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 it