A long (90 bp) direct repeat and several alternating purine/pyrimidine sequences are also present in the 5' flanking region . A 736-bp sequence of the 5' flanking region adjacent to the cap site of the human AFP gene shows a 61% similarity with the corresponding region of the mouse AFP gene . There are two Alu family sequences and two poly(dT-dG) repeats in the human AFP gene that show different distribution patterns from those in the mouse AFP gene.

Biochemistry, 1985 Apr 23, 24(9), 2219 - 27
Bacteriophage T7 E promoter: identification and measurement of kinetics of association with Escherichia coli RNA polymerase; Prosen DE et al.; The initiation point for transcription from the Escherichia coli RNA polymerase E promoter on bacteriophage T7 has been determined to be at 36 835 base pairs (92.22 T7 units) from the left end of T7 . The location was determined by RNA fingerprinting of a runoff transcription product . Kinetics of association for the E and the T7 A3 promoters were measured by using the abortive initiation assay for approach to steady-state turnover . The kinetic association constant, ka (=KBk2), for E was found to be over 10-fold slower than ka for A3 . For the E promoter, ka = 1.2 X 10(6) M-1 s-1 . For A3, we report ka greater than or equal to 4 X 10(7) M-1 s-1 . This difference is due mostly to a 10-fold difference in the initial equilibrium constant, KB, for formation of the initial polymerase-promoter complex . The rate of isomerization, k2, of the initial complex to the open polymerase-promoter complex for the E promoter was only 2-fold slower than k2 for the A3 promoter . Various numerical methods for calculation of the kinetic parameters are discussed and compared . We argue that a nonlinear analysis provides the most reliable means of data analysis.

Biochemistry, 1985 Apr 23, 24(9), 2262 - 8
Base-pairing properties of N4-methoxydeoxycytidine 5'-triphosphate during DNA synthesis on natural templates, catalyzed by DNA polymerase I of Escherichia coli; Reeves ST et al.; N4-Methoxydeoxycytidine 5'-triphosphate (mo4dCTP) was synthesized by reaction of dCTP with methoxyamine and then purified by high-performance liquid chromatography (HPLC) and used to analyze the specificity of mo4dCMP incorporation during polymerization on natural templates, catalyzed by DNA polymerase I of Escherichia coli . Elongation of synthetic 5'-32P-labeled primers, annealed to single-stranded DNA of bacteriophage M13, was carried out in the presence of only three of the four normal dNTPs; then, reaction products were displayed by high-resolution gel electrophoresis and visualized by autoradiography . By measuring primer elongation in each of the four "minus" reactions with and without added mo4dCTP, we examined the specificity of mo4dCMP incorporation at different positions along the M13 template . The results of this experimental approach indicated that (i) mo4dCTP is utilized most readily (although at low efficiency) in place of dTTP during DNA synthesis, (ii) the analogue can also replace dCTP during primer elongation, although at barely detectable efficiency, and (iii) the ease at which both mo4C.A and mo4C.G pairs are formed during DNA synthesis on natural templates is markedly influenced by the nucleotide sequence of the template.

J Mol Biol, 1985 Apr 20, 182(4), 509 - 17
The replication of bacteriophage P4 DNA in vitro . Partial purification of the P4 alpha gene product; Krevolin MD et al.; A soluble enzyme system has been prepared from a phage P4-infected Escherichia coli strain that supports the replication of exogenous, supercoiled P4 DNA . This DNA synthesis in vitro depends upon the four deoxyribonucleotides and ATP, but is enhanced about four- to fivefold by the presence of other ribonucleotides . E . coli DNA polymerase III holoenzyme, the E . coli single-strand DNA binding protein, and the partially purified P4 alpha gene product are required for replication in vitro . Rifamycin does not inhibit P4 replication in vitro . Since the P4 alpha gene codes for a rifamycin-resistant RNA polymerase (Barrett et al., 1983), and since P4 DNA replication is independent of the host primase (Bowden et al., 1975), we believe the alpha gene product is functioning as a P4-specific DNA primase.

J Mol Biol, 1985 Apr 20, 182(4), 567 - 78
Inhibition of the type I restriction-modification enzymes EcoB and EcoK by the gene 0.3 protein of bacteriophage T7; Bandyopadhyay PK et al.; The gene 0.3 protein of bacteriophage T7 is a potent inhibitor of the restriction-modification enzymes EcoB and EcoK, both in vivo and in vitro . We have analyzed the ability of purified 0.3 protein to inhibit different steps in the reactions of EcoB and EcoK with DNA . Most of our experiments were done with EcoK, but selected tests with EcoB indicate that the two enzymes are affected by 0.3 protein in the same way . Purified 0.3 protein binds tightly to free enzyme, apparently to one of the small subunits, and prevents it from binding to DNA . If EcoK is allowed to form specific recognition complexes with unmodified DNA before 0.3 protein is added, relatively low levels of 0.3 protein prevent the nuclease activity that would otherwise appear upon addition of ATP, but considerably higher levels are needed to prevent formation of filter-binding complexes or ATPase activity . This, together with other results, suggests that the binding site for 0.3 protein is protected in recognition complexes and in the early stages of the ATP-stimulated reactions, but that it becomes accessible again before cleavage of the DNA, perhaps after the translocation step . If added after the nuclease reaction is substantially over, 0.3 protein has little effect on ATPase activity, and indeed, the subunit having the binding site for 0.3 protein apparently dissociates from the enzyme-DNA complex . The methylase activity of EcoK on hemi-methylated recognition sites is strongly inhibited by 0.3 protein added at any stage of the reaction.

J Mol Biol, 1985 Apr 20, 182(4), 519 - 27
Bacteriophage P4 DNA replication . Location of the P4 origin; Krevolin MD et al.; An electron microscopic examination of replicating bacteriophage P4 DNA molecules has revealed theta-type structures that replicate bidirectionally from a single origin . Many replicating P4 DNA molecules also contain long (2000 bases) single-strand DNA regions at the growing fork that are deployed in a trans configuration, which supports the concept of continuous leading strand and discontinuous lagging strand syntheses . The position of the P4 origin was localized by the use of a plasmid complementation test for replication in vivo, as well as by labeling of DNA replicating in vitro in the presence of a chain-terminating inhibitor . During this study we discovered a second site on the P4 genome which is essential for replication, and we have named it crr (cis region required for replication) . The site is located at least 3300 bases from the origin but appears to be required for the initiation of DNA replication in vivo as well as in vitro.

Virology, 1985 Apr 15, 142(1), 1 - 11
Terminal sequences of the bacteriophage phi 6 segmented dsRNA genome and its messenger RNAs; Szekeres M et al.; The ends of the three dsRNA genome segments (L, M, and S) of bacteriophage phi 6 (strand separated and/or intact) and the 5' ends of the middle and small single-strand messenger RNAs have been sequenced by base-specific partial enzymatic digestion . Terminal sequences for the large and middle dsRNA strands extend about 60 bases . The three dsRNA segments have 18 homologous bases at the left end except for position 2, which differs in the L segment . A 17-base homology defines the right ends of L and M dsRNAs and probably S dsRNA as well . The 5' ends of middle and small messenger RNAs are identical to the corresponding viral (+) strands.

Virology, 1985 Apr 15, 142(1), 98 - 111
Transcription of Bacillis subtilis plasmid pBD64 and expression of bacteriophage SPO1 genes cloned therein; Curran JF et al.; Plasmid pBD64, a vector which is useful for cloning in Bacillis subtilis (T . J . Gryczan, A . G . Shivakumar, and D . Dubnau (1980), J . Bacteriol . 141, 246-253), has at least three substantial transcription units . Two of these include the single EcoRI, XbaI, and BamHI sites, while the other includes the single BglII site . Each of these transcripts was synthesized in the counterclockwise direction, relative to the pBD64 restriction map . No transcripts were detected in the opposite direction . Infection by bacteriophage SPO1 caused a substantial decrease in each of these transcripts . No new pBD64 transcripts were detected during SPO1 infection . Various SPO1 genes, cloned at several of these pBD64 sites, were tested for expression by observing their capacity to complement SPO1 mutants . Several middle and late genes were expressed substantially, regardless of the orientation in which the fragments were inserted . Since transcription from the vector could cause expression only in one orientation, this argues that the necessary transcription originated at SPO1 promoters, and, thus, that SPO1 middle and late promoters can be active in thymine-containing DNA.

Science, 1985 Apr 12, 228(4696), 149 - 54
Molecular cloning of the complementary DNA for human tumor necrosis factor; Wang AM et al.; Tumor necrosis factor (TNF) is a soluble protein that causes damage to tumor cells but has no effect on normal cells . Human TNF was purified to apparent homogeneity as a 17.3-kilodalton protein from HL-60 leukemia cells and showed cytotoxic and cytostatic activities against various human tumor cell lines . The amino acid sequence was determined for the amino terminal end of the purified protein, and oligodeoxyribonucleotide probes were synthesized on the basis of this sequence . Complementary DNA (cDNA) encoding human TNF was cloned from induced HL-60 messenger RNA and was confirmed by hybrid-selection assay, direct expression in COS-7 cells, and nucleotide sequence analysis . The human TNF cDNA is 1585 base pairs in length and encodes a protein of 233 amino acids . The mature protein begins at residue 77, leaving a long leader sequence of 76 amino acids . Expression of high levels of human TNF in Escherichia coli was accomplished under control of the bacteriophage lambda PL promoter and gene N ribosome binding site.

Nucleic Acids Res, 1985 Apr 11, 13(7), 2559 - 68
alpha-Putrescinylthymine and the sensitivity of bacteriophage phi W-14 DNA to restriction endonucleases; Miller PB et al.; The modified base alpha-putrescinylthymine (putT) in phi W-14 DNA blocks cleavage of the DNA by 17 of 32 Type II restriction endonucleases . The enzymes cleaving the DNA do so to widely varying extents . The frequencies of cleavage of three altered forms of the DNA show that putT blocks recognition sites either when it occurs within the site or when it is in a sequence flanking the site . The blocking is dependent on both charge and steric factors . The charge effects can be greater than the steric effects for some of the enzymes tested . All the enzymes cleaving phi W-14 DNA release discrete fragments, showing that the distribution of putT is ordered . The cleavage frequencies for different enzymes suggest that the sequence CAputTG occurs frequently in the DNA . Only TaqI of the enzymes tested appeared not to be blocked by putT, but it was slowed down . TaqI generated fragments are joinable by T4 DNA ligase.

Nucleic Acids Res, 1985 Apr 11, 13(7), 2227 - 40
Cleavage within an RNase III site can control mRNA stability and protein synthesis in vivo; Panayotatos N et al.; We report that processing at a cloned bacteriophage T7 RNase III site results in strong stabilization of the mRNA relative to the full-length transcript . In contrast, processing by RNase III of the bacteriophage lambda int transcript leads to rapid degradation of the messenger . It is proposed that the mode of cleavage within the RNase III site determines mRNA stability . Single cleavage leaves part of the phage T7 RNase III site in a folded structure at the generated 3' end and stabilizes the upstream mRNA whereas double cleavage at the lambda int site removes the folded structure and accelerates degradation . In addition, the processed transcript is as active a messenger as the unprocessed one and can direct protein synthesis for longer times . This increased efficiency is accompanied by a proportional (3-4 fold) increase in protein levels . In contrast, processing at the lambda int site reduces Int synthesis . Thus, processing may either stabilize mRNA and stimulate gene expression or destabilize a messenger and prevent protein synthesis . The end result appears to be determined by the mode of cleavage within the RNase III site.

J Biol Chem, 1985 Apr 10, 260(7), 4484 - 91
The uvsX protein of bacteriophage T4 arranges single-stranded and double-stranded DNA into similar helical nucleoprotein filaments; Griffith J et al.; The bacteriophage T4 uvsX gene codes for a DNA-binding protein that is important for genetic recombination in T4-infected cells . This protein is a DNA-dependent ATPase that resembles the Escherichia coli recA protein in many of its properties . We have examined the binding of purified uvsX protein to single-stranded DNA (ssDNA) and to double-stranded DNA (dsDNA) using electron microscopy to visualize the complexes that are formed and double label analysis to measure their protein content . We find that the uvsX protein binds cooperatively to dsDNA, forming filaments 14 nm in diameter with an apparently helical axial repeat of 12 nm . Each repeat contains about 42 base pairs and 9-12 uvsX protein monomers . In solutions containing Mg2+, the uvsX protein also binds cooperatively to ssDNA . The filaments that result are 14 nm in diameter, show a 12-nm axial repeat, and they are nearly identical in appearance to the filaments that contain dsDNA . In the filaments formed along ssDNA, each axial repeat contains about 49 DNA bases and 9-12 uvsX monomers . Both the filaments formed on the ssDNA and dsDNA show a strong tendency to align side-by-side . T4 gene 32 protein also binds cooperatively to ssDNA and interacts both physically and functionally with uvsX protein . However, when gene 32 and uvsX proteins were added to ssDNA together, no interaction between the two proteins was detected.

J Biol Chem, 1985 Apr 10, 260(7), 4468 - 77
The phi 80 and P22 attachment sites . Primary structure and interaction with Escherichia coli integration host factor; Leong JM et al.; Although the lambdoid bacteriophage phi 80 and P22 possess site-specific recombination systems analogous to bacteriophage lambda, they have different attachment (att) site specificities . We have identified and determined the nucleotide sequences of the att sites of phi 80 and P22 and have examined the interaction of these sites with purified Escherichia coli integration host factor (IHF) . The sizes of the homologous core regions of the att sites vary greatly: P22 has a 46-base pair core, while phi 80 and lambda have 17- and 15-base pair cores, respectively . The core sequences of the three phage show no significant homology, although dispersed regions of homology in arm sequences indicate that the three phage att sites are related . All three att sites have a high A + T composition, and restriction fragments carrying these sites migrate anomalously upon polyacrylamide gel electrophoresis . IHF binds to a site to the left of the common core in the phi 80 and P22 phage att sites (attP) and to a site to the right of the core in P22 attP and attB (the bacterial att site) . In the lambda system, IHF interacts with three regions on attP (designated H1, H2, and H') and none on attB (Craig N., and Nash, H.A . (1984) Cell 39, 707-716) . Alignment of the IHF sites of all three phage results in a consensus sequence for IHF binding, Pyr-AANNNNTTGATAT . Among the three phage, the number of IHF sites differs; however, the location and orientation of the binding sites in relation to the respective core regions are well conserved . An IHF site analogous to lambda H2 is present in both phi 80 and P22 attP, while a site analogous to lambda H' is present in P22 attP . This conservation suggests that IHF plays a very similar role in the site-specific recombination pathways of all three phage, and that the flanking arm sequences are necessary for phi 80 and P22 attP function, as is the case for lambda attP function . These structural similarities presumably reflect a conservation of the mechanism of site-specific recombination for the three phage.

Biochemistry, 1985 Apr 9, 24(8), 1920 - 8
Membrane protein conformational change dependent on the hydrophobic environment; Wilson ML et al.; Two conformational states of the coat protein of the filamentous bacteriophage M13 have been detected in detergent solution by using magnetic resonance techniques . When 3-fluorotyrosine is incorporated in place of the two tyrosine residues in the protein, four 19F nuclear magnetic resonance signals are observed, two for each conformer of the protein . The equilibrium between the two forms can be modulated by pH, temperature, and detergent structure . The rate of interconversion of the isomers is rapid on the minutes time scale but is slow relative to the T1 relaxation time of the fluorine resonances of approximately 50 ms . The conformational change between the conformers results in the perturbation of a basic residue in the protein such that this group has a pKa of approximately 9.5 in one state which shifts to 10.5 or more in the other conformational state . The temperature dependence of the equilibrium suggests an enthalpy difference of about 10 kcal/mol which is offset by entropy to give nearly zero free energy difference between the states at pH 8.3 in deoxycholate solution at room temperature . This suggests a substantial reorganization of the noncovalent interactions defining the two conformational states . The conformational equilibrium is strongly dependent on detergent structure and the presence of phospholipid in the detergent micelle . The results are not consistent with a strong, specific lipid binding to the protein but appear to be consistent with a more general effect of the overall micelle structure on the conformational state of the protein.

Biochemistry, 1985 Apr 9, 24(8), 2077 - 86
Nucleotide sequence of the gene for the gamma chain of human fibrinogen; Rixon MW et al.; A human genomic DNA library was screened for the gene coding for the gamma chain of fibrinogen by using a human cDNA for the gamma chain as a hybridization probe . The gene was identified in three overlapping recombinant lambda bacteriophage, and its sequence, including the immediate 5' and 3' flanking regions, was determined . The DNA sequence analysis revealed the presence of 10 exons coding for 411 amino acids present in the mature protein and a signal sequence of 26 amino acids . Two 30 base pair (bp) direct repeats of 93% identity were found 468 bp upstream from the transcription initiation site . The DNA sequence of the gene for the gamma chain of human fibrinogen showed considerable sequence homology with a partial sequence reported for the gene for the gamma chain of rat fibrinogen.

J Mol Biol, 1985 Apr 5, 182(3), 431 - 41
Three-dimensional structure of unstained, frozen-hydrated extended tails of bacteriophage T4; Lepault J et al.; Unsupported, unstained frozen-hydrated extended tails of bacteriophage T4 have been studied by cryo-electron microscopy . Their three-dimensional structure has been reconstructed after correlation and averaging of the information from different particles . While the reconstructions of hydrated tails show all the features found by conventional electron microscopy, they are characterized by an open structure . Individual subunits constituting the axial repeat cannot be outlined unambiguously, as the density connectivity is sensitive to the phase-contrast transfer function effects . In order to minimize these effects, we found that the best data set for three-dimensional reconstruction is composed of layer-lines corrected for the phase-contrast transfer function and an uncorrected equator.

J Mol Biol, 1985 Apr 5, 182(3), 367 - 81
Protein structure by solid state nuclear magnetic resonance . Residues 40 to 45 of bacteriophage fd coat protein; Cross TA et al.; The three-dimensional structure of part of the coat protein in the filamentous bacteriophage fd is described by nuclear magnetic resonance (n.m.r.) . Residues 40 to 45 are in a somewhat distorted alpha-helix . This n.m.r . approach for determining protein structure relies on the spectral manifestations of chemical shift and heteronuclear dipolar couplings in a symmetrical assembly of protein subunits oriented parallel to the applied magnetic field . The angles between individual peptide linkages and the filament axis of the virion constitute the basic source of structural information . These angles are directly related to x, y, z co-ordinates for describing the protein structure.

Science, 1985 Apr 5, 228(4695), 91 - 3
Thermosensitivity of a DNA recognition site: activity of a truncated nutL antiterminator of coliphage lambda; Peltz SW et al.; Antitermination is an important transcriptional control . In bacteriophage lambda, the presence of the nut antiterminators between the promoters and terminators results in relatively unhindered transcription when the lambda N gene product and necessary host factors are supplied . This antitermination system has been rendered thermosensitivity by modification of the nut site . A fragment of lambda DNA {74 base pairs (bp) in length}that contained the 17-bp nutL core sequence, but lacked the 8-bp boxA sequence, was cloned in a pp-N-tL1-galK plasmid between the pp promoter and gene N . This fragment mediated antitermination of transcription at 30 degrees C, as measured by assaying galK gene expression in Escherichia coli . At 42 degrees C, however, antitermination at the lambda tL1 terminator was abolished . Antitermination at 42 degrees C was restored by replacing the 74-bp nutL fragment with longer sequences containing both nutL and boxA or by cloning a synthetic boxA sequence ahead of the 74-bp nutL fragment . Thus, efficient antitermination required both boxA and the 17-bp nutL core, with the latter becoming conditionally defective when the boxA sequence was deleted.

Genetics, 1985 Apr, 109(4), 633 - 59
Mechanisms of spontaneous and induced frameshift mutation in bacteriophage T4; Streisinger G et al.; Frequencies of spontaneous and proflavine-induced frameshift mutations increased dramatically as a function of the number of reiterated base pairs at each of two sites in the lysozyme gene of bacteriophage T4 . At each site, proflavine induces addition mutations more frequently than deletion mutations . We confirm that the steroidal diamine, irehdiamine A, induces frameshift addition mutations . At sites of reiterated bases, we propose that base pairing is misaligned adjacent to a gap . The misaligned configuration is stabilized by the stacking of mutagen molecules around the extrahelical base, forming a sandwich . Proflavine induces addition mutations efficiently at a site without any reiterated bases . Mutagenesis at such sites may be due to mutagen-induced stuttering of the replication complex.

J Virol, 1985 Apr, 54(1), 86 - 91
Isolation and partial characterization of a bacteriophage T5 mutant unable to induce thymidylate synthetase and its use in studying the effect of uracil incorporation into DNA on early gene expression; Swart WJ Jr et al.; A mutant of phage T5 which is unable to induce thymidylate synthetase was isolated . T5 thy mutants synthesized less DNA than did wild-type T5, and the burst size of progeny phage was correspondingly reduced two- to threefold in thy+ Escherichia coli . No DNA or progeny phage were made in E . coli thy hosts grown in the absence of exogenous thymine . When the T5 thy mutation was recombined with a T5 dut mutation (unable to induce dUTPase), replication resulted in progeny which contained significant amounts of uracil in their DNA, and these phage failed to produce plaques unless the plating host was deficient in uracil-DNA glycosylase . T5 phage containing various amounts of uracil in their DNA were prepared and used to determine the effect of uracil on the induction of the early enzyme dTMP kinase . The presence of uracil in the parental DNA increased the rate of induction of this enzyme by about 2.5-fold . The T5 thy gene was mapped and is located near the T5 frd gene on the B region of the T5 genome.

J Virol, 1985 Apr, 54(1), 233 - 5
Analysis of spontaneous deletion mutants of satellite bacteriophage P4; Raimondi A et al.; Spontaneous deletion mutants of satellite bacteriophage P4 have been isolated and characterized . All of the deletions analyzed that were between 850 and 1,700 base pairs long are within the region nonessential for P4 lytic development; some of them cover the cII or the gop locus.

Can J Biochem Cell Biol, 1985 Apr, 63(4), 237 - 42
In vitro concatemerization of bacteriophage T7 DNA: role of DNA synthesis and gene 6 exonuclease; Lee DD et al.; The replication of bacteriophage T7 DNA in vivo proceeds via the synthesis of complex concatemeric intermediates which are joined via the 160 base pair terminal redundancies at either end of the phage chromosome . To gain some insight into the mode of generation of these structures, we have examined the role of DNA synthesis in the formation of concatemeric bacteriophage T7 DNA in vitro . Incubation of mature T7 DNA with T7-infected cell extracts and a deoxynucleoside {32P}triphosphate resulted in the incorporation of significant radioactivity into the DNA . Highest levels of incorporation were at the termini of the DNA and decreased toward the middle of the molecule . Incorporation was dependent upon the presence of the activity of the gene 6 exonuclease and correlated with the generation of concatemeric DNA . A model explaining the role of exonucleolytic degradation and DNA synthesis in the generation of concatemeric DNA is presented.

Proc Natl Acad Sci U S A, 1985 Apr, 82(7), 1901 - 5
Cloning, nucleotide sequence, and overexpression of the bacteriophage T4 regA gene; Adari HY et al.; The bacteriophage T4 regA gene codes for a regulatory protein that controls the expression of a number of T4 early genes, apparently at the level of translation . Restriction fragments containing the regA structural gene have been cloned into phage M13, and the nucleotide sequence has been determined . Translation of the DNA sequence predicted that regA protein contains 122 amino acids, with a Mr of 14,620 . A DNA fragment carrying 85% of the coding sequence of regA has been cloned into the phage lambda leftward promoter PL expression vector pAS1, and a high level of truncated regA protein was produced by nalidixic acid induction . Protein chemical studies of the truncated regA protein gave results consistent with the nucleotide sequence of the regA gene . Subsequently, an intact regA gene was cloned into plasmid pAS1 and overexpressed . The regA protein produced in this way regulates the level of T4 45 and 44 proteins when their corresponding genes are carried on the same plasmid as the regA gene.

J Bacteriol, 1985 Apr, 162(1), 147 - 54
Mutations in bacteriophage lambda repressor that prevent RecA-mediated cleavage; Gimble FS et al.; In this paper, we report on the isolation and sequence analysis of mutations that confer an induction-deficient phenotype to lambda repressor . A total of 16 different mutations, which occur at 13 different sites in the repressor gene, have been characterized . For most of the mutant lysogens, frequencies of spontaneous induction in a recA+ strain were reduced dramatically in comparison with those for a wild-type phage, and these mutant lysogens showed little or no prophage induction after UV irradiation . The immunity properties of cells containing the mutant repressors show that all of the mutants but one exhibit operator-binding properties indistinguishable from wild-type repressor.

Eur J Biochem, 1985 Apr 1, 148(1), 127 - 34
Purification and some of the functions of the products of bacteriophage T4 recombination genes, uvsX and uvsY; Yonesaki T et al.; The nonessential T4 genes uvsX and uvsY are involved in DNA repair and general recombination . Using newly isolated amber mutants of these genes, we have identified the gene products (gp) by sodium dodecyl sulfate (SDS)/polyacrylamide gel electrophoresis . Their relative molecular masses are 39 000 and 16 000, respectively . In the normal wild-type infection process they are produced early but not late in infection . Their synthesis continues for a longer period when DNA synthesis is blocked . We have developed procedures to isolate these gene products at a purity of more than 95% for gpuvsX and at 70% for gpuvsY, as judged by SDS/polyacrylamide gel electrophoresis and staining with Coomassie brilliant blue dye . The purification procedures suggest that these products may be membrane proteins . Using both an agarose gel assay and electron microscopy, we find that the product of the gene uvsX catalyzes the assimilation of a linear single-stranded fd DNA fragment into superhelical double-stranded fd DNA (RFI) . The reaction requires ATP and Mg2+ besides substrate DNAs and uvsX protein . The T4 uvsX protein therefore is similar to the Escherichia coli recA protein in molecular size and function, but differs in antigenic property.

Can J Biochem Cell Biol, 1985 Apr, 63(4), 243 - 8
In vitro recombination of bacteriophage T7 DNA: further characterization of the reaction using plasmid DNA; Lee D et al.; We have used a plasmid which contains a cloned fragment of T7 DNA to study the properties of general recombination of phage T7 in vitro . It was shown that T7-infected cell extracts promote recombination by the exchange of double strands of DNA . While both products of these double-strand exchanges were detected, we were unable to show that they were formed during a single recombination event.

Proc Natl Acad Sci U S A, 1985 Apr, 82(7), 2087 - 91
DNA sequences at the ends of the genome of bacteriophage Mu essential for transposition; Groenen MA et al.; We have determined the minimal DNA sequences at the ends of the genome of bacteriophage Mu that are required for its transposition . A mini-Mu was constructed on a multicopy plasmid that enabled the manipulation of the DNA sequences at its ends without affecting the genes essential for transposition . The genes A and B, which were cloned outside the ends of the mini-Mu on the same plasmid, were both needed for optimal transposition . In our experimental system the predominant end products of the transposition are cointegrates both in the presence and in the absence of B . Two regions ending approximately 25 and 160 bp from the left end and one ending approximately 50 bp from the right end appear to be essential for optimal transposition . Overlapping with these regions, a 22-base-pair sequence was recognized with the consensus Y-G-T-T-C-A-Y-T-N-N-A-A-R-Y-R-C-G-A-A-A-A, where Y and R represent any pyrimidine and purine, respectively . At the left end these sequences occur as direct repeats; at the right end this sequence is inverted with respect to those at the left end.

Mol Cell Biol, 1985 Apr, 5(4), 831 - 8
Isolation of duplicated human c-src genes located on chromosomes 1 and 20; Parker RC et al.; The oncogene (v-src) of Rous sarcoma virus apparently arose by transduction of the chicken gene known as c-src(chicken) . We isolated DNA fragments representative of two src-related loci from recombinant DNA bacteriophage libraries of the human genome . One of these loci, c-src1(human), appeared to direct the synthesis of a 5-kilobase polyadenylated RNA that presumably encodes pp60c-src(human) . Probes specific for the other locus, c-src2(human), did not hybridize to polyadenylated RNA prepared from a variety of human cell lines . Partial nucleotide sequence determinations of the loci demonstrated that c-src1(human) is highly related to chicken c-src and that c-src2(human) is slightly more divergent . The sequences imply that the final two coding exons of each human locus are identical in length to those of chicken c-src and that the location of an amber stop codon is unchanged in all three loci . c-src1(human) has been mapped to chromosome 20, and the second locus is located on chromosome 1 . We conclude that c-src1(human) is the analog of c-src(chicken) and that the duplicated locus, c-src2(human), may also be expressed.

Mol Biol Rep, 1985 Apr, 10(3), 129 - 36
Interaction in vitro of the neurofilament triplet proteins from porcine spinal cord with natural RNA and DNA; Traub P et al.; Neurofilaments were isolated from porcine spinal cord and separated into their subunit proteins (68 Kd NFP, 145 Kd NFP, 200 Kd NFP) by ion exchange chromatography on DEAE-cellulose in 6 M urea . The individual proteins were reacted with total rRNA from Ehrlich ascites tumor cells and the reaction products analysed by sucrose gradient centrifugation at low ionic strength and in the presence of EDTA . All three proteins interacted with rRNA with a preference for 18S rRNA . Competition experiments with native and heat-denatured calf thymus DNA showed that the affinities of the 68 Kd and 145 Kd NFPs were considerably higher for denatured DNA than for rRNA and that native DNA was only a weak competitor . The binding of the 200 Kd NFP to rRNA was unaffected by native and by denatured DNA . When denatured DNA was reacted with a mixture of the 68 Kd and 145 Kd NFPs, the two proteins interacted independently with the nucleic acid, giving rise to two different populations of deoxyribonucleoprotein particles . This segregation is the result of the cooperative interaction of the neurofilament proteins with single-stranded DNA . It could not be observed with rRNA or bacteriophage MS2 RNA . The results clearly show that the 68 Kd and 145 Kd NFPs are single-stranded RNA- and DNA-binding proteins, whereas the 200 Kd NFP seems to be only a single-stranded RNA-binding protein.

Nucleic Acids Res, 1985 Mar 25, 13(6), 1923 - 38
Optimizing the expression in E . coli of a synthetic gene encoding somatomedin-C (IGF-I); Buell G et al.; Double-stranded DNA encoding the human hormone somatomedin-C (SMC) has been synthesized . This synthetic gene has been inserted into a plasmid bearing the strong leftward promoter (PL) of bacteriophage lambda and expressed in E . coli . Codons for the N-terminal region of SMC which maximized the hormone's synthesis were chosen in an SMC-lac z fusion assay . The amounts of SMC accumulated in E . coli were influenced by mutations at two chromosomal loci, lon and htpR.

J Mol Biol, 1985 Mar 20, 182(2), 249 - 63
New control elements of bacteriophage T4 pre-replicative transcription; Pulitzer JF et al.; Bacteriophage T4 pre-replicative genes are transcribed, by Escherichia coli RNA polymerase, in two alternative modes: an early mode and a middle mode . Middle mode transcription is under the control of at least one viral protein, pmotA . We have identified two additional viral genes, motB and motC, that map in the dispensable region of the T4 genome, between genes 39 and 56 . pmotB and pmotC are diffusible factors which provide an alternative to the motA dependent mode of middle transcription of many T4 genes . Deletions of motB and motC are in fact lethal only in combination with a motA mutant . motB controls one of the alternative modes of transcription of the rIIA gene . When motA or motB are missing, transcription of rIIA is quantitatively unaffected; when both are missing the transcription rate drops by about 75% . Control of transcription of the tRNA gene cluster is more complex . Transcription of subcluster 2 is maximally reduced (70%) only by deletions that, besides motB, cut out an adjacent region . We guess that this adjacent region codes for an additional control element, which we call motC . The motB gene is situated in a 750-base region between the left end-points of del(39-56)-1 and -4.

Eur J Biochem, 1985 Mar 15, 147(3), 581 - 6
The ribonuclease-III-processing site near the 5' end of an RNA precursor of bacteriophage T4 and its effect on termination; Gurevitz M et al.; Infection of RNase III- (rnc) Escherichia coli cells with bacteriophage T4 delta 27, a deletion mutant missing seven out of the ten genes in the tRNA transcription unit, results in the accumulation of a tRNA precursor (10.5-S RNA) that contains the sequences of tRNAGln, tRNALeu and species 1 RNA {Pragai and Apirion (1981) J . Mol . Biol . 153, 619-630} . In vitro studies, using partially purified RNase III or cell extracts and 10.5-S RNA as substrate, have revealed a cleavage site at the 5' side of the molecule . A computerized secondary structure suggests that the RNase III cleavage site can be placed in a small bulge which could be part of a duplex structure and is adjacent to A-A-G and its complementary sequence U-U-U in the same relative relationships found for most RNase III cleavage sites were the adjacent sequences are (A-A-G/U-U-C) . Under normal processing conditions (presence of RNase III) the 3' end of the processed intermediate precursors, 10.1-S and p2Sp1 RNAs, is C-U-U-(U1-2)-UOH, which is determined by a stem and loop structure that could serve as a rho-independent termination signal site . However, in the absence of RNase III, the accumulated 10.5-S precursor RNA does not terminate at the same site and its 3' end is shifted a few nucleotides downstream . Thus, RNase III, besides playing a role in processing of 10.5-S RNA, also affects the termination of that molecule, even though both sites, the RNase III cleavage site and the termination site, are about 390 nucleotides apart.

Biochem Biophys Res Commun, 1985 Mar 15, 127(2), 540 - 5
One- and two- dimensional 15N/1H NMR of filamentous phage coat proteins in solution; Bogusky MJ et al.; High resolution 15N NMR studies of proteins in solution can be performed efficiently by combining the use of isotopically enriched proteins and pulse sequences that generate polarization transfer from protons and result in two-dimensional heteronuclear chemical shift correlation spectra . The coat proteins of the filamentous bacteriophages fd and Pf1 solubilized in detergent micelles give one- and two- dimensional NMR spectra with resolved resonances for nearly all of the nitrogen sites in the proteins . The resonances from the amide sites with slowly exchanging protons can be obtained as a subset of the resonances of all amide sites by comparing the spectra of proteins in D2O and H2O solutions at pH = 4.0.

Nucleic Acids Res, 1985 Mar 11, 13(5), 1517 - 28
Two new restriction endonucleases DraII and DraIII from Deinococcus radiophilus; Grosskopf R et al.; In addition to recently characterized DraI (1), two new Type II restriction endonucleases, DraII and DraIII, with novel site-specificities were isolated and purified from Deinococcus radiophilus ATCC 27603 . DraII and DraIII recognize the hepta- and nonanucleotide sequences (sequence in text) The cleavage sites within both strands are indicated by arrows . The recognition sequences were established by mapping of the cleavage sites on pBR322 (DraII) and fd109 RF DNA (DraIII) . The sequence specifities were confirmed by computer-assisted restriction analyses of the generated fragment patterns of the sequenced DNA's of the bacteriophages lambda, phi X174 RF, M13mp8 RF and fd109 RF, the viruses Adeno2 and SV40, and the plasmids pBR322 and pBR328 . The cleavage positions within the recognition sequences were determined by sequencing experiments.

J Biol Chem, 1985 Mar 10, 260(5), 3197 - 206
Initiation of DNA replication at the primary origin of bacteriophage T7 by purified proteins . Initiation of bidirectional synthesis; Fuller CW et al.; Replication of bacteriophage T7 DNA initiates in vivo at an origin located 15% of the distance from the genetic left end of the chromosome . Bidirectional DNA synthesis from this site results in complete replication of the chromosome . The combination of T7 RNA polymerase, T7 DNA polymerase, and T7 gene 4 protein initiates DNA synthesis in vitro within the cloned origin sequence (Fuller, C . W., and Richardson, C . C . (1985) J . Biol . Chem . 260: 3185-3196) . DNA synthesis is primed by T7 RNA polymerase transcripts, and proceeds in the same direction (rightward) as transcription to yield partially replicated Y-form DNA molecules . The DNA product of in vitro synthesis (Y-form DNA) has been characterized by electron microscopic, sedimentation, and gel electrophoretic analyses . These studies show that Y-form DNA is the product of unidirectional replication of both leading and lagging strands from the origin to the right-hand end of the template . The inclusion of either Escherichia coli single-stranded DNA-binding protein or the functionally similar T7 gene 2.5 protein results in marked stimulation of bidirectional synthesis . Studies using purified Y-form DNA provide direct evidence that this species is an intermediate in the complete replication of the linear template . Purified Y-form DNA is converted to linear DNA in a reaction catalyzed by T7 DNA polymerase, T7 gene 4 protein, and single-stranded DNA-binding protein . Y-form DNA is a competent, transient intermediate during the bidirectional replication of linear DNA molecules and DNA-binding protein is essential to initiate leftward synthesis.

J Biol Chem, 1985 Mar 10, 260(5), 3173 - 7
An endonuclease specific for single-stranded DNA selectively damages the genomic DNA and induces the SOS response; Panayotatos N et al.; A plasmid carrying the bacteriophage T7.3 endonuclease gene under the control of the lacUV5 promoter could be maintained in the transcriptionally active state only in recA+ strains . In recA- strains, endonuclease induction resulted in extensive degradation of the genomic DNA and cell death . In sharp contrast, the plasmid DNA remained intact in the supercoiled form . In recA+ strains, the recA protein levels were increased and the SOS functions of the host were activated, as shown by measurements of recA protein synthesis and prophage induction . These results indicate that in normal undisrupted and non-irradiated cells, enzymatic nucleolytic damage can induce the SOS response and can be controlled by the DNA repair system of the host . In addition, the higher sensitivity of the genomic DNA to the single-strand-specific endonuclease relative to the plasmid suggests that the two molecules differ in their physiological states and most likely in their degree of single-stranded content.

J Biol Chem, 1985 Mar 10, 260(5), 3116 - 25
Influence of template primary and secondary structure on the rate and fidelity of DNA synthesis; Hillebrand GG et al.; High resolution gel electrophoresis was used to monitor the successive addition of dNMP residues onto the 3'-OH ends of discrete 5'-32P-primers, during DNA synthesis on natural templates . Resulting autoradiographic banding patterns revealed considerable variation in the relative rates of incorporation at different positions along the template . The pattern of "pause sites" along the template was unique for each of three different DNA polymerases (polymerase I (the "large fragment" form of Escherichia coli), T4 polymerase (encoded by bacteriophage T4), and AMV polymerase (DNA polymerase of avian myeloblastosis virus} . Most pause sites were not caused by attenuation of polymerization at regions of local secondary structure in the template . Assays of the accuracy of incorporation at different positions along the template (in which elongation was monitored in the presence of only 3 of the 4 2'-deoxynucleoside 5'-triphosphates) strongly suggested that the relative fidelity of DNA synthesis catalyzed by different polymerases depends on the position on the template at which the comparison is made . Primer-templates were constructed that permitted comparison of elongation during synthesis on a single-stranded template with that during polymerization through a double-stranded region (wherein elongation required concomitant displacement of a strand annealed adjacent to the 5'-32P-primer) . Although strand displacement DNA synthesis catalyzed by polymerase I occurred approximately ten times more slowly than synthesis in the same region of a single-stranded viral template, most of the pause sites were the same in the presence or absence of "tandem" primer . Electrophoretic assays of the fidelity of DNA synthesis suggested that an increased tendency toward misincorporational "hotspots" occurred when elongation required concomitant strand displacement.

J Biol Chem, 1985 Mar 10, 260(5), 3185 - 96
Initiation of DNA replication at the primary origin of bacteriophage T7 by purified proteins . Site and direction of initial DNA synthesis; Fuller CW et al.; In vivo, T7 DNA replication is initiated 15% of the distance from the genetic left end of the chromosome . This site, the primary origin of replication, consists of a 200-base pair (bp) intergenic segment from 14.5 to 15.0% within which are located two tandem T7 RNA polymerase promoters (phi 1.1A and phi 1.1B) followed by a 61-bp AT-rich (79% A + T) region . A fragment of T7 DNA containing the primary origin has been inserted into plasmids in order to facilitate studies on initiation in vitro . Initiation of DNA synthesis can be reconstituted using T7 RNA polymerase, T7 DNA polymerase, and T7 origin-containing plasmid DNAs . DNA synthesis is stimulated greatly by the T7 gene 4 protein, an enzyme that has helicase and primase activities . When T7 gene 4 protein is present, replication primarily yields partially replicated Y-form molecules as observed by electron microscopy . Synthesis is unidirectional and the branches of the Y-form molecules are uniform in size, with the branch point of the Y located at the origin . Using restriction enzyme analysis, DNA synthesis has been shown to proceed in the same direction (rightward with respect to the T7 genetic map) as transcription from the two promoters located at the origin . Initiation of DNA synthesis in the opposite direction requires the addition of a single-stranded DNA-binding protein (Fuller, C.W., and Richardson, C.C . (1985) J . Biol . Chem . 260, 3197-3206) . The initial products of DNA synthesis have been analyzed by polyacrylamide gel electrophoresis . These DNAs have 10 to 60 ribonucleotides covalently linked to their 5' termini . These RNA primers arise by transcription from each of the two promoters, phi 1.1A and phi 1.1B, located within the primary origin.

J Biol Chem, 1985 Mar 10, 260(5), 2662 - 9
Amplification and purification of the bacteriophage Mu encoded B transposition protein; Chaconas G et al.; The A and B proteins encoded by the temperate bacteriophage Mu are involved in the high efficiency DNA transposition reaction which is the distinguishing feature of this phage . The genes encoding these early proteins were cloned in an expression vector under the control of the bacteriophage lambda leftward promoter . Under optimal conditions gpB was overproduced to account for 15% of the total cellular protein . The protein was purified to near homogeneity as determined by silver staining . Sequence analysis of the N terminus confirmed the identity of the purified protein as gpB . Proteolytic processing of the B protein does not occur at the amino terminus; the terminal methionine residue is quantitatively deformylated . The protein, which was found to be basic and a general DNA binding protein, was insoluble at low ionic strength in the absence, but not in the presence, of DNA . The B protein also displayed a tendency to aggregate at high ionic strength where it was soluble in the absence of DNA . In addition, the protein was characterized as to its amino acid composition and extinction coefficient at 280 nm . The purified protein is active in a soluble in vitro transposition-replication system.

J Mol Biol, 1985 Mar 5, 182(1), 179 - 82
A protein structure from nuclear magnetic resonance data . lac repressor headpiece; Kaptein R et al.; A procedure is described to determine the three-dimensional structure of biomolecules from nuclear magnetic resonance data . This procedure combines model building with a restrained molecular dynamics algorithm, in which distance information from nuclear Overhauser effects is incorporated in the form of pseudo potentials . The method has been applied to the N-terminal DNA-binding domain or headpiece (amino acid residues 1 to 51) of the lac repressor from Escherichia coli, for which no crystal structure is available . The relative orientation of the three helices of the headpiece is similar to that of the three homologous helices found in the cI repressor of bacteriophage lambda.

Z Naturforsch {C}, 1985 Mar-Apr, 40(3-4), 234 - 41
Small-angle X-ray and light scattering studies on the influence of Mg2+ ions on the structure of the RNA from bacteriophage MS2; Ribitsch G et al.; The influence of Mg2+ ions on the secondary and tertiary structure of the RNA from bacteriophage MS2 was investigated by small-angle X-ray scattering and light scattering and by sedimentation experiments . The analysis of the outer part of the X-ray scattering curve obtained at low temperature in the absence of Mg2+ yielded a cross-section radius of gyration of 0.88 nm and a mass per unit length of 1720 g mol-1 nm-1 . Very similar values for these parameters, which refer to the secondary structure of the RNA molecule, were also derived from the X-ray scattering curves obtained in the presence of different amounts of Mg2+ (0.07 to 1 ions per nucleotide) . On the contrary, the inner part of the X-ray scattering curves turned out to be highly dependent on the Mg2+ concentration: the cross-section radius of gyration and the mass per unit length, which were determined from the scattering curves at small angles as parameters related to the tertiary structure of the RNA, amounted to 3.11 nm and 4000 g mol-1 nm-1, respectively, in the absence of Mg2+ and increased significantly upon raising the concentration of Mg2+ . The increase of these structural parameters was found to be accompanied by a decrease of the overall radius of gyration (as revealed indirectly by X-ray scattering and directly by light scattering measurements) and by an increase of the sedimentation coefficient . The results from the investigations of the RNA at low temperature clearly establish the existence of double-stranded structures down to very low Mg2+ concentrations as well as the occurrence of Mg2+ induced changes of the tertiary structure.(ABSTRACT TRUNCATED AT 250 WORDS)

J Virol, 1985 Mar, 53(3), 871 - 8
Mutational mechanisms by which an inactive replication origin of bacteriophage M13 is turned on are similar to mechanisms of activation of ras proto-oncogenes; Kim MH et al.; M13 viral strand synthesis is initiated by nicking of the viral strand of the duplex replicative form by the M13 gene II initiator protein at a specific site within a sequence of about 40 base pairs having dyad symmetry . Efficient replication of the M13 viral strand also requires the presence of an adjacent sequence of ca . 100 base pairs . Together these sequences constitute the minimal origin for M13 viral strand synthesis . A pBR322 derivative having a 182-base-pair insert of M13 DNA contains a functional M13 viral strand origin and, when provided with M13 gene functions in trans, replicates under conditions nonpermissive for the parent plasmid . Chimeric plasmids containing deletions within the sequence flanking the viral strand origin are unable to replicate under these conditions . We isolated spontaneous mutants of M13 based on their ability to activate replication of such plasmids . The mutations found in these strains, as well as several produced by oligonucleotide-directed mutagenesis, all result in the substitution of any of at least four different amino acids for a specific glycine residue near the amino-terminal end of the initiator protein . Other studies have shown that overproduction of the wild-type initiator protein also restores replication . These alternate mechanisms are discussed in terms of their striking similarity to the mechanisms of activation of the ras proto-oncogenes which can be activated either by increased expression of the wild-type protein or by substitution of any of several amino acids for a glycine residue near the amino terminus.

Proc Natl Acad Sci U S A, 1985 Mar, 82(6), 1648 - 52
Isolation and point of action of a factor from Escherichia coli required to reconstruct translation; Ganoza MC et al.; To study the mechanism of translation we have attempted to reconstruct the process from purified components . Protein synthesis was programmed by the RNAs of wild-type or amber mutants of bacteriophages f2 or MS2 . Translation programmed by MS2 or f2am3 RNA does not occur using ribosomes, precharged aminoacyl-tRNAs, and the sum of the purified proteins involved in initiation (initiation factors; IF-1, IF-2, and IF-3), propagation (elongation factors; EF-Tu, EF-Ts, and EF-G) and termination (release factors; RF-1 or RF-2) of protein synthesis . The requirement for a protein called W was demonstrated . Protein W was purified free of all translation factors, activating enzymes, and other proteins such as the RR, "rescue," and EF-P implicated in translation . The stimulation of propagation by W depended on the position of the amino acid residue to be added in the synthesis of the NH2-terminal hexapeptide of the coat protein . In the reconstructed system, with the sum of all translation factors but in the absence of W, only dipeptides and smaller quantities of tripeptides were synthesized under the direction of f2am3 RNA . W stimulated the synthesis of the hexapeptide, fMet-Ala-Ser-AspNH2-Phe-Thr directed by this RNA . In addition, W stimulated ejection of non-cognate tRNAs that bind to ribosomal particles.

Infect Immun, 1985 Mar, 47(3), 713 - 8
Molecular cloning and expression of Chlamydia trachomatis major outer membrane protein antigens in Escherichia coli; Stephens RS et al.; DNA obtained from Chlamydia trachomatis (serovar L2) was partially digested with DNase I and inserted into the beta-galactosidase gene of bacteriophage lambda gt11 . Seven recombinants were selected that produced immunoreactive fusion proteins which were detected with anti-C . trachomatis rabbit serum . One recombinant, designated lambda gt11/L2/33, reacted with various monoclonal antibodies that recognize species-, subspecies-, and type-specific determinants on the chlamydial major outer membrane protein (MOMP) . Immunoblot analysis of a lambda gt11/L2/33 lysogen revealed a fusion protein that expressed a approximately 15,000-dalton carboxyl-terminal peptide of the chlamydial MOMP . This moiety of the MOMP possesses epitopes responsible for each of the unique reactivities demonstrated by anti-MOMP monoclonal antibodies . The lambda gt11/L2/33 recombinant contained a 1.1-kilobase DNA insert which hybridized to DNA isolated from each of the 15 C . trachomatis serovars.

J Virol, 1985 Mar, 53(3), 935 - 43
A vaccinia virus DNase preparation which cross-links superhelical DNA; Lakritz N et al.; Multiple DNA-dependent enzyme activities have been detected in highly purified preparations of a single-strand-specific nuclease from vaccinia virus . These enzyme preparations were extensively purified and characterized by using superhelical DNAs as substrates . In particular, the nuclease activity was monitored by the extent of conversion of supercoiled closed duplex DNA (DNA I) to nicked circular DNA (DNA II), which could subsequently be converted to duplex linear DNA (DNA III) by prolonged incubation with the enzyme . DNA species which were not substrates for the enzyme included relaxed closed duplex DNA, DNA II which had been prepared by nuclease S1 treatment or by photochemical nicking of DNA I, and DNA III . With plasmid pSM1 DNA as substrate, the extent of cleavage of DNA I to DNA II was found to increase with superhelix density above a threshold value of about -0.06 . The linear reaction products were examined by gel electrophoresis after restriction enzyme digestion of the DNAs from plasmids pSM1 and pBR322 and of the viral DNAs from bacteriophage phi X174 (replicative form) and simian virus 40, and the map coordinate locations of the scissions were determined . These products were further examined by electron microscopy and by gel electrophoresis under denaturing conditions . Electron micrographs taken under partially denaturing conditions revealed molecules with terminal loops or hairpins such as would result from the introduction of cross-links at the cutting sites . These species exhibited snapback renaturation . The denaturing gel electrophoresis experiments revealed the appearance of new bands at locations consistent with terminal cross-linking . With pSM1 and pBR322 DNAs, this band was shown to contain DNA that was approximately twice the length of a linear single strand . The terminal regions of the cross-linked linear duplex reaction products were sensitive to nuclease S1 but insensitive to proteinase K, suggesting that the structure is a hairpin loop not maintained by a protein linker . A similar structure is found in mature vaccinia virus DNA.

Arch Biochem Biophys, 1985 Mar, 237(2), 465 - 76
Gene structure and nucleotide sequence for rat cytochrome P-450c; Hines RN et al.; Two clones from rat genomic libraries that contain the entire gene for rat cytochrome P-450c have been isolated . lambda MC4, the first clone isolated from an EcoR1 library, contained a 14-kb insert . A single 5.5-kb EcoR1 fragment from lambda MC4, the EcoR1 A fragment, hybridized to a partial cDNA clone for the 3' end of the cytochrome P-450c mRNA . This fragment was sequenced using the dideoxynucleotide chain termination methodology with recombinant M13 bacteriophage templates . Comparison of this sequence with the complete cDNA sequence of cytochrome P-450MC {Yabusaki et al . (1984) Nucleic . Acids Res . 12, 2929-2938} revealed that the EcoR1 A fragment contained the entire cytochrome P-450c gene with the exception of a 90-bp leader sequence . The gene sequence is in perfect agreement with the cDNA sequence except for two bases in exon 2 . A second genomic clone, lambda MC10, which was isolated from a HaeIII library, contains the missing leading sequence as well as 5' regulatory sequences . The entire gene is about 6.1 kb in length with seven exons separated by six introns, all of the intron/exon junctions being defined by GT/AG . Amino- and carboxy-terminal information are contained in exons 2 and 7, respectively . These exons contain the highly conserved DNA sequences that have been observed in other cytochrome P-450 species . Potential regulatory sequences have been located both 5' to the gene as well as within intron I . A comparison of the coding information for cytochrome P-450c with the sequence of murine cytochrome P3-450 and rat cytochrome P-450d revealed a 70% homology in both the DNA and amino acid sequence, suggesting a common ancestral gene . Genomic blot analyses of rat DNA indicated that the 3-methylcholanthrene-inducible family of cytochrome P-450 isozymes is more limited in number compared to the phenobarbital-inducible isozymes . Cross-hybridization studies with human DNA suggest a high degree of conservation between rat cytochrome P-450c and its human homolog although gross structural differences do exist between the two genes.

Clin Exp Immunol, 1985 Mar, 59(3), 673 - 8
Antibody to bacteriophage phi X 174 synthesized by cultured human peripheral blood lymphocytes; Bohnsack J et al.; Following immunization of human subjects with the antigen bacteriophage phi X 174, concomitant with the rise in serum antibody, cells appear in the circulation which in vitro, without antigen stimulation, synthesize antibody of the same class as serum antibody in most subjects studied . This function is inhibited by puromycin or irradiation, is independent of T cells and occurs within the first 36-72 h of culture . Such cells are found only in recently immunized subjects . Peripheral blood mononuclear cells (PBM) from all immunized subjects synthesize more antibody to phi X 174 in vitro if antigen is present during cell culture; none was synthesized by antigen containing PBM cultures from unimmunized subjects . This antigen-induced antibody response is T cell and antigen dose-dependent and inhibited by puromycin or irradiation . Following primary immunization the antibody synthesized in vivo and in vitro is IgM . Following secondary immunization IgG antibody is immediately detected in vivo but in vitro antigen-induced antibody continues to be IgM for at least 3 months . IgG antibody then appears: once this class switch occurs, in vitro antigen-induced IgG antibody can be demonstrated in cultured PBM of subjects for many years, without further booster immunization.

J Virol, 1985 Mar, 53(3), 807 - 13
Expression of the cloned bacteriophage phi X174 A* gene in Escherichia coli inhibits DNA replication and cell division; Colasanti J et al.; The A* gene of bacteriophage phi X174 has been cloned into the inducible expression vector pCQV2 under conditions allowing its lethal action to be controlled by the lambda cI857 repressor . Upon induction of expression, DNA synthesis in Escherichia coli carrying the recombinant plasmid is severely inhibited; however, these same cells permit beta-galactosidase induction at a rate similar to that observed in control cells at the inducing (for A*) temperature . Cells in which A* is expressed form filaments and produce more RecA protein, indicating at least a partial induction of the SOS response; however, there is no evidence of damage to the bacterial chromosome . It appears that the A* protein has as one function the inhibition of cell division and DNA replication but not transcription or protein synthesis during phage infection.

J Virol, 1985 Mar, 53(3), 1008 - 11
Subcellular localization of lethal lysis proteins of bacteriophages lambda and phiX174; Altman E et al.; The gene products of the lethal lysis genes S and E of the bacteriophages lambda and phiX174, respectively, were shown to be associated primarily with inner membrane material by isopycnic sucrose gradient centrifugation of lysates of infected cells . A small amount of each polypeptide appeared to be in the outer membrane fraction.

Mol Biol (Mosk), 1985 Mar-Apr, 19(2), 496 - 504
{Comparative analysis of recombinant bacteriophages containing the sequences of the chromosomal ceruloplasmin gene in the rat}; Shvartsman AL et al.; Three clones carrying the sequences of ceruloplasmin gene were isolated from the library of EcoRI fragments of rat chromosomal DNA cloned in Charon 4A vector . These clones were identified using, i) plaque hybridization with {32P} cDNA transcribed from highly purified rat ceruloplasmin (Cp) mRNA; ii) blot hybridization of the restriction fragments of recombinant DNAs with Cp cDNA and iii) hybridization selection of Cp mRNA followed by its cell-free translation . Oligo (dT)-primed cDNA transcripts of Cp mRNA having different length as well as cloned Cp cDNA isolated from rat liver cDNA library were used as hybridization probes for the study of mRNA-coding segments of Cp gene . The length of inserts in recombinant DNAs varied from 7.5 up to 12.3 megadaltons . EcoRI-fragments of Cp gene were mapped within recombinant DNA and their homology to each other was studied.

Biochimie, 1985 Mar-Apr, 67(3-4), 301 - 7
Recognition of damaged regions in DNA by oligopeptides and proteins; Toulme JJ et al.; The binding of various damaged DNAs to the single-strand binding protein coded for by gene 32 from bacteriophage T4, on the one hand, and of oligopeptides containing tryptophan and lysine residues, on the other hand, is described . These molecules exhibit a higher affinity for modified DNA than for native DNA in so far as modification results in a local destabilization of the double-stranded structure of the nucleic acid . Stacking interactions between aromatic amino acids and nucleic acid bases appear to play a crucial role in the recognition of destabilized regions induced by chemical agents (carcinogens and antitumor drugs) . These interactions confer to the peptide lysyl-tryptophyl-lysine an endonucleolytic activity specific for apurinic sites . From results obtained with such oligopeptides a model for the active sites of Ap-endonucleases is proposed which could account for the strategy used by the denV endonuclease from phage T4 during the first step of excision repair of pyrimidine dimers in DNA . The effect of the overall conformation of modified DNA on repair efficiency is discussed.

J Biol Chem, 1985 Feb 25, 260(4), 2281 - 7
Nucleotide-dependent binding of the gene 4 protein of bacteriophage T7 to single-stranded DNA; Matson SW et al.; The gene 4 protein of bacteriophage T7 is a multifunctional enzyme that catalyzes (i) the hydrolysis of nucleoside 5'-triphosphates, (ii) the synthesis of tetraribonucleotide primers at specific recognition sequences on a DNA template, and (iii) the unwinding of duplex DNA . All three activities depend on binding of gene 4 protein to single-stranded DNA followed by unidirectional 5' to 3' translocation of the protein (Tabor, S., and Richardson, C . C . (1981) Proc . Natl . Acad . Sci . U.S.A . 78, 205-209) . Binding of gene 4 protein to single-stranded DNA, assayed by retention of DNA-protein complexes on nitrocellulose filters, is random with regard to DNA sequence . Although gene 4 protein does not bind to duplex DNAs, the presence of a 240-nucleotide-long single-stranded tail on a 7200-base pair duplex DNA molecule is sufficient for gene 4 protein to cause retention of the DNA on a filter . The binding reaction requires, in addition to MgCl2, the presence of a nucleoside 5'-triphosphate, but binding is not dependent on hydrolysis; nucleoside 5'-diphosphate will substitute for nucleoside 5'-triphosphate . Of the eight common nucleoside triphosphates, dTTP promotes optimal binding . The half-life of the gene 4 protein-DNA complex depends on both the secondary structure of the DNA and on whether or not the nucleoside 5'-triphosphate cofactor can be hydrolyzed . Using the nonhydrolyzable nucleoside 5'-triphosphate analog, beta,gamma-methylene dTTP, the half-life of the gene 4 protein-DNA complex is greater than 80 min . In the presence of the hydrolyzable nucleoside 5'-triphosphate, dTTP, the half-life of the gene 4 protein-DNA complex using circular M13 DNA is at least 4 times longer than that observed using linear M13 DNA.

Nucleic Acids Res, 1985 Feb 25, 13(4), 1103 - 18
Single stranded DNA SP6 promoter plasmids for engineering mutant RNAs and proteins: synthesis of a 'stretched' preproparathyroid hormone; Mead DA et al.; The intergenic region of bacteriophage f1 has been subcloned into the bacteriophage SP6 promoter plasmids, pSP64 and pSP65, in both orientations . Coinfection of E . coli with these SP6 promoter/phage f1 chimeric plasmids and the interference resistance phage, IR1, results in the replication and secretion of the pSP6.f1 plasmids as single stranded DNA . Bovine preProPTH cDNAs in both the native form and a form containing an insertion of 117 base pairs in the protein coding region have been inserted in these plasmids . The RNA transcribed from the SP6.f1/preProPTH cDNA constructs was efficiently translated in the wheat germ or reticulocyte cell free systems without addition of a 7-methylguanosine cap to the RNA . In the presence of dog pancreatic or chicken oviduct microsomal membranes, conversion of the resultant pre-proteins to pro-proteins was observed . Confirmation of the "mutated" preProPTH cDNA was determined by dideoxyribonucleotide DNA sequencing of single stranded plasmid DNA . These vectors are suitable for the efficient biosynthesis of large amounts of single or double stranded DNA, and translationally active RNA . The combined properties of single stranded DNA replication and the SP6 promoter simplify the engineering of mutant RNAs and their corresponding proteins . In addition, single stranded DNA or RNA corresponding to either complementary strand may be synthesized as nucleic acid hybridization probes.

Nucleic Acids Res, 1985 Feb 25, 13(4), 1193 - 208
The extent of DNA sequence required for a functional bacterial attachment site of phage lambda; Mizuuchi M et al.; We have investigated the extent of DNA sequence required to form a bacterial attachment site (attB) that functions in bacteriophage lambda integration . A DNA fragment carrying attB of Escherichia coli was trimmed, recloned and tested for recombination proficiency . We found that the common core sequence plus the adjoining 4-bp sequences of both the B and B' arms are required for full activity, while plasmids with an even shorter attB sequence retain some capacity to function as attB in vivo . We also found that the nonspecific DNA that is joined to the required attachment site sequence does not significantly influence the rate of the recombination reaction.

J Biol Chem, 1985 Feb 25, 260(4), 2501 - 8
A human opal suppressor tRNA gene and pseudogene; O'Neill VA et al.; A human DNA library, cloned in bacteriophage lambda, was screened with an opal suppressor tRNA probe . Two genes were isolated, subcloned into pBR322, and sequenced . One is a normal opal suppressor tRNA gene 87 nucleotides in length without intervening sequences . It has a TCA anticodon demonstrating that the mature tRNA reads the termination codon UGA . The 5' internal control region for transcription has two extra nucleotides compared to the consensus sequence for eucaryotic tRNA genes, while the 3' internal control region is normal . This gene differs from a previously sequenced chicken opal suppressor serine tRNA gene (Hatfield, D., Dudock, B., and Eden, F . (1983) Proc . Natl . Acad . Sci . U.S.A . 80, 4940-4944) only at position 11 . The second human gene appears to be a pseudogene truncated near the 3' end, since in the cloverleaf form of the mature tRNA there are three noncomplementary bases in the acceptor stem . The two human genes have a high degree of homology and, excluding the truncated 3' terminus of the pseudogene, differ in only two positions . The flanking sequences of the pseudogene are about 90% homologous to the consensus sequence of the human Alu family of repeated sequences . This gene appears to have been inserted between two adjacent Alu family members.

Biochim Biophys Acta, 1985 Feb 20, 824(2), 140 - 5
Temperature-sensitive translation of MS2 bacteriophage RNA; Armstrong-Major J et al.; A comparison was made of bacteriophage MS2 RNA translation in infected Escherichia coli cells and in a defined cell-free system . A number of temperature-sensitive mutants were used as hosts for viral RNA translation at permissive and restrictive temperatures . The amount of viral coat protein synthesis was determined after gel electrophoresis of proteins from the cell lysates . These results were compared to those obtained with cell-free translation assays conducted with ribosomes isolated from the same mutants . Compared with control cells, a reduced activity in vivo and in vitro was found for each mutant examined at elevated temperatures . A good correlation between the two types of translational assays was observed . These findings are discussed in terms of the translational defects known to be a characteristic of some of these mutant strains.

J Mol Biol, 1985 Feb 20, 181(4), 479 - 86
Eclipse kinetics as a probe of quaternary structure in bacteriophage phi X174; Incardona NL et al.; The extracellular form of bacteriophage phi X174 consists of single-stranded DNA within an icosahedral capsid, which has short spikes at each of its vertices . Each spike is composed of gene G and H proteins, while the capsid itself consists of gene F protein . Since several molecules of gene H protein are injected into the cell along with the DNA, specific protein--protein and DNA--protein interactions must be broken when the genome exits and leaves an intact capsid structure at the receptor site . To demonstrate this we examined the eclipse (DNA ejection) reaction with two types of phi X174 mutants . The first contains missense mutations in a capsid or spike protein gene, and the second involves insertions or deletions in non-coding regions of the DNA . Using an improved procedure, the eclipse rate in vivo of the eclipse mutants Fcs70 has been redetermined over a larger temperature range than in previous studies . The three- to fivefold decrease in rate between 37 degrees C and 25 degrees C is due to an increase in both the enthalpy and entropy of activation when compared to the wild-type values of these kinetic parameters . This missence mutation also confers an increase in virus stability in 2 to 3 M-urea . In contrast to this, inserting 163 bases into the length of DNA packaged within the phi X174 capsid does not lead to a detectable change in eclipse rate over the same temperature range . yet this insertion into the J--F intercistronic region imparts a significant decrease in virus stability in urea . These results suggest that a specific set of non-covalent interactions is involved in phi X174 DNA ejection . This is supported by the small (50%), but significant, increase in eclipse rate that occurs when 27 bases are deleted from the J--F intercistronic region . The latter effect must be base-sequence-specific since no change in rate is observed when only seven of the 27 bases are deleted . Thus, the kinetics of the phi X174 eclipse reaction can be used as a sensitive probe of quaternary structure by correlating the change in reaction rate with alterations in amino acid and base sequences in the structural components of the virus.

J Mol Biol, 1985 Feb 20, 181(4), 517 - 31
Effect of spermidine on the conformation of bacteriophage MS2 RNA . Electron microscopy and computer modeling; Jacobson AB et al.; The structure of single-stranded RNA from the bacteriophage MS2 has been examined by electron microscopy in the presence of the polyamine spermidine . The molecules are found in two alternate conformations . The first of these can be characterized as a cruciform structure composed of three large loops approximately 500 to 700 nucleotides in size . The interior of the molecule has extensive base-paired regions which connect distant regions of the molecule; the farthest being 2500 nucleotides apart . In the second conformation, the molecules appear rod-like . Two of the large loops disappear, and these regions form, instead, extensive long-range helices . Computer modeling has been employed to explore the base-pairing potential of the sequence of bacteriophage MS2 RNA . Double-stranded regions identified by electron microscopy are shown to occur in local G + C-rich stretches of the RNA . Detailed models have been calculated for two regions of long-range contact . One of these includes the ribosome-binding site for the viral coat protein gene . The results are discussed in the context of the known role of RNA structure in the regulation of viral gene expression.

FEBS Lett, 1985 Feb 11, 181(1), 69 - 73
Folding of single-stranded DNA on the histone octamer; Caffarelli E et al.; A complex between the single-stranded DNA of the bacteriophage M13 and the histone octamer was analyzed by electron microscopy, low-angle X-ray diffraction and nuclease analysis . The morphology and the diffraction pattern of the complex strongly resemble those of the nucleosome . These results, as well as the finding of a protected DNA fragment about 100 nucleotides long following single-stranded DNA specific nuclease digestion, indicate that 'a nucleosome-like' complex can be formed between single-stranded DNA and the histone octamer . Competition experiments suggest that under physiological conditions the histone octamer is transferred from single- to double-stranded DNA.

J Biol Chem, 1985 Feb 10, 260(3), 1711 - 22
Purification and properties of the bacteriophage T4 gene 61 RNA priming protein; Burke RL et al.; The bacteriophage T4 gene 61 protein is required, together with the gene 41 protein and single-stranded DNA, for the synthesis of the pentaribonucleotides that are used as primers for the start of each new Okazaki DNA fragment during T4 DNA replication . Using this priming activity as an assay, we have purified the 61 protein to essential homogeneity in milligram amounts . The priming activity was identified with the product of T4 gene 61 by using two-dimensional polyacrylamide gel electrophoresis to compare all of the T4-induced proteins in wild-type and mutant infections; the purified protein co-migrates with the only detectable protein missing in a 61- mutant infection . The purified 61 protein is shown to bind to the T4 helix-destabilizing protein (gene 32 protein) and to both single-stranded and double-stranded DNA . We have failed to detect any ribonucleotide polymerizing activity in either the 61 protein or the 41 protein alone; both the 61 and 41 proteins must be present to observe any synthesis of oligoribonucleotides.

J Biol Chem, 1985 Feb 10, 260(3), 1832 - 5
Cloning of the A gene of bacteriophage Mu and purification of its product, the Mu transposase; Craigie R et al.; The bacteriophage Mu transposase (the Mu A gene product), which is absolutely required for both integration of Mu and replicative transposition during the lytic cycle, has been overproduced by cloning the gene on a plasmid under the control of the phage lambda PL promoter . The protein has been purified to near homogeneity from the lysate of heat-induced cells of a strain carrying the plasmid . The purified protein is active as judged by its ability to complement Mu A- cell extracts for supporting Mu transposition in a cell-free reaction.

Nature, 1985 Feb 28-Mar 6, 313(6005), 818 - 9
Domain of E . coli DNA polymerase I showing sequence homology to T7 DNA polymerase; Ollis DL et al.; Escherichia coli contains three DNA polymerases that differ in their size, ability to interact with accessory proteins and biological function . Monomeric DNA polymerase I (Pol I) has a relative molecular mass (Mr) of 103,000 (103K) and is involved primarily in the repair of damaged DNA and the processing of Okazaki fragments; polymerase II is of Mr 120K, and polymerase III has a Mr of 140K, is responsible for the replication of the DNA chromosome and is just one of several proteins that are required for replication . DNA polymerases from bacteriophage as well as those of eukaryotic viral and cellular origin also differ with respect to their size and the number of associated proteins that are required for them to function in replication . However, the template-directed copying of DNA is identical in all cases . The crystal structure of the large proteolytic fragment of Pol I shows that it consists of two domains, the larger of which contains a deep crevice whose dimensions are such that it can bind duplex DNA . The T7 polymerase consists of two subunits, the 80K gene 5 protein and the host-encoded 12K thioredoxin of E . coli . We show here that there is an amino acid sequence homology between at least eight polypeptide segments that form the large cleft in the Klenow fragment and polypeptides in T7 DNA polymerase gene 5 protein, suggesting that this domain evolved from a common precursor . The parts of the Pol I and T7 DNA polymerase molecules that bind the DNA substrate appear to share common structural features, and these features may be shared by all of these varied DNA polymerases.

J Mol Biol, 1985 Feb 5, 181(3), 373 - 82
Bacteriophage P2 late promoters . II . Comparison of the four late promoter sequences; Christie GE et al.; The late genes of bacteriophage P2 are clustered into four transcription units . We have reported the transcription initiation sites for two of the late messenger RNAs, encoding genes QP and ONMLKRS . We have now located the 5' ends of the two remaining late mRNAs . The first gene in the VJHG transcription unit has been located by DNA sequence determination of the single nucleotide change in a V amber mutant . Location of the first gene in the FETUD transcription unit has been inferred from the DNA sequence . The 5' ends of the mRNAs for these two transcription units were located by protection of end-labeled restriction fragments in RNA-DNA hybrids from digestion with nuclease S1 . Similar protection of hybrids using RNA that had been 5' end-labeled with {alpha-32P}GTP and guanylyl transferase confirmed that these 5' termini resulted from initiation of transcription . The DNA sequences preceding the P2 late transcription starts are different from the Escherichia coli promoter consensus sequences at -10 and -35, consistent with the apparent requirement for phage-encoded proteins in the regulation of P2 late gene expression . The four P2 late promoters do share sequence homologies in the -10 and -35 regions, however, and several additional homologies further upstream . P2 late gene expression also appears to involve negative regulation by a product of the ONMLKRS gene cluster . When cells are infected with P2 polar O amber mutants, a marked increase in the levels of proteins encoded by the other three gene clusters is observed . This increase is reflected in the amounts of late mRNAs, suggesting that RNA synthesis is normally repressed or that late mRNAs are more labile in the presence of a gene product from the ONMLKRS transcription unit . Satellite phage P4 induced P2 late gene expression without the usual requirement for P2 DNA replication . The 5' ends of the P2 late mRNAs are the same during P4 transactivation as during normal P2 late gene expression . Thus, the regulation of P2 late gene expression by P4 does not involve altered promoter selection.

J Mol Biol, 1985 Feb 5, 181(3), 351 - 62
Mechanism of strand cleavage and exchange in the Cre-lox site-specific recombination system; Hoess RH et al.; The bacteriophage P1 recombinase Cre mediates site-specific recombination between loxP sites . The loxP site consists of two 13 base-pair inverted repeats separated by an eight base-pair spacer region . When DNA containing the loxP site is incubated with Cre, specific cleavages occur within the spacer region, creating a six base-pair staggered cut . The cuts are centered on the axis of dyad symmetry of the loxP site, resulting in a 5' protruding terminus: 5' A decreases T-G-T-A-T-G C 3' T A-C-A-T-A-C increases G . At the point of cleavage, Cre becomes covalently attached to a 3' PO4, and produces a free 5' OH . A series of experiments were carried out in which a radioactively labeled loxP site is recombined with an unlabeled loxP site to locate the point at which strand exchange takes place during recombination . The points of strand exchange coincide with the sites at which Cre cleavage of the DNA backbone had been detected.

J Appl Bacteriol, 1985 Feb, 58(2), 231 - 3
A note on a membrane filtration method for the concentration and enumeration of bacteriophages from water; Purdy RN et al.; A membrane filtration method is described for the recovery and enumeration of bacteriophage from water . The method is conveniently used in the field and requires no complex or expensive equipment.

Chem Biol Interact, 1985 Feb-Apr, 53(1-2), 89 - 97
Repair of benzo{a}pyrene diol epoxide damaged bacteriophage T7 DNA determined by survival of phage made by in vitro packaging; Van Houten B et al.; DNA from bacteriophage T7 was treated with benzo{a}pyrene diol epoxide (BPDE) and the number of covalently bound adducts per T7 genome was determined . BPDE treated T7 DNA was then incubated in an in vitro DNA packaging system so as to form infective T7 phage . The observed reduced survival of these phage measured with Escherichia coli uvrA- indicator bacteria showed that the BPDE treated DNA was in fact utilized by the in vitro packaging system and that the resulting phage contained DNA damage caused by in vitro exposure to BPDE . T7 DNA damage by BPDE was also incubated in an in vitro DNA repair system that used partially purified uvrABC proteins from E . coli . Alkaline sucrose gradient analysis demonstrated that nicks were introduced into the damaged DNA and that these incisions were repaired to yield nearly intact DNA molecules of about the size of a T7 genome . Encapsulation of the repaired DNA with the packaging system yielded phage that showed higher survival than the unrepaired control when plated on uvrA- indicator bacteria.

J Virol, 1985 Feb, 53(2), 667 - 71
Isolation of different bacteriophages using the LamB protein for adsorption on Escherichia coli K-12; Charbit A et al.; Ten phages which use the LamB protein for adsorption have been isolated from sewage waters . Nine have a shape similar to lambda and require only the LamB protein for adsorption . One has a shape similar to T phages and can use either the LamB or the OmpC protein . Preliminary characterization by a number of criteria showed that at least nine of these phages were different and also differed from other known phages which use the LamB protein, such as lambda, 21, and K10.

J Bacteriol, 1985 Feb, 161(2), 799 - 802
Thioredoxin is the bacterial protein encoded by fip that is required for filamentous bacteriophage f1 assembly; Lim CJ et al.; Escherichia coli mutants defective in the fip gene contain reduced levels of thioredoxin activity . F' derivatives of trxA mutants failed to support f1 growth, although suppressors of an amber trxA mutation restored both thioredoxin activity and the Fip+ phenotype . Plasmids carrying the trxA gene restored the Fip+ phenotype to fip strains.

J Bacteriol, 1985 Feb, 161(2), 563 - 6
Repressor for the sn-glycerol-3-phosphate regulon of Escherichia coli K-12: cloning of the glpR gene and identification of its product; Schweizer H et al.; The glpR gene encoding the repressor for the glp regulon of Escherichia coli was cloned from a library of HindIII DNA fragments established in bacteriophage lambda . Phages harboring glpR were isolated by selection for sn-glycerol-3-phosphate dehydrogenase function encoded by glpD, which is adjacent to glpR on the E . coli linkage map . Restriction endonuclease analysis and recloning of DNA fragments localized glpR to a 3-kilobase-pair EcoRI-SalI segment of DNA . Strains exhibiting constitutive expression of the glp operons were strongly repressed after introduction of multicopy plasmids containing the glpR gene . Analysis of proteins labeled in minicells harboring either glpR+ recombinant plasmids or a glpR::Tn5 derivative showed that the glpR gene product is a protein with an apparent molecular weight of 33,000.

Proc Natl Acad Sci U S A, 1985 Feb, 82(4), 1084 - 8
Altered Cro repressors from engineered mutagenesis of a synthetic cro gene; Eisenbeis SJ et al.; A portion of the gene coding for the Cro repressor protein of bacteriophage lambda has been chemically synthesized, incorporating base pair changes that generate restriction endonuclease sites without altering the amino acid coding sequence . These restriction endonuclease sites were used to remove small segments of the synthetic cro gene and the segments were replaced with duplexes carrying desired mutations . Altered Cro proteins produced by mutants constructed in this manner were then assayed for binding to lambda operator OR3 in vivo . Mutations directed into the region of the cro gene encoding the alpha-3 helix produced altered Cro proteins with a range of affinities for operator DNA . These changes suggest which amino acids play an important role in Cro-OR3 complex formation.

Proc Natl Acad Sci U S A, 1985 Feb, 82(4), 1074 - 8
A bacteriophage T7 RNA polymerase/promoter system for controlled exclusive expression of specific genes; Tabor S et al.; The RNA polymerase gene of bacteriophage T7 has been cloned into the plasmid pBR322 under the inducible control of the lambda PL promoter . After induction, T7 RNA polymerase constitutes 20% of the soluble protein of Escherichia coli, a 200-fold increase over levels found in T7-infected cells . The overproduced enzyme has been purified to homogeneity . During extraction the enzyme is sensitive to a specific proteolysis, a reaction that can be prevented by a modification of lysis conditions . The specificity of T7 RNA polymerase for its own promoters, combined with the ability to inhibit selectively the host RNA polymerase with rifampicin, permits the exclusive expression of genes under the control of a T7 RNA polymerase promoter . We describe such a coupled system and its use to express high levels of phage T7 gene 5 protein, a subunit of T7 DNA polymerase.

Proc Natl Acad Sci U S A, 1985 Feb, 82(4), 1040 - 4
Interaction of the lambda site-specific recombination protein Xis with attachment site DNA; Yin S et al.; Nuclease protection experiments show that Xis protein of bacteriophage lambda specifically binds attachment (att) site DNA . The region of Xis binding, present in both the phage att site and the right prophage att site, extends from position -102 to position -62 in the P arm . The sequence of this region, the positions of purines protected by Xis against methylation, and the binding of Xis to a resected att site indicate the presence of two binding sites . The postulated recognition elements, contained in 13-base-pair direct repeats separated by 7 base pairs, are situated on the same face of the DNA helix . Protection experiments performed with DNase I suggest that the DNA wraps around (or along the surface of) the bound Xis protein . The Xis binding data presented here establishes that Xis, like the other two proteins involved in lambda site-specific recombination, interacts specifically with att DNA . This rules out that class of models in which the profound effects of Xis on the directionality of site-specific recombination are mediated solely through protein-protein interactions or modification of another protein . In addition, nuclease protection experiments with pairwise combinations of the proteins show that Xis and integration host factor (IHF), or Xis and Int, can bind simultaneously to either the phage or right prophage att sites, and the DNA sequences protected are the sum of those protected with each protein alone . It is therefore unlikely that the effect of Xis on the direction of recombination is exerted by directly blocking the binding of Int or IHF to one or more of their respective binding sites.

J Virol, 1985 Feb, 53(2), 708 - 9
Gene K of bacteriophage phi X174 codes for a protein which affects the burst size of phage production; Gillam S et al.; Site-directed mutagenesis has been used to produce a T----A change at nucleotide 70 of phi X174 genome . This generates an am codon, TAG, in the gene K reading frame without affecting the amino acid, leucine, encoded by the overlapping gene A . The gene K mutant produces small plaques on su- hosts . It has an identical latent period, but a more reduced burst size than that of the wild-type phi X174 . The reduced burst size in the gene K mutant suggests that the gene K protein, although not essential, has a role in increasing infectivity by increasing the burst size three- to sixfold.

J Virol, 1985 Feb, 53(2), 695 - 7
Bacteriophage phi X174 A protein cleaves single-stranded DNA and binds to it covalently through a tyrosyl-dAMP phosphodiester bond; Sanhueza S et al.; The phi X174 A protein cleaves single-stranded DNA and binds covalently to the 5'-phosphorylated end . To determine the nature of the covalent linkage and the amino acid involved, we used the A protein to cleave DNA synthesized in vitro with {alpha-32P}dATP to form the complex of A protein covalently linked to single-stranded DNA . The complex was then digested with DNase I, and the 32P-labeled A protein was isolated by electrophoresis on polyacrylamide gels . The isolated complex was treated extensively with trypsin, and the released peptide-oligonucleotide complexes were incubated with formic acid and diphenylamine (Burton reaction) . The Burton reaction caused a transfer of the labeled phosphate from dAMP to the peptide . The labeled phosphopeptides were isolated and hydrolyzed, revealing a linkage of the phosphate to a tyrosine . These results indicate that the A protein cleaves single-stranded DNA and binds covalently to the 5'-phosphorylated terminus by a tyrosyl-dAMP phosphodiester bond.

Mol Gen Mikrobiol Virusol, 1985 Feb, (2), 17 - 21
{Effectiveness of incorporation of spermine-condensed viral DNA into liposomes . Interaction of liposomes with cells}; Kislina OS et al.; DNA from bacteriophage lambda or monkey adenovirus type 7 have been condensed with spermine and included into three types of liposomes: ethereal, obtained by inverted phases technique and by Ca++ fusion . Compact DNA form presents a tor with 100 nm external diameter and 430 nm width . It can be included into 100 nm or larger liposomes . Total preparation contains 15-18% of the required liposomes . Liposomal fractions with included DNA were separated from empty liposomes by step gradient of ficoll 400 . Liposomal fraction having included DNA contains 15-18% of common lipid . Liposomal interaction with monkey cell cultures has been studied.

Virology, 1985 Feb, 141(1), 162 - 6
Nucleotide sequence and structure of integrated bovine leukemia virus long terminal repeats; Derse D et al.; Bovine leukemia virus (BLV) proviruses, harbored by the productively infected fetal lamb kidney (FLK-BLV) cell line, were cloned in bacteriophage lambda L47 . The nucleotide sequence of the proviral long terminal repeats (LTR) with flanking cell and virus DNA have been determined . The BLV LTR is 531 bp in length and is bounded by the dinucleotides 5'-TG...CA-3', which are part of a 3-bp inverted repeat . The integrated provirus is flanked by 6-bp direct repeats of cellular DNA . A tRNApro primer binding site is present starting 2 bp downstream of the 5' LTR . In addition to sequencing integrated proviral DNA clones, the nucleotide sequence of a cDNA clone, representing the 3' end of genomic viral RNA, was determined; thus revealing the RNA polyadenylation site and R:U5 boundary within the LTR . Unlike most other retroviruses, a consensus polyadenylation signal, "AATAAA," is not located proximal to the BLV polyadenylation site . The RNA initiation site, defining the U3:R boundary, was located in the BLV LTR by S1 nuclease mapping . This site is approximately 25 bp downstream of an A + T-rich region which probably encompasses a Goldberg-Hogness ("TATAA") box and about 90 bp downstream of a potential "CCAAT" box . The BLV LTR possesses a U3 region of 204 bp, an unusually long R region of 241 bp, and a U5 region of 86 bp.

DNA, 1985 Feb, 4(1), 39 - 49
A new method for purifying lambda DNA from phage lysates; Helms C et al.; A new method for preparing small quantities of lambda DNA from phage lysates has been developed . The protocol is based on the concentration and purification of bacteriophage particles from crude lysates using small DEAE-cellulose columns . This chromatographic step gives an absolute separation of the lambda DNA from the cellular nucleic acids and a 20-fold enrichment relative to the major soluble proteins in crude lysates, while effecting a 10-fold concentration of the phage . Final deproteinization and concentration of the lambda DNA is achieved by conventional precipitation steps . The lambda DNA produced by this method is shown to be nondegraded, biologically active, and an excellent substrate for restriction enzymes . A detailed protocol is provided for starting with individual plaques and using the method to obtain purified DNA from large numbers of lambda clones.

DNA, 1985 Feb, 4(1), 23 - 31
Use of avian retroviral-bovine growth hormone DNA recombinants to direct expression of biologically active growth hormone by cultured fibroblasts; Kopchick JJ et al.; A variety of recombinant DNA molecules were constructed in which an avian retroviral long terminal repeat (LTR) was ligated to the bovine growth hormone (bGH) gene . The retroviral LTR was derived from a plasmid clone of a Schmidt Ruppin B strain of Rous sarcoma virus while the bGH gene was subcloned from a lambda bacteriophage genomic library . Using a transient eukaryotic expression assay system, recombinant plasmid constructs were screened for their ability to direct expression and secretion of bGH . One such plasmid DNA construct, termed pBGH-4, was found to be active in the production of bGH . Stable mouse fibroblast cell lines were generated containing pBGH-4 DNA integrated into the mouse cell genome . Many of these mouse cell lines express and secrete bGH . One line, L-Pd lambda-BGH4-13, was found to secrete bGH at a rate of 75 micrograms per 5 X 10(6) cells per 24 hr . Bovine growth hormone derived from this cell line is biologically active.

EMBO J, 1985 Feb, 4(2), 519 - 26
Translational initiation frequency of atp genes from Escherichia coli: identification of an intercistronic sequence that enhances translation; McCarthy JE et al.; The c, b and delta subunit genes of the Escherichia coli atp operon were cloned individually in an expression vector between the tac fusion promoter and the galK gene . The relative rates of subunit synthesis directed by the cloned genes were similar in vitro and in vivo and compared favourably with the subunit stoichiometry of the assembled proton-translocating ATP synthase of E . coli in vivo . The rate of synthesis of subunit c was at least six times that of subunit b and 18 times that of subunit delta . Progressive shortening of the long intercistronic sequence lying upstream of the subunit c gene showed that maximal expression of this gene is dependent upon the presence of a sequence stretching greater than 20 bp upstream of the Shine-Dalgarno site . This sequence thus acts to enhance the rate of translational initiation . The possibility that similar sequences might perform the same function in other operons of E . coli and bacteriophage lambda is also discussed . Translation of the subunit b cistron is partially coupled to translation of the preceding subunit c cistron . In conclusion, the expression of all the atp operon genes could be adjusted to accommodate the subunit requirements of ATP synthase assembly primarily by means of mechanisms which control the efficiency of translational initiation and re-initiation at the respective cistron start codons.

J Biomol Struct Dyn, 1985 Feb, 2(4), 821 - 9
The conformation of the complex of the helix destabilizing protein GP32 of bacteriophage T4 and single stranded DNA; Scheerhagen MA et al.; The conformation of single stranded polynucleotides is changed specifically upon binding of the helix destabilizing protein of bacteriophage T4 (GP32) . On the basis of circular dichroism (CD) and absorption experiments it is shown that denaturing conditions and the binding of oligopeptides can not induce the altered conformation . On the contrary, according to the current CD and absorption theory, the optical properties of the complex can be explained by a specific, regular conformation, characterized by an appreciable tilt of the bases (less than or equal to -10 degrees) and either a small rotation per base or a small helix diameter . This conformation agrees nicely with the increase of the base-base distance in the complex as determined in solution by electric field induced birefringence measurements . Our calculations show that also the model proposed by Alma (Ph.D . Thesis Catholic University Nijmegen, The Netherlands (1982)) for the complex of the helix destabilizing protein of bacteriophage fd, in which the helix diameter is large and the bases are almost parallel to the helix axis, would agree with the CD- and absorption spectra of the GP32-complex . For the latter protein this model would have to be modified with regard to the axial increment of the bases which is much larger in the GP32-complexes.

J Virol, 1985 Feb, 53(2), 430 - 9
Bacteriophage T4 unf (=alc) gene function is required for late replication in the presence of plasmid pR386; Herman RE et al.; The bacteriophage T4 unf gene, known to be involved in the arrest of transcription from cytosine-containing DNA, is unessential except in Escherichia coli strains containing plasmid pR386 . Comparative genetic and biochemical analyses of parameters of unf+ and unf- phage growth in host cells isogenic except for the presence or absence of plasmid pR386 have shown that unf gene function is required for late phage DNA synthesis in the presence of the plasmid . Shutoff of host DNA, RNA, and protein syntheses, degradation of host DNA, adsorption, injection, and early phage DNA, RNA, and protein syntheses all occurred with normal or near-normal kinetics in unf- infections, even in the presence of the plasmid . The switch from early to late protein synthesis occurred in plasmid pR386-containing cells infected with unf+ or unf- phage . However, this switchover was slow in both cases and may be slower in unf- infections than in unf+ infections . Net incorporation of {3H}thymidine terminated at about 30 min after infection of pR386-containing cells with unf- phage at 30 degrees C . Alkaline sucrose gradient studies of the intracellular pools of replicative DNA in unf-infected plasmid pR386-containing cells indicated that this DNA is not detectably nickel or cleaved at the time that DNA synthesis aborts . The addition of chloramphenicol subsequent to early enzyme synthesis prevented the arrest of DNA synthesis in plasmid-containing cells infected with unf-phage.

Virology, 1985 Jan 30, 140(2), 364 - 7
Relating cistrons and functions in bacteriophage PM2; Canelo E et al.; An approach to correlate functions with cistrons in bacteriophage PM2 is presented . Two of the temperature-sensitive mutants obtained differed from the wild type in heat sensitivity and four differed in host range . Comparison of the electrophoretic patterns of DNA from cells infected at the restrictive temperature with those obtained in wildtype infection revealed that viral DNA bands were absent with three additional mutants . Complementation analysis assigned the four host range mutants to cistron I and the three mutants defective in DNA synthesis to cistron IV . Recombination frequencies were used to locate cistrons III and IV on the partial genetic map of PM2.

Virology, 1985 Jan 30, 140(2), 313 - 27
A phi 80 function inhibitory for growth of lambdoid phage in him mutants of Escherichia coli deficient in integration host factor . I . Genetic analysis of the Rha phenotype; Mozola MA et al.; Bacteriophage phi 80 and lambda-phi 80 hybrid phage of the type lambda (QSR)80, in which the rightmost 10% of the lambda genome is replaced by corresponding phi 80 material, are unable to grow lytically in himA and hip/himD mutants of Escherichia coli K12 at 32 degrees . The genetic element responsible for the growth defect, rha, has been mapped to the (QSR)80 region and was located more precisely by restriction enzyme and DNA heteroduplex analysis of mutations that result in loss of the Rha phenotype . Such an Rha mutant carrying a 1.5-kb deletion beginning 0.58 kb from the right end of the chromosome and extending leftward locates the rha locus at least in part within this region of (QSR)80 . In addition, a substitution derivative of lambda (QSR)80 was isolated which does not exhibit the Rha phenotype . In this phage, lambda-80hy95, the right half of the (QSR)80 region is replaced by DNA homologous to the 95-100% segment of lambda . In mixed infections in the himA42 host at 32 degrees, lambda + does not complement lambda (QSR)80 for growth and the burst size of the coinfecting lambda + is reduced in comparison to that in a single infection . Deletion mutants of lambda (QSR)80 that grow normally in himA42 at 32 degrees in single infections are inhibited for growth in mixed infections with lambda (QSR)80 . These results suggest the existence of a trans-acting function which inhibits phage growth in the absence of HimA or Hip/HimD function . It is likely that the rha gene either encodes that function or indirectly controls its action.

Nucleic Acids Res, 1985 Jan 25, 13(2), 605 - 16
The nucleotide sequences of the tail fiber gene 36 of bacteriophage T2 and of genes 36 of the T-even type Escherichia coli phages K3 and Ox2; Riede I et al.; Genes 36 have been cloned from phage T2 and the T-even type phages K3 and Ox2 . The products of these genes are part of the long tail fibers of the phages, they form the proximal moiety of the distal half fiber . The genes have been sequenced, the nucleotide sequence of gene 36 of phage T4 is known (Oliver, D.B . & Crowther, R.A . (1981) J.Mol.Biol . 153, 545-568) . Comparison of the deduced amino acid sequences of the four proteins revealed a surprising pattern . These sequences can be divided into two highly conserved and one very variable region . The former consist of about 60 NH2-terminal and 70 CO2H-terminal residues flanking the variable middle region of about 100 residues . Thus, an identical and unique morphology can be formed by a number of different primary structures . It is proposed that the conserved areas are involved in binding of the proteins to the neighboring products of genes 35 and 37 and that this function has put constraints on the variability of the primary protein structure . The overall amino acid composition of the proteins is rather similar; the codon usage is that known for phage T4 . The intercistronic region between genes 35 and 36 consisting of 62 base pairs and containing a presumed terminator for g35 transcription and the 'late type' promoter for transcription of genes 36, 37, and 38, is almost completely identical in the four phages.

J Biol Chem, 1985 Jan 25, 260(2), 1280 - 6
A gene encoding rat cholecystokinin . Isolation, nucleotide sequence, and promoter activity; Deschenes RJ et al.; The gene for rat cholecystokinin (CCK) was isolated from a rat genomic DNA library . The transcription unit spans 7 kilobases and is interrupted by two introns . The initiator methionine codon lies 2 bases into exon 2; therefore, exon 1 is a noncoding exon . The transcription initiation site was determined using avian myeloblastosis reverse transcriptase, a cDNA primer, and mRNA isolated from a rat medullary thyroid carcinoma . A "TATA"-like sequence precedes the transcription initiation site at position -34 . The polyadenylation site for the gene was mapped by a nuclease protection assay using a cRNA generated by transcription of the exon 3 region of the CCK gene with SP6 bacteriophage RNA polymerase . The sequence AT-TAAA is found 22 bases 5' to the site determined to be the polyadenylation addition site . Two regions of simple repetitive DNA occur within the CCK lambda clone, one within intron 2 and the other 4 kilobases 3' to the gene . Sequence analysis of the repetitive element 3' distal to the gene revealed two copies of the sequence 5'-(AC)n-3', where n is 22 and 25 . A 114-base pair sequence of predominantly repeating purine-pyrimidine nucleotides separates these two d(AC) repeats . Transcriptional control elements were investigated by fusing regions of the CCK gene to the structural gene encoding chloramphenicol acetyltransferase . Promoter activity was determined by transfecting COS-7 cells with plasmids containing the gene fusions, followed by determining chloramphenicol acetyltransferase activity in cellular extracts . The region necessary for expression of the CCK gene fusions in COS-7 cells is within 144 bases 5' to the initiation of transcription.

J Mol Biol, 1985 Jan 20, 181(2), 211 - 30
The OR control system of bacteriophage lambda . A physical-chemical model for gene regulation; Shea MA et al.; A quantitative model has been developed for processes in the bacteriophage lambda that control the switchover from lysogenic to lytic modes of growth . These processes include the interactions of cI repressor and cro proteins at the three DNA sites of the right operator, OR, the binding of RNA polymerase at promoters PR and PRM, the synthesis of cI repressor and cro proteins, and the degradative action of recA during induction of lysis . The model is comprised of two major physical-chemical components: a statistical thermodynamic theory for relative probabilities of the various molecular configurations of the control system; and a kinetic model for the coupling of these probabilities to functional events, including synthesis of regulatory proteins cI and cro . Using independently evaluated interaction constants and rate parameters, the model was found capable of predicting essential physiological characteristics of the system over an extended time . Sufficiency of the model to predict known physiological properties lends credence to the physical-chemical assumptions used in its construction . Several major physiological characteristics were found to arise as "system properties" through the non-linear, time-dependent, feedback-modulated combinations of molecular interactions prescribed by the model . These include: maintenance of the lysogenic state in the absence of recA-mediated cI repressor degradation; induction of lysis and the phenomenon of subinduction; and autogenous negative control of cro . We have used the model to determine the roles, within the composite system, of several key molecular processes previously characterized by studies in vitro . These include: co-operativity in cI repressor binding to DNA; interactions between repressors and RNA polymerase (positive control); and the monomer-dimer association of cI repressor molecules . A major role of cI repressor co-operativity is found to be that of guaranteeing stability of the lysogenic state against minor changes in cI repressor levels within the cell . The role of positive control seems to be that of providing for a peaked, rather than monotonic, dependence of PRM activity on cI repressor level, while permitting PR activity to be a step function . The model correlates an immense body of studies in vivo and in vitro, and it makes testable predictions about molecular phenomena as well as physiological characteristics of bacteriophage lambda . The approach developed in this study can be extended to include more features of the lambda system and to treat other systems of gene regulation.

J Mol Biol, 1985 Jan 20, 181(2), 187 - 97
Extent of sequence homology required for bacteriophage lambda site-specific recombination; Bauer CE et al.; Bacteriophage lambda integration and excision occur by reciprocal recombination within a 15-base homologous core region present in the recombining attachment (att) sites . Strand exchange within the core occurs at precise nucleotide positions, which define an overlap region in which the products of recombination contain DNA strands derived from different parents . In order to define the role of sequence homology during recombination we have constructed point mutations within the core and assayed their effects in vivo and in vitro on site-specific recombination . Two of the mutations are located at position -3 of the core, which is one base-pair outside of the overlap region where strand exchange occurs . These mutations do not affect integrative or excisive recombination, thereby suggesting that homology outside the overlap region is not required for recombination . Two other mutations are located at position -2 of the core, which is one base-pair within the overlap region . These mutations show severely depressed integrative and excisive recombination activities in vitro and in vivo when recombined against wild-type att sites . However, the -2 mutations show normal recombination activity when recombined against att sites containing the homologous mutation, thereby suggesting that homology-dependent DNA interactions are required within the overlap region for effective recombination . In vitro recombination between homoduplex attP sites and heteroduplex attB sites demonstrated that the DNA interactions require only one strand of the attB overlap region to be homologous to attP in order to promote recombination.

Biochemistry, 1985 Jan 15, 24(2), 408 - 12
Inhibition of enzymic incision of thymine dimers by covalently bound 2-{N-{(deoxyguanosin-8-yl)acetyl}amino}fluorene in deoxyribonucleic acid; Duker NJ et al.; The effects of DNA adducts of the carcinogen 2-{N-(acetoxyacetyl)amino}fluorene on enzymic incision of thymine dimers was investigated . Escherichia coli DNA labeled with {3H}thymidine was reacted with the carcinogen . Thymine dimers were then introduced into the modified DNA by irradiation with monochromatic 254-nm light in the presence of the photosensitizer silver nitrate . This DNA containing both types of damages, mainly 2-{N-{(deoxyguanosin-8-yl)acetyl}fluorene and thymine dimers, was then used as substrate for pyrimidine dimer-DNA glycosylase, purified from E . coli infected by bacteriophage T4 . Activity was assayed by measuring release of free labeled thymine after photoreversal of the enzyme-reacted DNA by 254-nm light . The Vmax of the enzyme was decreased when it was reacted with the extensively arylamidated substrate . This inhibition of incision of pyrimidine dimers was increased with the number of carcinogen-DNA adducts, although no enzymic activity against modified guanines was present . Therefore, carcinogen-modified purine moieties can interfere with initiation of excision repair of ultraviolet-induced pyrimidine dimers . This suggests an indirect pathway by which modified DNA bases can be mutagenic.

Virology, 1985 Jan 15, 140(1), 80 - 90
Alterations of the bacteriophage T7 and T3 DNA packaging pathway in Escherichia coli mutant TSN B; Serwer P et al.; Data previously obtained indicate that, during assembly of the related bacteriophages T7 and T3, a DNA-free procapsid (capsid I) is produced and that subsequently capsid I: (1) binds to a longer than mature (concatemeric) DNA and then becomes structurally altered to a particle isolated as a capsid (capsid II) physically resembling the mature bacteriophage capsid more than the procapsid (initiation phase of packaging), (2) draws DNA to its interior (entry phase of packaging), (3) participates in cutting the concatemeric DNA to mature size . It was found that, after infection of Escherichia coli mutant tsnB (selected for a deficiency in plating T7; M . Chamberlin {1974}, J . Virol . 14, 509-516), T7 and T3 capsid I is assembled at a rate not significantly different from its rate of assembly in the wild-type host . However, the conversion of capsid I to capsid II was slowed in E . coli tsnB, suggesting that the tsnB mutation interferes with the initiation of DNA packaging . Although some T3 and T7 DNA enters capsids and is cut to mature size in the tsnB mutant, the data further suggest that the entry rate of DNA into capsid II is lower in the tsnB mutant than it is in an unaltered host . T7 capsid II-concatemeric DNA complexes accumulate during infection of the tsnB mutant . These observations suggest that use of the tsnB mutant as a host will simplify studies of bacteriophage T7 and T3 DNA packaging.

Science, 1985 Jan 11, 227(4683), 140 - 6
Mutation in LDL receptor: Alu-Alu recombination deletes exons encoding transmembrane and cytoplasmic domains; Lehrman MA et al.; The molecular size of the plasma LDL (low density lipoprotein) receptor synthesized by cultured fibroblasts from a patient with the internalization-defective form of familial hypercholesterolemia (FH 274) was smaller by 10,000 daltons than the size of the normal LDL receptor . The segment of the gene encoding the truncated portion of the FH 274 receptor was cloned into bacteriophage lambda . Comparison of the nucleotide sequences of the normal and FH 274 genes revealed a 5-kilobase deletion, which eliminated the exons encoding the membrane-spanning region and the carboxyl terminal cytoplasmic domain of the receptor . The deletion appeared to be caused by a novel intrastrand recombination between two repetitive sequences of the Alu family that were oriented in opposite directions . The truncated receptors lack membrane-spanning regions and cytoplasmic domains; they are largely secreted into the culture medium, but a small fraction remains adherent to the cell surface . The surface-adherent receptors bind LDL, but they are unable to cluster in coated pits, thus explaining the internalization-defective phenotype.

J Biol Chem, 1985 Jan 10, 260(1), 663 - 71
An alkyl imidate labeling study of the organization of phospholipids and proteins in the lipid-containing bacteriophage PR4; Davis TN et al.; The structure of the lipid-containing bacteriophage PR4 was studied using two alkyl imidates, ethyl acetimidate (EAI), a reagent permeant to lipid bilayers and isethionyl acetimidate (IAI), which is impermeant to membranes . The virion is an icosahedral particle consisting of a protein coat surrounding a membrane of phospholipid and protein which in turn encloses the DNA genome . Upon exposure to the permeant reagent, EAI, 50% of the phosphatidylethanolamine (PE) molecules reacted rapidly (half-life less than 10 min) . A similar fraction of the PE also reacted with IAI, the impermeant reagent . The remaining half of the PE molecules reacted slowly with EAI (half-life of 80 min) and failed to react with IAI . All of the phage proteins reacted with both EAI and IAI (except a DNA-associated protein which reacted only with EAI) . These labeling results indicate that the phage membrane consists of a lipid bilayer and that at least a portion of each phage protein (except the DNA-associated protein) is exposed on the external face of the lipid bilayer . Several of the membrane proteins could be cross-linked either to the phage membrane PE after EAI treatment or to phage phosphatidylglycerol after periodate treatment . The major structural protein of the phage was readily cross-linked to PG but failed to cross-link to PE suggesting that the protein specifically interacts with PG.

FEBS Lett, 1985 Jan 7, 179(2), 221 - 4
Hydrodynamic studies of a DNA-protein complex . Elongation of single stranded nucleic acids upon complexation with the gene 32 protein of phage T4 deduced from electric field-induced birefringence experiments; Scheerhagen MA et al.; Short DNA and RNA fragments complexed with the helix destabilizing protein of bacteriophage T4, GP32, have been studied in solution by electric birefringence and circular dichroism . The birefringence of the complexes is positive and the magnitude indicates that the DNA and RNA fragments become linear and rigid upon protein binding . The field free decay is biphasic . On the basis of a rigid rod approximation the slow relaxation time leads to a base-base distance along the helix axis in the complex from 4.3 to 5.6 A, an elongation of at least 50% compared to single-stranded DNA.

J Mol Biol, 1985 Jan 5, 181(1), 27 - 39
Nucleotide sequence and genetic organization of the genome of the N-specific filamentous bacteriophage IKe . Comparison with the genome of the F-specific filamentous phages M13, fd and f1; Peeters BP et al.; The nucleotide sequence and genetic organization of the genome of the N-specific filamentous single-stranded DNA phage IKe has been established and compared with that of the F-specific filamentous phages M13, fd and f1 (Ff) . The IKe DNA sequence comprises 6883 nucleotides, which is 476 (475) nucleotides more than the nucleotide sequence of the Ff genome . The data indicate that IKe and Ff have evolved from a common ancestor (overall homology approx . 55%) and that their genomes contain ten homologous genes, the order of which is identical . Similar to Ff, the major coat protein and the gene III-encoded pilot protein of IKe are synthesized via precursor molecules . The extent of homology between the genes of IKe and Ff differs significantly from one gene to another . Genes that code for viral capsid proteins are less homologous than genes whose products are involved in the processes of DNA replication and phage morphogenesis . During evolution, large nucleotide sequence rearrangements have occurred in the gene (gene III) whose product is needed for the attachment of the virion to the conjugative pili of the host cell, suggesting that these rearrangements have led to phages with different host specificities . Extensive nucleotide sequence homology was noted between the structural elements involved in DNA replication and phage morphogenesis, indicating that the mechanisms involved in DNA replication and morphogenesis are highly conserved.

J Mol Biol, 1985 Jan 5, 181(1), 111 - 21
Fine structure of a membrane anchor domain; Davis NG et al.; We describe a detailed deletion analysis of the anchoring domain of a model membrane protein . Removal of the 23 contiguous uncharged amino acids from the carboxy terminus of the bacteriophage fl gene III protein (pIII) converts it from an integral membrane protein to a secreted periplasmic form . Deletions that remove six or fewer residues of the hydrophobic core result in no diminution of the protein's capacity to anchor in the membrane . Longer deletions into this hydrophobic domain gradually destablize the protein-membrane association . pIII derivatives with over half of the hydrophobic core deleted retain substantial residual anchor function . The basic residues, arginine and lysine, which provide a carboxy-terminal boundary for this domain, can be deleted without loss of anchoring capacity.

J Mol Biol, 1985 Jan 5, 181(1), 85 - 91
"N" transcription antitermination proteins of bacteriophages lambda, phi 21 and P22; Franklin NC; Comparison is made among the amino acid sequences of three transcription antitermination proteins, based upon the DNA sequences of their genes in bacteriophages lambda, phi 21 and P22 . The three proteins are all small (about 100 amino acids), hydrophilic and basic, but otherwise show little homology . A basic region near the amino terminus has several amino acid positions common to all three proteins and is the locus of mutations that alter six different amino acid positions inactivating the lambda N protein . A less basic region near the center is the locus of three mutations affecting the interaction of lambda N with host nusA protein . The N gene of phi 21 has an amino terminus more like that of P22, and a carboxy terminus clearly related to that of lambda.

J Mol Biol, 1985 Jan 5, 181(1), 75 - 84
Conservation of genome form but not sequence in the transcription antitermination determinants of bacteriophages lambda, phi 21 and P22; Franklin NC; Comparisons are made among DNA sequences upstream from terminators in both leftwards and rightwards early operons of related coliphages lambda, phi 21 and P22 . These sequences include both left and right determinants of response to phage-coded antitermination proteins, "N", as well as the N structural genes themselves . Despite almost total disparity of DNA sequence, the three genomes can be discerned to include the same elements in the same order and spacing: downstream from the early left promoter are sequentially a site of recognition for host nusA protein, a dyad symmetry "nut" essential for N function in lambda, overlapping sites for processing of the transcript by RNAase III and then the N structural genes; downstream from the cro gene on the right are sites of nusA recognition and nut dyad symmetries homologous to those on the left . Because the N proteins of lambda, phi 21 and P22 do not for the most part complement each other, a specific site of N recognition has been postulated for each N-responding operon . The nut dyad symmetry qualifies as such a site, since the loop of the left dyad in lambda is marked by mutations that block N function leftwards, and since DNA sequences here show close homology between the loops of left and right dyads for each phage, but less if not little homology for different phages.

Nature, 1985 Jan 3-9, 313(5997), 64 - 7
Polypeptide ligation occurs during post-translational modification of concanavalin A; Carrington DM et al.; Lectins are proteins with multivalent carbohydrate-binding sites, which confer the ability to agglutinate . The seeds of legumes are particularly rich in lectins, for example, concanavalin A (Con A) comprises up to 15% of the protein in the cotyledons of jack bean (Canavalia ensiformis) seeds . The amino acid sequences of Con A and several other legume lectins have been partially or fully determined, and comparison of these sequences from different species reveals a circular homology (Fig . 1A); rearrangements within the genome have been suggested to explain this . We report here that the circular homology displayed by Con A is due to a post-translational transposition and ligation within the initial polypeptide . This type of modification has not been reported previously for eukaryotes, although it has been suggested to occur in bacteriophage lambda.

Nucleic Acids Symp Ser, 1985, (16), 201 - 4
Studies on antitumor drugs targeting DNA: photosensitive DNA cleavage of copper-camptothecin; Kuwahara J et al.; In combination with copper(II) ion and 365 nm-light, anti-tumor alkaloid camptothecin produced remarkable DNA strand scission . The DNA sequencing analysis revealed considerably random nucleotide sequence cleavage . The present DNA breakage reaction was strongly inhibited by catalase and bathocuproine, but not by superoxide dismutase, mannitol, and 1,4-diazabicyclo-{2.2.2}octane . The camptothecin-Cu(II)-UV light system also clearly induced bacteriophage-inactivation which is associated with the DNA degradation . On the basis of the experimental results, the reaction mechanism for the present DNA cleavage has been discussed.

Gene, 1985, 38(1-3), 119 - 29
Characterization and sequencing of the region containing gene N, the nutL site and tL1 terminator of bacteriophage phi 80; Tanaka S et al.; We have cloned the early region of the major leftward operon of bacteriophage phi 80 DNA and determined the nucleotide sequence of the PL-nutL-tL1 segment . The genes and sites equivalent to lambda gene N, antiterminator nutL, and terminator tL1 were found in this region by functional tests using the recombinant plasmids carrying the transcriptional units Ptrp-tL1-galK, Ptrp-nutL-tL1-galK and PlacUV5-N . Sequence homologies were observed between the boxA of the phage 21 nutR site and the inverted repeat of the nut core in the nutL-equivalent site of phi 80 . The N-equivalent gene of phi 80 codes for a protein with 98 amino acids . It is highly basic as is the lambda N protein . Recombinant plasmids carrying the phi 80-PL promoter and nut-equivalent site of phi 80 are lethal to the Escherichia coli host cell, but can be stably maintained in the presence of the phi 80-cI repressor.

Biosystems, 1985, 18(1), 3 - 14
Computer modeling studies of the structure of a repressor; Anderson WF et al.; Due to advances in molecular biology the DNA sequences of structural genes coding for proteins are often known before a protein is characterized or even isolated . The function of a protein whose amino acid sequence has been deduced from a DNA sequence may not even be known . This has created greater interest in the development of methods to predict the tertiary structures of proteins . The a priori prediction of a protein's structure from its amino acid sequence is not yet possible . However, since proteins with similar amino acid sequences are observed to have similar three-dimensional structures, it is possible to use an analogy with a protein of known structure to draw some conclusions about the structure and properties of an uncharacterized protein . The process of predicting the tertiary structure of a protein relies very much upon computer modeling and analysis of the structure . The prediction of the structure of the bacteriophage 434 cro repressor is used as an example illustrating current procedures.

Aust J Biol Sci, 1985, 38(1), 13 - 22
Three-dimensional structure of goose-type lysozyme from the egg white of the Australian black swan, Cygnus atratus; Isaacs NW et al.; The egg white of C . atratus contains two forms of lysozyme, a 'chick-type' which is similar to that found in the egg white of the domestic hen, and a 'goose-type' similar to that found in the egg white of the Embden goose . The molecular structure of the goose-type lysozyme has been determined at a resolution of a 2.8 A by X-ray crystallographic analysis . The structure consists of two domains linked by a long stretch of alpha-helix . In all, there are seven helical segments in the structure . While there is no amino acid sequence homology with either hen egg-white or bacteriophage T4 lysozymes, there are portions of the structure where the folding of the main chain is similar to that found in portions of either hen egg-white lysozyme or T4 lysozyme or both . In particular, there is a consistency of structure in the arrangement of acid groups in the catalytic site . G-o plots calculated for this structure and for the bacteriophage T4 lysozyme structure show that both have similar 'modules' of structure with boundaries occurring at structurally equivalent positions . Three of the common boundaries are equivalent structurally to three of the four module boundaries observed in G-o plots of hen egg-white lysozyme . The variation in the position of the remaining boundary may be related to differences in substrate binding.

Eur Biophys J, 1985, 11(3), 195 - 201
Time resolved fluorescence of bacteriophage Pfl DNA binding protein and its complex with DNA; Greulich KO et al.; The DNA binding protein of the filamentous bacteriophage Pfl exhibits fluorescence from a single tryptophan residue . The location of the emission maximum at 340 nm ist quite common for proteins, but the single lifetime of 7.8 ns is one of the longest yet reported . Protein fluorescence is quenched more efficiently by Cs+ than by I-; the Trp is located in a partially exposed pocket, in the vicinity of a negative charge . In the native complex of the binding protein with Pfl DNA the fluorescence emission maximum is at 330 nm, indicating a more apolar environment for Trp 14 . The native nucleoprotein complex exhibits a similar fluorescence lifetime (6.5 ns) and an approximately equal fluorescence yield, indicating the absence of Trp-DNA stacking . The tryptophan in the complex is virtually inaccessible to ionic quenchers, and thus appears to be buried . Fluorescence depolarisation measurements have been used to examine the rotational mobility of the tryptophan in the protein and in the nucleoprotein complex . In the protein alone a single rotational correlation time (phi) of approximately 19 ns is observed, corresponding to rotation of the entire dimeric molecule; in the native nucleoprotein complex with Pfl DNA, a phi of approximately 500 ns is observed, corresponding to a rigid unit of at least 50 subunits . In neither case does the tryptophan exhibit any detectable flexibility on the subnanosecond time scale.

EMBO J, 1985 Jan, 4(1), 257 - 64
Genes 55, alpha gt, 47 and 46 of bacteriophage T4: the genomic organization as deduced by sequence analysis; Gram H et al.; The nucleotide sequence of T4 genes 55, alpha gt, 47 and 46 was determined by a combination of 'classical' procedures and a shotgun approach . Small DNA fragments generated by frequent cleavage with restriction enzymes or by sonication of restriction fragments were cloned in phage M13 vectors and sequenced by the dideoxy method . The positions of the genes were determined by marker rescue between the corresponding T4 amber mutants and the cloned T4 DNA fragments used in the sequencing experiments . The sequence gives an insight into the organization of this 7.1-kb early region of the T4 genome and shows that genetically 'silent' portions within this region are not void of genetic information.

Basic Life Sci, 1985, 31, 409 - 23
The biochemical basis of 5-bromouracil- and 2-aminopurine-induced mutagenesis; Goodman MF et al.; We describe in vitro measurements of heteroduplex base mispaired intermediates involving 5-bromouracil and 2-aminopurine in A X T----G X C and G X C----A X T transition mutation pathways . For the case of 2-aminopurine, 2-aminopurine X cytosine mispairs are formed at a much higher frequency than adenine X cytosine mispairs in either transition pathway . For the case of 5-bromouracil, at least a 40-fold increase in 5-bromouracil X guanine mispairs are observed over thymine X guanine mispairs but only in the G X C----A X T pathway . In the A X T----G X C pathway, mispairs involving 5-bromouracil are formed 2.5-fold more frequently to those involving thymine suggesting perhaps that 5-bromouracil may exhibit substantially different base-pairing behavior depending on whether it is present as a template base or as a deoxyribonucleosides triphosphate substrate . The effect of the base analogs on dNTP pool size perturbations is discussed . A measurement of dNTP pools in 2-aminopurine mutagenized bacteriophage T4-infected cells is presented . An approximate eight-fold expansion in common dNTP pools is observed in a ts L141 antimutator genetic background compared to wild type T4 43+ and ts L56 mutator backgrounds . The effects of distorted dNTP pools on mutagenesis will be considered.

Gene, 1985, 37(1-3), 31 - 6
Bacteriophage T4, a new vector for the expression of cloned genes; Shub DA et al.; The amino-terminal portion of the T4 rIIB gene has been fused to the coding sequence of a truncated lacZ gene from Escherichia coli, giving rise to a fusion protein with beta-galactosidase activity . The 3192-bp rIIB-lacZ gene fusion was transferred into phage T4, and enzymatically active protein was produced after phage infection . T4 may be a useful expression vector in special circumstances, in particular for proteins whose accumulation in E . coli is limited by sensitivity to proteases.

C R Acad Sci III, 1985, 301(6), 277 - 82
{Constraints on base sequences in a polynucleotide: I . Significance of the degeneration of the code}; Henaut A et al.; The statistical study of polynucleotide sequences constituting the genes of E . coli, bacteriophages lambda and T7 reveals that constraints act upon nucleic acids (DNA or RNA) and contribute to determine the choice between the synonymous codons . The existence of synonymous codons seems to be the way of satisfying these constraints, keeping the possibility of specifying a large variety of polypeptides . At least in the case of amino acids with a small number of codons, these constraints are strong enough to influence the primary structure of proteins.

Gene, 1985, 40(2-3), 241 - 6
In vivo transfer of chromosomal mutations onto multicopy plasmids by transduction with bacteriophage P1; Liljestrom P et al.; A technique is presented by which chromosomal mutations may be efficiently transferred onto chimeric multicopy plasmids in vivo . The technique employs the transduction of plasmids using bacteriophage P1 as vector . The utility of this method was demonstrated by cloning a chromosomal ompR mutation of Escherichia coli K-12 . The high-frequency transduction of the chimeric plasmid appeared to be dependent on its integration into the chromosome by homologous recombination . The results also suggest that the plasmid was transduced as part of the chromosome and resolved from its integrated state in the recipient cell, resulting in a high yield of mutant plasmid segregants.

Gene, 1985, 38(1-3), 275 - 6
The Escherichia coli strain JM105 contains partial supE activity; Reha-Krantz LJ; The Escherichia coli JM105 strain was constructed as a sup0 strain to facilitate the cloning of selected recombinants (Yanisch-Perron et al., 1985) . In our work with bacteriophage T4, we observed that several T4 am mutants could grow on JM105 . To characterize the suppressor activity of JM105, we tested the growth of several T4 am mutants on a variety of E . coli suppressor-containing strains.

Zentralbl Mikrobiol, 1985, 140(4), 309 - 15
The effects of the virulence plasmid ColV, I-K94 on the survival of Escherichia coli in sewage effluent; Rowbury RJ; Escherichia coli 1829 and its mucoid and non-mucoid ColV, I-K94+ derivatives (1829 ColVMuc and 1829 ColVNM) were tested for survival in final sewage effluent (FE) at 20 degrees C . All three strains survived well for 24 h; in some experiments there was also good survival at 48 h but in others there was marked loss of viability . There were only small differences between the strains in their ability to survive in FE i.e . neither ColV, I-K94 nor the mucoid character consistently increased or reduced survival markedly . To assess which factors in FE influence survival, the three strains were also tested in heated FE, filtered FE and chloroform-treated FE . All three strains survived well in heated FE at 20 degrees C but 1829 ColVNM, lost viability much more markedly than the other two strains in filtered FE or chloroform-treated FE . In spite of the poor nutrient level in FE, there was a marked increase, in bacteriophage number in those cultures incubated in chloroform treated FE.

Mol Gen Genet, 1985, 199(3), 481 - 5
Isolation of transfer-negative nif-plasmids (pCE1) and their integration into the chromosome of Escherichia coli with the help of phage Mu; Gottfert M et al.; Strain JC5466 of Escherichia coli K12 harbouring the nitrogen fixation plasmid pCE1 was lysogenized with bacteriophage Mu cts, followed by partial induction and infection with bacteriophage PRD1 . This made it possible to obtain transfer-defective derivatives of pCE1, carrying Mu prophage . These derivatives could be mobilized by using the helper plasmid pME400 and it was possible to segregate the helper plasmid from the donor plasmid in the transconjugants . By incubating the strains 302 and 328 at 42 degrees C, for induction of Mu prophage, derivatives with different plasmid contents could be obtained such as strains without plasmids, some with smaller or larger plasmids and others possessing plasmids without any visible alteration in size . Integration of the nitrogen-fixation (nif) genes into the chromosomes of the strains without plasmids and those containing a smaller plasmid, was confirmed by Southern hybridization using radioactive nifKDH DNA . Conjugation assays have shown that the plasmid is integrated into the chromosome as a unit but that it can also be excised.

Gene, 1985, 34(2-3), 263 - 8
A new strategy to create ordered deletions for rapid nucleotide sequencing; Misra TK; A method is described for generating ordered deletions using previously published techniques but a new strategy . This method is simpler than the published ones and has many advantages . Target DNA is cloned in both orientations into one of the unique restriction enzyme sites adjacent to the complementary region of the commercially available primers in bacteriophage M13 . Ordered unidirectional deletions are created using BAL 31 nuclease and religating into M13 vector DNA without the need of purifying BAL 31-digested DNA from a gel.

Gene, 1985, 34(1), 63 - 72
Expression of the bacteriophage P1 cin recombinase gene from its own and heterologous promoters; Huber HE et al.; The cin recombinase of bacteriophage P1, a protein that catalyses site-specific DNA inversions, has been identified and its structural gene has been cloned under the control of different promoters . One of the DNA sequences used for the site-specific recombination, cixL, overlaps with the 3' end of the gene, but we show that the presence of this site does not affect cin gene expression from strong promoters . To assay cin activity we have constructed plasmids that carry antibiotic resistance genes within the invertible segment that are transcribed from promoters outside the segment . DNA inversion switches on or off genes for chloramphenicol or kanamycin resistance . These tester plasmids are used to study cin-mediated DNA inversion both in vivo and in vitro.

Dev Biol Stand, 1985, 59, 3 - 9
Molecular cloning and a stable amplification of the DNA molecules heavily methylated at CpG sequences using a new E . coli cell system (GC3); Darai G et al.; Fish lymphocystis disease virus (FLDV) DNA is heavily methylated in CpG sequences and therefore the amplification of recombinant DNA of FLDV inserted into a bacterial plasmid vector and/or a bacteriophage system poses severe problems . The problem was solved using a newly constructed and developed bacterial system (E . coli GC-3 system), which allowed the amplification of foreign DNA material heavily methylated at CpG sequences . E . coli GC-3 (Gc-1, rifd) is derived from E . coli GC-1 (K 12, hsd-, MDU) after transformation with phage lambda drifd 18 Hind III-B-fragment . A defined and complete gene library of the FLDV DNA sequences was established by insertion of FLDV DNA fragments (Eco RI, Eco RI/Bam HI, Eco RI/Hind III) into the corresponding restriction sites of bacterial plasmid vector pAT 153 . Bacterial colonies harbouring recombinant plasmids were individually identified by digestion of the recombinant plasmid DNA with different restriction enzymes and screened by hybridizing plasmid DNA to viral DNA . The analyses revealed that sequences representative for the complete viral genome were cloned.

J Gen Microbiol, 1985 Jan, 131 ( Pt 1), 135 - 44
The location of a mutation affecting ribosomal protein synthesis by Escherichia coli; Butler PD et al.; A mutation in a strain of Escherichia coli 15 produced ribosomes by an abnormal pathway that caused the accumulation of 47S ribonucleoprotein particles . The mutation was transferred to strains of E . coli K12 by transductions with bacteriophage P1cam and was at about 82 min, between cysE and pyrE and rather closer to the latter . The location and the physiological properties of the mutant suggested that the mutation was in the rpmB,G transcription unit and affected the synthesis of ribosomal proteins L28 and L33.

J Virol, 1985 Jan, 53(1), 185 - 91
Posttranscriptional modulation of bacteriophage P22 scaffolding protein gene expression; Casjens S et al.; The bacteriophage P22 late operon contains 2 genes whose products are required for cell lysis and 13 genes whose products are involved in the morphogenesis of the phage particle . This operon is under the positive control of the phage gene 23 product and is thought to have a single promoter . The expression of one of these late genes, the scaffolding protein gene, is autogenously modulated independently from the remainder of the late genes . When unassembled, scaffolding protein turns down the rate of synthesis of additional scaffolding protein, and when it is assembled into phage precursor structures, it does not . Experiments presented here show (i) that the mRNA from the scaffolding protein gene is functionally threefold more stable when most of the scaffolding protein is assembled than when it is unassembled and (ii) that no new promoter near the scaffolding protein gene is activated at the high level of synthesis . These data support the model that this autogenous modulation occurs at a posttranscriptional level . We also observed that another message, that of coat protein, appears to become increasingly stable with time after phage infection.

J Virol, 1985 Jan, 53(1), 174 - 9
Assembly-controlled autogenous modulation of bacteriophage P22 scaffolding protein gene expression; Casjens S et al.; In the assembly of bacteriophage P22, precursor particles containing two major proteins, the gene 5 coat protein and the gene 8 scaffolding protein, package the DNA molecule . During the encapsidation reaction all of the scaffolding protein molecules are released intact and subsequently participate in further rounds of DNA encapsidation . We have previously shown that even though it lies in the center of the late region of the genetic map, the scaffolding protein gene is not always expressed coordinately with the remainder of the late proteins and that some feature of the phage assembly process affects its expression . We present here in vivo experiments which show that there is an inverse correlation between the amount of unassembled scaffolding protein and the rate of scaffolding protein synthesis and that long amber fragments of the scaffolding protein can turn down the synthesis of intact scaffolding protein in trans . These results support a model for scaffolding protein regulation in which the feature of the assembly process which modulates the rate of scaffolding protein synthesis is the amount of unassembled scaffolding protein itself.

Mol Gen Mikrobiol Virusol, 1985 Jan, (1), 33 - 4
{Acetobacter methanolicus--a new organism for genetic studies}; Sattler K et al.; A new bacterial strain is described belonging to Acetobacter methanolicus species . It is of industrial value as a producer of protein and methanol products . The strain is acidophile and this feature comprises a conspicuous technological advantage . The results of bacteriophage and cell interactions are reported . They might be potentially useful for elaboration of the transduction technique for the strain . The collection of mutants was obtained including those utilizing methanol, having auxotrophic markers as well as streptomycin and rifampicin resistances . The transfer of plasmids RSF1010 and R68 to Acetobacter methanolicus from other bacteria has been demonstrated.

Biosystems, 1985, 18(1), 23 - 45
A simulation of T4 bacteriophage assembly and operation; Thompson RL et al.; A model is presented for the self-assembly and operation of a bacteriophage comparable with the T4 bacteriophage that infects Escherichia coli . The model treats protein molecules as simple units obeying the principle free energy minimization, and exhibiting the properties of quasi-equivalence and conformational switching . A computer program incorporating the model has been developed . The results of simulation using this program are presented.

Gene, 1985, 33(3), 279 - 84
Isolation of a human tissue-type plasminogen-activator genomic DNA clone and its expression in mouse L cells; Browne MJ et al.; We have isolated a cDNA clone corresponding to a substantial portion of the human tissue-type plasminogen activator (t-PA) protein . It encodes almost all of the protein B chain and part of the 3' untranslated region . We have used this clone to screen bacteriophage lambda and cosmid libraries of human genomic DNA . Several related genomic clones were isolated . One of these, a cosmid clone, carried approx . 40 kb of human DNA . Mapping experiments indicate that the region containing the protein-coding exons is approx . 20 kb in length . The cosmid, containing the t-PA gene and the aminoglycosyl-3'-phosphotransferase dominant-selection marker, was introduced into mouse L cells . Approximately half of the transformants were shown to produce human t-PA . We demonstrated that the fibrinolytic t-PA activity could be specifically quenched by anti-t-PA antibody and that the recombinant t-PA was of similar size (by SDS-polyacrylamide gel electrophoresis) to the t-PA produced by the human Bowes melanoma cell line . Our results suggest that the cosmid clone carries the whole t-PA coding region together with the regulatory elements necessary for its expression.

Mol Gen Genet, 1985, 199(3), 427 - 33
Gene fusions to the ptsM/pel locus of Escherichia coli; Palva ET et al.; We have constructed gene fusions between ptsM/pel and lacZ . These fusions affect both phenotypes assigned to the ptsM/pel locus (at 40 min), namely, no growth on mannose or glucosamine and inhibition of the penetration of bacteriophage lambda DNA, as well as that of other lambdoid phages such as Hy-2 . Since the lacZ gene fusions are insertion mutations that abolish target gene function by disrupting the linear contiguity of the gene, it would appear that ptsM and pel are either the same gene or two genes within the same operon . Several size classes of these ptsM/pel-lacZ fusions have been isolated and the corresponding hybrid proteins are associated with the cytoplasmic membrane of Escherichia coli . This is consistent with the proposal that ptsM/pel codes for Enzyme II of the phosphotransferase transport system (PTS) specific for mannose, glucosamine, fructose and glucose . However, we have also identified Tn10 insertion mutations that confer a Man- phenotype but have no effect on the Pel phenotype . Complementation analysis indicates that the Tn10 insertions and the lacZ gene fusions are in different genes . Both of these genes are involved in mannose uptake . This suggests that the locus at 40 min can be subdivided into two genes whose products are required for mannose uptake and that only one of these is involved in the penetration of lambda DNA.

Eur Biophys J, 1985, 12(2), 87 - 95
1H NMR study of the interaction of bacteriophage lambda Cro protein with the OR3 operator . II . Assignment of the non-exchangeable proton resonances of the OR3 operator; Hahn KD et al.; The 17 base pair operator OR3 oligonucleotide, which is the preferential binding site for the Cro repressor of phage lambda, was studied by two-dimensional NMR spectroscopy . A sequential assignment procedure based on two-dimensional Nuclear Overhauser Effect (NOESY) and scalar coupling correlated (COSY) NMR spectroscopy, together with the knowledge of the oligodesoxynucleotide sequence, made it possible to assign the non-exchangeable base protons and the H1' and the H2'-H2" sugar protons of the OR3 operator DNA . The pattern of the observed NOE connectivities is consistent with a right-handed helical DNA structure . The base and sugar proton assignments provide the necessary information for further studies of the OR3 operator - Cro repressor interaction.

Mol Gen Genet, 1985, 198(3), 491 - 6
Integration of bacteriophages lambda and phi 80 in wild-type Escherichia coli at secondary attachment sites . II . Genetic structure and mechanism of polylysogen formation for lambda, phi 80 and the lambda att80 hybrid; Kholodii GY et al.; The frequency of occurrence and the genetic structure of polylysogens were studied for phages lambda, phi 80 and lambda att80 . In the case of lambda, frequency of polylysogenization is high (0.20 to 0.41) with a tandem integration of prophages at the primary att site (att lambda) . With phi 80 and lambda att80, this frequency is about 10 times lower, and usually one prophage becomes integrated at the primary att site (att80-I) while another (sometimes two others) integrates at one of the secondary sites . At least four secondary att80 sites have been found in wild-type Escherichia coli, two of which (near the his and tolC loci) are preferred . The frequency of secondary integration of phi 80 and lambda att80 does not differ significantly in the wild-type host and in that deleted for the primary att site (0.041 and 0.045, respectively, among surviving cells at an MOI of 10) . Homoimmune superinfection has revealed a constitutive cI-independent expression of the phi 80 int gene in the prophage state . The only phi 80 tandem detected proved to be unstable . With the phi 80int- mutant, we observed stabilization of phi 80 tandems; as a consequence, their frequency of occurrence during coinfection with phi 80int+ was up to the lambda level and no nontandem insertions were found . A model is proposed for the phi 80 and lambda att80 nontandem integration.

J Mol Appl Genet, 1985, 3(1), 18 - 25
Purification of hybrid beta-galactosidase proteins encoded by phi X174 E phi lacZ and Escherichia coli prlA phi lacZ: a general method for the isolation of lacZ fusion polypeptides produced in low amounts; Struck DK et al.; A facile immunoaffinity chromatography method is described for the purification of lacZ fusion gene products . The method is general for any molecule antigenically related to beta-galactosidase and involves only a single step . We report its use to purify the products of lacZ fusions with a bacteriophage gene, phi X174E, and an Escherichia coli chromosomal gene, prlA . The hybrid protein products of both of these genes are membrane bound and present in very low molar amounts with respect to total cellular protein . Evidence is presented that substrate-affinity chromatography is not applicable to the isolation of low-level fusion proteins such as these.

Mol Gen Genet, 1985, 199(1), 64 - 9
Involvement of DNA polymerase III in UV-induced mutagenesis of bacteriophage lambda; Brotcorne-Lannoye A et al.; It has been proposed that the mutation fixation processes stimulated by SOS induction result from an induced infidelity of DNA replication (Radman 1974) . The aim of this study was to determine if mutator mutations in the E . coli DNA polymerase III might affect UV-induced mutagenesis . Using a phage lambda mutation assay which can discriminate between targeted and untargeted mutations, we show that the polC74 mutator mutation (Sevastopoulos and Glaser 1977) primarily affects untargeted mutagenesis, which occurs in a recA1 genetic background and is amplified in the recA+ genetic background . The polC74 mutation also increases the UV-induced mutagenesis of the bacterial chromosome . These results suggest that DNA polymerase III is involved in the process of UV-induced mutagenesis in E . coli.

Gene, 1985, 33(2), 235 - 9
Comparison of left-end DNA sequences of bacteriophages Mu and D108; Bukhari AI et al.; The nucleotide sequences of the left ends of bacteriophage Mu DNA and that of its close relative D108 have been determined . The first 100 bp of phages Mu and D108 are substantially the same except for an octanucleotide change from bp 53 to 61 and other small interspersed base-pair changes from bp 61 to 200 . The first five host nucleotides preceding the host-phage junction are generally, but not always, G + C-rich and these five nucleotides display no obvious consensus sequence . Both phages Mu and D108 share striking similarity in their end DNA sequences to the end sequences of the newly described Escherichia coli movable genetic element IS30.

J Virol, 1985 Jan, 53(1), 340 - 2
Mapping of transcription terminators of bacteriophages phi X174 and G4 by sequence analysis; Brendel V; An algorithm to locate transcription terminators (V . Brendel and E . N . Trifonov, Nucleic Acids Res . 12:4411-4427, 1984) was applied to the genomes of bacteriophages phi x174 and G4 . The proposed sites are similarly located in phiX and G4 and fit with transcript lengths previously observed in vivo and in vitro.

J Virol, 1985 Jan, 53(1), 180 - 4
Bacteriophage P22 tail protein gene expression; Adams MB et al.; We have found that mutations which block bacteriophage P22 head assembly at or before the DNA packaging stage (1-, 2-, 3-, 5-, and 8-) cause up to a 20-fold increase in the amount of tail (gene 9) protein made during infection . This correlation seems strong enough to warrant consideration of a control mechanism in which the failure to package DNA per se causes a large increase in the synthesis of tail protein . Our results indicate that one of the repressors required for maintenance of lysogeny, the mnt gene product, may be partially responsible for this phenomenon.

Mol Gen Mikrobiol Virusol, 1985 Jan, (1), 9 - 12
{Different effect of mutations in Escherichia coli K12 dna-genes on the transposition of Tn5- and Tn10-elements}; Il'ina TS; The effect of mutations in dnaA(dnaA46), dnaG(dnaG3), dnaC (dnaC1 and dnaC2) and dnaB genes on transposition of two transposons, Tn5 and Tn10, from bacteriophage lambda genome into the chromosome of host cells has been studied . Transposition was performed at permissive temperatures for the mutant recipients . The mutations in dnaA, dnaC, dnaG genes were shown to decrease the transposition of Tn10 for some orders of magnitude as compared with transposition registered in wild type cells . Independence of Tn5 transposition of the above mentioned genes was demonstrated, providing evidence on the different modes of transposition of these two Tn-elements.

Gene, 1985, 40(2-3), 325 - 9
Formation of small circular DNA molecules via an in vitro site-specific recombination system; Hoess R et al.; The Cre-lox site-specific recombination system of bacteriophage P1 has been used to investigate the role of DNA flexibility in recombination . We have determined that a minimal distance of 82 bp must separate two loxP sites located on the same DNA molecule to allow these sites to undergo intramolecular recombination with one another . As a result of recombination, DNA circles as small as 116 bp have been produced . IN addition, we have demonstrated that the nuclease BAL 31 recognizes distortions in the DNA helix resulting from the formation of small DNA circles whose length is not a multiple of the helical repeat.

Gene, 1985, 40(2-3), 273 - 84
Nucleotide sequence and genome organization of bacteriophage S13 DNA; Lau PC et al.; The complete sequence of bacteriophage S13 DNA has been determined . The molecule has 5386 nucleotides and differs from phi X174 by 87 transitions and 24 transversions . All the proteins, A,A*,B,C,D,E,F,G,H, J and K found in phi X174 are also present in S13 . Due to changes in the H/A intergenic region of S13, the start of an additional protein, A', has been identified . Genes F and H coding for the capsid and spike proteins, respectively, are the least conserved in comparison to phi X174 . Many of the silent changes, as well as some amino acid changes, are found in the same nucleotide sequence positions in phage G4, confirming the interrelationship between the three phages.

Gene, 1985, 40(1), 151 - 5
Plasmids supplying the Q-qut-controlled gam function permit growth of lambda red- gam- (Fec-) bacteriophages on recA- hosts; Crouse GF; A plasmid, pgam, has been constructed which expresses the phage lambda gene, gam, under the control of the lambda late promoter, p'R, contained in a form of a p'R-qut-t'R1 module . Lambda red- gam-, which normally do not grow on recA- hosts, are able to grow on recA- hosts containing pgam, because their Q function can turn on the gam gene expression . This facilitates cloning with lambda red- gam- vectors in recA- hosts.

Mol Gen Genet, 1985, 201(2), 357 - 9
The nature of ompA mutants of Escherichia coli K12 exhibiting temperature-sensitive bacteriophage resistance; Morona R et al.; A class of ompA mutants of Escherichia coli, exhibiting temperature-sensitive resistance towards phages using the OmpA protein as receptor, was analysed . The mutants produce detectable levels of the protein at 42 degrees C but not at 30 degrees C (Manning and Reeves 1976) . They were found to have a deletion (one isolate) or insertions (three isolates) upstream of the coding part of the ompA gene . Several previously characterized mutants possessing insertions or a deletion in the non-translated 5' area of the gene also exhibited a similar temperature-sensitive phage resistance . This cold-sensitive phenotype is explained in terms of the recent discovery that the stability of ompA mRNA is regulated by the rate of cell growth (Nilsson et al . 1984).

Mol Gen Genet, 1985, 201(2), 269 - 76
Cloning and characterization of the prs gene encoding phosphoribosylpyrophosphate synthetase of Escherichia coli; Hove-Jensen B; The gene, prs, encoding phosphoribosylpyrophosphate (PRPP) synthetase of Escherichia coli was isolated from a library of E . coli genes cloned in the bacteriophage lambda D69 vector . A strain with a temperature-lethal defect in PRPP synthetase, (prs-2), was used as the host and cloning was performed by lysogenic complementation . The prs gene resided on a 5.6 kilobase-pair (kbp) DNA fragment generated by hydrolysis with restriction endonuclease BamHI . The nearby gene pth, encoding peptidyl-tRNA hydrolase, was also on this fragment . Subcloning of the fragment in the multi-copy plasmid pBR322 and subsequent deletion of parts of the insert resulted in a 1.7 kbp DNA fragment containing the entire prs gene . Bacterial strains harbouring prs-bearing plasmids showed up to 50-fold increased PRPP synthetase activity . The PRPP synthetase subunit was identified by analysis of plasmid-harbouring minicells and the subunit molecular mass established as 33,000 daltons . Analysis, by the minicell procedure, of plasmids with deletions extending into the prs gene established the direction of transcription as counterclockwise . A putative leader sequence of approximately 400 bp preceded the coding sequence . By deletion analysis and by cloning fragments of this leader sequence in a galK expression vector it was found to contain the prs promoter as well as a potential transcription termination site.

Gene, 1985, 39(1), 55 - 9
Cloning of mini-mu bacteriophage in cosmids: in vivo packaging into phage lambda heads; de Mendoza D et al.; A technique has been developed that permits the packaging of mini-Mu-carrying cosmids into phage lambda heads . This procedure has several advantages over packaging into Mu helper capsids: the amounts of DNA to be packaged can be increased, the packaging efficiency is improved, and the stability of transducing lysates is high.

Gene, 1985, 36(1-2), 131 - 41
Plasmid vectors designed for the analysis of transcription termination signals; Honigman A et al.; We have constructed synthetic operons in which two genes (cat and lacZ or cat and galK) were placed in tandem under the control of the bacteriophage lambda oLpL operator and promoter . Restriction sites were introduced between the promoter and the proximal cat gene or between the cat and lacZ or galK genes . In the latter case, introduction of a transcriptional terminator between the two structural genes should affect only the distal gene . Thus, following induction, the expression of the cat gene serves as an internal control, compensating for changes due to plasmid copy number or possible decrease in transcription initiation . We used these plasmids to select a lambda DNA fragment which includes the N-unresponsive tJ transcriptional terminator . This DNA fragment was inserted between the cat and galK genes . Enzymatic assays of these two gene activities following induction indicate that transcripts initiated at the pL promoter under N+ conditions terminate at tJ between the two genes . S1-nuclease analysis showed that these transcripts terminate at several sites in the tJ region . Similar results were obtained whether the host cells were RNaseIII+ or RNaseIII- . As a control, we showed a complete antitermination of the lambda t'I terminator under similar conditions, indicating that a sufficient amount of the N gene product is made from one N gene copy to suppress terminators carried on multicopy plasmids.

Mol Gen Genet, 1985, 201(1), 107 - 14
Rearrangements between the operators in the bacteriophage lambda; Stephenson FH et al.; The left operator mutant lambda v2s develops poorly during infection as a result of constitutive expression of the left operon . A revertant of lambda v2s, designated lambda iri, was found to contain an inversion of the cI region with the inversion endpoints to be within the lambda operators OL and OR . Formation of the inversion is facilitated by a translocation of right operator OcR mutant sequence to the left operator in lambda v2s . The inversion in lambda iri positions wild-type OR sequence at OL returning control of the left operon to repression by the lambda cro repressor.

Gene, 1985, 37(1-3), 229 - 39
High-level production of the EcoRI endonuclease under the control of the pL promoter of bacteriophage lambda; Botterman J et al.; Escherichia coli strains overproducing the EcoRI restriction endonuclease have been constructed, using lambda pL promoter expression vectors . In a first step we constructed endRI::lacZ gene fusions by fusing the N-terminal part of the endRI gene with a lacZ gene fragment, whereafter the hybrid gene was positioned randomly under the control of the pL promoter to optimize the level of expression . These plasmids direct the synthesis of large amounts of fusion protein approaching 30% of the total cellular protein content . In most cases the overproduced protein forms enzymatically inactive intracellular aggregates . The position of the promoter in front of the hybrid gene had little effect on the level of expression, except in fusions directly affecting the ribosome-binding site (RBS) . In a second step, several of these promoter-gene configurations were used to reconstruct the intact endRI gene in appropriate hosts producing EcoRI methylase and cI-coded repressor . The levels of EcoRI endonuclease overproduction were similar to that obtained for the corresponding fusion protein, despite the fourfold difference in protein size . Intracellular precipitation was also observed with the overproduced EcoRI endonuclease.

Peptides, 1985, 6 Suppl 1, 95 - 102
Vasoactive intestinal peptide: expression of the prohormone in bacterial cells; DeLamarter JF et al.; A complementary DNA (cDNA) to the messenger RNA for the preprohormone of human vasoactive intestinal peptide (VIP) has been isolated and characterized . This cDNA extends from 65 bases 5' of the AUG translation start codon through the entire 3' untranslated region . Using this cDNA we have constructed expression plasmids which allow the synthesis of 120 out of the 150 amino acids of the prohormone in E . coli . This portion of the prohormone gene was either fused to a segment of a bacteriophage structural gene or expressed alone . When expression was induced the fusion protein constituted 15% of the total bacterial cell protein while the prohormone alone was 5% . Both proteins are recognized by antiserum raised against porcine VIP . They provide protein to study the precursor-product relationship of the hormone plus the possibility of identifying cryptic regulatory peptides contained within the prohormone.

Mol Gen Genet, 1985, 199(3), 476 - 80
An electron microscopic heteroduplex study of the sequence relations between the bacteriophages LP52 and theta; Storchova H et al.; The genomes of the phylogenetically related but morphologically distinct bacteriophages LP52 and theta (theta) were compared by electron microscopic heteroduplex analysis . The heteroduplex maps were aligned with known restriction maps . In the heteroduplices of LP52 DNA (63.8 kb) with the DNA of the lytic phage theta c (65.9 kb) the tracts of homologous DNA cover about 50% of the genome length and are interspaced by four large and ten smaller non-base-paired regions . The largest block of non-homologous DNA (18.9 kb), represents the right-hand end and there is an unmatched piece of DNA at the left-hand end as well . Most of the heterology is due to substitution resulting in the conservation of the total length of DNA; the three insertions/deletions amount to less than 3.2% of the genome length . Heteroduplices between the DNAs of phage LP52 and the temperate phage theta 1 (65.0 kb) resembled those of LP52:theta c except for the absence of minor loops . Heteroduplex theta c:theta 1 displayed about 9% heterology in seven separate loops which coincided with sections of diversity on the restriction maps; 4.8% of theta 1 DNA did not hybridize with either theta c or LP52 DNA.

Mol Gen Genet, 1985, 199(3), 465 - 70
Recombination in the lambda repressor gene: evidence that very short patch (VSP) mismatch correction restores a specific sequence; Lieb M; The mutation am6 in the cI gene of bacteriophage lambda is identified as a C----T transition in a 5'CCATGG sequence . In four-factor crosses of am6 with nearby mutations in cI, the frequencies of cI+ recombinants are much higher than expected from the physical distances . A very short patch (VSP) mismatch repair system is presumed to recognize am6/am+ mispairs in the heteroduplexes that accompany recombination between the outside markers . Mutation am6 is corrected to am+; correction of am+ to am6 was not detected . Clear-plaque mutation 1-1 in cI is a T----C transition in a 5'CTTGG sequence, resulting in the sequence 5'CCATGG . When 1-1 was crossed with nearby mutations in gene cI, there were no excess cI+ recombinants, which would result from repair of CCTGG (1-1) to CTTGG (cI+) . However, in crosses of cI+ phages with mutation 1-1, there was an excess of cI- recombinants, indicating that cI+ was repaired to 1-1 . Preferential repair does not require adenine or cytosine methylation: when repairing a mismatch, the VSP repair system apparently identifies specific mispaired bases by sequence alone.

Basic Life Sci, 1985, 30, 33 - 44
Mechanisms of transposition in bacteria; Berg DE; The transposable elements of bacteria are diverse in size, functional arrangement, DNA sequence, and in their modes of transposition . We review here data suggesting that the kanamycin-resistance transposon Tn5 moves without replicating (conservative transposition), but the ampicillin-resistance transposon Tn3 is duplicated when it transposes, and that both the chloramphenicol-resistance transposon Tn9 and bacteriophage Mu are replicated in some events but not in others . A model is presented in which conservative and replicative transposition are alternative branches of a single pathway.

Mol Gen Genet, 1985, 198(3), 509 - 13
The novel gene(s) ARD of plasmid pKM101: alleviation of EcoK restriction; Belogurov AA et al.; The host-controlled K restriction of unmodified phage lambda was 10-100-fold alleviated in the wild-type strain E . coli K12, carrying plasmid pKM101 of incompability group N . pKM101-mediated release of K restriction was also observed in lexA-, recA-, and recB- strains of E . coli K12 . By restriction mapping Tn5 insertions in pKM101, which reduced pKM101-mediated alleviation of restriction, were shown to be located within the BglIIB fragment approximately 11 kb anticlockwise from the RI site of pKM101 . We have termed the gene(s) promoting the alleviation of K restriction of phage lambda ard (alleviation of restriction of DNA) . It was shown (1) that ard function affected only the EcoK restriction system and not the EcoB, EcoRI, EcoRIII, or EcoPI systems, (2) ard gene(s) did not mediate EcoK type modification of lambda DNA and did not increase the modification activity of the EcoK system in a way similar to that observed with gene ral of bacteriophage lambda.

Gene, 1985, 34(2-3), 147 - 53
Cloning of cDNAs coding for rat hepatic microsomal UDP-glucuronyltransferases; Jackson MR et al.; Radioiodinated, affinity-purified, anti-UDP-glucuronyltransferase (UDPGT) antibodies have been used to isolate cDNAs coding for UDPGT(s) from a rat liver cDNA library cloned in the expression vector bacteriophage lambda gt11 . The sizes of ten cloned cDNAs range from 0.3-2.1 kb . The identity of the cDNAs was confirmed by the hybrid-select translation and immunochemical analyses . Restriction mapping indicates that two classes of cDNA coding for different UDPGT mRNAs have been cloned.

Gene, 1985, 34(1), 9 - 15
Cloning and expression of genes for lysozyme and a 20-kDal protein of colitis bacteriophage; Vasavada HA et al.; BamHI fragments of colitis phage DNA were cloned in pBR322 DNA, and the recombinant clones carrying the lysozyme gene were identified by lysozyme activity . The inserted DNA was 1.2 kb long and when expressed in minicells it produced lysozyme and a 20-kDal protein . Colitis-phage-specific mRNAs which hybridized to the insert were 0.5 kb and 0.7 kb long and were translated into lysozyme and a 20-kDal protein, respectively, in a cell-free system derived from rice embryos . They were transcribed as monocistronic mRNAs using the internal promoters present on the inserted DNA.

Gene, 1985, 33(3), 367 - 71
A novel deletion found during cloning of a synthetic palindromic DNA; Goodchild J et al.; A 212-bp palindromic DNA comprising two copies of the left end of bacteriophage Mu was assembled from chemically synthesized oligonucleotides and inserted into plasmid pUC9 . When cloned and propagated in Escherichia coli, the palindrome was found to be unstable and was generally lost . However, in a few cases, a precise, asymmetric deletion of one half of the insert was observed . This pattern of deletion suggests that the symmetry axis region of the palindrome was involved as recognition site in the deletion process.

Gene, 1985, 33(3), 363 - 5
The DNA between Rz and cosR in bacteriophage lambda is nonessential; Hernandez VJ et al.; Near the right end of phage lambda DNA, between gene Rz and the cos site, are 2050 bp of apparently non-coding DNA . We have cloned a lambda DNA fragment containing this DNA into a plasmid and constructed a deletion, omega l, extending from a site within the Rz gene to a site about 560 bp from cos . This deletion could be recombined into viable lambda phage at a frequency equal to that observed for the undeleted sequence . Recombinant phage lambda carrying the omega l deletion were demonstrated to have the same burst size and kinetics of phage production as undeleted lambda . The omega l deletion can be used to extend the capacity of lambda cloning vectors and to provide a region for the insertion of heterologous DNA which should exhibit controllable high level expression from the lambda late promoter, p'R.

Gene, 1985, 33(3), 351 - 5
Nucleotide sequence of a major class-III phage-T3 RNA-polymerase promoter located at 98.0% of phage-T3 genetic map; Sarkar P et al.; The entire nucleotide sequence of a 409-bp HincII fragment, located within the MboI-E fragment on bacteriophage T3 DNA and containing a major class-III T3 RNA polymerase promoter positioned at 98% on the standard T3 genetic map, has been determined . Alignment of this class-III promoter with previously determined T3 RNA polymerase promoters, with start points of transcription (+1) in register, indicates high degree of sequence conservation between position -16 to +6 among all T3 RNA polymerase promoters . The conserved portion of the (-) strand sequence is 5'-A-TA-T-AT-A-C-C-C-T-C-A-C-T-A-A-A-G-G-G-A---3' . This fragment also contains an open reading frame (ORF) with a translational start codon located at position +146 which is preceded by a potential ribosome binding site (RBS) . There is more than 70% amino acid-sequence homology between the deduced sequences of the -NH2 terminal region of this putative T3 phage protein and the corresponding protein coded by bacteriophage T7 (protein of T7 gene 19.5).

Gene, 1985, 33(3), 341 - 9
A plasmid cloning system utilizing replication and packaging functions of the filamentous bacteriophage fd; Geider K et al.; DNA cloning vectors were developed which utilize the replication origin (ori) of bacteriophage fd for their propagation . These vectors depend on the expression of viral gene 2 that was inserted into phage lambda, which in turn was integrated into the host genome . The constitutive expression of gene 2 in the host cells is sufficient for the propagation of at least 100 pfd plasmids per cell . In addition to the fd ori, the pfd vectors carry various antibiotic-resistance genes and unique restriction sites . Some of these vectors have no homologies to commonly used pBR plasmids or to lambda DNA . The nucleotide sequence of the vectors can be deduced from published sequences . Large DNA inserts can be stably propagated in pfd vectors; these are more stable than similar DNA fragments cloned in intact genomes of filamentous bacteriophage . Inclusion of phage sequences required for efficient phage packaging and infection with a helper phage resulted in formation of phage particles containing single-stranded plasmid genomes . Growth at 42 degrees C without selective pressure results in loss of pfd plasmids.

Mol Gen Genet, 1985, 199(1), 123 - 32
Physical characterization of the genome of the Myxococcus xanthus bacteriophage MX-8; Stellwag E et al.; We have constructed a restriction map for the genome of bacteriophage MX-8 from Myxococcus xanthus using the enzymes PvuII, MboI, and EcoRI . The phage genome size, as determined by restriction analysis, is 51.7 +/- 0.6 Kb . Double digestions, redigestions of isolated fragments, and crossed-contact hybridization of partial digestion products show that the restriction map is circular . Restriction analysis and Southern hybridization show that the phage DNA molecules are packaged sequentially from a concatemer starting from a specific site which we have mapped . The DNA molecules have an average terminal redundancy of approximately 8% and are circularly permuted over at least 40% of the genome.

J Gen Microbiol, 1985 Jan, 131 ( Pt 1), 129 - 34
Bacteriophage P1 derivatives unaffected in their growth by a large inversion or by IS insertions at various locations; Iida S et al.; Several plaque-forming phage P1 derivatives carrying DNA rearrangements associated with IS elements are described . They have IS1, IS3 and IS5 inserted in four distinct locations, all of which are non-essential regions for phage P1 propagation . One derivative carries a genome segment, inverted relative to the one in the P1 wild-type genome, between two inverted copies of IS1 . The inverted DNA segment spans about 23 kb of the 90 kb long P1 genome and it includes the invertible C segment . This phage is as viable as an isomeric P1 which carries the relevant segment in its original orientation . These results are discussed with regard to the genome organization of phage P1.

Gene, 1985, 33(1), 103 - 19
Improved M13 phage cloning vectors and host strains: nucleotide sequences of the M13mp18 and pUC19 vectors; Yanisch-Perron C et al.; Three kinds of improvements have been introduced into the M13-based cloning systems . (1) New Escherichia coli host strains have been constructed for the E . coli bacteriophage M13 and the high-copy-number pUC-plasmid cloning vectors . Mutations introduced into these strains improve cloning of unmodified DNA and of repetitive sequences . A new suppressorless strain facilitates the cloning of selected recombinants . (2) The complete nucleotide sequences of the M13mp and pUC vectors have been compiled from a number of sources, including the sequencing of selected segments . The M13mp18 sequence is revised to include the G-to-T substitution in its gene II at position 6 125 bp (in M13) or 6967 bp in M13mp18 . (3) M13 clones suitable for sequencing have been obtained by a new method of generating unidirectional progressive deletions from the polycloning site using exonucleases HI and VII.

J Gen Virol, 1985 Jan, 66 ( Pt 1), 55 - 68
Analysis of bovine cytomegalovirus genome structure: cloning and mapping of the monomeric polyrepetitive DNA unit, and comparison of European and American strains; Ehlers B et al.; The genome of bovine cytomegalovirus (BCMV) strain 66-P-347 consists of double-stranded, linear DNA with a size of 144 +/- 6 kb . It contains polyrepetitive DNA (prDNA) segments like five other BCMV strains . The restriction patterns of the prDNA of all six strains are very similar and indicate that monomeric prDNA units are either 1950 bp (class I and Ia), 2350 bp (class II) or 2750 bp (class III) in size . The complete unit sequence of strain 66-P-347 (class II) was cloned in bacteriophage vector M13mp7 and mapped by the restriction enzymes EcoRI, BamHI, Bg/I, NaeI, SstII and PstI . From these results the restriction maps of the prDNA of the other five strains were deduced . The 400 bp differences in size between the three prDNA classes are a consequence of the appearance of an internal 200 bp sequence being present one-, three- or fivefold in head-to-tail formation . Hybridization of 35S-labelled recombinant phage DNA to Southern blots with DNA of the different strains leads to the conclusion that prDNA units are present as multimers in tandem formation at both genomic termini in the same orientation . The number of terminal repeat units varies between individual molecules of a strain, but the actual terminal sequences are identical.

J Bacteriol, 1985 Jan, 161(1), 314 - 20
Molecular cloning of the phenylalanine ammonia lyase gene from Rhodosporidium toruloides in Escherichia coli K-12; Gilbert HJ et al.; A genomic library of Rhodosporidium toruloides DNA was constructed in bacteriophage lambda 1059 . Recombinant phage containing phenylalanine ammonia lyase (PAL) gene sequences were identified by using 32P-labeled cDNA to partially purified PAL mRNA . The PAL gene was subcloned on an 8.5-kilobase PstI DNA restriction fragment into pUC8 to generate the recombinant plasmid pHG2 . A restriction map of the PAL gene, together with its flanking regions, was constructed . Northern hybridization analysis of R . toruloides RNA with a restriction fragment encoding part of the PAL gene indicates that PAL mRNA is 2.5 kilobases in length . A single-stranded DNA hybridization probe was constructed and used to quantitate PAL mRNA levels in R . toruloides grown under different physiological conditions . PAL mRNA levels paralleled changes in functional PAL mRNA and antigen . These data are consistent with control of PAL expression being at the level of transcription.

J Bacteriol, 1985 Jan, 161(1), 219 - 22
Leucine tRNA family of Escherichia coli: nucleotide sequence of the supP(Am) suppressor gene; Thorbjarnardottir S et al.; We describe the cloning and the DNA sequence of an amber suppressor allele of the Escherichia coli leuX (supP) gene . The suppressor allele codes for a tRNA with anticodon CUA, presumably derived by a single base change from a CAA anticodon . The mature coding sequence of the leuX gene is preceded by a putative Pribnow box sequence (TATAAT) and followed by a termination signal . The sequence of the leuX-coded tRNA is compared with the sequences of the four remaining tRNALeu isoacceptors of E . coli and with two tRNALeu species from bacteriophage T4 and T5 . The conserved nucleotides in these seven tRNAs recognized by E . coli leucyl-tRNA synthetase are located mainly in the aminoacyl stem and in the D-stem/loop region.

J Bacteriol, 1985 Jan, 161(1), 179 - 82
Release of respiratory control in Escherichia coli after bacteriophage adsorption: process independent of DNA injection; Letellier L et al.; Adsorption of phages T4, T5, and BF23 to previously starved Escherichia coli cells triggered the immediate release of respiratory control . A similar stimulation of respiration was induced after T4 ghost attachment, showing that this process was independent of the mechanism of DNA injection . Rather, this change in the respiratory rate was related to the transient depolarization of the cytoplasmic membrane also induced after phage and ghost adsorption . Both processes were suppressed by addition of ethylene glycol-bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid in the case of T4 (phage and ghosts) but not of T5 and BF23 . The increase in respiratory rate observed after phage adsorption was of a magnitude similar to that induced by protonophores . Since other treatments that depolarize the membrane without a massive proton influx did not increase the rate of respiration of starved bacteria with the same efficiency, these results suggest that phage adsorption induced an entry of protons into the cell cytoplasm.

J Virol, 1985 Jan, 53(1), 296 - 8
Involvement of DNA gyrase in bacteriophage T7 growth; Steck TR et al.; We have found that the burst size of bacteriophage T7 was decreased in two Escherichia coli temperature-sensitive gyrase mutants incubated at the restrictive temperature . This reduction in burst size indicates that gyrase may be required for T7 growth.

J Virol, 1985 Jan, 53(1), 192 - 7
Autoregulation of the bacteriophage P22 scaffolding protein gene; Wyckoff E et al.; During the formation of each bacteriophage P22 head, about 250 molecules of the product of gene 8, scaffolding protein, coassemble with and dictate correct assembly of the coat protein into a proper shell structure . At approximately the time that DNA is inserted inside the coat protein shell, all of the scaffolding protein molecules leave the structure . They remain active and participate in several subsequent rounds of shell assembly . Previous work has shown that scaffolding protein gene expression is affected by the head assembly process and has generated the hypothesis that unassembled scaffolding protein negatively modulates the expression of its own gene but that it lacks this activity when complexed with coat protein in proheads . To test this model, a P22 restriction fragment containing the scaffolding and coat protein genes was cloned under control of the lac promoter . These cloned genes were then expressed in an in vitro DNA-dependent transcription-translation reaction . The addition of purified scaffolding protein to this reaction resulted in reduced scaffolding protein synthesis relative to coat and tail protein synthesis to an extent and at a protein concentration that was consistent with the observed reduction in vivo . We conclude that scaffolding protein synthesis is autoregulated and that scaffolding protein is the only phage-coded protein required for this process . In addition, these experiments provide additional evidence that this autoregulation is posttranscriptional.

Curr Genet, 1985, 10(2), 139 - 45
The endpoints of an inversion in wheat chloroplast DNA are associated with short repeated sequences containing homology to att-lambda; Howe CJ; The endpoints of an inversion in wheat chloroplast DNA are shown to be associated with copies of a short repeated sequence . Recombination across the repeats in an inverted configuration may have been responsible for the inversion, although they are currently in a direct orientation owing to a second inversion . The repeated sequence contains an element homologous to the core of the bacteriophage lambda att-site, which can function as such in vivo.

Gene, 1985, 40(1), 39 - 46
Deletion and fusion analysis of the phage phi X174 lysis gene E; Maratea D et al.; The lysis gene, E, of bacteriophage phi X174 has been subjected to deletion and gene fusion analysis . C-terminal deletions of as few as 17 of the 91 codons inactivate the cloned E gene, which in its intact form can cause lysis of the host cell . Fusion of lacZ to deletion joints at the 59th codon or beyond apparently restores lethal and lytic competence to the respective E deletion alleles, whereas a fusion at the 23rd codon remains non-lethal . The lethal E phi lacZ fusions are also lethal to a mutant, designated slyD, which was isolated as a spontaneous E . coli mutant resistant to the expression of the intact E gene . slyD appears to be linked to rpsE . The data are interpreted in terms of a model in which E-mediated lethality requires oligomerization of the E gene product . Calculations based on the beta-galactosidase activity accumulated by the time of lethal action of E phi lacZ suggest that fewer than 1000 molecules of E gene product are required for lysis and probably fewer than 100 are required for loss of host viability.

Mol Gen Genet, 1985, 201(2), 329 - 33
Weigle reactivation and mutagenesis of bacteriophage lambda in lexA(Def) mutants of E . coli K12; Calsou P et al.; The SOS response in UV-irradiated bacteria enhances the survival and mutagenesis of infecting damaged bacteriophage lambda . In a lexA(Def) strain, SOS bacterial genes are fully derepressed by an inactivating mutation in the LexA repressor gene . We tested several lexA(Def) derivative strains for their capacity to constitutively promote high survival and mutagenesis of irradiated lambda . We showed that UV irradiation of the lexA(Def) host bacteria is still necessary for optimal efficiency of both these SOS functions, which are dependent on the umuC gene product and an activated form of RecA protein.

Gene, 1985, 39(1), 71 - 6
Regulation and expression of the bacteriophage mu mom gene: mapping of the transactivation (dad) function to the C region; Hattman S et al.; Expression of the bacteriophage Mu mom gene is under tight regulatory control . One of the factors required for mom gene expression is the trans-acting function (designated Dad) provided by another Mu gene . To facilitate studies on the signals mediating mom regulation, we have constructed a mom-lacZ fusion plasmid which synthesizes beta-galactosidase only when the Mu Dad transactivating function is provided . lambda pMu phages carrying different segments of the Mu genome have been assayed for their ability to transactivate beta-galactosidase expression by the fusion plasmid . The results of these analyses indicated that the Dad transactivation function is encoded between the leftmost EcoRI site and the lys gene of Mu; this region includes the C gene, which is required for expression of all Mu late genes . Cloning of an approx . 800-bp fragment containing the C gene produced a plasmid which could complement MuC- phages for growth and could transactivate the mom-lacZ fusion plasmid to produce beta-galactosidase . These results suggest that the C gene product mediates the Dad transactivation function.

Gene, 1985, 39(1), 61 - 70
The mom gene of bacteriophage mu: a unique regulatory scheme to control a lethal function; Kahmann R et al.; The mom gene of bacteriophage Mu encodes a DNA modification function which converts adenine to acetamido adenine in a sequence-specific manner . The mom gene itself is subject to a complex regulation: gene expression requires methylation by the Escherichia coli Dam methylase of specific sites upstream of the mom promoter and transactivation of the promoter by a Mu gene product . The requirement for transactivation can be overcome when mom is transcribed from foreign promoters . When cloned into various sites in pBR322, the mom gene is always found in an orientation where transcription from vector promoters is excluded . The productive orientation is lethal to the cell . This effect is mediated by the concerted action of the mom gene product and the product of gene com (control of mom, previously termed ORF-x) whose coding region overlaps the 5-coding region of the mom gene . When mom is expressed from its own promoter, internal deletions in com completely abolish expression of the mom gene . Fragments lacking the 5' end of com can be cloned downstream of constitutive plasmid promoters . The com gene product itself is not lethal to the cell . The region encoding mom has been cloned in pL expression vectors . The mom gene product, a peptide of 27 kDal, has been visualized on gels . Efficient expression of Mom from pL requires gene com . A fusion between MS-2 polymerase and com has been generated . The fusion product is made in large amounts, whereas the mom gene product is not overproduced although the gene is present on the same transcriptional unit.(ABSTRACT TRUNCATED AT 250 WORDS)

Gene, 1985, 38(1-3), 253 - 8
High-level synthesis of the phage lambda outer-membrane protein from the cloned lom gene; Munn AL et al.; A 2.7-kb KpnI-EcoRI fragment carrying the lom gene of bacteriophage lambda has been cloned into plasmid pPR42 and recloned into the SmaI site of pUC9 . Large quantities of Lom were seen in outer-membrane (OM) preparations of strains carrying the latter clone and its derivatives . The reading frame of lom was identified as ORF206a . The protein was not demonstrably associated either covalently or non-covalently with the peptidoglycan layer of the cell envelope.

Gene, 1985, 35(3), 313 - 20
A CII-responsive promoter within the Q gene of bacteriophage lambda; Stephenson FH; A site within the phage lambda Q gene shares homology with the CII-activated promoters, pE and pI, and is oriented in the direction opposite to that of Q gene transcription . A DNA fragment containing this site can serve as a template for CII-activated transcription in vitro . To ask if this presumptive CII control site functions as a CII-activated promoter in vivo, a restriction fragment containing this promoter has been cloned on a plasmid so that synthesis of beta-galactosidase will be under its control . When CII protein is supplied in trans from a compatible plasmid, this promoter, designated PaQ, is activated to produce beta-galactosidase . A promoter positioned within the Q gene which can be activated by CII protein to initiate transcription in an anti-sense direction should result in an interference with Q gene expression, enhancing CII regulation of late functions, and adding to the list of known CII controls on the lysogenic response.

Dev Biol Stand, 1985, 61, 265 - 71
Bordetella pertussis filamentous hemagglutinin gene: molecular cloning of a potential coding sequence; Reiser J et al.; We have constructed an expression library of Bordetella pertussis DNA sequences, cloned in Escherichia coli . Random DNA fragments were inserted into the beta-galactosidase gene of bacteriophage lambda gt11 and plaques were screened with antibodies directed against filamentous hemagglutinin . The antigen-positive clone obtained expressed B . pertussis sequences as polypeptides fused to beta-galactosidase . The fusion polypeptides reacted both with antibodies to beta-galactosidase and with antibodies to filamentous hemagglutinin . The positive clone contains B . pertussis DNA sequences as determined by specific hybridization to Bordetella DNA.

Adv Exp Med Biol, 1985, 188, 31 - 47
Biosynthesis of rat preprosomatostatin; Goodman RH et al.; The biologically active forms of somatostatin, somatostatin-14 (SS-14) and somatostatin-28 (SS-28) arise by post-translational cleavage of prosomatostatin . Prosomatostatin in turn is derived from a larger precursor, preprosomatostatin . We have previously reported the structure of a complementary DNA molecule encoding rat preprosomatostatin . The nucleotide sequence of this cDNA indicated that SS-14 and SS-28 are located at the carboxy-terminus of a 116 amino acid precursor . At the amino-terminus of the precursor is a hydrophobic region characteristic of a leader or pre-sequence . Sequential Edman degradations of cell-free translation products synthesized in the presence of microsomal membranes indicate that preprosomatostatin is cleaved within the endoplasmic reticulum to form prosomatostatin, a precursor of 92 amino acids . To begin to elucidate the factors which regulate the expression of the rat somatostatin gene, we have determined the sequence of the gene isolated from recombinant bacteriophage libraries . The gene spans 1.2 kilobases in length and is interrupted within the coding sequence of prosomatostatin by a single intron of 630 bases . A variant of the Goldberg-Hogness promotor, TTTAAA, is located 31 bases upstream from the transcriptional start point . A repetitive sequence was identified in the 5' region of the gene within 650 bases of the promoter . The nucleotide sequence of this region reveals an alternating GT sequence 42 bases in length characteristic of DNA with Z-forming potential . Such sequences are thought to influence the expression of other eukaryotic genes.

Acta Virol, 1985 Jan, 29(1), 73 - 8
Isolation and partial characterization of temperature bacteriophages from enteropathogenic strains of Escherichia coli 0111:K58; Sezzano P et al.; We report studies on ten strains of Escherichia coli 0111:K58 isolated from children with acute diarrhea . Our results show that these E . coli strains do not produce the pathogenic factors of enterotoxic E . coli (ETEC) and are lysogenic for phages belonging to two groups that differ for host range, kinetics of thermal inactivation, antigenicity and morphology . These data support the hypothesis that these phages may in vivo contribute to reduction of the number of common E . coli strains by lytic infection favouring the development of the enteropathogenic strain of E . coli.

J Bacteriol, 1985 Jan, 161(1), 411 - 6
Entry exclusion determinant(s) of IncN plasmid pKM101; Winans SC et al.; pKM101 renders its host a poor recipient in conjugal matings with genetically distinguishable derivatives of itself . The gene(s) primarily responsible for this, denoted eex, is located in between genes required for both conjugal transfer and sensitivity to donor-specific bacteriophage, although it itself is not necessary for transfer . A gene linked to, or coincident with, the region needed for vegetative plasmid replication also inhibited establishment of related plasmids under certain conditions . Construction of an operon fusion between eex and the Escherichia coli lac promoter has shown that this gene is transcribed in a clockwise fashion on the circular map of pKM101 . To date, we have not been able to visualize a protein product(s) of the eex gene(s).

Cancer Surv, 1985, 4(3), 493 - 516
Translesion DNA synthesis: polymerase response to altered nucleotides; Strauss BS; A system for the determination of the specificity of incorporation opposite lesions during DNA synthesis past damaged bases has been used as a model of mutation . The system, based on the dideoxynucleotide sequence method, uses lesions in the template strand as chain terminators . As a first approximation, such lesions constitute non-instructive sites in the DNA . DNA synthesis catalysed by bacteriophage T4 DNA polymerase terminates one nucleotide 3' to the lesion on the template strand . With other polymerases, synthesis may terminate opposite the lesion . The details of termination site depend on the enzyme, the metal ion (Mg2+ or Mn2+), the lesion and the particular nucleotide sequence . The sequence effect for termination on normal templates has two components, one ascribable to secondary structure, the other intrinsic to the sequence itself . DNA molecules terminated before lesions may be used as substrates in 'second stage' reactions in which elongation can be detected on the addition of particular dNTPs (deoxynucleoside triphosphates) to a reaction mixture . The specificity of elongation depends on the polymerase, on the activity of the 3'--greater than 5' editing nuclease and on the particular lesion . DNA polymerases have a preference for the addition of purines, particularly adenine, opposite non-instructional sites . This preference suggests an explanation for the specificity of base substitution mutations: treatments that produce non-instructional sites from purines will lead to transversions, treatments that affect pyrimidines lead to transitions . Even though a base is added opposite a lesion, further elongation may be rate limiting . Whether or not elongation occurs is dependent on the sequence 5' to the lesion on the template strand . The interactions of the factors affecting bypass: polymerase, lesion and sequence, may well result in an idiosyncratic behaviour for each mutable site.

Mol Gen Genet, 1985, 198(2), 304 - 8
Modulation of gene expression in Escherichia coli infected with single-stranded bacteriophage phi X174; Ghosh A et al.; Synthesis of tryptophanase, D-serine deaminase and alkaline phosphatase in Escherichia coli C was repressed as the result of infection with the single-stranded DNA bacteriophage phi X174 . However, the degree of repression differed, the more catabolite-sensitive the operon was, the more severe was the repression . For the catabolite-sensitive enzymes it was found that cyclic adenosine 3'5' monophosphate (cyclic AMP or cAMP) was unable to release or reduce the phage-induced inhibition . Experiments with amber mutants of phi X174 revealed that A, product of cistron A, was responsible for the inhibition . The cistron A product probably acted at the level of transcription . The possible role of A in the observed modulation of gene expression is discussed.

Nucleic Acids Symp Ser, 1985, (16), 197 - 200
Bleomycin cleaves DNA depending on DNA primary, secondary, and tertiary structures; Ueda K et al.; The cleavage by bleomycin-Fe(II) complex in the presence of dithiothreitol was investigated by using 3'- or 5'-end-labeled DNA containing the region of the bacteriophage G4 origin of complementary strand synthesis as substrates . Bleomycin cleaved single-stranded DNA substrates preferentially at inverted repeat sequences, which potentially form stem-and-loop structures, in addition to the primary sequence specificity previously reported . DNA sequences preferentially cleaved in the double-stranded substrate were resistant when they lay outside the stem regions . These results suggest the formation of three predicted stem-and-loop structures and other possible secondary structures near the replication origin . Changes of the degree of bleomycin-induced DNA cleavage in a NaCl concentration between 0 and 50 mM suggest that a subtle change of ionic conditions within the double helix, or of DNA conformation, or of both, may occur at 0-50 mM NaCl . Bleomycin appears to be a useful reagent for analyzing secondary and tertiary structures of DNA.

Ultramicroscopy, 1985, 16(3-4), 436 - 50
Correlation of surface topography of metal-shadowed specimens with their negatively stained reconstructions; Buhle EL Jr et al.; We present a comparison of surface reconstructions from three different freeze-dried and unidirectionally metal-shadowed specimens (i.e . bacteriophage T4 polyheads, crystalline actin sheets and nuclear pore complexes) with two- or three-dimensional reconstructions of the same specimens when prepared by negative staining . Based on these and many published results, the following conclusions have been reached: With a "cooperative" specimen (e.g . the polyheads), the surface reconstruction computed from a metal-shadowed replica compares favorably with two- or three-dimensional reconstructions obtained from the same specimen after negative staining at the 3-4 nm resolution level . This relatively "poor" level at which the surface topographies of the two preparations can be compared appears to be set by a "practical" resolution limit (i.e . of distinct and reproducible structural detail) of metal replicas of biological specimens, despite the appearance of weak higher-order diffraction spots (i.e . corresponding to 2-3 nm) . While in some cases (e.g . the crystalline actin sheets) the surface reliefs of metal replicas may bear little resemblance to the actual structure under investigation, the replicas may still contain sufficient features to establish the polarity or handedness of the structure (i.e., the "top" and "bottom" surfaces of a crystalline sheet) . Information from negatively stained specimens is usually complementary with information from freeze-dried and metal-shadowed specimens . However, there are artifacts in both techniques, and we present an example with the nuclear pore complex, where these techniques yield confusing results.

Gene, 1985, 35(3), 249 - 58
Cloning and overexpression of Moloney murine leukemia virus reverse transcriptase in Escherichia coli; Kotewicz ML et al.; A pBR322-derived expression vector, plasmid pKD1, was constructed containing the strong leftward promoter (pL) of bacteriophage lambda, the ribosome-binding site (RBS) of the cII gene of lambda, and a unique downstream NdeI restriction site for construction of an ATG initiation codon . The section of the pol gene of Moloney murine leukemia virus (M-MLV) that codes for reverse transcriptase (RT) was cloned into the NdeI site of this vector generating the plasmid pRT103 . Upon thermal induction, enzymatically active RT was expressed in Escherichia coli {pRT103} . The identity of this activity was confirmed by its template specificity and its sensitivity to inhibition by immunoglobulin G (IgG) prepared against authentic murine RT . RT represented 20% of the newly synthesized protein in these cells 20 min after induction.

J Mol Biol, 1984 Dec 25, 180(4), 837 - 63
DNA injection proteins are targets of acridine-sensitized photoinactivation of bacteriophage P22; Bryant JL Jr et al.; Viruses and other nucleoprotein complexes are inactivated on exposure to white light in the presence of acridine and related dyes . The mechanism is thought to involve generation of singlet oxygen or related species, but the actual molecular targets of the inactivating event have not been well defined . We have re-examined the mechanism of dye-sensitized photoinactivation taking advantage of the well characterized bacteriophage P22 . Though the inactivated phage absorb to their host cells, the cells are not killed and genetic markers cannot be rescued from the inactivated phage . These observations indicate that the chromosome is not injected into the host cell . However, the DNA of the damaged particles shows no evidence of double-stranded breaks or crosslinking . The DNA injection process of P22 requires three particle-associated proteins, the products of genes 7, 16 and 20 . Gp16, which can act in trans during injection, is inactivated in the killed particles . Sodium dodecyl sulfate/polyacrylamide gel analysis reveals that gp16, gp7 and gp20 are progressively covalently damaged during photoinactivation . However, this damage does not occur in particles lacking DNA, indicating that it is DNA-mediated . Similar findings were obtained with acridine orange, acridine yellow, proflavin and acriflavin . These results indicate that the actual targets for inactivation are the DNA injection proteins, and that the lethal events represent absorption of photons by acridine molecules stacked in a region of DNA closely associated with the injection proteins.

J Mol Biol, 1984 Dec 25, 180(4), 865 - 80
Mutations that alter the DNA binding site for the bacteriophage lambda cII protein and affect the translation efficiency of the cII gene; Place N et al.; The efficiency of translation of the cII gene of bacteriophage lambda is greatly reduced by the cII3059 mutation, a GUU----GAU (Val----Asp) change in the second cII codon . Mutations in the third and fourth codons of the cII gene, called ctr mutations, reverse this translation deficiency . Lambda cII3059 ctr-1, which has a GCA----ACA (Ala----Thr) change in the fourth cII codon, produces about half the normal level of cII activity in liquid cultures, and lambda cII3059 ctr-2 and lambda cII3059 ctr-3, which have identical CGT----CGC changes in the third codon, produce normal levels of cII activity in liquid culture . Since the cII protein of ctr-3 has the same primary sequence as that of lambda cII3059, the cII- phenotype of lambda cII3059 can be explained entirely by the deficiency of translating cII mRNA . We propose that ctr mutations increase translation efficiency by destabilizing a stable stem structure which can be formed by cII mRNA . The ctr mutations lie in an overlapping regulatory region which contains, in addition to sequence elements that influence the rate of cII translation, a region to which cII protein binds to activate transcription from the PRE promoter . The ctr-1 mutation alters the cII recognition sequence from 5'-T-T-G-C-N6T-T-G-C-3' to 5'-T-T-G-C-N6T-T-G-T-3', but has no effect on PRE activity . Since a C----T change in the first (5'-proximal) T-T-G-C sequence (to yield 5'-T-T-G-T-N6T-T-G-C) greatly lowers cII binding affinity, cII protein must not recognize the two T-T-G-C sequences in an identical manner.

J Biol Chem, 1984 Dec 25, 259(24), 15069 - 77
Termination sites of the in vitro DNA synthesis on single-stranded DNA photosensitized by promazines; Merville MP et al.; Bacteriophage phi X174 and M13 mp9 single-stranded DNA molecules were primed either with restriction fragments or synthetic primers and irradiated with near UV light in the presence of promazine derivatives . These DNAs were used as template for in vitro complementary chain synthesis by Escherichia coli DNA polymerase I large fragment . Chain terminations were observed by denaturing polyacrylamide gel electrophoresis of the synthesis products and localized by comparison with a standard dideoxy sequencing pattern . More than 90% of the chain terminations were mapped exactly one nucleotide before a guanine residue . In addition, photoreaction was shown to occur more predominantly with guanine residues localized in single-stranded parts of the genome . The same guanine residues could also be damaged when the reaction was performed, in the dark, in the presence of the artificially generated promazine cation radicals . Using the BamHI-SmaI adaptor (5'GATCCCCGGG-3'), it was shown that the guanine alteration was a covalent addition of the promazine, or of a cation radical photodegradation product, on the guanine moiety . Kinetics of chlorpromazine photoaddition on single-stranded and double-stranded DNAs were determined.

J Biol Chem, 1984 Dec 25, 259(24), 15425 - 32
Bacteriophage T4 gene 44 DNA polymerase accessory protein . Sequences of gene 44 and its protein product; Spicer EK et al.; Bacteriophage T4 gene 44 protein is a DNA polymerase accessory protein which is required for T4 DNA replication . We have isolated the gene for 44 protein from a previously constructed lambda-T4 hybrid phage (Wilson, G . G., Tanyashin, V . I., and Murray, N . E . (1977) Mol . Gen . Genet . 156, 203-214) . We report here the nucleotide sequence of gene 44 and about 60 nucleotides 5' upstream from its coding region, which is immediately adjacent to gene 45 . We have also purified 44 protein from T4-infected cells and submitted it to extensive protein chemistry characterization . Thus, considerable portions of the protein sequence predicted from the DNA sequence were confirmed by direct protein sequencing of peptides or by matching amino acid compositions of purified peptides . A total of 84% of the predicted amino acids was confirmed by the protein data . These studies indicate that gene 44 codes for a polypeptide containing 319 amino acids, with a calculated Mr = 35,371 . The coding region of gene 44 is preceded by a potential regulatory region containing sequences homologous to the Escherichia coli (-10) RNA polymerase binding region and to a conserved sequence at -25 to -30 found in other T4 middle genes . In addition, there are sequence similarities in the translation initiation regions of genes 44, 45, and rIIB, all of which are subject to regulation by regA protein.

J Mol Biol, 1984 Dec 25, 180(4), 947 - 60
Ribonucleoprotein organization of eukaryotic RNA . XXXI . Structure of the U1 small nuclear ribonucleoprotein; Lin WL et al.; A small nuclear ribonucleoprotein, U1 snRNP, has been implicated in mRNA processing . In this investigation sites of protein binding on U1 RNA were mapped by nuclease protection and RNA sequencing . Partially purified human U1 snRNP was sequentially digested with Escherichia coli RNAase III and S1 nuclease . The resistant ribonucleoprotein fragments were deproteinized, preparatively hybridized to the U1 RNA--complementary DNA strand of a human U1 gene cloned in bacteriophage M13, and displayed by electrophoresis . The nuclease-resistant U1 RNA fragments were between 23 and 63 nucleotides in length . Most of these fragments were not obtained when protein-free U1 RNA was similarly digested, whereas others were obtained in low yield from U1 RNA and much higher yield from U1 snRNP . RNA sequencing of the fragments revealed that the protein-protected sites in U1 snRNP correspond to base-paired stems I and II, loop a, and portions of stems III and IV (secondary structure nomenclature of Branlant et al., 1981) . Single, "bulged" pyrimidines are present within the protein-covered helical regions of stems I and III . Most interestingly, the single-stranded 5' end of U1 RNA, implicated in mRNA splicing, was also highly protected by protein . These results demonstrate that the great majority of U1 RNA is covered by protein in U1 snRNP . The association of protein with the 5' end of U1 RNA is in agreement with recent evidence that snRNP proteins potentiate the binding of this region of U1 RNA with pre-mRNA splice sites.

EMBO J, 1984 Dec 20, 3(13), 3055 - 62
Cloned single- and double-stranded DNA copies of potato spindle tuber viroid (PSTV) RNA and co-inoculated subgenomic DNA fragments are infectious; Tabler M et al.; A set of monomeric and oligomeric potato spindle tuber viroid (PSTV) specific DNA forms representing complete DNA copies of the circular PSTV RNA genome were constructed and cloned in plasmid pBR322 and bacteriophage M13 . Both single- and double-stranded PSTV DNAs are capable of initiating viroid replication in mechanically inoculated tomato plants where it normally proceeds via the RNA-RNA pathway without DNA being involved . All dimeric and higher multimeric forms were infectious irrespective of their polarity in the case of single-stranded DNA and regardless of their orientation in the vector DNA in the case of double-stranded DNA . The vector-inserted monomeric PSTV DNA units were also found to be infectious but of low specific infectivity which was increased when these monomers had been excised . Even two subgenomic DNA fragments, representing together the 359 nucleotides of the PSTV RNA genome, initiated the synthesis of viroid RNA progeny when co-inoculated although each fragment by itself is non-infectious . These results are discussed with respect to the infectivity previously observed with certain cloned DNAs of conventional RNA and DNA viruses.

EMBO J, 1984 Dec 20, 3(13), 3049 - 53
Study of the genetic organisation of a plant viral RNA genome by in vitro expression of a full-length DNA copy; Vos P et al.; The genetic approach for elucidating functions encoded by RNA plant viruses has been hampered by the lack of methods to select desired mutants following random mutagenesis . An alternative might be to copy RNA genomes into DNA and use methods for site-directed mutagenesis to modify specific regions of the DNA copy . Transcription of the DNA copy will subsequently produce viral RNA with desired mutations . We have constructed a full-length DNA copy of the smaller of the two cowpea mosaic virus (CPMV) RNAs, referred to as M RNA . The DNA copy was positioned downstream from the promoter of bacteriophage SP6 and using SP6 RNA polymerase, this copy and two derivatives of it containing a specific deletion and insertion, respectively, have been transcribed into RNA molecules which are efficiently translated in rabbit reticulocyte lysates . The results obtained show that the subsequent in vitro transcription and translation of DNA copies may be a powerful tool to unravel the genetic properties of viral RNA genomes.

EMBO J, 1984 Dec 20, 3(13), 3353 - 8
Different Z DNA forming sequences are revealed in phi X174 RFI by high resolution darkfield immuno-electron microscopy; Revet B et al.; The specific interaction between left-handed Z DNA sequences in negatively supercoiled bacteriophage phi X174 replicative form I (RFI) DNA and anti-Z DNA immunoglobulin G (IgG) was investigated by high resolution darkfield immuno-electron microscopy . DNA-antibody complexes were formed and maintained under optimal binding conditions, purified by column chromatography, and visualized after uranyl acetate staining without using aldehyde fixation, shadowing, or second antibody . Bivalent anti-Z DNA IgGs bound to RFI molecules, thus forming intramolecular bridges . They could also oligomerize separate molecules by intermolecular linking of Z DNA sequences . At relatively low ionic strength and low temperature, high affinity anti-Z IgG was retained at certain loci even after restriction endonuclease cleavage of the DNA . In these cleaved molecules some superhelices could be visualized in the loops generated by the bivalent IgG . To our knowledge this is the first example of polypeptide stabilization of local superhelical strain in a cut molecule . Z DNA sequences in phi X174 RFI DNA were mapped . Alternating tracts of purines and pyrimidines starting at nucleotides 763, 1027, 1714, 2146, 2363, 3504, 4161, 4911 and 5345 occur within the nine different anti-Z IgG binding sites which were expressed with varying frequencies (53-3%) on the molecules . Usually, a limited number of sites (generally less than or equal to 2) exists on any one molecule . The formation of multiple Z sites (at the extracted superhelix density) in a given molecule is probably non-cooperative due to relaxation of torsional stress by the B----Z transition . Z sites occur in several different genes, including regions where transcription is attenuated and, in one case, in front of a promoter of transcription.

J Mol Biol, 1984 Dec 15, 180(3), 399 - 416
Nucleotide sequence of bacteriophage T4 gene 23 and the amino acid sequence of its product; Parker ML et al.; We have determined the nucleotide sequence of gene 23 of bacteriophage T4 by the methods of Maxam and Gilbert and of Sanger . The identities of approximately 80% of the amino acid residues of the major capsid protein which is encoded by gene 23 were determined additionally by Edman degradation of the intact protein and its peptides . Fifteen gene 23 amber mutation sites have been located within the sequence, and the 3' transcription termination site for genes 21, 22 and 23 has been identified.

J Biol Chem, 1984 Dec 10, 259(23), 14339 - 42
A site in a tRNA precursor that can be processed by the whole RNase P enzyme but not by the RNA alone; Subbarao MN et al.; A precursor molecule for 10 Sb RNA, the RNA moiety of the RNA processing enzyme RNase P, was purified, characterized for enzymatic activity, and compared to 10 Sb RNA and to RNase P . In these studies the K RNA, a dimeric precursor of tRNAGln-tRNALeu, coded by bacteriophage T4, was used as a substrate . This precursor contains two RNase P cleavage sites, one at each 5' end of the two tRNAs . The precursor 10 Sb and 10 Sb RNAs have the capacity to cleave the precursor tRNA molecule but only at the 5' end of tRNALeu, not at the 5' end of tRNAGln . Even when a substrate was prepared that contained only one site for RNase P (the one next to tRNAGln), this substrate was not cleaved by the RNA alone while the whole enzyme was effective in processing this substrate . The possible function of the protein of RNase P in the enzymatic reaction is discussed.

J Biol Chem, 1984 Dec 10, 259(23), 14829 - 34
Nucleotide sequence and expression of the Escherichia coli dapB gene; Bouvier J et al.; The Escherichia coli dapB gene encodes dihydrodipicolinate reductase . This enzyme is part of the diaminopimelate-lysine pathway, and its synthesis is repressed by lysine . The dapB gene was cloned into pBR322 from a transducing lambda bacteriophage, its complete nucleotide sequence established, and the transcriptional start localized . The DNA sequence predicts that the dapB gene codes for a 273-amino acid polypeptide, Mr 28,798 . No attenuation-type sequence can be found between the mRNA start and the coding sequence . The dapB promoter signals appear to be weak as compared to RNA polymerase consensus sequences . Nevertheless an efficient in vivo synthesis of beta-galactosidase was obtained when the lac operon was inserted in vitro in the dapB gene, downstream of the dapB regulatory signals . Further studies were performed on an in-frame gene fusion constructed in vitro between the dapB and the lacZ genes . They indicated that repression by lysine is exerted on a DNA region restricted to a 153-base pair fragment with only 102 nontranscribed nucleotides . Finally, dapB gene expression showed a gene dosage effect which suggests that it is not controlled by an element present in limiting amounts in the cell.

J Mol Biol, 1984 Dec 5, 180(2), 283 - 300
A functional domain of bacteriophage lambda terminase for prohead binding; Frackman S et al.; Terminase is a multifunctional protein complex involved in DNA packaging during bacteriophage lambda assembly . Terminase is made of gpNul and gpA, the products of the phage lambda Nu1 and A genes . Early during DNA packaging terminase binds to lambda DNA to form a complex called complex I . Terminase is required for the binding of proheads by complex I to form a DNA: terminase: prohead complex known as complex II . Terminase remains associated with the DNA during encapsidation . The other known role for terminase in packaging is the production of staggered nicks in the DNA thereby generating the cohesive ends . Lambdoid phage 21 has cohesive ends identical to those of lambda . The head genes of lambda and 21 show partial sequence homology and are analogous in structure, function and position . The terminases of lambda and 21 are not interchangeable . At least two actions of terminase are involved in this specificity: (1) DNA binding; (2) prohead binding . The 1 and 2 genes at the left end of the 21 chromosome were identified as coding for the 21 terminase . gp1 and gp2 are analogous to gpNu1 and gpA, respectively . We have isolated a phage, lambda-21 hybrid 33, which is the product of a crossover between lambda and 21 within the terminase genes . Lambda-21 hybrid 33 DNA and terminase have phage 21 packaging specificity, as determined by complementation and helper packaging studies . The terminase of lambda-21 hybrid 33 requires lambda proheads for packaging . We have determined the position at which the crossover between lambda DNA and 21 DNA occurred to produce the hybrid phage . Lambda-21 hybrid 33 carries the phage 21 1 gene and a hybrid phage 2/A gene . Sequencing of lambda-21 hybrid 33 DNA shows that it encodes a protein that is homologous at the carboxy terminus with the 38 amino acids of the carboxy terminus of lambda gpA; the remainder of the protein is homologous to gp2 . The results of these studies define a specificity domain for prohead binding at the carboxy terminus of gpA.

Gene, 1984 Dec, 32(1-2), 161 - 70
Chromosomal arrangement of the duck alpha-globin genes and primary structure of the embryonic alpha-globin gene pi; Erbil C et al.; A recombinant lambda Charon 4A bacteriophage, D alpha G-1, carrying the genes coding for the duck embryonic (pi') and adult (alpha A, alpha D) alpha-like globins was isolated from a previously constructed duck DNA recombinant library . The three globin genes are transcribed from the same DNA strand and are arranged in the order of their expression during development: 5'-pi'-alpha D-alpha A-3' . We have determined the complete nucleotide sequence of the duck pi'-globin gene, including the flanking regions . Due to the unusual length of intron 1 (963 bp) and intron 2 (568 bp) the 2167-bp duck pi'-globin gene is by far the largest among all known mammalian or avian alpha- and beta-globin genes . For instance, the duck pi'-globin gene introns are almost twice as long as those of the chicken pi'-globin genes . A surprisingly high degree of nucleotide sequence homology (88%) has been found for the 5' flanking region (positions -1 to -223) of the duck and chicken pi'-globin gene.

Gene, 1984 Dec, 32(1-2), 135 - 40
Pseudogene IFN-alpha L: removal of the stop codon in the signal sequence permits expression of active human interferon; Fuke M et al.; Biologically active interferon (10(6)-10(7) units/liter) was produced in Escherichia coli from modified human alpha interferon (IFN-alpha) pseudogene L . IFN-alpha pseudogene L has a stop codon in the signal peptide coding region . The region that contains the stop codon was replaced with the corresponding region of another human IFN-alpha gene, WA, that does not have a stop codon and was previously engineered for expression by fusion to the M13mp11 lac promoter . The interferon L fusion product was induced with IPTG after infecting E . coli JM103 with the M13 bacteriophage that contained the modified human IFN-alpha pseudogene L . Hence, the IFN-alpha L mature interferon coding sequence, which is not identical to any other alpha-interferon gene, has been conserved for active interferon coding information.

Virology, 1984 Dec, 139(2), 283 - 94
Regulation of transcription in the segment of the bacteriophage T4 genome containing three of the head protein genes: plasmid-specific and phage chromosome-specific effects; Kassavetis GA et al.; The activity of the phage T4 late promoters, P22, P23, and P24, is regulated by the gene 33, 45, and 55 products and, through the gene 43 product, by DNA replication . The accumulation of RNA of opposing polarity, with 5' ends mapping to Q22 and Q23, as described by T . Elliott, G . A . Kassavetis, and E . P . Geiduschek (Virology, 1984, 260-282), is subject to the same regulation . When located on a plasmid, the P23 promoter escapes the gene 43 product dependence.

DNA, 1984 Dec, 3(6), 457 - 68
Primary structure of the human renin gene; Hardman JA et al.; The gene encoding human renin has been isolated on two overlapping clones from a bacteriophage lambda library of human DNA . The entire gene spans about 12,000 bp and contains 10 exons separated by 9 intervening sequences . The gene structure is similar to that of human pepsinogen in terms of overall size, homology in the coding regions, position of introns, and sizes of the exons, suggesting that the two genes are evolutionarily related . However, a novel exon coding for only three amino acids was detected that is not present in the pepsinogen gene and whose amino acids are also not found in mouse renin . Although the nucleotide sequence of the 5'-flanking DNA differs from that of the pepsinogen gene, in both cases this region contains a structure of almost perfect dyad symmetry which immediately precedes the TATA box and may have functional importance . Furthermore, sequences resembling the putative consensus sequence for glucocorticoid regulation of gene expression are located approximately 200 and 300 bp upstream from the gene . The overall structural anatomy suggests that the human renin gene evolved by mechanisms that include a duplication of exon segments, particularly those containing the codons for the catalytically important aspartate residues, together with the insertion of other exon and flanking DNA structures . An analysis of human chromosomal DNA demonstrates that there is only one gene with high homology to human renin.

J Bacteriol, 1984 Dec, 160(3), 1191 - 5
Bacteriophage T5 gene A2 protein alters the outer membrane of Escherichia coli; Snyder CE Jr; Evidence for changes in Escherichia coli envelope structure caused by the bacteriophage T5 gene A2 protein was obtained by the use of mutant bacteriophages, envelope fractionation procedures, electrophoretic analysis, and in vitro binding studies with purified gene A2 protein . The results suggested that the T5 gene A2 protein perturbs the host envelope as it functions to promote DNA transfer.

J Virol, 1984 Dec, 52(3), 1028 - 31
Genetic analysis of dar, uvsW, and uvsY in bacteriophage T4: dar and uvsW are alleles; Wu JR et al.; A genetic study of the T4 dar (DNA arrested synthesis restoration) mutations was performed by two- and three-factor crosses . The dar mutations restore the viability of gene 59 mutants . Mapping studies of the dar mutations indicated that the dar gene extended over 16 map units . This high recombination frequency is attributed to an increased level of recombination in the dar region . Two other mutations, uvsY and uvsW, known to be located in the vicinity of dar, were studied . These studies indicated that the uvsY and dar mutations were located in separate genes but that dar and uvsW were allelic . The genes are ordered as follows: gene 24, dar(uvsW), uvsY, and gene 25 in clockwise order.

J Virol, 1984 Dec, 52(3), 1009 - 10
Multiplicity reactivation of bacteriophage T7 inactivated by methyl methanesulfonate; Karska-Wysocki B et al.; Experiments were conducted to determine whether phage T7 treated with methyl methanesulfonate used multiplicity reactivation to repair alkylation lesions . This type of repair was found to be operative at high multiplicities in actively growing wild-type Escherichia coli B cells.

Jpn J Exp Med, 1984 Dec, 54(6), 249 - 53
Distribution of "intrastrand annealing" sequences in lambda bacteriophage genome; Yamamoto K et al.; Our computer programme which is originally developed for calculating distribution of folding structures in single stranded nucleic acids was applied for analysis of lambda phage DNA . We found that we have to choose an optimum condition of calculation depending upon what kind of structures we are looking for, for example secondary structure of transcripts or clover-leaf structures in the double stranded DNA molecules . In one calculation condition, the lambda genome was divided into two distinct regions, i.e., capsid genes with high folding probabilities . In this condition, the clover-leaf structure previously indicated in the replication origin was not detected . Calculation in the condition which included annealings with higher free energies (lower stabilities) revealed the presence of the clover-leaf structure in the DNA replication origin in the middle of the gene O.

Biochem Int, 1984 Dec, 9(6), 799 - 806
Bacteriophage phi X 174 A protein binds in vitro to the phage phi X 174 DNA by a phosphodiester bond via a tyrosine residue; Zolotukhin AS et al.; Nuclease digestion of the phi X 174 A* protein covalent complex with /P32/ - phi X 174 phage DNA results in the transfer of 32p-label to the protein . Acid hydrolysis of the 32p-labelled A* protein yields (4)0-phosphotyrosine.

Virology, 1984 Dec, 139(2), 251 - 9
Purification and properties of gene 18 product of bacteriophage T3; Hamada K et al.; Two noncapsid proteins of T3 and T7 phage, the products of gene 18(gp18) and gp19, are required for DNA packaging . By using in vitro complementation for DNA packaging as an assay system, T3 gp18 was purified to near homogeneity from an extract prepared cells infected with a mutant of gene 19(19- extract) . The purified gp18 consisted of a single polypeptide having a molecular weight of 10,000, and was eluted as dimers and higher multimers from Sephadex G-75 columns . T7 gp18 was purified by the same procedures as that for T3 gp18 and behaved in the same manner as T3 gp18 throughout all purification steps . Gp18 from either T3 or T7 phage complemented both T3 and T7 18- extract for DNA packaging . These results indicate that, in contrast to gp19 {H . Fujisawa and M . Yamagishi (1981) Prog . Clin . Biol . Res . 64, 239-252}, gp18 does not have specificity for T3 or T7 DNA during the in vitro packaging reaction . T3 gp18 was purified from extract containing functional gp19 . The gp18 copurified with the gp19 activity . Gp18 and gp19 activities were stable when they were copurified but were unstable when purified separately . These results suggest that gp18 and gp19 function as a complex in the DNA packaging process . The gp18-gp19 preparation had a prohead-stimulated, DNA-dependent ATPase activity.

Mutat Res, 1984 Dec, 129(3), 311 - 7
Genetic analysis of clear-plaque mutations induced in bacteriophage lambda by 9-aminoacridine; Pons FW; Clear-plaque mutations were induced in the cI and cII genes of lambda by treating lysogenic cells with 9-aminoacridine (9AA) . Mapping of the mutations revealed that there were two hot spots for 9AA mutagenesis in cI, and one strong hot spot in cII . The hot spots in cI mapped close to 1 of the 3 runs of 4 G/C base-pairs and near the only run of 5 G/Cs, respectively, in this gene . Of 36 cI mutations tested, at most one mapped near a run of 6 A/T base-pairs . By analogy, the sequence responsible for the strong hot spot in cII may be the run of 6 G/Cs in this gene.

Genetics, 1984 Dec, 108(4), 773 - 94
Coupling with packaging explains apparent nonreciprocality of Chi-stimulated recombination of bacteriophage lambda by RecA and RecBC functions; Kobayashi I et al.; Chi (chi, 5'-GCTGGTGG) is a recombinator in RecA- and RecBC-mediated recombination in Escherichia coli . In vegetative recombination between two bacteriophage lambda strains, one with and the other without Chi (a+ chi +b- X a- chi 0b+), the chi-containing recombinant (a- chi +b-) is less abundant than the non-chi-containing recombinant (a+ chi 0b+) . Previously this was taken was evidence for nonreciprocality of chi-stimulated exchange . This inequality, however, is now seen to result from an event at cos (lambda's packaging origin) that both activates Chi and initiates DNA packaging . An event at rightward cos leads to activation of leftward chi on the same chromosome for an exchange to its left . From the resulting circulating dimer (--cos-a+- chi 0-b+-cos-a-- chi +-b- --), the cos that activated chi is more likely to be used for rightward packaging initiation than is the cos from the other parent . Consistent with this coupling model is "biased packaging" in lambda carrying two cos sites per monomer genome . When their maturation is dependent on dimerization by chi-stimulated exchange, the phage particles result more often from packaging from the cos that activates chi than from packaging from the other cos . Since Chi activation and packaging can be uncoupled, we infer that some early and reversible step in packaging activates chi . A strong candidate for this step is a double-strand break at cos that provides an oriented entry site for a recombinase.

J Virol, 1984 Dec, 52(3), 966 - 72
An accessory role for Escherichia coli integration host factor: characterization of a lambda mutant dependent upon integration host factor for DNA packaging; Bear SE et al.; Bacteriophage lambda grows lytically on Escherichia coli defective for integration host factor, a protein involved in lambda site-specific recombination and the regulation of gene expression . We report the characterization of a mutant, lambda cos154, that, unlike wild-type lambda, is defective for growth in integration host factor-defective E . coli . The cis-dominant mutation in lambda cos154 is a single base pair change in a region of hyphenated dyad symmetry close to the lambda left cohesive end; this mutation prevents DNA packaging . We propose the following two alternative roles for this site in lambda DNA packaging: (i) to bind an E . coli accessory protein required in the absence of integration host factor or (ii) to bind the phage-encoded terminase protein that is essential for DNA packaging.

J Virol, 1984 Dec, 52(3), 892 - 6
Construction and properties of a ribosome-binding site mutation in gene E of phi X174 bacteriophage; Gillam S et al.; Oligodeoxyribonucleotide mutagenesis has been used to produce a G----A mutation at nucleotide 557 of the phi X174 genome . This changes the ribosome-binding sequence GAGG of gene E to GAAG without affecting the amino acid, glutamine, encoded by the overlapping gene D . The phi X174rb(E)557 mutant does not lyse infected Escherichia coli C and therefore results in the accumulation of a large number intracellular mature phage particles . Thus, the mutation inactivates production of the gene E lytic product, presumably by blocking translation of gene E, without affecting other phage functions.

Immunology, 1984 Dec, 53(4), 827 - 36
Adjuvant effect of bacterial LPS and/or alum precipitation in responses to polysaccharide and protein antigens; Seppala IJ et al.; A conjugate of a hapten (NIP) and a strongly antigenic protein chicken gamma globulin (CGG), when injected in soluble form into mice, induced weak primary responses, as weak as responses induced by conjugates of NIP (or other haptens) to polysaccharides Ficoll or alpha (-1-6) dextran . Mean concentrations of anti-hapten antibodies on day 14 varied within the range of 37-105 micrograms/ml (C57BL mice) or 14-38 micrograms/ml (CBA mice) . The NIP-protein conjugate administered in alum-precipitated form induced 100 times higher primary antibody responses . Alum-precipitation of NIP-Ficoll made it a modestly stronger antigen than soluble NIP-Ficoll . When lipopolysaccharide (LPS) was injected together with any of the soluble antigens, the mice produced plenty of anti-hapten antibodies regardless of whether the antigen was hapten-polysaccharide or hapten-protein conjugate . Concentrations on day 14 varied from around 400 micrograms/ml to approximately 1600 micrograms/ml . LPS had a similar adjuvant effect on antidextran responses . LPS alone induced a polyclonal immunoglobulin production, and the immunoglobulin produced included 'anti-NIP' or 'anti-dextran' detectable in the solid phase antibody assay . These 'antibodies' induced by LPS alone were almost totally mercapto-ethanol-sensitive and poorly detectable by Farr assay or the bacteriophage assay . The response to the LPS+antigen combination was specific for the antigen and included both mercapto-ethanol-sensitive and resistant antibodies.

Gene, 1984 Dec, 32(3), 419 - 26
Regulation of Mu transposition . II . The escherichia coli HimD protein positively controls two repressor promoters and the early promoter of bacteriophage Mu; Goosen N et al.; Two leftward Pc promoters for the repressor gene of bacteriophage Mu have been localized by fusions of the promoter region to the structural galK gene and by S1 nuclease mapping . Transcription initiated at the left-end-proximal promoter (Pc-1) starts 23 bp ahead of the c gene . The second promoter (Pc-2) is located 200 bp from the translation start codon of gene c . The RNA initiated from Pc-2 overlaps 35 bp with the rightward transcript from the early Mu promoter (Pe) . The expression from Pe and both repressor promoters is positively regulated by the Escherichia coli HimD (Hip) protein, probably acting as a subunit of the integration host factor (IHF) . Two overlapping sequences matching the consensus for the IHF binding site (ihf) are found between Pe and Pc-1.

Gene, 1984 Dec, 32(1-2), 217 - 24
A new selective phage cloning vector, lambda 2001, with sites for XbaI, BamHI, HindIII, EcoRI, SstI and XhoI; Karn J et al.; An improved bacteriophage lambda cloning vector, lambda 2001, has been constructed . The phage includes a 34-bp polylinker oligonucleotide which introduces cleavage sites for XbaI, SstI, XhoI, EcoRI, HindIII and BamHI, and can accommodate 10-kb to 23-kb fragments . Inserts that destroy the BamHI or XhoI cloning sites may be recovered by excision at flanking sites in the polylinker sequence . Insertion of foreign DNA into lambda 2001 generates phage with a Spi- phenotype . The recombinant phage are able to grow on P2 lysogens but the parental vector phages are not . In the course of this work, the polylinker sequence was also introduced into M13mp8 . This produced a new vector, M13mp12, with cloning sites for EcoRI, SmaI, XbaI, SstI, XhoI, BamHI, and HindIII.

EMBO J, 1984 Dec 1, 3(12), 2863 - 71
Regulation of a new bacteriophage T4 gene, 69, that spans an origin of DNA replication; Macdonald PM et al.; We have determined the DNA sequence and transcription patterns in a 3-kb segment (between 15 and 18 kb on the standard phage T4 map) spanning an origin of DNA replication . A new gene, 69, spans this origin . Gene 69 codes for two overlapping proteins that share a common C-terminal segment . Defective DNA replication in an appropriate amber mutant shows that at least the larger of the two proteins is required for efficient T4 DNA replication . The two proteins coded by gene 69 are expressed from different transcripts that are under different regulation . The smaller protein, gp69*, can be expressed immediately from an Escherichia coli-like promoter, whereas expression of the larger protein, gp69, must be delayed since its middle promoter requires T4 coded proteins, most likely gp mot, for activation . We discuss the possible significance of two overlapping proteins in the assembly of replisomes . Gene 69 is bracketed by the non-essential early gene dam (DNA adenine methylase) and the late gene soc (small outer capsid protein) . Transcripts through this region are interdigitated in a complex pattern, which reveals all elements that are thought to be important in regulation of pre-replicative and post-replicative T4 genes.

Virology, 1984 Dec, 139(2), 260 - 82
The complex pattern of transcription in the segment of the bacteriophage T4 genome containing three of the head protein genes; Elliott T et al.; A detailed analysis of the temporal pattern of transcription in the gene 22-24 region of bacteriophage T4 has been made . Each of these three late genes has its own promoter, activated during the late phase of viral development . There is also late transcription of the opposite DNA strand across two of these late promoters . The 5' ends of these two sets of convergent transcripts have been mapped to sites designated Q22 and Q23 . Middle (T4 gene mot product-dependent) transcription was found opposing the third late promoter, P24 . An early promoter was mapped to the region between genes 23 and 24.

DNA, 1984 Dec, 3(6), 437 - 47
Chromogenic immunodetection of human serum albumin and alpha-L-fucosidase clones in a human hepatoma cDNA expression library; de Wet JR et al.; A human hepatoma cDNA library was constructed in lambda gt11, a bacteriophage vector that was designed to express cDNA-encoded antigenic determinants in Escherichia coli . The cDNA expression library contained approximately 8 X 10(6) recombinant phages with an average insert size of 780 bp . The feasibility of using a chromogenic immunodetection procedure to isolate cDNA clones was proved by isolating a human serum albumin (HSA) cDNA clone . An approximately 1.0-kb cDNA clone was then isolated by screening the library with rabbit anti-human alpha-L-fucosidase antibodies . The identity of the alpha-L-fucosidase cDNA clone was confirmed by DNA sequence analysis and a comparison of the predicted amino acid sequence to the amino acid sequences of human alpha-L-fucosidase tryptic peptides.

Cell, 1984 Dec, 39(3 Pt 2), 691 - 8
Length determination in bacteriophage lambda tails; Katsura I et al.; We have isolated viable mutants of bacteriophage lambda that have in-frame deletions in gene H, which codes for a minor tail protein . They produce correspondingly smaller but active gene H protein products and assemble shorter-tailed phage particles . The deficiency in tail length for each mutant corresponds to the calculated shortening of the gene H protein caused by the deletion . These results show that the H protein determines tail length and argue strongly for a scheme in which the H protein is a ruler or template that measures length during tail assembly.

Cell, 1984 Dec, 39(3 Pt 2), 663 - 73
The major promoter element of rRNA transcription in yeast lies 2 kb upstream; Elion EA et al.; Conventional genetic analysis of the transcription of rDNA in yeast is precluded because the genes are highly reiterated . As an alternative strategy to determine which sequences modulate transcription of pre-rRNA, a series of artificial rRNA genes containing a fragment of DNA from E . coli bacteriophage T7 were introduced into the yeast Saccharomyces cerevisiae . Correct transcription of the artificial genes was observed . Three regions of ribosomal spacer are found to affect transcription of rRNA . Sequences within 210 bp of the 5' terminus of 35S rRNA support low levels of transcription, but at multiple initiation points . Sequences from -210 to -2230 direct correct initiation and increase somewhat the efficiency of transcription . Most striking is that sequences from -2230 to -2420 stimulate transcription 15-fold . The function of this major promoter element is absolutely orientation-dependent but relatively independent of position . Its activity is blocked when an rRNA transcription termination sequence is placed between it and the site of initiation.

Proc Natl Acad Sci U S A, 1984 Dec, 81(23), 7510 - 4
Identification of a retrovirus-like repetitive element in human DNA; Mager DL et al.; We describe a 5- to 6-kilobase-pair repetitive family in human DNA . One member of this family is linked to the beta-globin gene cluster and is close to the 3' breakpoints of three different naturally occurring deletions involving this gene cluster . Sequence analysis indicates that this element includes terminal direct repeats of 415 base pairs that exhibit the features of long terminal repeats (LTRs) of retroviruses . A potential histidine tRNA primer binding site occurs just 3' to the 5' direct repeat . This retrovirus-like element interrupts a member of the Kpn I family of repeated DNA and is bracketed by a 5-base-pair directly repeated sequence . When attempts are made to clone the element in bacteriophage, homologous recombination between the LTR-like sequences is very frequently observed . Copy number estimates by two methods indicate that the element is repeated 800-1000 times in the human genome . We term this Homo sapiens family of retrovirus-like elements having a histidine tRNA primer binding site the hsRTVL-H family.

Proc Natl Acad Sci U S A, 1984 Dec, 81(23), 7373 - 7
Identification and characterization of a new transcriptional termination factor from Escherichia coli; Briat JF et al.; We have identified and partially purified an activity from Escherichia coli that enhances transcription termination at the bacteriophage T7 early terminator when cloned on the plasmid pAR1707 . The factor also causes the transcript to be terminated at a site several nucleotides earlier than in its absence . The resulting 3' OH ends of the transcripts are identical to those found in vivo by S1 nuclease mapping . From this we conclude that the factor we have identified is probably responsible for determination of the 3' OH ends of T7 RNA transcripts in vivo . This factor does not act by processing a preformed RNA transcript, nor is it replaced by rho protein or nusA or nusB proteins . Therefore, it appears to be a new transcription termination factor, and we have designated it "tau factor." Elucidation of its role in transcription in E . coli will depend on its purification to homogeneity and further studies of its properties.

Cell, 1984 Dec, 39(2 Pt 1), 395 - 404
Mechanism of transposition of bacteriophage Mu: polarity of the strand transfer reaction at the initiation of transposition; Mizuuchi K; The distribution of newly synthesized DNA strands in the transposition products of bacteriophage Mu made in an in vitro system has been analyzed . The results support a model in which all Mu transpositions are initiated by a pair of strand transfer reactions that attach the 3' ends of Mu DNA to 5' protruding staggered ends of the target DNA . Joining of these ends produces a pair of structures similar to replication forks at the ends of the Mu DNA . Successful initiation of replication at either one or both ends, followed by a round of semiconservative replication, results in formation of a cointegrate structure . When the intermediate structure fails to replicate, breakage of the junctions between the Mu sequence and the vector sequence derived from the donor molecule can lead to a simple insert with a pair of gaps at the 5' ends of the Mu DNA . Evidence for a gap repair process that completes the simple insertion process has been obtained.

Cell, 1984 Dec, 39(2 Pt 1), 387 - 94
Site-specific recognition of the bacteriophage Mu ends by the Mu A protein; Craigie R et al.; The Mu A protein binds site-specifically to the ends of Mu DNA . Two blocks of protection against nuclease are seen at the left (L) end; the right (R) end exhibits one continuous block of protection . We interpret the nuclease protection pattern and sequence data as evidence for three Mu A protein binding sites at each end of Mu . Both the L and R ends have one site close to the terminus; each end also has two additional sites that differ in location between the L and R ends . The Mu A protein protection patterns on the L ends of Mu and the closely related phage D108 are, despite many interspersed sequence differences in one of the protected regions, essentially identical . We show that the A proteins of Mu and D108 can function, at different efficiencies, interchangeably on the Mu and D108 L ends in vivo . Purified Mu repressor, in addition to its primary binding in the operator region, also binds less strongly to the Mu ends at the same sites as the Mu A protein . This affinity of Mu repressor for DNA sites recognized by the Mu A protein may play a role as a second level of control of transposition by the repressor.

J Virol, 1984 Dec, 52(3), 905 - 12
Nucleotide sequence of an immediate-early frog virus 3 gene; Willis D et al.; We have used "gene walking" with synthetic oligonucleotides and M13 dideoxynucleotide sequencing techniques to obtain the complete coding and flanking sequences of the gene encoding a major immediate-early RNA (molecular weight, 169,000) of frog virus 3 . R-loop mapping of the cloned XbaI K fragment of frog virus 3 DNA with immediate-early RNA from infected cells showed that an RNA of approximately 500 to 600 nucleotides (the right size to code for the immediate-early viral 18-kilodalton protein of unknown function) hybridized to a region within 100 base pairs of one end of the XbaI K fragment; no evidence for splicing was observed in the electron microscope or by single-strand nuclease analysis . Further restriction mapping narrowed the location of the gene to the XbaI end of a 2-kilobase-pair XbaI-Bg/II fragment, which was bidirectionally subcloned into the bacteriophage pair mp10 and mp11 for sequencing . Mung bean nuclease mapping was used to identify both the 5' and the 3' ends of the mRNA . The 5' end mapped within an AT-rich region 19 base pairs upstream from two in-phase AUG start codons that were immediately followed by an open reading frame of 157 amino acids . Another AT-rich sequence was found at -29 base pairs from the 5' end of the mRNA start site; this sequence may function as a TATA box . The 3' end of the message displayed considerable microheterogeneity, but clearly terminated within a third AT-rich region 50 to 60 base pairs from the translation stop codon . The eucaryotic polyadenylic acid addition signal (AATAAA) was not present, a finding to be expected since frog virus 3 mRNA is not polyadenylated . Both the single-stranded mp10 clone of the XbaI-Bg/II fragment and a 15-base oligonucleotide complementary to the region flanking the two AUG translation start codons inhibited translation of the immediate-early 18-kilodalton protein in vitro, confirming the identity of the sequenced gene . As the regulatory sequences of this gene did not resemble those of known eucaryotic genes or of the cytoplasmic vaccinia virus, we conclude that frog virus 3 has evolved unique signals for the initiation and termination of transcription.

J Virol, 1984 Dec, 52(3), 846 - 56
Physical mapping and complete nucleotide sequence of the denV gene of bacteriophage T4; Radany EH et al.; Phage T4 deletion mutants that are folate analog resistant (far) and contain deletions in the region of the T4 genome near denV have been isolated previously . We showed that one of these mutants (T4farP12) expressed normal denV gene activity, whereas another mutant (T4farP13) was defective in the denV gene . The rII-distal (right) physical endpoints of these deletions defined the limits of the interval in which the rII-proximal (left) endpoint of the denV gene should be located . The deletion endpoints were identified by restriction and Southern hybridization analyses of phage derivatives containing deoxycytidine instead of hydroxymethyldeoxycytidine in their DNAs . The results of these analyses localized the rII-proximal (left) end of the denV gene to a region between 62.4 and 64.3 kilobases on the T4 physical map . denV+ phage resulted from marker rescue with two of five denV- alleles tested, using plasmids containing a 1.8-kilobase fragment from this region or a 179-base-pair terminal fragment derived from it . Sequencing of the 179-base-pair fragment from wild-type DNA showed a 130-base-pair open reading frame with its termination codon at the rII-proximal end . Confirmation that this open reading frame is part of the denV coding sequence was obtained by identifying a TAG amber codon in the homologous DNA derived from a denV amber mutant strain . This mutant strain rescued the denV+ allele from plasmids containing the wild-type sequence . An adjacent overlapping restriction fragment was also cloned, permitting determination of the remaining denV gene sequence . Based on these results, the 3' end of the coding region of the denV locus was mapped to kilobase position 64.07 on the T4 physical map, and the 5' end was mapped to position 64.48.

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