J Virol, 1984 Dec, 52(3), 822 - 7 Construction of bacteriophage luminal diameterX174 mutants with maximum genome sizes; Russell PW et al.; The bacteriophage phi X174 strain ins6 constructed previously was used to investigate the maximum genome size that could be packaged into the icosahedral phage without concomitant loss of phage viability . The J-F intercistronic region of ins6, which already contains an insert of 117 base pairs with a unique PvuII site, was enlarged further by insertion of HaeIII restriction fragments of the plasmid pBR322 into that PvuII site . By using a biochemical approach for the site-specific mutagenesis as well as selection of mutant genomes, a series of mutants was isolated with genomes of up to 5,730 nucleotides, 6.4% larger than that of the wild-type DNA . Phages with genomes larger than 5,550 nucleotides were highly unstable and were rapidly outgrown by spontaneously occurring deletion mutants . The data predict that genomes of at least 6,090 nucleotides could be constructed and, most likely, packaged, but the resulting phages would not grow well . We speculate that the volume of the phage capsid is not the limiting factor of genome size or is not the only limiting factor. J Virol, 1984 Dec, 52(3), 727 - 33 Interaction with nucleic acids and stimulation of the viral DNA polymerase by the herpes simplex virus type 1 major DNA-binding protein; Ruyechan WT et al.; The interaction of the herpes simplex virus type 1 (HSV-1) major DNA-binding protein, infected-cell polypeptide 8 (ICP8), with nucleic acids has been examined by a filter-binding assay and electron microscopy . Filter-binding assays done over a broad pH range indicated that the optimum pH for the protein-DNA interaction is approximately 7.6 . Heat inactivation studies showed that ICP8 is stable at temperatures up to 40 degrees C with a rapid loss of binding activity on incubation at 45 degrees C and above . Competition binding experiments have established the following relative affinities of ICP8 for the following nucleic acids: single-stranded HSV-1 DNA congruent to bacteriophage fd DNA greater than polyriboadenylate much greater than double-stranded HSV-1 DNA congruent to d(pCpT)5 . Observation of negatively stained ICP8-single-stranded DNA complexes indicated that ICP8 binds along the length of the DNA in a regular repeating fashion . The average width of these complexes is 9.3 +/- 0.8 nms . Finally . Finally, addition of purified ICP8 to HSV-1 DNA polymerase reactions resulted in a stimulation of the viral polymerase activity. Nucleic Acids Res, 1984 Nov 26, 12(22), 8667 - 84 Nucleotide sequence of the essential region of bacteriophage P4; Lin CS; Nucleotide sequence of one-third of the genome of coliphage P4 has been obtained and mutations virl, epsilon am104, cI405, sidl, and delta 35 identified . The epsilon gene likely encodes a 10 kd protein with epsilon am104 being located at the beginning of the gene . cI405, a proposed repressor gene mutation, is located in a sequence capable of coding for a 15 kd protein . A new class of P4 mutations, ash, is located in the neighborhood of cI405 . Two TATA-like sequences are mapped 5' to this cI (ash) sequence . Virl is possibly a promoter-up mutation and is located near or within the replication origin, which is about 400 bp long and AT rich . A sidl mutation is amber that shortens the sid protein by 9 amino acids . The delta gene may encode a 17 kd protein and appears to be coupled with the sid gene translationally . In the 5' side of the sid gene a sequence of CACAAT is the best TATA-like sequence . Sequences of two possible genes that are previously unrecognized and part of the alpha and psu genes are also identified. Nucleic Acids Res, 1984 Nov 26, 12(22), 8627 - 38 The nucleotide sequence of the B gene of bacteriophage Mu; Miller JL et al.; Bacteriophage Mu is a highly efficient transposon which requires the products of the Mu A and B genes in order to transpose at a normal frequency . We have determined the nucleotide sequence of the B gene as well as that of the A-B intergenic region upstream of B . The protein product of the gene contains 312 amino acids and has a predicted molecular weight of 35,061 . As expected, there do not appear to be any potential promoter sequences in the intergenic region prior to the gene, but it is preceded by a strong Shine-Dalgarno sequence . The intergenic region does not contain any obvious transcription termination sequences . The frequency of optimal codon usage is similar to that for other transposon and phage genes, and the amino acid composition is comparable to that of an "average" E . coli protein . A region near the amino terminus of the protein resembles the highly conserved bihelical fold which is involved in DNA contact and sequence specific recognition in a number of DNA binding proteins. J Biol Chem, 1984 Nov 25, 259(22), 13851 - 6 Structure of murine complement component C3 . I . Nucleotide sequence of cloned complementary and genomic DNA coding for the beta chain; Lundwall A et al.; The nucleotide sequence coding for the beta chain of murine C3 was determined from cloned cDNA and genomic DNA fragments . Sonicated subfragments were randomly inserted into the bacteriophage M13 and sequenced using the dideoxynucleotide technique . Each nucleotide was sequenced on average six times in these studies . The derived amino acid sequence includes a signal peptide and a tetra-arginine sequence between the beta and alpha subunits in the precursor polypeptide prepro-C3 . Together with the accompanying report (Wetsel, R.A., Lundwall, A., Davidson, F., Gibson, T., Tack, B.F., and Fey, G.H . (1984) J . Biol . Chem . 259, 13857-13862), this paper completes the analysis of the coding sequences for the prepro-C3 polypeptide . The derived molecular weight of the unglycosylated beta chain (642 amino acids) is 70,641 . The sequences of the first two introns in the murine C3 gene and of the 5'-flanking 106 nucleotides are also reported . The 5'-flanking region contains a TATA consensus sequence in agreement with an earlier report (Wiebauer, K., Domdey, H., Diggelmann, H., and Fey, G.H . (1982) Proc . Natl . Acad . Sci . U . S . A . 79, 7077-7081), presumed to be involving in regulating the expression of the C3 gene . A striking feature of the derived sequence was that only 3 cysteine residues were found, all located in the C-terminal part of the polypeptide chain . No carbohydrate attachment sites were predicted in the beta chain. J Biol Chem, 1984 Nov 25, 259(22), 14038 - 43 DNA-dependent ATPase activity associated with phage P22 gene 12 protein; Wickner S; The product of bacteriophage P22 gene 12 is known from genetic experiments to be essential for phage DNA replication . The P22 12 protein has been purified to near homogeneity from Escherichia coli lysogenic for lambda-P22 hybrid phage containing the replication genes of P22 . The protein has a subunit molecular weight of 46,000 . The purified protein contains ATPase activity that is stimulated by single-stranded DNA . The ATPase is poorly stimulated by double-stranded DNA . All four ribonucleoside triphosphates are hydrolyzed; none of the deoxynucleoside triphosphates are hydrolyzed . In addition, the P22 12 protein binds to single-stranded DNA in the presence of ATP . Studies of oligonucleotide synthesis by P22 12 protein in conjunction with E . coli dnaG primase are presented in the succeeding paper (Wickner, S . (1984) J . Biol . Chem . 259, 14044-14047). J Mol Biol, 1984 Nov 25, 180(1), 21 - 40 Structure of circular copies of the 412 transposable element present in Drosophila melanogaster tissue culture cells, and isolation of a free 412 long terminal repeat; Shepherd BM et al.; We have isolated, from Drosophila melanogaster tissue culture cells, extrachromosomal circular forms of the transposable element 412, and have cloned some of them in bacteriophage lambda . A total of 24 clones have been analysed in detail by restriction and heteroduplex mapping . Seventeen clones are virtually identical, and contain complete 412 elements with one copy of the long terminal direct repeat (LTR) . The remaining seven clones are all different and contain various rearrangements . Four have deletions, two have some 412 sequence substituted by other DNA and one has both an inversion and a deletion . The clone containing the inversion has two LTRs in inverted orientation and separated by a few thousand bases of 412 DNA . The base sequences of the two LTRs in this clone, and of the LTR in one of the 17 clones containing complete elements are very similar to that of the 481 base-pair LTR of a genomic 412 element . We have found no evidence, in either cloned or uncloned material, for 412 elements with two LTRs as a tandem direct repeat . We have found that there are several "free" 412 LTRs in genomic DNA from D . melanogaster strains Canton S and Oregon R, and from D . melanogaster tissue culture cells . We have cloned and sequenced one of these free LTRs . It is 475 base-pairs long and is flanked by a direct repeat four base-pairs long . This sequence differs from that of the 481 base-pair repeat at 16 places including a ten base deletion. Philos Trans R Soc Lond B Biol Sci, 1984 Nov 13, 307(1131), 179 - 87 The expression of Plasmodium falciparum bloodstage antigens in Escherichia coli; Brown GV et al.; A library of cDNA clones expressing proteins of the asexual blood stages of a Papua New Guinean isolate of Plasmodium falciparum (isolate FCQ27/PNG (FC27} was constructed in the bacteriophage vector lambda gt11-Amp3 . In an in situ colony immunoassay, human serum was used to identify colonies producing natural immunogens . Sera from donors of defined clinical status, or reactive to a defined subset of natural immunogens were used to identify clones of particular interest (for example, clones reacting with convalescent but not with acute serum or clones expressing the isolate specific S-antigen of FC27) . Antisera raised by immunizing mice and rabbits with cloned antigens were used to characterize the P . falciparum proteins corresponding to the antigen-positive clones . Nucleotide sequence analysis of an antigen found on the surface of cells infected with ring stage parasites revealed an unusual sequence coding for eight, four and three amino acid repeats rich in acidic amino acids . The discussion centres on the use of cloned antigens as tools for the analysis of the host-protective immune response and selection of candidate vaccine molecules. Nucleic Acids Res, 1984 Nov 12, 12(21), 8085 - 96 Identification, physical map location and sequence of the denV gene from bacteriophage T4; Valerie K et al.; The denV gene from bacteriophage T4, which codes for endonuclease V, a small DNA repair enzyme, has been cloned and identified by an approach combining DNA sequencing and genetics, independent of the phenotypic effect of the cloned gene . Appropriate DenV+ and DenV- deletion mutants were mapped physically to define precisely a region encompassing the denV gene . This region was sequenced in order to identify a protein-coding sequence of the correct size for the denV gene (400-500 bp) . Finally, identification was confirmed by sequencing the corresponding fragments cloned from four genetically and phenotypically well-characterized denV mutants . The denV gene is located at 64 kb on the T4 genome, adjacent to the ipII gene, and codes for a basic protein of 138 amino acids with a deduced molecular weight of 16,078. J Biol Chem, 1984 Nov 10, 259(21), 13292 - 7 The effect of a bacteriophage T4-induced polypeptide on host RNA polymerase interaction with promoters; Malik S et al.; After infection of Escherichia coli with bacteriophage T4, the host RNA polymerase acquires several small phage-induced polypeptides (Stevens, A . (1974) Biochemistry 13, 493-503) and its alpha subunits get ADP-ribosylated by a virus-specific enzyme (Zillig, W., Mailhammer, R., Skorko, R., and Rohrer, H . (1977) Curr . Top . Cell . Regul . 12, 263-271) . The modified polymerase displays changed enzymatic properties including sensitivity to increased salt concentration and a higher transition temperature of open promoter complex formation (promoter melting temperature) . In order to assess the role of individual modifications in the changed enzyme properties, we isolated RNA polymerase from cells infected with T4 mutant defective in the ADP-ribosylating enzyme . We also purified one of the associated polypeptides, the 15,000-dalton protein which is invariably present in stoichiometric amounts in different RNA polymerase preparations . In an in vitro transcription system using T4 DNA as template, we demonstrate that the 15-kDa protein is the cause of the elevated promoter melting temperature and can induce this property when added to host RNA polymerase . We also show that the increased salt sensitivity of T4-modified polymerase is primarily the result of ADP-ribosylation of its alpha subunits. Science, 1984 Nov 9, 226(4675), 694 - 6 Shiga-like toxin-converting phages from Escherichia coli strains that cause hemorrhagic colitis or infantile diarrhea; O'Brien AD et al.; Escherichia coli K-12 acquired the ability to produce a high titer of Shiga-like toxin after lysogenization by either of two different bacteriophages isolated from a highly toxinogenic Escherichia coli O157:H7 strain that causes hemorrhagic colitis . One of these phages and another Shiga-like toxin-converting phage from an Escherichia coli O26 isolate associated with infantile diarrhea were closely related in terms of morphology, virion polypeptides, DNA restriction fragments, lysogenic immunity, and heat stability, although a difference in host range was noted . These phages are currently the best-characterized representatives from a broader family of Shiga-like toxin-converting phages. J Mol Biol, 1984 Nov 5, 179(3), 415 - 30 Gene 68, a new bacteriophage T4 gene which codes for the 17K prohead core protein is involved in head size determination; Keller B et al.; We have identified the gene for a major component of the prohead core of bacteriophage T4, the 17K protein . The gene, which we call gene 68, lies between genes 67 and 21 in the major cluster of T4 head genes . All of the genes in this region of the T4 genome have overlapping initiation and termination codons with the sequence T-A-A-T-G . We present the DNA sequence of the gene and show that it codes for a protein containing 141 amino acids with an acidic amino-terminal half and a basic carboxyl terminus . Antibodies prepared against the 17K protein were used to show that it is cleaved by the phage-coded gp21 protease during head maturation and that most of the protein leaves the head after cleavage . A frameshift mutation of the gene was constructed in vitro and recombined back into the phage genome . The mutated phages had a drastically reduced burst size and about half of the particles produced were morphologically abnormal, having isometric rather than prolate heads . Thus, the 17K protein is involved in head shape determination but is only semi-essential for T4 growth. J Mol Biol, 1984 Nov 5, 179(3), 565 - 9 On the presence of guanosine phosphate in the tail of bacteriophage T4; Serysheva II et al.; Treatment of gp18, a biologically active monomer of the structural protein of the bacteriophage T4 contractile sheath, with 0.6 M-HClO4 leads to the release of GDP, GMP and inorganic phosphate . Each gp18 molecule is shown to carry three atoms of phosphorus . In the isolated protein preparation, gp18 and the nucleoside phosphate are in equimolar relation . It is suggested that in the native sheath-protein subunit, GDP and inorganic phosphate are united as GTP. J Mol Biol, 1984 Nov 5, 179(3), 351 - 65 Long range base-pairing in the leftward transcription unit of bacteriophage lambda . Characterization by electron microscopy and computer-aided sequence analysis; Edlind TD et al.; Restriction fragments of bacteriophage lambda DNA corresponding to the major leftward transcription unit were purified, denatured to form single-stranded DNA, self-annealed, and examined by electron microscopy . Three intrastrand stem and loop secondary structures were observed reproducibly and the locations of the paired regions were determined . A method for computer-aided sequence analysis of these regions is presented and used to identify sets of base-pairings likely to account for the observed structures . One loop observed within gene Ea47 is postulated to involve pairing of sequences which include the polypeptide initiation and termination codons . Another loop is postulated to involve pairing of sequences in gene int with sequences located in the gam-cIII region . A third loop appears to involve sequences in and to the right of gene Ea22 paired with sequences located in the bet-gam region . A general discussion of base-pairing which gives rise to long range interactions is presented along with possible effects of the postulated models on gene expression. FEBS Lett, 1984 Nov 5, 177(1), 115 - 8 The catabolite gene activation system of E . coli may be directly involved in regulation of bacteriophage lambda development; Glukhov IL et al.; The primary structure of bacteriophage lambda DNA has been searched for the presence of consensus CAP binding sites . Four putative CAP binding sites have been found on the lambda genome, indicating that the catabolite gene activation system of E . coli may be directly involved in the regulation of lambda development . Molecular mechanisms of putative cAMP-CAP-mediated stimulation of lysogenic and lytic responses are discussed. Eur J Biochem, 1984 Nov 2, 144(3), 571 - 6 Analysis of the different molecular forms of penicillin-binding protein 1B in Escherichia coli ponB mutants lysogenized with specialized transducing lambda (ponB+) bacteriophages; Rojo F et al.; Penicillin-binding protein (pbp) 1b, the main DD-transpeptidase/transglycosylase of Escherichia coli, is normally present in the cell in three molecular forms alpha, beta and gamma, differentiated by their mobility in sodium dodecyl sulfate/polyacrylamide gel electrophoresis . The three molecular forms are enzymatically active in vitro and their relative amounts are kept fairly constant in most labelling experiments with radioactive beta-lactam antibiotics . In this paper, we have analyzed the expression of ponB (mrcB), the structural gene for pbp 1b, and the relation among the three forms of pbp 1b in ponB strains lysogenyzed by lambda 540 (ponB+) recombinant bacteriophages . Our data indicate that ponB is transcribed anti-clockwise on the E . coli chromosome and suggest that pbp 1b alpha is the first membrane-bound form of pbp 1b able to bind labelled beta-lactams, and is the precursor of pbp 1b beta which is, in turn, the precursor of pbp 1 beta gamma. Mutat Res, 1984 Nov, 129(2), 149 - 52 Bacteriophage T4 particles are refractory to bisulfite mutagenesis; Ripley LS et al.; Bisulfite-induced deamination of cytosine produces uracil, a thymine analog reported to be mutagenic both in vitro and in vivo . Although deamination of cytosine in DNA should produce G:C----A:T transitions, treating bacteriophage T4 particles with 0.9 M bisulfite at pH 5 at 37 degrees C produced no more mutations than did the equivalent buffer without bisulfite . Lack of bisulfite mutagenicity is fully consistent with the reported resistance of 5-substituted cytosines to bisulfite-induced deamination, since T4 DNA contains glucosylated 5-hydroxymethylcytosine . However, bisulfite also failed to induce mutations in T4 particles whose DNA contained unmodified cytosine . The lack of mutagenesis persisted in E . coli hosts deficient in uracil glycosylase, an enzyme expected to participate in the repair of the putative bisulfite-generated uracil . Cytosine in T4 DNA may be largely protected from bisulfite attack within phage particles. J Bacteriol, 1984 Nov, 160(2), 818 - 21 Abnormal motility and fruiting behavior of Myxococcus xanthus bacteriophage-resistant strains induced by a clear-plaque mutant of bacteriophage Mx8; Ruiz-Vazquez R et al.; Myxococcus xanthus mutants resistant to a clear-plaque derivative of phage Mx8 were isolated . A significant fraction of the mutants, easily recognizable by their colony morphology, were induced by the presence of the phage and may correspond to low-frequency lysogens . They were all defective in cell motility and showed the same nonfruiting phenotype under starvation conditions. Genetics, 1984 Nov, 108(3), 523 - 32 Genetic mapping of genes required for motility in Caulobacter crescentus; Ely B et al.; Mutations in more than 30 genes affect motility in Caulobacter crescentus . We have determined the chromosomal map locations for 27 genes involved in flagellar morphogenesis (fla), three genes involved in flagellar function (mot), and three genes that have a pleiotropic effect on both motility and bacteriophage resistance (ple) . Three multigene clusters have been detected at widely separated chromosomal locations, but in addition, there are 12 fla and mot genes that are found at eight additional sites scattered around the C . crescentus chromosome . Thus, there is more scatter of genes involved in flagellar structure and function than has been observed in other bacterial systems. Mutat Res, 1984 Nov, 129(2), 153 - 64 Azaserine: further evidence for DNA damage in Escherichia coli; Williams-Hill DM et al.; Azaserine causes DNA damage in stationary-phase cells . In our investigation of this damage, we used strains of Escherichia coli differing in repair capabilities to study azaserine-induced DNA damage, detected as DNA strand breaks by sucrose gradient sedimentation techniques . Reduced sedimentation in alkaline and neutral sucrose gradients indicated the presence of both alkali-labile sites and in situ strand breaks . Azaserine induced DNA single-strand breaks (SSBs) abundantly in all but the recA strain, in which SSBs were greatly reduced . Treatment of purified DNA with azaserine from bacteriophages T4 and PM2 produced no detectable SSBs . Several other studies also failed to detect DNA damage induced directly by azaserine . Increased levels of beta-galactosidase were induced in an E . coli strain possessing a rec::lac fusion, providing further evidence for azaserine induction of the recA gene product . In addition, azaserine induced adaptation against killing but not against mutagenesis in wild-type E . coli strain. J Virol, 1984 Nov, 52(2), 344 - 9 Identification of bacteriophage T4D gene products 26 and 51 as baseplate hub structural components; Kozloff LM et al.; Products of two bacteriophage T4D genes, 26 and 51, both known to be essential for the formation of the central hub of the phage tail baseplate, have been partially characterized chemically, and their biological role has been examined . The gene 26 product was found to be a protein with a molecular size of 41,000 daltons and the gene 51 product a protein of 16,500 daltons . The earlier proposal (L . M . Kozloff and J . Zorzopulos, J . Virol . 40:635-644), from observations of a 40,000-dalton protein in labeled hubs, that the gene 26 product is a structural component of the baseplate, has been confirmed . The gene 51 product, not yet detected in phage particles, appears from indirect evidence also to be a structural component of the baseplate hub . These current conclusions about the gene 26 and 51 products are based on properties of T4 mutant particles containing altered gene 26 or 51 products and include (i) changes in heat lability, (ii) changes in adsorption rates, and (iii) changes in plating efficiencies on different hosts, and with the results of previous isotope incorporation experiments indicate that T4 particles contain three copies of the gene 26 product and possibly one or at most two copies of the gene 51 product . Properties of these mutant particles indicate that the gene 26 product, together with the other hub components such as the gene 28 product, plays a critical role in phage DNA injection into the host cell, whereas the 51 product seems essential in initiating baseplate hub assembly. Eur J Immunol, 1984 Nov, 14(11), 1069 - 72 Cytotoxic T cell responses to haptenated cells . IV . Requirements for in vivo priming; Haas W et al.; Hapten-specific cytotoxic T cells (CTL) can be generated in cultures containing mouse spleen cells and hapten-coupled syngeneic stimulator cells . A response to sparsely hapten-coupled stimulator cells is only obtained with responder cells from immunized H-2k mice . Immunization was effective with hapten coupled to syngeneic, allogeneic or xenogeneic nucleated cells or membranes thereof . Hapten-coupled erythrocytes, bacteriophages or soluble proteins did not induce CTL precursors (CTL-P) nor responses of other lymphocytes which would interfere with the response of CTL-P . The results show that antigen presentation to CTL-P is very efficient in vivo . Haptens could be presented to and recognized by CTL-P only if coupled to surface membranes of nucleated cells. Gene, 1984 Nov, 31(1-3), 103 - 8 Nucleotide sequence of the gene, protein purification and characterization of the pSC101-encoded tetracycline resistance-gene-repressor; Unger B et al.; The nucleotide sequence of the pSC101-encoded tetracycline repressor gene (tetR) was confirmed . The deduced amino acid sequence is compared to that of other repressor proteins . To overproduce the repressor protein, tetR was placed under the control of bacteriophage lambda promoter pL . Tet repressor protein was purified to homogeneity and shown to bind specifically to two tet operators and also to tetracycline (Tc) . The inducer function of Tc is demonstrated by the loss of the specific binding between the tet operator DNA and the Tet repressor-Tc complex. J Biochem Biophys Methods, 1984 Nov, 10(1-2), 25 - 34 Use of a cloned bacteriophage gene to disrupt bacteria; Henrich B et al.; A plasmid, pUH51, was constructed, which contains the lysis gene E of bacteriophage phi X174, subjected to the regulatory region of the lac operon, as well as the lac repressor gene . This plasmid can readily replicate in any strain of E . coli and mediates lysis of the bacteria after induction of the cloned phi X174 gene E . Taking advantage of these properties, plasmid pUH51 was used as a tool for gentle disruption of E . coli . At cell concentrations below 5 X 10(10)/ml, the efficiency of this method, as measured by release of beta-galactosidase from the cells, exceeded the efficiency of conventional methods for cell breakage. Proc Natl Acad Sci U S A, 1984 Nov, 81(22), 7180 - 4 Evidence for inclusion of regions of nonhomology in heteroduplex products of bacteriophage lambda recombination; Lichten M et al.; Total intracellular DNA was isolated from replication-restricted bacteriophage lambda crosses in which the infecting parents were heteroallelic for wild-type and deletion mutant alleles . This DNA was examined for the presence of heteroduplex DNA molecules that contained wild-type sequences in one strand and deletion-mutant sequences in the other . Molecules hybrid for a 689-nucleotide deletion in the immunity region of lambda were detected at significant levels only in crosses in which both the red recombination system of lambda and the rec recombination system of Escherichia coli were active . Molecules hybrid for a 1300-nucleotide deletion in the central portion of the lambda genome were detected at significant levels in DNA isolated from both red+ and red- crosses in which recA function was present. Virology, 1984 Nov, 139(1), 97 - 108 The role of gene O protein in the replication of bacteriophage lambda; Erdile L et al.; The role of the product of gene O of bacteriophage lambda in phage DNA replication was examined by shifting cells infected with an Ots mutant to the nonpermissive temperature after incubation at the permissive temperature . Thymidine incorporation after the temperature shift exhibits biphasic kinetics, with rapid synthesis immediately after the shift and slower synthesis 2-15 min after the shift . Following a shift to the nonpermissive temperature early in infection, the proportion of replicative intermediates decreases substantially and sigma-structures are favored for preservation . When the shift is done late in infection, the proportion of replicative intermediates remains the same . The average length of single-stranded regions at the branch points increases after a shift to the nonpermissive temperature . Most of the counts which are incorporated after the temperature shift are incorporated into strands which are longer than unit length . These results favor a model in which lambda O protein is required for the initiation of replication, but at least some elongation can continue in the absence of O . It is possible that O protein plays a role in elongation of the lagging strand at replicative forks . This model suggests a way to regulate the transition between theta and sigma replication which occurs as lambda infection proceeds. Mol Biol (Mosk), 1984 Nov-Dec, 18(6), 1590 - 6 {Weakening of bacteriophage lambda EcoK DNA restriction in the presence of plasmid pKM101 ard+ . I . General characteristics and genetic localization}; Zavil'gel'skii GB et al.; The host-controlled K-restriction of unmodified phage lambda is ten to hundred-fold alleviated in the E . coli K12 strain, carring plasmid pKM101 of N-incompatibility group . By restriction mapping Tn5 insertion in pKM101, which reduced pKM101-mediated alleviation of K-restriction, was shown to by located within BglII-B-fragment approximately 9 kb anticlockwise from the EcoRI-site of pKM101 . We have termed the gene(s) promoting the alleviation of K-restriction ARD (Alleviation of Restriction of DNA) . It was shown that (i) plasmid pKM101-mediated alleviation of K-restriction did not depend on bacterial genes LexA, RecBC, umuC and plasmid gene muc; (ii) ard gene did not mediate EcoK type modification of DNA and did not enhance the modification activity of EcoK system in a way similar to that observed with RAL gene of phage lambda . Action of Ard gene of plasmid pKM101 is highly specific: alleviation of restriction of DNA lambda takes place only in K-strains of E . coli and is practically absent in B-strains and also in E . coli strains which have restricting enzymes of 11 type, EcoRI and EcoRIII. J Ultrastruct Res, 1984 Nov, 89(2), 165 - 78 Specific labeling of protein domains with antibody fragments; Buhle EL Jr et al.; Monovalent antibody Fab fragments, prepared from antisera raised against two different types of crystalline arrays made of either intact, or a proteolytic fragment of bacteriophage T4 major capsid protein, gp23*, were employed to stoichiometrically label different gp23* protein domains on the outer surface of a tubular variant (i.e., "polyheads") of bacteriophage T4 capsids . Computer filtrations of both negatively stained and freeze-dried/metal-shadowed specimens permitted approximate mapping of the Fab binding sites within the capsomere of the polyheads. Nature, 1984 Oct 25-31, 311(5988), 721 - 6 Resolution of synthetic att-site Holliday structures by the integrase protein of bacteriophage lambda; Hsu PL et al.; Site-specific recombination of the bacteriophage lambda genome into and out of the host bacterial genome is postulated to involve the formation of Holliday structure intermediates by reciprocal single-strand exchanges . Synthetic analogues of the predicted recombination intermediates are resolved in vitro by the protein product of the lambda int gene . Some of the structural features and reaction conditions for this genetic recombination can now be defined. J Biol Chem, 1984 Oct 25, 259(20), 12933 - 8 Effects of the bacteriophage T4 dda protein on DNA synthesis catalyzed by purified T4 replication proteins; Jongeneel CV et al.; The T4 bacteriophage dda protein is a DNA-dependent ATPase and DNA helicase that is the product of an apparently nonessential T4 gene . We have examined its effects on in vitro DNA synthesis catalyzed by a purified, multienzyme T4 DNA replication system . When DNA synthesis is catalyzed by the T4 DNA polymerase on a single-stranded DNA template, the addition of the dda protein is without effect whether or not other replication proteins are present . In contrast, on a double-stranded DNA template, where a mixture of the DNA polymerase, its accessory proteins, and the gene 32 protein is required, the dda protein greatly stimulates DNA synthesis . The dda protein exerts this effect by speeding up the rate of replication fork movement; in this respect, it acts identically with the other DNA helicase in the T4 replication system, the T4 gene 41 protein . However, whereas a 41 protein molecule remains bound to the same replication fork for a prolonged period, the dda protein seems to be continually dissociating from the replication fork and rebinding to it as the fork moves . Some gene 32 protein is required to observe DNA synthesis on a double-stranded DNA template, even in the presence of the dda protein . However, there is a direct competition between this helix-destabilizing protein and the dda protein for binding to single-stranded DNA, causing the rate of replication fork movement to decrease at a high ratio of gene 32 protein to dda protein . As shown elsewhere, the dda protein becomes absolutely required for in vitro DNA synthesis when E . coli RNA polymerase molecules are bound to the DNA template, because these molecules otherwise stop fork movement (Bedinger, P., Hochstrasser, M., Jongeneel, C.V., and Alberts, B . M . (1983) Cell 34, 115-123). J Biol Chem, 1984 Oct 25, 259(20), 12925 - 32 Purification and characterization of the bacteriophage T4 dda protein . A DNA helicase that associates with the viral helix-destabilizing protein; Jongeneel CV et al.; A DNA-dependent ATPase found in crude preparations of the phage T4 gene 32 protein, shown to be the product of the nonessential T4 dda gene, has been purified to apparent homogeneity and free of nucleases . The dda protein hydrolyzes ATP or dATP to the respective nucleoside diphosphates, in a reaction that is completely dependent on the presence of DNA . DNA in a single-stranded form is strongly preferred and there is little effect of differences in strand length or base composition . We show that the dda protein is the DNA helicase previously studied by Krell et al . (Krell, H., Durwald, H., and Hoffmann-Berling, H . (1979) Eur . J . Biochem . 94, 387-395); it can unwind extensive stretches of double-stranded DNA very rapidly, appearing to move with a 5'-3' polarity relative to the single DNA strand to which it initially binds . The reaction is highly distributive, indicating that the dda protein is continuously dissociating and reassociating with the DNA being unwound . The T4 gene 32 protein, a single-strand-binding, helix-destabilizing protein, competes with the dda protein for binding to single-stranded DNA . Consequently, it seems to inhibit rather than to promote the helicase reaction . The other known T4-encoded DNA helicase, the gene 41 protein, has little effect on the helicase activity of the dda protein . These results are relevant to the suspected role of the dda protein in phage T4 DNA replication, as well as to its possible role in phage genetic recombination. J Biol Chem, 1984 Oct 25, 259(20), 12724 - 32 Purification and properties of Int-h, a variant protein involved in site-specific recombination of bacteriophage lambda; Lange-Gustafson BJ et al.; Under physiological conditions, integration of lambda DNA into the Escherichia coli chromosome requires the direct participation of only two proteins, the viral int gene product and E . coli integration host factor (IHF) . A variant of the int gene has been isolated that permits integrative recombination in cells mutant for one of the two subunits of IHF (Miller, H.I., Mozola, M.A., and Friedman, D.I . (1980) Cell 20, 721-729) . In the present work, we have purified Int-h, the product of this variant gene . In contrast to the wild-type int gene product (Int+), which produces almost no recombinants in the absence of IHF, purified Int-h protein sponsors reduced but significant levels of integrative recombination in the absence of any E . coli supplement . This shows that the int gene encodes all the information necessary for the elementary steps in recombination and implies that IHF functions as an accessory protein . When supplemented by IHF, recombination promoted by Int-h resembles that promoted by Int+ in kinetics, stoichiometry of Int and IHF, and nature of the recombinant product . Under these conditions, Int-h uses supercoiled DNA more effectively than nonsupercoiled DNA as a substrate for recombination, as does Int+ . However, in the absence of IHF, Int-h recombines supercoiled and nonsupercoiled substrates identically, indicating that IHF is an important part of the mechanism that senses the supercoiled state of the substrate DNA during recombination . A surprising difference in recombination carried out by Int-h in the presence or absence of IHF concerns the degree to which sites on the same circle recombine with one another as opposed to sites on sister molecules . In the presence of IHF, Int-h favors intramolecular recombination, as does Int+ . However, in the absence of IHF, Int-h almost exclusively promotes intermolecular recombination. Nature, 1984 Oct 11-17, 311(5986), 580 - 1 Transposition without duplication of infecting bacteriophage Mu DNA; Harshey RM; Most models of DNA transposition invoke replication of the transposable element, but it is not clear whether a 'co-integrate' is an obligatory intermediate in the pathway leading to the production of simple insertions during transposition . Such an intermediate can be accounted for only by a replicative transposition scheme . Bacteriophage Mu is a temperate phage that can either lysogenize or lyse its host, and it encodes at least two modes of transposition as judged by the end-products generated by the process . During the lytic development of the integrated prophage, co-integrates are the predominant end-products; transposition is coupled to replication during this phase . A small number of simple insertions are also produced during the lytic growth, but during transposition from the infecting phage into the host chromosome, simple insertions are the main end-products . Conditions can be found where the choice between the two kinds of end-products depends on a delicate balance between the essential transposition functions encoded by Mu . Experiments have suggested that the simple insertions which arise during transposition from the infecting phage may do so without Mu DNA replication . Here I demonstrate using an infecting phage with completely methylated DNA, a dam- (DNA adenine methylase) host and a combination of restriction enzymes that can cut either fully methylated or unmethylated DNA but not hemi-methylated DNA, that transposition of the phage DNA into the host chromosome does not involve a duplication of its DNA . This result may also have significance for other transposons that do not appear to go through a co-integrate intermediate during transposition. J Biol Chem, 1984 Oct 10, 259(19), 11651 - 3 A multiple mutant of Escherichia coli lacking the exoribonucleases RNase II, RNase D, and RNase BN; Zaniewski R et al.; A multiple mutant strain of Escherichia coli containing mutations affecting the exoribonucleases, RNase II, RNase D, and RNase BN, and also the endonuclease, RNase I, was constructed by P1-mediated transduction . Extracts of the mutant strain were lacking the aforementioned RNase activities . The multiple mutant displayed normal growth in both rich and minimal media at a variety of temperatures, recovered from starvation essentially as the wild-type parent, and could support the growth of a variety of bacteriophages . In addition, RNA synthesis was normal and no precursor RNA accumulation was observed . The properties of the mutant strain indicate that the three exoribonucleases are not essential for the viability of E . coli . The implications of these findings to our understanding of RNA processing and degradation are discussed. Zh Mikrobiol Epidemiol Immunobiol, 1984 Oct, (10), 74 - 5 {Intracellular neutralization of bacteriophage T4 by antiphagic serum}; Tsutsaeva AA et al.; Specific antiserum, introduced into the spheroplasts of Escherichia coli B infected with bacteriophage T4, has been shown to neutralize phage particles formed within the cells. Biosci Rep, 1984 Oct, 4(10), 885 - 95 Sequence analysis of a PM2-DNA anti-Z-IgG-binding region; Miller FD et al.; An anti-Z-antibody-binding region between PM2-DNA map units 0.05 and 0.18, containing approx . 25% of the bound PM2 antibody molecules (1,2) has been sequenced . Analysis of this PM2 DNA sequence from map units 0.00 to 0.175 demonstrates that alternating purine/pyrimidine tracts capable of adopting the left-handed conformation are present within this antibody-binding region . Longer (GC)n-rich tracts are clustered together and comprise seven alternating purine/pyrimidine-rich areas (48%-84%) ranging from 19 to 142 nucleotides in length . The DNA located between these alternating purine/pyrimidine-rich areas exhibit a low level (0%-19%) of this sequence arrangement . There is a very strong correlation between the alternating purine/pyrimidine-rich areas and the anti-Z-DNA-IgG-binding sites . Nucleotides 1461-1583 of the PM2-DNA genome encode the bacteriophage capsid protein IV . One of the PM2 left-handed sites is located within this protein-coding sequence; a B-to-Z transition within this site may be involved in protein-IV gene regulation in vivo. Microbiologica, 1984 Oct, 7(4), 323 - 9 Plasmid-mediated inducible reactivation of an actinophage; Misuraca F; The inducible Weigle-Reactivation (WR) of UV-irradiated bacteriophage VP5 has been examined in Streptomyces coelicolor A3(2) with and without the mutagenesis-enhancing SCP1 plasmid . A higher inducible reactivation was observed in SCP1+ strains ("free" plasmid) than in SCP1- strains and in NF strains (SCP1 integrated) . The efficiency of the plasmid-mediated and cellular repair processes have been examined. Radiat Res, 1984 Oct, 100(1), 1 - 15 The use of viscoelastometry to determine survival curves for intact genomes; Lange CS et al.; Viscoelastometry enables one to determine both size (Mr) and number concentration (L1) of intact genome molecules in solution . Comparison of four parameters, corrected for shear stress, as functions of 60Co gamma-ray dose showed that (1) the principal retardation time (tau 11,0) remained constant, indicating that intact genomes (bacteriophage T4c) were being measured; (2) the principal recoil (gamma 11,r,0) decreased with dose directly proportionately to (and determining) L1; (3) both the total recoil (gamma r,0) and the recoil area (Ar,0), under conditions of high solvent viscosity decreased with dose almost as sensitively as gamma 11,r,0 . The DNAD37 was 540 +/- 25 Gy and the biological PFUD37 was 410.1 +/- 4.5 Gy yielding 75.9 +/- 3.6% of inactivating events explicable by one double-strand break (DSB) per genome . This value is comparable to Freifelder's {Virology 36, 613-619 (1981)} value of 86% for phage T4r48+, and the Frankenberg-Schwager et al . (Br . J . Cancer, in press) value of 0.84 DSB/cell/lethal event in diploid mutant rad54-3 yeast under conditions restrictive for DSB repair . Therefore, T4c may be an excellent model system for DNA damage repair studies with relevance to pro- and eukaryotes . Viscoelastometry, working near its lower size limit, provided precise estimates of the proportion of genomes lacking DSBs . It is not subject to the molecular deformation upper size limitations of the other biophysically understood size measurement methods (e.g., sedimentation rotor speed dependence) . Therefore, it should be the method of choice for the study of genomes larger than those of the T even bacteriophages. Mutat Res, 1984 Oct, 141(2), 75 - 82 Preferential repair of nuclear matrix associated DNA in xeroderma pigmentosum complementation group C; Mullenders LH et al.; The distribution of ultraviolet-induced DNA repair patches in the genome of xeroderma pigmentosum cells of complementation group C was investigated by determining the molecular weight distribution of repair labeled DNA and prelabeled DNA in alkaline sucrose gradients after treatment with the dimerspecific endonuclease V of bacteriophage T4 . The results were consistent with the data reported by Mansbridge and Hanawalt (1983) and suggest that DNA-repair synthesis in xeroderma pigmentosum cells of complementation group C occurs in localized regions of the genome . Analysis of the spatial distribution of ultraviolet-induced repair patches in DNA loops attached to the nuclear matrix revealed that in xeroderma pigmentosum cells of complementation group C repair patches are preferentially situated near the attachment sites of DNA loops at the nuclear matrix . In normal human fibroblasts we observed no enrichment of repair-labeled DNA at the nuclear matrix and repair patches appeared to be distributed randomly along the DNA loops . The enrichment of repair-labeled DNA at the nuclear matrix in xeroderma pigmentosum cells of complementation group C may indicate that the residual DNA-repair synthesis in these cells occurs preferentially in transcribing regions of the genome. Mol Biochem Parasitol, 1984 Oct, 13(2), 173 - 85 Expression of Taenia taeniaeformis antigens in Escherichia coli; Bowtell DD et al.; Two important features of infection of mice with larvae of Taenia taeniaeformis are the ready demonstration of host protective antibodies and the ability to immunize susceptible strains of mice against first infection using crude parasite preparations . Candidate immunogens in established larvae and the invasive oncosphere have been identified by immunoprecipitation of radiolabeled parasite proteins with host-protective antibodies . To overcome the difficulties associated with purification of these antigens from parasite material, the alternative strategy of expressing parasite proteins in Escherichia coli has been adopted . Double stranded DNA complementary to mRNA from 28 day old liver larvae was inserted into the beta-galactosidase gene of the bacteriophage lambda Amp 3 . Some recombinants express a fusion protein with additional parasite-encoded epitopes located at the C-terminal end of the beta-galactosidase protein . Four clones that reacted with antibodies in an E . coli colony immunoassay were selected for detailed characterization . Analysis of lysates of the selected clones by SDS-PAGE and Western blotting revealed that each clone produced an abundant fusion protein that reacted specifically with a hyperimmune anti-oncosphere serum . Sibling analysis revealed that the four antiserum-positive clones encoded three immunologically-distinct parasite antigens . The identity of the native protein of larvae encoded by one clone (designated TA10) was an abundant antigen of Mr 70,000 . This approach allows the assessment of antigens expressed in E . coli as vaccines in susceptible strains of mice by direct immunization and challenge and thus the development of a model defined-antigen vaccine against a larval cestode parasite. Genetics, 1984 Oct, 108(2), 305 - 17 Identification of the bacteriophage T4 unf ( = alc) gene product, a protein involved in the shutoff of host transcription; Herman RE et al.; The introduction of plasmid pR386 into E . coli cells renders them restrictive to the growth of phage T4 unf ( = alc) mutants . This system has been used to isolate Unf+ revertants, which, along with the mutant parental strains, have been used to identify the unf gene product by two-dimensional gel electrophoresis . Synthesis of the unf gene product, a polypeptide of just over 18,000 daltons in size, begins within 1 min after infection and terminates at about 12 min after infection at 30 degrees . Gene dosage experiments suggest that the unf protein functions catalytically. Genetics, 1984 Oct, 108(2), 291 - 304 Identification and characterization of the alc gene product of bacteriophage T4; Kutter E et al.; Bacteriophage T4 infection rapidly and almost completely inhibits transcription of host and other phage DNAs . Two processes have been implicated to date in this inhibition: (1) ADP ribosylation of the alpha subunits of the RNA polymerase, involving gpalt (which is injected with the phage DNA) and, later, gpmod; and (2) the action of the T4 alc/unf gene product, synthesized immediately after infection . The latter unfolds the host genome and also blocks transcription of cytosine-containing DNA . Here, we describe the identification on two-dimensional polyacrylamide gels of gpalc/unf, the more precise mapping of the gene and the identification and analysis of the appropriate DNA sequence from an Unf+ alc mutant. J Bacteriol, 1984 Oct, 160(1), 354 - 9 Increased permeability and subsequent resealing of the host cell membrane early after infection of Escherichia coli with bacteriophage T1; Keweloh HW et al.; The addition of T1 to cells growing at 37 degrees C in a minimal medium at 0.4 mM Mg2+ rapidly induced an irreversible loss of K+ and Mg2+ and uptake of Na+ by the cells . Both the ATP pool of the cells and the transmembrane proton motive force were reduced . These cells did not lyse from within, since viral DNA replication and the maturation of the 36,000-molecular-weight phage head protein were inhibited . By contrast, cells lysed when infected at 5.4 mM Mg2+ . In these cells, T1 initially induced K+ efflux and Na+ influx and lowered the cytoplasmic ATP concentration . After a few minutes, the cation gradients and ATP pool were restored to levels close to that of control cells . At 5.4 mM Mg2+, the shutoff of host protein synthesis was delayed and coincided with the restoration of the ATP pool . In an ATP synthase-negative mutant, infection with T1 did not affect the cytoplasmic ATP concentration but inhibited host protein synthesis with the same rate as it did in wild-type cells. J Bacteriol, 1984 Oct, 160(1), 347 - 53 Permeability changes in the cytoplasmic membrane of Escherichia coli K-12 early after infection with bacteriophage T1; Keweloh H et al.; The nature of the bacteriophage T1-induced changes in the permeability of the cytoplasmic membrane of Escherichia coli K-12 was investigated . At 20 degrees C and with glucose as a substrate, the addition of one bacteriophage per cell induced a complete and irreversible loss of K+ ions (single-hit phenomenon) . K+ loss was compensated by an uptake of Na+, Li+, or choline by the cell, depending on which of these ions was the major cation in the medium . T1 depolarized the cells and inhibited 86Rb+-K+ exchange across the cytoplasmic membrane . The loss of K+ occurred independently of the Mg2+ concentration in the medium . By contrast, at low but not at high Mg2+ concentrations, T1 caused efflux of Mg2+ which in turn caused inhibition of respiration and a decrease of delta pH. Biochem J, 1984 Oct 1, 223(1), 23 - 9 The role of bivalent ions in the inactivation of bacteriophage phi X174 by lipopolysaccharide from Escherichia coli C; Rowatt E; The need for Ca2+ in the inactivation of bacteriophage phi X174 by lipopolysaccharide from Escherichia coli C was confirmed . Ca2+ could be replaced almost completely by Na+, but the concentration of Na+ needed was greater by more than an order of magnitude . Other bivalent ions caused inactivation in the same way as Ca2+, and the degree of inactivation varied according to the ion . At 50% inactivation of bacteriophage, the relation between the concentrations of NaCl and of bivalent or tervalent ions (Mx+) fitted the conception that NaCl was neutralizing electrostatic repulsion between virus and lipopolysaccharide by an ionic-strength effect: that is, log{Mx+} varies inversely with square root{NaCl} . The variation in effect of bi- and ter-valent ions and the low concentration needed show that this is not an ionic-strength effect but likely to involve binding to more than one site. Proc Natl Acad Sci U S A, 1984 Oct, 81(20), 6432 - 6 Kinetic analysis of mutations affecting the cII activation site at the PRE promoter of bacteriophage lambda; Shih MC et al.; Abortive initiation and run-off transcription assays were used to study the effects of cy mutations on activation of the phage lambda PRE promoter by cII gene product . Six point mutations in the repeated T-T-G-C sequences that flank the -35 consensus region of PRE decreased the apparent affinity of the promoter for cII protein by factors of 4-16 relative to the wild-type affinity . Kinetic analyses of transcription initiation in the presence and absence of cII protein demonstrated that five of the six mutations did not significantly affect the intrinsic interaction of RNA polymerase with PRE . Thus, these mutations differ from other cy mutations, including those in the -35 consensus region, which affect the formation of polymerase-PRE closed complexes or the isomerization of closed complexes to open complexes but do not affect the binding of cII protein . A sixth T-T-G-C mutation, cy3001, may affect intrinsic initiation by RNA polymerase as well as cII binding. Gene, 1984 Oct, 30(1-3), 63 - 8 High-sensitivity S1 mapping with single-stranded {32P}DNA probes synthesized from bacteriophage M13mp templates; Burke JF; A method is described by which high-specific-activity single-stranded (ss) {alpha-32P}DNA of a defined size complementary to sequences cloned into bacteriophage M13 is synthesized . The ss DNA template is annealed with a universal sequencing primer, the primer extended with DNA polymerase I Klenow fragment and the DNA duplex cut at a unique site 5' to the multiple cloning sites in the M13 phage . The reaction products are denatured and the ss alpha-32P probe fragment complementary to the cloned sequence is separated from the template by electrophoresis . The utility of such probes for S1 mapping is shown by mapping the 3' ends of transcripts from a mutant Drosophila heat-shock gene . The method described here is up to 300 times more sensitive than conventional S1 mapping techniques. Gene, 1984 Oct, 30(1-3), 41 - 6 Regulation of Mu transposition . I . Localization of the presumed recognition sites for HimD and Ner functions controlling bacteriophage Mu transcription; Goosen N et al.; The Escherichia coli HimD function (also known as Hip) is essential for Mu development (Miller and Friedman, 1977) . We show that the role of HimD is to stimulate early transcription of Mu DNA, probably by acting as a subunit of integration host factor (IHF) and binding at a site located approx . 70 bp upstream from the start of the early transcription . HimD-independent phages were isolated . These mutant phages carry a promoter-up mutation in the Pribnow-box of the early promoter . Early Mu transcription is negatively regulated by the repressor (c gene product) and the Ner proteins . Mutants were isolated which are insensitive to the overproduction of Ner by multicopy plasmids, which normally inhibits Mu development . The mutations that map close to the startpoint of the early transcription reveal a structure which is presumably the Ner recognition site. Gene, 1984 Oct, 30(1-3), 33 - 9 Tandem repeated DNA in an intergenic region of herpes simplex virus type 1 (Patton); Umene K et al.; When the entire US region of HSV-1 (Patton) was cloned as an EcoRI fragment in bacteriophage lambda gtWES, the BamHI B6B5 fragment was observed to vary in size among independent isolates {Umene and Enquist, Gene 13 (1981) 251-268} . This fragment polymorphism also occurred in DNA of HSV-1 single plaque isolates . We report here that this heterogeneity is due to variation in copy number of a 15-bp tandem repeat of sequence 5'-CCACTCCCCACCCAC-3', which apparently lies in an intergenic region of the HSV-1 DNA. Cell, 1984 Oct, 38(3), 851 - 60 Antitermination of E . coli rRNA transcription is caused by a control region segment containing lambda nut-like sequences; Li SC et al.; We have localized the antitermination system involved in E . coli ribosomal RNA transcription and compared it with antitermination in the lamboid bacteriophages . In vivo experiments with gene-fusion plasmids were used to examine the ability of specific areas of the rrnG control region to convert an ordinary transcription complex into antitermination transcription complex . A 67 bp restriction fragment immediately following the rrnG P2 promoter decreased transcription termination about 50% . This fragment contains box A-, box B-, and box C-like sequences similar to those in lambda nut loci . It also caused transcripts from lac and hybrid trp-lac promoters to read through a transcription terminator . Translation through the 67 bp segment or reversal of its orientation resulted in complete loss of antitermination activity . We conclude that the E . coli ribosomal RNA operons possess an antitermination system similar to that used by the bacteriophage lambda. J Bacteriol, 1984 Oct, 160(1), 112 - 21 Measurement of in vivo expression of the recA gene of Escherichia coli by using lacZ gene fusions; Weisemann JM et al.; A recA-lacZ protein fusion was constructed in vivo by using bacteriophage Mu dII301(Ap lac) . The fusion contained the promoter and first 47 codons of the recA mutant, as determined by DNA sequence analysis . The fusion was cloned and used to construct a recA-lacZ operon fusion at the same site within the recA gene . These fusions were introduced into the Escherichia coli chromosome at the lambda attachment site either as complete or cryptic lambda prophages . Synthesis of beta-galactosidase from these fusions was inducible by UV radiation . As the UV dose was increased, induction became slower and persisted for a longer period of time . At low doses of UV radiation, more beta-galactosidase was produced in a uvrA mutant than in a wild-type strain; however, at high doses, no induced synthesis of beta-galactosidase occurred in a uvrA mutant . recA+ strains carrying either the protein or operon fusion on a multicopy plasmid showed reduced survival after UV irradiation . This UV sensitivity was not exhibited by strains containing a single copy of either fusion, however; hence, the fusions provide a reliable measure of recA expression. Biochem Biophys Res Commun, 1984 Sep 28, 123(3), 1019 - 26 A 3' to 5' exonuclease activity is associated with phage 029 DNA polymerase; Watabe K et al.; Bacteriophage 029 produces its own DNA polymerase which is encoded by gene 2 {Watabe, K . and Ito, J . (1983) Nucleic Acid Res . 11, 8333} . This 029 DNA polymerase has been purified by phospho-cellulose, DEAE-cellulose, double-stranded DNA cellulose chromatography and glycerol gradient centrifugation . An exonuclease activity associated with the DNA polymerase was found through all the steps of the purification . This nuclease preferably degrades single-stranded DNA from the 3' to the 5' terminus direction, suggesting that the enzyme plays a role for proofreading during DNA replication . While DNA polymerase activity isolated from cells infected with temperature sensitive mutant of gene 2 is thermolabile, the nuclease activity is not significantly reduced at the restrictive temperature. J Mol Biol, 1984 Sep 25, 178(3), 629 - 51 Immunity repressor of bacteriophage P2 . Identification and DNA-binding activity; Lundqvist B et al.; The product of gene C of the temperate bacteriophage P2, the immunity repressor, can be detected as a unique band eluting from phosphocellulose columns at 0.12 M-potassium phosphate when differentially labelled with a radioactive amino acid: the band is absent when phages that either have lost gene C through deletion or carry a suppressor-sensitive mutation in the gene are used . The repressor in its monomeric form is about 11,000 in molecular weight . At near physiological salt concentrations, the form predominantly recovered is the dimer . In filter-binding assays, the partially purified repressor binds wild-type P2 DNA strongly . It does not bind DNA of P2 vir94, a deletion that removes all the genetic elements involved in the regulation of lysogeny; it also does not bind, or binds inefficiently, DNA of P2 vir3, a mutation in the operator that controls the early replicative functions of P2 . At the concentrations employed, the dimer is the active form in binding . The P2 repressor clearly differs in several features from the well-studied immunity repressor of bacteriophage lambda. Biochemistry, 1984 Sep 25, 23(20), 4665 - 75 Kinetics and mechanism of dissociation of cooperatively bound T4 gene 32 protein-single-stranded nucleic acid complexes . 2 . Changes in mechanism as a function of sodium chloride concentration and other solution variables; Lohman TM; The dissociation kinetics of bacteriophage T4 coded gene 32 protein-single-stranded nucleic acid complexes have been examined as a function of monovalent salt concentration, temperature, and pH in order to investigate the details of the dissociation of cooperatively bound protein . Fluorescence stopped-flow techniques were used, and irreversible dissociation was induced by a combination of {NaCl} jumps and mixing with excess nucleic acid competitor . This made it possible to directly investigate the irreversible dissociation process over a wide range of NaCl concentrations {e.g., from 50 mM to 0.60 M for the gene 32 protein-poly(A) complex}, in the absence of reassociation . Over the entire salt range, the only dissociable species observed is the singly contiguously bound gene 32 protein which dissociates from the ends of protein clusters . However, the {NaCl} dependence of the dissociation rate constant suggests that two competing pathways exist for dissociation of cooperatively bound gene 32 protein from the ends of protein clusters . At high monovalent salt concentrations, dissociation is dominated by a single-step process, with log ke/log {NaCl} = 6.5 +/- 0.5; i.e., the dissociation rate constant increases with increasing NaCl concentration due to the uptake of approximately six monovalent ions upon dissociation . This indicates that singly contiguous protein dissociates directly into solution . However, at much lower {NaCl} the data suggest that gene 32 protein, when bound at the end of a protein cluster, dissociates by first sliding off the end to form a noncooperatively bound intermediate which subsequently dissociates . A quantitative model which incorporates the sliding pathway {Berg, O . G., Winter, R . B., & von Hippel, P . H . (1981) Biochemistry 20, 6929-6948} in the dissociation mechanism fits the data reasonably well and suggests that noncooperatively bound monomers of gene 32 protein may be capable of one-dimensional translocation along single-stranded nucleic acids as suggested by independent kinetic data on the association reaction {Lohman, T . M., & Kowalczykowski, S . C . (1981) J . Mol . Biol . 152, 67-109} . It is also observed that both the absolute dissociation rate constant for T4 gene 32 protein and its salt dependence are sensitive to the average molecular weight and polydispersity of the nucleic acid sample used . This is a general phenomenon exhibited by proteins that bind to nucleic acids in a highly cooperative manner. Biochemistry, 1984 Sep 25, 23(20), 4656 - 65 Kinetics and mechanism of dissociation of cooperatively bound T4 gene 32 protein-single-stranded nucleic acid complexes . 1 . Irreversible dissociation induced by sodium chloride concentration jumps; Lohman TM; The dissociation kinetics of cooperatively bound bacteriophage T4 gene 32 protein from a variety of single-stranded homopolynucleotides has been investigated by stopped-flow techniques . Irreversible dissociation of the complexes was induced by rapidly increasing the salt concentration and monitoring the increase in tryptophan fluorescence upon dissociation of the gene 32 protein . The dependence of the apparent dissociation rate constant on initial fractional saturation of the nucleic acid lattice as well as the observation of zero-order kinetics when the lattice is initially fully saturated with protein indicates that dissociation occurs only from the ends of protein clusters and not from doubly contiguous molecules . The data for the entire time course are quantitatively fit by a kinetics model specifying irreversible dissociation of only singly contiguously bound protein {Lohman, T.M . (1983) Biopolymers 22, 1697-1713} . This model is used to extract molecular rate constants for the dissociation of isolated, singly contiguously and doubly contiguously bound protein . It is also shown that the polynucleotide specificity observed for the cooperative binding constant, K omega, and the cooperativity itself are intrinsic properties of the dissociation rate of the various complexes. J Mol Biol, 1984 Sep 25, 178(3), 699 - 709 Isolation and characterization of precursors in bacteriophage T4 baseplate assembly . III . The carboxyl termini of protein P11 are required for assembly activity; Plishker MF et al.; The assembly activity and electrophoretic mobility of a T4 bacteriophage baseplate protein, P11, have been found to be affected by digestion with the proteases trypsin, subtilisin and carboxypeptidase Y . Analysis of the trypsin limit-digestion product of P11 by sodium dodecyl sulfate/polyacrylamide gel electrophoresis and size analysis by high performance liquid chromatography indicate that there is a decrease of approximately 5000 in the molecular weight of the P11 molecule or a loss of 2500 in Mr from each of the gp11 subunits of the dimer . During protease treatment P11 demonstrates a time-dependent loss in the ability to interact with the baseplate protein P10 to form the P(10/11) complex, the first assembly intermediate of the T4 baseplate 1/6th arm . Similar treatments of the P(10/11) complex indicate that P11 in the complex is not affected by these proteases . Concomitant with the loss of assembly activity is a change in the electrophoretic mobility of P11 on non-denaturing polyacrylamide gels from a single band to a series of more mobile bands suggesting sequential loss of positive charge . P11 assembly activity is completely lost after removal of the first positive charge . These results suggest that the carboxyl termini of the two gp11 subunits of the P11 molecule are involved in the interaction of P11 with P10 to form the P(10/11) complex . Analysis of the portion of gp11 removed by carboxypeptidase Y demonstrates that there are up to 13 aliphatic and aromatic carboxyl-terminal amino acids. Nucleic Acids Res, 1984 Sep 25, 12(18), 7269 - 82 Primary structural comparison of RNA-dependent polymerases from plant, animal and bacterial viruses; Kamer G et al.; Possible alignments for portions of the genomic codons in eight different plant and animal viruses are presented: tobacco mosaic, brome mosaic, alfalfa mosaic, sindbis, foot-and-mouth disease, polio, encephalomyocarditis, and cowpea mosaic viruses . Since in one of the viruses (polio) the aligned sequence has been identified as an RNA-dependent polymerase, this would imply the identification of the polymerases in the other viruses . A conserved fourteen-residue segment consisting of an Asp-Asp sequence flanked by hydrophobic residues has also been found in retroviral reverse transcriptases, a bacteriophage, influenza virus, cauliflower mosaic virus and hepatitis B virus, suggesting this span as a possible active site or nucleic acid recognition region for the polymerases . Evolutionary implications are discussed. Nucleic Acids Res, 1984 Sep 25, 12(18), 7035 - 56 Efficient in vitro synthesis of biologically active RNA and RNA hybridization probes from plasmids containing a bacteriophage SP6 promoter; Melton DA et al.; A simple and efficient method for synthesizing pure single stranded RNAs of virtually any structure is described . This in vitro transcription system is based on the unusually specific RNA synthesis by bacteriophage SP6 RNA polymerase which initiates transcription exclusively at an SP6 promoter . We have constructed convenient cloning vectors that contain an SP6 promoter immediately upstream from a polylinker sequence . Using these SP6 vectors, optimal conditions have been established for in vitro RNA synthesis . The advantages and uses of SP6 derived RNAs as probes for nucleic acid blot and solution hybridizations are demonstrated . We show that single stranded RNA probes of a high specific activity are easy to prepare and can significantly increase the sensitivity of nucleic acid hybridization methods . Furthermore, the SP6 transcription system can be used to prepare RNA substrates for studies on RNA processing (1,5,9) and translation (see accompanying paper). J Biol Chem, 1984 Sep 25, 259(18), 11571 - 5 Isolation of gram quantities of EcoRI restriction and modification enzymes from an overproducing strain; Cheng SC et al.; Structural genes for EcoRI restriction endonuclease and modification methylase have been inserted into the plasmid vector pKC30 (Shimatake, H., and Rosenberg, M . (1981) Nature (Lond.) 292, 128-132) downstream from the bacteriophage lambda pL promoter . Upon induction of pL expression in strains producing a thermolabile lambda cI857 repressor, synthesis of EcoRI polypeptides is enhanced to the extent that after 4 h they represent several per cent of the total cell protein . Purification of activities overproduced in this manner yields preparations of endonuclease and methylase which appear identical to those obtained from conventional sources, with overall yields corresponding to 0.5 to 0.9 g of each enzyme/kg of cell paste. J Mol Biol, 1984 Sep 15, 178(2), 191 - 207 Plasmid mode of propagation of the genetic element P4; Deho G et al.; The satellite bacteriophage P4, in the presence of a helper phage, can enter either the lytic or the lysogenic cycle . In the absence of the helper, P4 can integrate in the bacterial chromosome . In addition, the partially immunity-insensitive mutant P4 vir1 can be maintained as a plasmid . We have found that in the absence of the helper, P4 wt also can be maintained as a plasmid, and that both P4 wt and P4 vir1 have two options for their intracellular propagation: a repressed-integrated or a derepressed-high copy number plasmid mode of maintenance . In the repressed mode, the P4 wt genome is only found integrated into the bacterial chromosome, while the P4 vir1 is found also as a low copy number plasmid coexisting with the integrated P4 vir1 genome . The clones carrying P4 in the derepressed-high copy number plasmid state are obtained at low frequency (0.3%) upon infection with P4 wt, while the vir1 mutation increases this frequency about 300-fold . Such clones can be distinguished easily because of their typical colony morphology (rosettes), due to the presence of filamentous cells . Filamentation of the bacterial host suggests that the presence of derepressed P4 genomes in high copy number interferes with the normal cell division mechanism . The derepressed clones are rather stable, but may revert spontaneously to the repressed state . Spontaneous transition from the repressed to the derepressed state was not observed; however, it can be induced by P2 or P4 vir1 superinfection of P4 wt and P4 vir1 lysogens or by growing the P4 vir1 lysogens up to the late exponential phase . The ability of P4 to choose either of two stable states and the potential to shift between these two modes of propagation indicate that the synthesis of the immunity repressor is regulated. J Mol Biol, 1984 Sep 15, 178(2), 481 - 6 Site-specific recombination by the bacteriophage P1 lox-Cre system . Cre-mediated synapsis of two lox sites; Hamilton DL et al.; The bacteriophage P1-encoded recombinase Cre forms a simple DNA-protein complex at the specific recognition site loxP . Furthermore, Cre is able to mediate a synaptic union of two loxP sites . When two loxP sites are on the same linear DNA molecule, Cre binds the two sites together to form a circular protein-DNA complex . These complexes can be resolved into a linear DNA molecule and a closed circular DNA molecule, the end products of site-specific recombination. J Mol Biol, 1984 Sep 15, 178(2), 137 - 53 Gene X of bacteriophage f1 is required for phage DNA synthesis . Mutagenesis of in-frame overlapping genes; Fulford W et al.; The gene II protein of bacteriophage f1 is a site-specific endonuclease required for initiation of phage viral strand DNA synthesis . Within gene II is another gene, X, encoding a protein of unknown function identical to the C-terminal 27% of the gene II protein, and separately translated from codon 300 (AUG) of gene II . By oligonucleotide mutagenesis, we constructed phage mutants in which this codon has been changed to UAG (amber) or UUG (leucine), and propagated them on cells carrying a cloned copy of gene X on a plasmid . The amber mutant makes no gene X protein, and cannot grow in the absence of the complementing plasmid; the leucine-inserting mutant can make gene X protein, and grows normally without the plasmid . Without gene X protein, phage DNA synthesis (particularly viral strand synthesis) is impaired . We discuss this finding in the context of other known in-frame overlapping genes (particularly genes A and A* of phage phi X174), many of which are also involved in the specific initiation of DNA synthesis, and suggest applications for the mutagenic strategy we employed. Nucleic Acids Res, 1984 Sep 11, 12(17), 6779 - 95 Site-specificity of abnormal excision: the mechanism of formation of a specialized transducing bacteriophage lambda plac5; Shpakovski GV et al.; Molecular mechanism of the specialized transducing bacteriophage lambda plac5 formation has been studied . Phage-bacterial DNA junctions in lambda plac5 DNA are localized and primary structure of regions of the abnormal excisional recombination leading to the phage formation is elucidated; the crossover region proved to be comparable with the central part of attP and attB sites (the core and the adjacent tetranucleotide) in length and degree of homology . Bacterial insert in lambda plac5 DNA is shown to end immediately after Z-Y spacer, the DNA not containing lacY gene segments . The data obtained led to the conclusion of site-specific (homologous) character of abnormal excision upon formation of lambda transducing bacteriophages . Possible mechanisms of the excision are discussed. J Biol Chem, 1984 Sep 10, 259(17), 10850 - 6 Photochemical cross-linking of the Escherichia coli single-stranded DNA-binding protein to oligodeoxynucleotides . Identification of phenylalanine 60 as the site of cross-linking; Merrill BM et al.; The single-stranded DNA-binding proteins from bacteriophage T4, F plasmid, Escherichia coli, and calf thymus can all be covalently cross-linked in vitro to thymine oligonucleotides by irradiating the respective protein-oligonucleotide complexes with ultraviolet light . More extensive studies on the E . coli single-stranded DNA-binding protein (SSB) indicate that this reaction is dependent upon both the length of the oligonucleotide and the dose of ultraviolet irradiation . Using anion-exchange and reverse-phase ion-pairing high-performance liquid chromatography we have isolated a specific cross-linked tryptic peptide comprising residues 57-62 of the SSB protein with the sequence valine-valine-leucine-phenylalanine-glycine-lysine . Solid-phase sequence analysis of the covalent {32P} p(dT)8-peptide complex indicates that phenylalanine 60 is the site of cross-linking . This amino acid is located within the general region of SSB (residues 1-115) that has previously been shown to contain the DNA-binding site (Williams, K . R., Spicer, E . K., LoPresti, M . B., Guggenheimer, R . A., and Chase, J . W . (1983) J . Biol . Chem . 258, 3346-3355) . The high-performance liquid chromatography purification procedure we have devised to isolate cross-linked peptide-oligonucleotide complexes should be of general applicability and should facilitate future structure/function studies on other nucleic acid-binding proteins. FEBS Lett, 1984 Sep 3, 174(2), 243 - 7 Spatial arrangement of the three alpha helices in the solution conformation of E . coli lac repressor DNA-binding domain; Zuiderweg ER et al.; The relative orientations of the 3 helices in the DNA-binding domain ('headpiece') of lac repressor have been determined using distance constraints obtained from 2-dimensional 1H nuclear Overhauser enhancement spectra . The relative orientations of its helices is similar to that of the central 3 helices in the DNA-binding domain of the lambda repressor of the bacteriophage lambda. Cell, 1984 Sep, 38(2), 361 - 9 Substituting an alpha-helix switches the sequence-specific DNA interactions of a repressor; Wharton RP et al.; It has been suggested that many DNA-binding proteins use an alpha-helix for specific sequence recognition . We have used amino acid sequence homologies to identify the presumptive DNA-recognition helices in two related proteins whose structures are unknown--the repressor and cro protein of bacteriophage 434 . The 434 repressor and cro protein each bind to three similar sites in the rightward phage 434 operator, OR, and they make different contacts in each binding site, as revealed by the chemical probe dimethyl sulfate . We substituted the putative recognition alpha-helix of 434 repressor with the putative recognition alpha-helix of 434 cro protein to create a hybrid protein named repressor* . The specific DNA contacts made by repressor* are like those of 434 cro protein. Radiat Res, 1984 Sep, 99(3), 562 - 72 Roles of copper and O(2) in the radiation-induced inactivation of T7 bacteriophage; Samuni A et al.; The effect of copper on the radiation damage induced in T7 bacteriophage has been investigated . The phages were gamma-irradiated and the effects of copper(II) ions in the presence of various additives and radical scavengers were examined in an attempt to better understand the effect of transition metal ions on the role of free radicals, particularly superoxide, in biological damage . The present work extends a study previously done on isolated enzyme to a whole biological entity . Copper(II) ions even at very low concentrations enhanced the lethal effect of radiation . This sensitization was observed in both the presence and the absence of oxygen . The effect of copper could be reverted by chelating agents such as EDTA or 1,10-phenanthroline . Hydrogen peroxide enhanced the sensitizing effect of copper, though little if any protection was provided by catalase or SOD . High molecular weight scavengers of free radicals in the presence of both copper(II) and hydrogen peroxide had no protective effect . (This is in contrast to metal-free systems where, although such scavengers are incapable of penetrating the phages, they protect them against inactivation.) These scavengers, without added H2O2, afforded only slight protection to the irradiated phages in the presence of Cu . Low molecular weight scavengers of free radicals reduced but did not eliminate the sensitizing effect of copper . The sensitizing effect of copper was also observed with other T-odd phages, but not with the T-even series . Copper(II) ions under similar experimental conditions did not sensitize T4 or T2 phages but rather had a protective effect . The results are interpreted in terms of a site-specific Fenton mechanism according to which the binding of the metal ion to the phages is a prerequisite for the occurrence of the biological damage . The results also indicate that most of the copper effect is endogenous . This is in accord with the failure of copper to sensitize the T-even phages, which differ by the rigidity and permeability of their outer coat structures. J Bacteriol, 1984 Sep, 159(3), 832 - 6 Regulation of transcription of the Escherichia coli phosphoenolpyruvate carboxykinase locus: studies with pck-lacZ operon fusions; Goldie H; Mutants of Escherichia coli containing genetic fusions of lacZ to the pck (phosphoenolpyruvate carboxykinase) locus were isolated by using Mu d(lacZ Ampr) bacteriophage . Synthesis of beta-galactosidase in these strains is regulated by cyclic AMP and glucose (catabolite repression) . Synthesis of beta-galactosidase by pck-lacZ fusions was induced in log-phase cells growing on gluconeogenic media, was repressed by glucose, and was also induced up to 100-fold at the onset of stationary phase in LB medium . This stationary-phase induction required cyclic AMP and some other unknown regulatory signal. Proc Natl Acad Sci U S A, 1984 Sep, 81(17), 5374 - 8 Replication of bacteriophage phi 29 DNA in vitro: the roles of terminal protein and DNA polymerase; Watabe K et al.; phi 29 DNA replication is initiated by the formation of a covalent complex between the viral-coded terminal protein and dAMP (TP-dAMP) . This initiation reaction system has been reconstituted from two phage-encoded proteins, the terminal protein and DNA polymerase . The phi 29 DNA polymerase was purified from phage-infected cells by using poly(dA) X p(dT)12-18 as an assay template . The purified polymerase has an apparent molecular mass of 68 kDa in its native form and it appears to function as a monomer . The terminal protein was purified to homogeneity from Escherichia coli cells harboring a cloned plasmid that contained a phi 29 gene 3 segment . The molecular mass of the purified terminal protein was about 30 kDa in both the denatured and the native form . The protein apparently functions as a monomer . When the terminal protein and DNA polymerase were incubated in the presence of dATP, Mg2+, and phi 29 DNA-protein as template, the terminal protein bound covalently to dAMP . This reaction did not require ATP . In addition, these two purified fractions catalyzed DNA chain elongation from both ends of phi 29 DNA, yielding the expected 9- to 12-base fragment when assayed in the presence of 2',3'-dideoxycytidine triphosphate . These results indicate that phi 29 DNA polymerase catalyzes formation of the terminal protein-dAMP complex and can also catalyze chain elongation at least 9-12 bases from both ends of phi 29 DNA. J Gen Microbiol, 1984 Sep, 130 ( Pt 9), 2339 - 46 Concentration of a major outer membrane protein at the cell poles in Escherichia coli; Begg KJ et al.; Autoradiography of cell envelope ghosts obtained from a strain of Escherichia coli which lacks two major outer membrane proteins has been used to demonstrate the polar concentration of another major outer membrane protein, ompA protein . The beta-lactam antibiotic cephalexin prevents the insertion of newly synthesized ompA protein into the poles but removal of the antibiotic allows the randomly dispersed protein to migrate to the polar and possibly the septal areas of the cell . Labelling of whole cells with bacteriophage K3 has confirmed a polar concentration of ompA protein. Virology, 1984 Sep, 137(2), 331 - 7 A new suppressor of mutations in the DNA repair-recombination genes of bacteriophage T4: sur; Wakem LP et al.; A new mutation designated sur was isolated as a suppressor of a mutation in the uvsX gene of T4 phage . Unlike the other suppressors of mutations in genes involved in DNA repair and recombination, sur has a wide range, suppressing both DNA repair and replication defects in mutations in genes uvsX, uvsY, 46, 47, and 59 . However, its suppressor functions may be confined to the uvsX-uvsY DNA repair pathway since sur did not suppress a mutation in the denV gene . The sur mutation results in an increased degradation of host DNA to an acid-soluble form, but this increase was blocked by a mutation in gene 46 (nuclease) indicating that the sur function is involved in an earlier step in the degradation of host DNA . This increased degradation of host DNA might be a reflection of a compensatory increase in an alternate DNA repair activity in the {sur} mutant. Virology, 1984 Sep, 137(2), 324 - 30 An analysis of DNA repair and recombination functions of bacteriophage T4 by means of suppressors: the role of das; Wakem LP et al.; Previous studies have indicated that the bacteriophage T4 das mutations partially suppressed the DNA replication defects in gene 46 and 47 mutations . Here it is shown that the das mutation also suppresses the DNA repair defects but not the DNA replication defects of the uvsX and uvsY mutations . In contrast, the das mutation suppressed both the DNA replication and repair defects of gene 46 and 47 mutations . These characteristics of das as well as those of the other suppressors, including uvsW(dar) and two new suppressors sur and uvsU have been used for the analysis of the DNA repair pathway . Based on the functions of these suppressors, a sequence in which the gene products in this pathway might act is suggested. Virology, 1984 Sep, 137(2), 305 - 13 Plasmid-phage recombination in T7 infected Escherichia coli; Stone JC et al.; Recombination between genetically marked T7 bacteriophage and plasmids containing inserts of T7 DNA has been studied in order to gain some insight into the phage recombination process . The results suggest that plasmid-phage recombination requires the products of T7 genes 3 (endonuclease), 4 (DNA primase), 5 (DNA polymerase), and 6 (exonuclease), as has been demonstrated previously for phage-phage recombination . Plasmid replication does not compensate for a complete block in phage polymerase synthesis, suggesting a direct role for this enzyme in recombination, rather than an indirect role, by means of producing replicative structures that are recombinogenic . In most respects, plasmid-phage recombination appears to be similar to phage-phage recombination . The participation of two autonomous, structurally dissimilar, homologues, however, might render certain aspects of the recombination process more amenable to analysis . As examples, the characterization of an apparent marker effect and the demonstration of genetic heterozygotes among the products of plasmid-phage recombination are presented. J Bacteriol, 1984 Sep, 159(3), 1072 - 3 Convenient transduction of recA with bacteriophage T4GT7; Plakidou S et al.; The generalized transducing phage T4GT7 grew well on recA strains of Escherichia coli and transduced recA into F- and Hfr strains of E . coli at high frequency. J Bacteriol, 1984 Sep, 159(3), 1047 - 52 Effect of bacteriophage P1 lysogeny on lipopolysaccharide composition and the lambda receptor of Escherichia coli; Tomas JM et al.; The outer membrane of Escherichia coli was altered as a consequence of lysogeny by bacteriophages P1 and P1 cmts . The predominant change was a reduction in the size of lipopolysaccharide to a heptose-deficient form . P1 cmts lysogens were still sensitive to several bacteriophages but were resistant to lambda vir . Neither whole cells nor solubilized outer membranes from P1 cmts lysogens were able to inactivate lambda vir, and 32P-labeled lambda vir was unable to adsorb to P1 cmts lysogens . P1 cmts lysogens were also affected in maltose transport . The level of periplasmic maltose-binding protein was reduced somewhat, but there was no significant reduction in the level of the outer membrane lambda receptor (LamB) . These membrane abnormalities were all corrected in strains cured of P1 cmts . It is suggested that P1 cmts affects lipopolysaccharide biosynthesis by a phage conversion mechanism and consequently the function of the lambda receptor. Plasmid, 1984 Sep, 12(2), 139 - 41 A physical map of pPH1JI and pJB4JI; Hirsch PR et al.; The antibiotic resistance plasmid pPH1JI was derived from two IncP plasmids, R751 and R1033 . The suicide vector for Tn5, pJB4JI, contains pPH1JI, bacteriophage Mu, and Tn5 . Restriction enzyme cleavage maps for pPH1JI and pJB4JI, and the antibiotic resistance levels determined by pPH1JI and its parent plasmids are presented . The relationships between pPH1JI and its parent plasmids, and pJB4JI, are discussed. Virology, 1984 Sep, 137(2), 338 - 46 The coupling of DNA repair-recombination functions with DNA replication in bacteriophage T4: a new DNA repair mutant; Wakem LP et al.; The requirement of DNA repair-recombination functions for T4 phage DNA replication has been known for some time but the underlying basis for this relationship has been unclear . This report is concerned with a new uv-sensitive gene {uvsU}, whose function appears to bridge these two major activities of DNA . The {uvsU} mutant fails to complement {uvsX} mutants but uvsU maps in a region distinct from uvsX . Furthermore, the uvsU mutation specifically suppressed the DNA replication defect but not the uv sensitivity of the uvsX mutation . The previously discovered uvsW gene, whose mutations suppress the DNA replication defects of gene 59, 46, and 47 mutations, seems to have an analogous role . As a possible explanation for these observations, it is suggested that the uvsW and uvsU gene products (gps) couple the DNA repair-recombination and replication functions by controlling the entry of DNA intermediates from the replication pool into the DNA repair-recombination pathway . Furthermore the suppression data are interpreted to suggest that the gps uvsW, 59, 46, and 47 function together . Similarly the gps uvsU and uvsX may form a functional unit. Proc Natl Acad Sci U S A, 1984 Sep, 81(17), 5561 - 5 Clones from the human gene complex coding for salivary proline-rich proteins; Azen E et al.; The salivary protein gene complex consists of a series of loci coding for related but distinct proline-rich proteins (PRPs) found chiefly in saliva . We have screened a library of human genomic DNA fragments in bacteriophage lambda Charon 4A with a PRP cDNA synthesized and cloned from rat parotid gland mRNA . Two phages (PRP1 and PRP2) hybridizing to the rat probe under moderately stringent conditions contain related but not identical DNAs . Preliminary nucleotide sequence data indicate that both DNAs include regions comprised of nearly identical tandemly repeated sequences, each able to code for about 21 amino acids . The decoded consensus repeat sequence is homologous to the repeating amino acid units found by others in human PRPs . This and other features demonstrate that these two clones are members of the PRP gene family . Polymorphic differences between the DNAs of different individuals were observed after probing digests of human genomic DNA with a HinfI fragment from PRP1 . These DNA polymorphisms reflect size differences, possibly caused by frequent unequal crossing-over between the repeated units in the PRP genes. Proc Natl Acad Sci U S A, 1984 Sep, 81(17), 5305 - 9 Expression of normal and transforming H-ras genes in Escherichia coli and purification of their encoded p21 proteins; Lacal JC et al.; The H-ras gene of the BALB murine sarcoma virus (BALB-MSV) was placed under the transcriptional control of the tightly regulated PL promoter of bacteriophage lambda in the expression vectors pEV-vrf-1 and pRC23 . Upon derepression of the PL promoter, large amounts (10-20% of total cellular protein) of the H-ras gene product p21 are synthesized in Escherichia coli . We constructed three H-ras gene expression vectors, designated pJCL-H5, pJCL-E30, and pJCL-33 . pJCL-H5 directs the synthesis of p21, a fusion protein whose four amino-terminal residues are replaced by eight amino acids coded for by plasmid sequences . The 13 5' coding nucleotides of the BALB-MSV H-ras gene missing in pJCL-H5 were regenerated in pJCL-E30 by inserting a pair of complementary synthetic oligodeoxynucleotides . As a result, pJCL-E30 encodes a p21 protein, p21T, of sequence identical to that of the transforming p21 protein of BALB-MSV . pJCL-33 is a derivative of pJCL-E30 in which the 12th codon, AAA, a lysine codon, was replaced by GGA, a glycine codon . Thus, pJCL-33 directs the synthesis of a p21 protein, p21N, whose sequence corresponds to that of a normal cellular p21 protein . We report the purification of H-ras p21 proteins to apparent homogeneity by a method involving solubilization with chaotropic agents followed by reverse-phase high-performance liquid chromatography. J Mol Biol, 1984 Aug 25, 177(4), 685 - 700 Interference between M13 and oriM13 plasmids is mediated by a replication enhancer sequence near the viral strand origin; Johnston S et al.; The origin of replication for the viral strand of bacteriophage M13 DNA is contained within a 507 base-pair intergenic region of the phage chromosome . The viral strand origin is defined as the specific site at which the M13 gene II protein nicks the duplex replicative form of M13 DNA to initiate rolling-circle synthesis of progeny viral DNA . Using in vitro techniques we have constructed deletion mutations in M13 DNA at the unique AvaI site which is located 45 nucleotides away on the 3' side of the gene II protein nicking site . This deletion analysis has identified a sequence near the viral strand origin that is required for efficient replication of the M13 genome . We refer to this part of the intergenic region as a "replication enhancer" sequence . We have also studied the function of this sequence in chimeric pBR322-M13 plasmids and found that plasmids carrying both the viral strand origin and the replication enhancer sequence interfere with M13 phage replication . Based upon these findings we propose a model for the mechanism of action of the replication enhancer sequence involving binding of the M13 gene II protein. J Biol Chem, 1984 Aug 25, 259(16), 10556 - 68 Analysis of bacteriophage phi X174 gene A protein-mediated termination and reinitiation of phi X DNA synthesis . II . Structural characterization of the covalent phi X A protein-DNA complex; Roth MJ et al.; In the preceeding paper (Brown, D . R., Roth, M . J., Reinberg, D., and Hurwitz, J . (1984) J . Biol . Chem . 259, 10545-10555), it was shown that following bacteriophage phi X174 (phi X) DNA synthesis in vitro using purified proteins, the phi X A protein could be detected covalently linked to nascent 32P-labeled DNA . This phi X A protein-{32P}DNA complex was the product of the reinitiation reaction . The phi X A protein-{32P}DNA complex could be trapped as a protein-32P-oligonucleotide complex by the inclusion of ddGTP in reaction mixtures . In this report, the structure of the phi X A protein-32P-oligonucleotide complex has been analyzed . The DNA sequence of the oligonucleotide bound to the phi X A protein has been determined and shown to be homologous to the phi X (+) strand sequence immediately adjacent (3') to the replication origin . The phi X A protein was directly linked to the 5' position of a dAMP residue of the oligonucleotide; this residue corresponded to position 4306 of the phi X DNA sequence . The phi X A protein-32P-oligonucleotide complex was exhaustively digested with either trypsin or proteinase K and the 32P-labeled proteolytic fragments were analyzed . Each protease yielded two different 32P-labeled peptides in approximately equimolar ratios . The two 32P-labeled peptides formed after digestion with trypsin (designated T1 and T2) and with proteinase K (designated PK1 and PK2) were isolated and characterized . Digestion of peptide T1 with proteinase K yielded a product which co-migrated with peptide PK2 . In contrast, peptide T2 was unaffected by digestion with proteinase K . These results suggest that the phi X A protein contains two active sites that are each capable of binding covalently to DNA . The peptide-mononucleotide complexes T1-{32P}pdA and T2-{32P}pdA were isolated and subjected to acid hydrolysis in 6.0 N HCl . In each case, the major 32P-labeled products were identified as {32P} phosphotyrosine and {32P}Pi . This indicates that each active site of the phi X A protein participates in a phosphodiester linkage between a tyrosyl moiety of the protein and the 5' position of dAMP. J Biol Chem, 1984 Aug 25, 259(16), 10545 - 55 Analysis of bacteriophage phi X174 gene A protein-mediated termination and reinitiation of phi X DNA synthesis . I . Characterization of the termination and reinitiation reactions; Brown DR et al.; The phi X174 (phi X) gene A protein-mediated termination and reinitiation of single-stranded circular (SS(c} phi X viral DNA synthesis in vitro were directly and independently analyzed . Following incubation together with purified DNA replication enzymes from Escherichia coli, ATP, {alpha-32P}dNTPs, and either the phi X A protein and phi X replicative form I (RF I) DNA, or the purified RF II X A complex, the phi X A protein was detected covalently linked to newly synthesized 32P-labeled DNA . Formation of the phi X A protein-{32P}DNA covalent complex required all the factors necessary for phi X (+) SS(c) DNA synthesis in vitro . Thus, it was a product of the reinitiation reaction and an intermediate of the replication cycle . Identification of this complex provided direct evidence that reinitiation of phi X (+) strand DNA synthesis involved regeneration of the RF II X A complex . Substitution of 2',3'-dideoxyguanosine triphosphate (ddGTP) for dGTP in reaction mixtures resulted in the formation of covalent phi X A protein 32P-oligonucleotide complexes; these complexes were trapped analogues of the regenerated RF II X A complex . They could not act catalytically due to the presence of ddGMP residues at the 3'-termini of the oligonucleotide moieties . Reaction mixtures containing ddGTP also yielded nonradioactive (+) SS(c) DNA products derived from circularization of the displaced (+) strand of the input parental template DNA . The formation of the phi X A protein-32P-oligonucleotide complexes and nonradioactive (+) SS(c) DNA were used to assay both reinitiation and termination reactions, respectively . Both reactions required DNA synthesis from the 3'-hydroxyl primer at nucleotide residue 4305 which was formed by cleavage of phi X RF I DNA by the phi X A protein . Elongation of this primer by 18, but not 11 nucleotides was sufficient to support each reaction . Reinitiation reactions proceeded rapidly and were essentially complete after 90 s . In contrast, when ddGTP was replaced with dGTP in reaction mixtures, DNA synthesis proceeded with linear kinetics for up to 10 min . These results suggested that in the presence of all four dNTPs, active templates supported more than 40 rounds of DNA synthesis. Nucleic Acids Res, 1984 Aug 24, 12(16), 6443 - 54 Gene A protein cleavage of recombinant plasmids containing the phi X174 replication origin; Fluit AC et al.; Synthetic oligonucleotides, DNA ligase and DNA polymerase were used to construct double-stranded DNA fragments homologous to the first 25, 27 or 30 b.p . of the origin of replication of bacteriophage phi X174 (nucleotides 4299-4328 of the phi X174 DNA sequence) . The double-stranded DNA fragments were cloned into the unique SmaI or HindIII restriction sites in the kanamycin-resistance gene of pACYC177 (AmpR, KmR) . Recombinant plasmids were picked up by colony hybridization . DNA sequencing showed that not only recombinant plasmids with the expected insert were formed, but also recombinant plasmids with a shorter insert . Recombinant plasmids with an insert homologous to the first 24, 25, 26, 27, 28 or all 30 b.p . of the phi X174 origin region were thus obtained . Supercoiled plasmids containing a sequence homologous to the first 27, 28 or 30 b.p . of the phi X174 origin region are nicked by the phi X174 gene A protein . However, the other supercoiled plasmids are not nicked by the phi X174 gene A protein . These results show that the first 27 b.p . of the phi X174 origin region are sufficient as well as required for the initiation step in phi X174 RF DNA replication, i.e . the cleavage by gene A protein. Nucleic Acids Res, 1984 Aug 24, 12(16), 6615 - 28 Site specific mutagenesis: insertion of single noncomplementary nucleotides at specified sites by error-directed DNA polymerization; Zakour RA et al.; We have utilized infidelity of DNA synthesis as a basis for site-directed mutagenesis . Both an endonuclease restriction fragment and a synthetic oligonucleotide were used as primers . DNA polymerase from bacteriophage T4 was used to elongate primer termini to a position immediately adjacent to two different preselected positions on phiX174 DNA templates . Then, the error-prone DNA polymerase from avian myeloblastosis virus was used to insert single non-complementary nucleotides at the designated positions at high efficiency . DNA sequence analysis confirmed that the mutant phage produced as a result of each site-specific mutagenesis reaction contained the nucleotide that was complementary to the one provided during the DNA copying reaction . The general applicability of this methodology to cloned DNAs will be discussed. Biochem Biophys Res Commun, 1984 Aug 16, 122(3), 960 - 5 Synthesis of rat transferrin in Escherichia coli containing a recombinant bacteriophage; Aldred AR et al.; Using mRNA from rat liver a cDNA library was constructed in lambda gt11Amp3 . Immunochemical screening identified 15 clones producing transferrin . The identity of two clones was confirmed by nucleotide sequencing, which also indicated a presegment rich in hydrophobic amino acids but lack of a prosegment in precursor transferrin . A 920 base pair insert in one clone corresponded to 84% of the N-terminal domain of transferrin, which was synthesized as a hybrid protein with bacterial beta-galactosidase . A 1540 base pair insert in another clone corresponded to the N-terminal plus 50% of the carboxy terminal domain of transferrin . The product of this clone possessed only antigenic properties of transferrin. Eur J Biochem, 1984 Aug 15, 143(1), 183 - 7 Selection of a cDNA clone which contains the complete coding sequence for the mature form of ornithine transcarbamylase from rat liver: expression of the cloned protein in Escherichia coli . Molecular cloning of rat ornithine transcarbamylase; McIntyre P et al.; A cDNA clone corresponding to the mature form of ornithine transcarbamylase (OTCase) was selected from a rat liver cDNA library constructed in bacteriophage lambda gt10 . OTCase clones were selected using a synthetic DNA probe of 15 bases corresponding to the 3' end of the OTCase mRNA {Horwich, A . L., Kraus, J.P., Williams, K., Kalousek, F., Konigsberg, W . & Rosenberg, L.E . (1983) Proc . Natl Acad . Sci . USA, 80, 4258-4262} . Putative OTCase clones were subcloned into the expression vector, pUC9, and the identity of inserts confirmed by colony immunoassay and by electrophoretic transfer of cloned proteins from sodium dodecyl sulphate/polyacrylamide gels to nitrocellulose filters followed by probing with monospecific anti-OTCase antibodies and 125I-labelled protein A . A clone corresponding to the full-length mature form of rat liver OTCase (plus 15 amino acids from Escherichia coli beta-galactosidase) was obtained and the identity of the clone was confirmed by comparison of the 5' sequence with a limited N-terminal amino acid sequence {Lusty, C., Jilka, R . L . & Nietsch, E . H . (1979) J . Biol . Chem . 254, 10030-10036} . A sequence discrepancy between the published sequence (Lusty et al.) and the sequence predicted from the cDNA structure is noted. J Biol Chem, 1984 Aug 10, 259(15), 9821 - 5 The chicken delta-crystallin gene family . Two genes of similar structure in close chromosomal approximation; Hawkins JW et al.; delta-Crystallin is a major protein product of the differentiated chicken lens . We have isolated two, non-allelic delta-crystallin genes using a recombinant bacteriophage/chicken genomic DNA library . There appear to be only these two delta-crystallin genes in the haploid chicken genome . Southern hybridization and R-loop analyses indicate that the two genes are oriented on the chromosome with similar 5'-3' polarity . delta 1, arbitrarily designated as the directionally 5' of the two genes, is 6.7 kilobases in length, while delta 2 is 9.2 kilobases . The two delta-crystallin genes are about 4.2 kilobases apart . Structurally, both genes are arranged in a similar and characteristic pattern of 17 exons/16 introns, as judged by electron microscopy . The delta-crystallin gene locus represents a simple model for the study of structural co-evolution and/or functional co-expression of two related genes within a developmentally modulated region of the genome. Nucleic Acids Res, 1984 Aug 10, 12(15), 5979 - 93 The bacteriophage T4 regA gene: primary sequence of a translational repressor; Trojanowska M et al.; The regA gene product of bacteriophage T4 is an autogenously controlled translational regulatory protein that plays a role in differential inhibition (translational repression) of a subpopulation of T4-encoded "early" mRNA species . The structural gene for this polypeptide maps within a cluster of phage DNA replication genes, (genes 45-44-62-regA-43-42), all but one of which (gene 43) are under regA-mediated translational control . We have cloned the T4 regA gene, determined its nucleotide sequence, and identified the amino-terminal residues of a plasmid-encoded, hyperproduced regA protein . The results suggest that the T4 regA gene product is a 122 amino acid polypeptide that is mildly basic and hydrophilic in character; these features are consistent with known properties of regA protein derived from T4-infected cells . Computer-assisted analyses of the nucleotide sequences of the regA gene and its three upstream neighbors (genes 45, 44, and 62) suggest the existence of three translational initiation units in this four-gene cluster; one for gene 45, one for genes 44, 62 and regA, and one that serves only the regA gene . The analyses also suggest that the gene 44-62 translational unit harbors a stable RNA structure that obligates translational coupling of these two genes. Nucleic Acids Res, 1984 Aug 10, 12(15), 6337 - 55 Analysis of the Escherichia coli proBA locus by DNA and protein sequencing; Deutch AH et al.; A 2.9 kb DNA fragment carrying the Escherichia coli proBA region, which encodes the first two enzymes of the proline biosynthetic pathway, was subcloned onto an expression plasmid carrying both the bacteriophage lambda PL promoter (lambda PL) and the lambda gene encoding a thermolabile cI repressor protein (cI857) . Derepression of the lambda PL promoter by thermal inactivation of the cI857 repressor protein resulted in the simultaneous overproduction of the proB (gamma-glutamyl kinase) and proA (gamma-glutamyl phosphate reductase) gene products . Nucleotide sequence analysis of the proBA locus allowed gene assignments consistent with the NH2 and COOH-terminal analyses and amino acid compositions of homogeneous preparations of the proB and proA proteins . The contiguous nature of the proB and proA genes suggests that the two genes constitute an operon in which proB precedes proA. FEBS Lett, 1984 Aug 6, 173(2), 351 - 6 The bond in the bacteriophage phi X174 gene A protein--DNA complex is a tyrosyl-5'-phosphate ester; van Mansfeld AD et al.; The bacteriophage phi X174 gene A protein cleaves the viral strand of the double-stranded replicative form (RF) DNA of the phage at a specific site, the origin . It leaves a free 3'-OH at nucleotide 4305 (G) of the phi X DNA sequence and binds covalently to the DNA . The nature and position of the covalent bond have been determined using the octadecadesoxyribonucleotide CAACTTG{32P}ATATTAATAAC . This octadecamer, which corresponds to nucleotides 4299-4316 of phi X viral DNA, is cleaved by gene A protein . Gene A protein is bound to the labelled phosphate via a tyrosyl residue, indicating that binding occurs to the nucleotide corresponding to 4306 (A) of the phi X viral DNA strand. J Mol Biol, 1984 Aug 5, 177(2), 295 - 311 T4 DNA polymerase . Rates and processivity on single-stranded DNA templates; Mace DC et al.; Three different methods have been used to determine the rate at which an individual bacteriophage T4 DNA polymerase molecule moves when synthesizing DNA on a single-stranded DNA template chain . These methods agree in suggesting an in vitro rate for this enzyme of about 250 nucleotides per second at 37 degrees C . This rate is close to the rate at which bacteriophage T4 replication forks move in vivo (about 500 nucleotides per second) . Comparison with the overall amount of DNA synthesis seen in in vitro reactions reveals that only a small fraction of the T4 DNA polymerase molecules present are synthesizing DNA at any one time . This is explicable in terms of the limite