==================================BSR44================================== 44. Genomic and C DNA library for human and animal parasites, especially against: nematodes; cestodes; bacteria. DNA synthesis enzymes of: DNA polymerase; Thymidine kinase. 1 UI - 87091566 AU - Hinton SM ; Freyer G TI - Cloning, expression and sequencing the molybdenum-pterin binding protein (mop) gene of Clostridium pasteurianum in Escherichia coli. AB - mop is the structural gene for the molybdenum-pterin binding protein, which is the major molybdenum binding protein in Clostridium pastuerianum. The mop gene was detected by immunoscreening genomic libraries of C. pastuerianum and identified by determining the nucleotide sequence of the cloned insert of clostridial DNA. The deduced amino acid sequence of an open reading frame proved to be identical to the first twelve residues of purified Mop. The DNA sequence flanking the mop gene contains promoter-like consensus sequences which are probably responsible for the expression of Mop in Escherichia coli. The deduced amino acid composition shows that the protein is hydrophobic, lacks aromatic and cysteine residues and has a calculated molecular weight of 7,038. The N-terminal amino acid sequence of Mop has sequence homology with DNA binding proteins. The pattern and type of residues in the N-terminal region suggest it forms the helix-turn-helix structure observed in DNA binding proteins. We propose that Mop may be a regulatory protein binding the anabolic source of molybdenum. MH - Amino Acid Sequence ; Bacterial Proteins/ANALYSIS/*GENETICS/PHYSIOLOGY ; Base Sequence ; Carrier Proteins/ANALYSIS/*GENETICS/PHYSIOLOGY ; Chromosome Mapping ; *Cloning, Molecular ; Clostridium/*GENETICS ; DNA-Binding Proteins/ANALYSIS ; DNA, Bacterial/ANALYSIS ; Escherichia Coli/*GENETICS ; *Genes, Bacterial ; Molybdenum/*METABOLISM ; Pteridines/ *METABOLISM ; Pterins SO - Nucleic Acids Res 1986 Dec 9;14(23):9371-80 2 UI - 87089093 AU - Takano K ; Nakabeppu Y ; Maki H ; Horiuchi T ; Sekiguchi M TI - Structure and function of dnaQ and mutD mutators of Escherichia coli. AB - The nucleotide sequences of the recessive dnaQ49 and the dominant mutD5 mutator were determined. The dnaQ49 mutator has a single base substitution in the dnaQ gene, thus causing one amino acid change, 96Val (GTG)----Gly (GGG), in the DnaQ protein (epsilon subunit of DNA polymerase III holoenzyme). The mutD5 mutator possesses two base substitutions in the same gene, resulting in two amino acid changes, 73Leu (TTG)----Trp (TGG) and 164Ala (GCA)----Val (GTA), which were designated the mutD52 and mutD51 mutations, respectively. Construction of chimaeric genes carrying one or two of these mutations revealed: either mutD51 or mutD52 alone causes the dominant mutator phenotype when present in a multi-copy plasmid; mutator phenotype when present in a low-copy plasmid; the dominant mutD51 mutator activity is suppressed by the dnaQ49 mutation when both mutations are present in the same gene. Based on these findings, we devised a model for the action of these mutators. MH - Amino Acid Sequence ; Base Sequence ; Drug Resistance, Microbial ; DNA Polymerase III/*GENETICS ; DNA Polymerases/*GENETICS ; Escherichia Coli/ DRUG EFFECTS/ENZYMOLOGY/*GENETICS ; *Genes, Bacterial ; *Genes, Dominant ; *Genes, Recessive ; *Genes, Structural ; *Mutation ; Plasmids ; Rifampin/PHARMACOLOGY ; Streptomycin/PHARMACOLOGY ; Support, Non-U.S. Gov't SO - MGG 1986 Oct;205(1):9-13 3 UI - 87089084 AU - Riede I TI - T-even type phages can change their host range by recombination with gene 34 (tail fibre) or gene 23 (head). AB - T-even type phages recognize their cellular receptors with the tip of their long tail fibres. The gene products involved in receptor recognition are proteins 37 and 38. While screening libraries of phage K3 with a probe of gene 38 from phage T2, a class of weakly hybridizing clones was found in addition to the expected clones of gene 38 of K3. One of these clones was identified as being from gene 23 of the phage which codes for the major head subunit; another clone originated from gene 34, which codes for the proximal half of the long tail fibres. Neither gene product 23 nor 34 is involved in receptor recognition. Phages can recombine with the DNA of the gene 23 and gene 34 clones and change the host range. MH - Base Sequence ; Cloning, Molecular ; DNA Restriction Enzymes ; Escherichia Coli/*GENETICS ; *Genes, Viral ; Mutation ; *Recombination, Genetic ; T-Phages/*GENETICS SO - MGG 1986 Oct;205(1):160-3 4 UI - 87060973 AU - Cox EC ; Horner DL TI - DNA sequence and coding properties of mutD(dnaQ) a dominant Escherichia coli mutator gene. AB - The mutD(dnaQ) gene in Escherichia coli codes for the epsilon subunit of the DNA polymerase pol III holoenzyme. Previous work has shown that this gene lies adjacent to the gene coding for RNase H (rnh). The two products are translated from diverging promoters. Here we report on the 1.6 kb (1 kb = 10(3) bases or base-pairs) sequence of the region coding for both genes, and the transcripts encoded by them. mutD codes for two transcripts, one of whose origins lies within the rnh structural gene. Both transcripts overlap and are complementary to a region of the rnh transcript. Thus, they can potentially form double-stranded helices with rnh. Of the two possible double-stranded structures, the shorter does not interfere with a likely rnh ribosome binding site, while the longer one does. We suggest that this unique organization may regulate rnh translation rates. MH - Amino Acid Sequence ; Base Sequence ; DNA Polymerase III/GENETICS ; DNA, Bacterial/*GENETICS ; Endoribonucleases/GENETICS ; Escherichia Coli/ ENZYMOLOGY/*GENETICS ; *Genes, Bacterial ; *Genes, Dominant ; Genes, Regulator ; *Mutation ; Support, U.S. Gov't, P.H.S. ; Transcription, Genetic SO - J Mol Biol 1986 Jul 5;190(1):113-7 5 UI - 87057033 AU - Stephens RS ; Mullenbach G ; Sanchez-Pescador R ; Agabian N TI - Sequence analysis of the major outer membrane protein gene from Chlamydia trachomatis serovar L2. AB - The structural gene for the major outer membrane protein (MOMP) from Chlamydia trachomatis was cloned and sequenced. A lambda gt11 recombinant (lambda gt11/L2/33) that contains a portion of the MOMP coding sequence was used to probe a lambda 1059 library constructed from DNA obtained from C. trachomatis serovar L2. Selected lambda 1059 recombinants were mapped with endonuclease restriction enzymes. The MOMP gene was mapped to the 5' end of a BamHI fragment of approximately 9 kilobases. Contiguous endonuclease restriction fragments identified within this region permitted the selection of specific fragments for subcloning and DNA sequencing. The MOMP gene consisted of a 1,182-base-pair open reading frame that encoded 394 amino acids and ended with three stop codons. The known amino-terminal amino acid was preceded by 22 amino acids whose sequence was compatible with a leader or signal sequence. The primary structure of MOMP determined from the translated DNA sequence demonstrated nine cysteine residues and a remarkably homogeneous distribution of charged and hydrophobic residues. MH - Amino Acid Sequence ; Bacterial Outer Membrane Proteins/*GENETICS ; Base Sequence ; Chlamydia trachomatis/*GENETICS ; *Genes, Bacterial ; Genes, Structural ; Phage Lambda/GENETICS ; Recombinant Proteins/GENETICS ; Support, Non-U.S. Gov't SO - J Bacteriol 1986 Dec;168(3):1277-82 6 UI - 87057025 AU - Roberts I ; Mountford R ; High N ; Bitter-Suermann D ; Jann K ; Timmis K ; Boulnois G TI - Molecular cloning and analysis of genes for production of K5, K7, K12, and K92 capsular polysaccharides in Escherichia coli. AB - With a DNA fragment from within the region encoding the transport functions for K1 production as a hybridization probe in Southern blot experiments, homologous DNA sequences were detected in the DNA from Escherichia coli strains producing K5, K7, K92, and K100 capsular polysaccharides. No homology with the laboratory strain LE392 was detected. The same DNA probe was used to prescreen cosmid libraries in LE392 by colony hybridization, as a rapid method to isolate clones encoding the genes for K5, K7, K12, and K92 antigen production. Clones carrying sequences homologous to the probe that also produced capsular material were identified by using polyclonal and monoclonal antibodies raised against the K antigen in question and K antigen-specific phages. By restriction enzyme mapping of the appropriate cosmid clones it was possible to align the genes for the production of different K antigens in terms of common restriction endonuclease cleavage sites. A DNA fragment encoding the postulated transport functions for K7 antigen production could complement deletion mutations in the transport functions for K1 antigen production. Thus the transport to the cell surface of chemically distinct polysaccharides may be by a common process. Analysis in E. coli of the proteins produced by plasmids carrying the likely transport functions for K1, K5, and K7 antigen production revealed that each region coded for a similar polypeptide. MH - Antigens, Bacterial/GENETICS ; Biological Transport ; Cloning, Molecular ; Cosmids ; DNA Restriction Enzymes ; DNA, Bacterial/GENETICS ; Escherichia Coli/*GENETICS/IMMUNOLOGY/METABOLISM ; *Genes, Bacterial ; Genetic Complementation Test ; Polysaccharides, Bacterial/*GENETICS ; Sequence Homology, Nucleic Acid ; Support, Non-U.S. Gov't SO - J Bacteriol 1986 Dec;168(3):1228-33 7 UI - 87056928 AU - Lindgren PB ; Peet RC ; Panopoulos NJ TI - Gene cluster of Pseudomonas syringae pv. "phaseolicola: controls pathogenicity of bean plants and hypersensitivity of nonhost plants [published erratum appears in J Bacteriol in 1987 Feb;169(2):928] AB - Loss of the ability of Pseudomonas syringae pv. "phaseolicola: NPS3121 to elicit a hypersensitive response on tobacco and other nonhost plants was associated with loss of pathogenicity on the susceptible host bean. Eight independent, prototrophic transposon Tn5 insertion mutants which had lost the ability to elicit a hypersensitive response on tobacco plants were identified. Six of these mutants no longer produced disease lesions on primary leaves of the susceptible bean cultivar Red Kidney and failed to elicit a hypersensitive response on the resistant bean cultivar Red Mexican and on the nonhost plants tomato, cowpea, and soybean. The two remaining mutants had reduced pathogenicity on Red Kidney bean and elicited variable hypersensitive responses on the other plants tested. Southern blot analysis indicated that each mutant carried a single independent Tn5 insertion in one of three EcoRI fragments of about 17, 7, and 5 kilobases. Marker exchange mutagenesis further supported the conclusion that the pleiotropic mutant phenotype was not associated with multiple Tn5 insertions. A genomic library of the wild-type strain was constructed in the cosmid vector pLAFR3. A recombinant plasmid, designated pPL6, that carried P. syringae pv. "phaseolicola: genomic sequences was identified by colony hybridization. This plasmid restored the wild-type phenotype to all but one mutant, suggesting that genes affected by the insertions were clustered. Structural analysis of pPL6 and the wild-type genome indicated that the 17- and 5-kilobase EcoRI fragments were contiguous in the strain NPS3121 genome. MH - Cloning, Molecular ; DNA Insertion Elements ; *Genes, Bacterial ; Legumes ; Mutation ; *Plant Diseases ; Plants/*MICROBIOLOGY ; Pseudomonas/ *GENETICS/PHYSIOLOGY ; Support, U.S. Gov't, Non-P.H.S. SO - J Bacteriol 1986 Nov;168(2):512-22 8 UI - 87055245 AU - Liu KY ; Manning JS TI - Identification of the thymidine kinase gene of infectious bovine rhinotracheitis virus and its function in Escherichia coli hosts. AB - In an attempt to understand the gene expression of the infectious bovine rhinotracheitis virus (IBRV), the viral thymidine kinase gene (tk), a well regulated viral gene has been chosen for this study. A cosmid library of IBRV has been constructed in Escherichia coli HB101 by cloning partially Sau3A-digested DNA fragments into a cosmid vector, pJB8. Recombinant cosmids were further analyzed by restriction digestions and by Southern blot hybridization. Results showed that this cosmid library comprised all of the IBRV genome with the exception of both termini. The individual recombinant cosmid clones were then used to transform E. coli tdk- mutant strains, Ky895 or C600tdk- for the selection of the IBRV tk gene. The clones able to grow on the selection plates containing 5-fluorouracil, uridine, thymidine and ampicillin were selected and further characterized. The physical location of the viral DNA inserts of one of the clones, pIBR5, was determined and the sequences complementing the tk activity have been isolated by subcloning. The plasmid, pIBRTK, was shown to grow on selection plates and therefore, retained the ability to complement the tk gene. The E. coli mutant strain C600tdk- harboring pIBRTK partially restores the tk activity by exhibiting a three and half fold increase in the level of the incorporation of [3H]thymidine into bacterial DNA over that of C600tdk- mutant. MH - Chromosome Deletion ; Cloning, Molecular ; Cosmids ; DNA Restriction Enzymes ; Escherichia Coli/*GENETICS ; *Genes, Structural ; *Genes, Viral ; Infectious Bovine Rhinotracheitis Virus/ENZYMOLOGY/*GENETICS ; Mutation ; Nucleic Acid Hybridization ; Support, U.S. Gov't, Non-P.H.S. ; Thymidine Kinase/*GENETICS ; Translation, Genetic SO - Gene 1986;44(2-3):279-85 9 UI - 87040775 AU - Flower AM ; McHenry CS TI - The adjacent dnaZ and dnaX genes of Escherichia coli are contained within one continuous open reading frame. AB - The dnaZ and dnaX loci of Escherichia coli have been genetically defined as separate genes, both of which are essential for DNA replication (1). The 2.1 kb region of DNA that complements mutations in both genes has a maximum coding capacity of approximately 80,000 daltons. Two protein products are produced from this region with molecular weights of 77,000 and 52,000 (2,3). We have sequenced a 2.7 kb fragment containing the dnaZ and dnaX genes and determined that it contains only one open reading frame of sufficient length to encode either of these proteins. This open reading frame may encode a protein of 71,147 daltons or of 68,451 daltons depending on which potential translational initiation codon is utilized. There are two transcriptional promoters preceding the gene as well as a ribosome binding site preceding the two potential initiation codons. Both the promoters and ribosome binding sites are predicted to be weak, perhaps contributing to the low expression of these genes. MH - Base Sequence ; Binding Sites ; DNA Polymerase III/*GENETICS ; DNA Polymerases/*GENETICS ; DNA-Binding Proteins/METABOLISM ; DNA, Bacterial/ GENETICS ; Escherichia Coli/*GENETICS ; Gene Expression Regulation ; Genes, Bacterial ; Genes, Structural ; Promoter Regions (Genetics) ; Ribosomes/METABOLISM ; Support, U.S. Gov't, P.H.S. ; Transcription, Genetic SO - Nucleic Acids Res 1986 Oct 24;14(20):8091-101 10 UI - 87036332 AU - Gaziev AI ; Fomenko LA ; Zakrzhevskaya DT ; Petrov SI TI - Proteins tightly bound to DNA and DNA-synthesizing activity of nucleoids from Escherichia coli. AB - Membrane-attached nucleoids were isolated from E. coli and separated from proteins by 2 M NaCl. Disintegration of such nucleoids by ultrasound and subsequent centrifugation resulted in the formation of two fractions: a sediment (fraction I) and a supernatant (fraction II). The protein:DNA ratio of fraction I was equal to 27 and was different from that to fraction II (2.6). More than 70% of the proteins not dissociating at 2 M NaCl and bound to DNA of both fractions were polypeptides with molecular weights (Mw) of 31,000-23,000 daltons (31-23 Kdal). After pulse labelling of the cells with [3H]-thymidine, the specific radioactivity of newly synthesized DNA associated with fraction I was shown to be considerably higher than that of fraction II. The analysis of DNA-synthesizing activities in fractions I and II showed that both nucleoid fractions contained DNA polymerase I. After dissolving the two fractions in 8 M urea - 0.15% sodium dodecylsulphate (SDS) they were chromatographed on hydroxyapatite. DNA-protein complexes were obtained that did not dissociate at 4 M guanidine X HCl - 5 M urea and 1% SDS. The main protein of the complexes was a 31 Kdal polypeptide tightly bound to DNA. MH - Bacterial Proteins/ANALYSIS/*METABOLISM ; Chromosomes, Bacterial/ *ANALYSIS ; Densitometry ; DNA Polymerases/METABOLISM ; DNA, Bacterial/ BIOSYNTHESIS/*METABOLISM ; Electrophoresis, Polyacrylamide Gel ; Escherichia Coli/*GENETICS/METABOLISM ; Peptides/ANALYSIS ; RNA, Bacterial/ANALYSIS SO - J Basic Microbiol 1986;26(4):211-8 11 UI - 87033769 AU - Nakai H ; Richardson CC TI - Dissection of RNA-primed DNA synthesis catalyzed by gene 4 protein and DNA polymerase of bacteriophage T7. Coupling of RNA primer and DNA synthesis. AB - Gene 4 protein and DNA polymerase of bacteriophage T7 catalyze RNA-primed DNA synthesis on single-stranded DNA templates. T7 DNA polymerase exhibits an affinity for both gene 4 protein and single-stranded DNA, and gene 4 protein binds stably to single-stranded DNA in the presence of dTTP (Nakai, H. and Richardson, C. C. (1986) J. Biol. Chem. 261, 15208-15216). Gene 4 protein-T7 DNA polymerase-template complexes may be formed in both the presence and absence of nucleoside 5'-triphosphates. The protein-template complexes may be isolated free of unbound proteins and nucleotides by gel filtration and will catalyze RNA-primed DNA synthesis in the presence of ATP, CTP, and the four deoxynucleoside 5'-triphosphates. RNA-primed DNA synthesis may be dissected into separate reactions for primer synthesis and DNA synthesis. Upon incubation of gene 4 protein with single-stranded DNA, ATP, and CTP, a primer-template complex is formed; it is likely that gene 4 protein mediates stable binding of the oligonucleotide to the template. The complex, purified free of unbound proteins and nucleotides, supports DNA synthesis upon addition of DNA polymerase and deoxynucleoside 5'-triphosphates. Association of primers with the template is increased by the presence of dTTP or DNA polymerase during primer synthesis. DNA synthesis supported by primer-template complexes initiates predominantly at gene 4 recognition sequences, indicating that primers are bound to the template at these sites. MH - Base Sequence ; DNA Polymerases/*METABOLISM ; *DNA Replication ; Escherichia Coli/*GENETICS/METABOLISM ; Kinetics ; Protein Binding ; Ribonucleotides/METABOLISM ; RNA, Viral/*GENETICS ; Support, Non-U.S. Gov't ; Support, U.S. Gov't, P.H.S. ; T-Phages/*GENETICS/METABOLISM ; Viral Proteins/*METABOLISM SO - J Biol Chem 1986 Nov 15;261(32):15217-24 12 UI - 87031593 AU - Martinez S ; Lopez P ; Espinosa M ; Lacks SA TI - Cloning of a gene encoding a DNA polymerase-exonuclease of Streptococcus pneumoniae. AB - A procedure was developed for cloning and characterizing genes that encode proteins with nuclease activity in the Streptococcus pneumoniae [pLS1] host/vector system. Clones are screened for nuclease activity by a DNase colony assay and the nucleases that they produce are characterized by detection of enzyme activity after sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The method was used to clone the gene encoding a DNA polymerase (Pol)-exonuclease of S. pneumoniae. The activity of this enzyme, the predominant DNA Pol of S. pneumoniae, is tenfold greater in cells carrying the multicopy recombinant plasmid than in cells without the plasmid. The enzyme corresponds to a 100-kDa polypeptide, and its properties are similar to PolI of Escherichia coli. A restriction map of the pSM22 plasmid containing the pneumococcal polA gene was obtained. The gene was transferred into Bacillus subtilis and E. coli, and it was expressed in both species. Its direction of transcription was determined by placement of the gene in both orientations in an E. coli hyperexpression plasmid. In one of the orientations the pneumococcal PolI enzyme was produced at a level 50-fold greater than normally found in S. pneumoniae, and it comprised 5% of the total protein. MH - Bacillus Subtilis/GENETICS ; *Cloning, Molecular ; DNA Polymerase I/ *GENETICS/METABOLISM ; DNA Polymerases/*GENETICS ; DNA Restriction Enzymes ; Escherichia Coli/GENETICS ; *Genes, Bacterial ; *Genes, Structural ; Plasmids ; Streptococcus pneumoniae/ENZYMOLOGY/*GENETICS ; Support, Non-U.S. Gov't ; Support, U.S. Gov't, Non-P.H.S. ; Support, U.S. Gov't, P.H.S. SO - Gene 1986;44(1):79-88 13 UI - 87016368 AU - Moore RE ; Davies MS ; O'Connell KM ; Harding EI ; Wiegand RC ; Tiemeier DC TI - Cloning and expression of a cDNA encoding a maize glutathione-S-transferase in E. coli. AB - The isolation and characterization of a family of maize glutathione-S-transferases (GST's) has been described previously. These enzymes are designated GSTs I, II and III based on size, substrate specificity and responsiveness to safeners. GST III has been shown to act on the herbicide alachlor as well as the commonly used substrate 1-chloro-2,4-dinitrobenzene (CDNB). Clones were isolated from a maize cDNA library in lambda gt10. Three clones contained the entire coding region for GST III. The sequences of these clones were consistent with the known amino terminal GST III protein sequence. Moreover, expression of one of these clones in E. coli resulted in a GST activity as measured with both CDNB and alachlor, proving that at least one of the clones encodes an active GST III species. With the enzyme expressed in E. coli it will become possible to study enzyme structure-function relationships ex planta. While a number of different GST proteins are present in maize tissue the GST III gene is present in single or low copy in the genome. MH - Amino Acid Sequence ; Base Sequence ; *Cloning, Molecular ; Corn/ ENZYMOLOGY/GENETICS ; DNA/*METABOLISM ; Escherichia Coli/*GENETICS ; *Genes, Structural ; Glutathione Transferases/*GENETICS ; Nucleic Acid Hybridization ; Plants/ENZYMOLOGY/*GENETICS SO - Nucleic Acids Res 1986 Sep 25;14(18):7227-35 14 UI - 87014500 AU - Weinberger S ; Sperling J TI - Characterization of Escherichia coli mutant strains deficient in AP DNA-repair synthesis. AB - Deficiency of apurinic/apyrimidinic (AP) DNA-repair enzymes in crude extracts of E. coli mutants was determined by following general and specific AP DNA-repair synthesis via nick translation in the presence of either all four dNTPs, or only one dNTP. We have shown that mutations either in DNA polymerase I or in AP endonucleases or in both, inhibit to different degrees the ability to repair AP DNA. The polA mutation totally abolishes the ability to perform both general and specific AP DNA repair, while the polAex mutation affects only general AP DNA repair. The xthA tight mutants, including the deletion mutant BW9101, can cope with small amounts of AP sites but hardly with high amounts of these lesions. In addition we have found that crude extracts of the xthA mutants degrade AP DNA by two modes: a nonspecific, and an AP-specific mode. These phenomena are common to all xth mutants and enabled us to discover this mutation. In contrast to the xth mutants so far isolated, BW2001 exhibits marked sensitivity to MMS and to X-ray irradiation. We found that this strain has a proficient DNA polymerase I but is absolutely deficient in AP endonucleases. We attribute its sensitivities to a secondary mutation at the structural gene of endonuclease IV. MH - Apurinic Acid/*GENETICS ; Cell-Free System ; DNA Polymerase I/DEFICIENCY/ GENETICS ; *DNA Repair ; Endodeoxyribonucleases/DEFICIENCY/*GENETICS ; Escherichia Coli/*GENETICS ; Mutation ; Polynucleotides/*GENETICS SO - Mutat Res 1986 Sep;166(2):123-34 15 UI - 87014124 AU - Birmingham VA ; Cox KL ; Larson JL ; Fishman SE ; Hershberger CL ; Seno ET TI - Cloning and expression of a tylosin resistance gene from a tylosin-producing strain of Streptomyces fradiae. AB - A gene conferring high-level resistance to tylosin in Streptomyces lividans and Streptomyces griseofuscus was cloned from a tylosin-producing strain of Streptomyces fradiae. The tylosin-resistance (Tylr) gene (tlrA) was isolated on five overlapping DNA fragments which contained a common 2.6 Kb KpnI fragment. The KpnI fragment contained all of the information required for the expression of the Tylr phenotype in S. lividans and S. griseofuscus. Southern hybridization indicated that the sequence conferring tylosin resistance was present on the same 5 kb SalI fragment in genomic DNA from S. fradiae and several tylosin-sensitive (Tyls) mutants. The cloned tlrA gene failed to restore tylosin resistance in two Tyls mutants derived by protoplast formation and regeneration, and it restored partial resistance in a Tyls mutant obtained by N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) mutagenesis. The tlrA gene conferred resistance to tylosin, carbomycin, niddamycin, vernamycin-B and, to some degree, lincomycin in S. griseofuscus, but it had no effect on sensitivity to streptomycin or spectinomycin, suggesting that the cloned gene is an MLS (macrolide, lincosamide, streptogramin-B)-resistance gene. Twenty-eight kb of S. fradiae DNA surrounding the tlrA gene was isolated from a genomic library in bacteriophage lambda Charon 4. Introduction of these DNA sequence into S. fradiae mutants blocked at different steps in tylosin biosynthesis failed to restore tylosin production, suggesting that the cloned Tylr gene is not closely linked to tylosin biosynthetic genes. MH - *Cloning, Molecular ; Drug Resistance, Microbial ; DNA Insertion Elements ; DNA Restriction Enzymes ; *Genes, Structural ; Leucomycins/BIOSYNTHESIS/ *PHARMACOLOGY ; Streptomyces/DRUG EFFECTS/*GENETICS ; Support, Non-U.S. Gov't ; Transformation, Bacterial SO - MGG 1986 Sep;204(3):532-9 16 UI - 87014104 AU - Leach D ; Lindsey J TI - In vivo loss of supercoiled DNA carrying a palindromic sequence. AB - Interest in the fate of long palindromic DNA sequences in E. coli has been kindled by the observation that their inviability is overcome in recBC sbcB strains and that these hosts permit the construction of DNA libraries containing long palindromic sequences present in the human genome. In this paper we show that a reduction in the level of intracellular supercoiled DNA occurs as the result of the presence of a 530 bp palindrome in bacteriophage lambda. This reduction occurs in Rec+ and recA strains but not in strains lacking exonucleases V and I (recBC sbcB). However, the DNA must be active (not repressed) for this reduction to be observed, since it is not seen in a Rec+ host lysogenic for phage lambda. These results argue against two hypotheses: firstly, that the palindrome causes inviability solely by interfering with packaging, and secondly, that it does so solely by interfering with recombination. Conversely, these results suggest that a feature of active monomeric DNA (probably its replication) is involved in inviability. MH - Base Sequence ; DNA, Single-Stranded/GENETICS/ULTRASTRUCTURE ; DNA, Superhelical/*GENETICS/ULTRASTRUCTURE ; Escherichia Coli/*GENETICS ; Genotype ; Lysogeny ; Phage Lambda/GENETICS ; Species Specificity ; Support, Non-U.S. Gov't SO - MGG 1986 Aug;204(2):322-7 17 UI - 87008594 AU - Kunkel TA ; Beckman RA ; Loeb LA TI - On the fidelity of DNA synthesis. Pyrophosphate-induced misincorporation allows detection of two proofreading mechanisms. AB - The effect of pyrophosphate on the fidelity of in vitro DNA synthesis has been examined. Pyrophosphate enhances misincorporation by Escherichia coli DNA polymerase I in copying phi X174 DNA. The increased misincorporation is directly proportional to the extent of inhibition of the rate of polymerization. In contrast, pyrophosphate is not detectably mutagenic with avian myeloblastosis virus DNA polymerase or DNA polymerases alpha and beta from animal cells, which lack associated proofreading activities. This suggests that increased misincorporation by pyrophosphate is not due to an increase in misinsertions by DNA polymerase, but rather due to inhibition of proofreading by pyrophosphate. However, the pyrophosphate-induced infidelity has a different specificity from, and is not competitive with, two experimental markers of 3'----5' exonuclease proofreading; i.e. the effects of the next nucleotide or the addition of deoxynucleoside monophosphates. These distinctive features suggest a second mode of proofreading susceptible to inhibition by pyrophosphate. This concept is discussed in relation to models for proofreading described in the literature. MH - Deoxyribonucleotides/METABOLISM ; DNA/GENETICS ; DNA Polymerases/ *METABOLISM ; DNA Replication/*DRUG EFFECTS ; DNA, Viral/GENETICS ; Escherichia Coli/DRUG EFFECTS/*GENETICS ; Kinetics ; Phage Phi X 174/ GENETICS ; Pyrophosphates/*PHARMACOLOGY ; Support, U.S. Gov't, Non-P.H.S. ; Support, U.S. Gov't, P.H.S. SO - J Biol Chem 1986 Oct 15;261(29):13610-6 18 UI - 87008431 AU - Suh JW ; Boylan SA ; Price CW TI - Gene for the alpha subunit of Bacillus subtilis RNA polymerase maps in the ribosomal protein gene cluster. AB - We isolated the gene encoding the alpha subunit of Bacillus subtilis RNA polymerase from a lambda gt11 expression vector library by using anti-alpha antibody as a probe. Four unique clones were isolated, one carrying a lacZ-alpha gene fusion and three carrying the entire alpha coding region together with additional sequences upstream. The identity of the cloned alpha gene was confirmed by the size and immunological reactivity of its product expressed in Escherichia coli. Further, a partial DNA sequence found the predicted NH2 terminus of alpha homologous with E. coli alpha. By plasmid integration and PBS1 transduction, we mapped alpha near rpsE and within the major ribosomal protein gene cluster on the B. subtilis chromosome. Additional DNA sequencing identified rpsM (encoding S13) and rpsK (encoding S11) upstream of alpha, followed by a 180-base-pair intercistronic region that may contain two alpha promoters. Although the organization of the alpha region resembles that of the alpha operon of E. coli, the putative promoters and absence of rpsD (encoding S4) immediately preceding the B. subtilis alpha gene suggest a different regulation. MH - Bacillus Subtilis/ENZYMOLOGY/*GENETICS ; Bacterial Proteins/GENETICS ; Base Sequence ; Chromosome Mapping ; Chromosomes, Bacterial ; Cloning, Molecular ; Escherichia Coli/GENETICS ; *Genes, Bacterial ; Operon ; Promoter Regions (Genetics) ; Ribosomal Proteins/*GENETICS ; RNA Polymerases/*GENETICS ; Support, Non-U.S. Gov't ; Support, U.S. Gov't, P.H.S. SO - J Bacteriol 1986 Oct;168(1):65-71 19 UI - 86223789 AU - Bhagwat AS ; Sohail A ; Roberts RJ TI - Cloning and characterization of the dcm locus of Escherichia coli K-12. AB - The dcm locus of Escherichia coli K-12 has been shown to code for a methylase that methylates the second cytosine within the sequence 5'-CC(A/T)GG-3'. This sequence is also recognized by the EcoRII restriction-modification system coded by the E. coli plasmid N3. The methylase within the EcoRII system methylates the same cytosine as the dcm protein. We have isolated, from a library of E. coli K-12 DNA, two overlapping clones that carry the dcm locus. We show that the two clones carry overlapping sequences that are present in a dcm+ strain, but are absent in a delta dcm strain. We also show that the cloned gene codes for a methylase, that it complements mutations in the EcoRII methylase, and that it protects EcoRII recognition sites from cleavage by the EcoRII endonuclease. We found no phage restriction activity associated with the dcm clones. MH - Base Sequence ; *Chromosome Mapping ; *Cloning, Molecular ; Cytosine/ METABOLISM ; DNA (Cytosine-5-)-Methyltransferase/GENETICS ; DNA Restriction Enzymes/METABOLISM ; Electrophoresis, Agar Gel ; Escherichia Coli/*GENETICS ; Genes, Bacterial ; Plasmids ; Support, Non-U.S. Gov't ; Support, U.S. Gov't, Non-P.H.S. SO - J Bacteriol 1986 Jun;166(3):751-5 20 UI - 86314305 AU - Jouanin L ; Tourneur J ; Tourneur C ; Casse-Delbart F TI - Restriction maps and homologies of the three plasmids of Agrobacterium rhizogenes strain A4. AB - Agrobacterium rhizogenes strain A4 is a virulent agropine-type strain possessing three plasmids: plasmid a (pArA4a, 180 kb) is not necessary for plant transformation, plasmid b (250 kb) is the root-inducing plasmid (pRiA4), and plasmid c (pArA4c) is a cointegrate of pArA4a and pRiA4. The total plasmid DNA (pArA4) of strain A4 was cloned in the cosmid pHSG262 and the library obtained was used to establish BamHI maps of the three plasmids. The plasmids a and Ri have an apparently identical region and a partly homologous region, and are different in the remaining regions including their origins of replication. Another agropine-type A. rhizogenes strain, HRI, bears only one plasmid, which is the Ri plasmid (pRiHRI). pRiHRI and pRiA4 present the same restriction maps for a great part, but are different in a region of 48 kb; however, this region of pRiHRI is found unmodified in pArA4a and may have a role in the virulence of the bacteria. The comparison between the restriction maps of the plasmids of strain A4 leads us to propose that the recombination event leading to pArA4c formation occurs within the identical regions of pArA4a and pRiA4. In addition, the comparison with the already established map of pRiHRI suggests that strain HRI could have been derived from a recombination event between the two homologous regions of pArA4c with subsequent loss of the smaller plasmid. MH - Agrobacterium/*GENETICS ; Chromosome Mapping ; Comparative Study ; DNA Restriction Enzymes/DIAGNOSTIC USE ; DNA, Bacterial/GENETICS ; Escherichia Coli/GENETICS ; Molecular Weight ; *Plasmids ; Sequence Homology, Nucleic Acid ; Support, Non-U.S. Gov't SO - Plasmid 1986 Sep;16(2):124-34 21 UI - 86311020 AU - Zakour RA ; Schaaper RM ; Glickman BW TI - Introduction, rescue and expression of plasmid genes in mammalian cells and Escherichia coli. AB - A shuttle-vector system is described for the study of mutational specificity in mammalian cells. Using a plasmid (pGKTK) carrying the E. coli galactokinase gene (gk) and the herpes simplex virus thymidine kinase gene (tk), we demonstrate the introduction of a foreign gene into the chromosome of a mammalian cell (TK- mouse fibroblasts) and its efficient rescue back into E. coli. This system makes use of two genes, each of which can expressed in both E. coli and mammalian cells, thereby permitting one marker to be the mutational target and the other to maintain stable integration in the host. In addition, expression of both genes in bacteria makes it possible to deletion map mutants to facilitate their sequencing. In the case of a putative single-copy transformant (T8), about half of the rescued plasmids are identical in size and restriction pattern to the original plasmid. Each of these expressed the tk gene, indicating the fidelity of the rescue system. MH - Animal ; Cell Line ; DNA Restriction Enzymes/DIAGNOSTIC USE ; Escherichia Coli/*GENETICS ; Galactokinase/GENETICS ; Gene Expression Regulation ; *Genetic Vectors ; Mice ; *Mutation ; *Plasmids ; Thymidine Kinase/ GENETICS ; Transformation, Genetic SO - Mutat Res 1986 Oct;163(1):3-13 22 UI - 86303021 AU - Aoki H ; Shiroza T ; Hayakawa M ; Sato S ; Kuramitsu HK TI - Cloning of a Streptococcus mutans glucosyltransferase gene coding for insoluble glucan synthesis [published erratum appears in Infect Immun 1986 Dec;54(3):931] AB - The gtfB gene coding for a glucosyltransferase (GTF) activity of Streptococcus mutans GS-5 was isolated on a 15.4-kilobase DNA fragment by using a lambda L47.1 gene library. The activity was catalyzed by gene products of 150 and 145 kilodaltons which reacted with antibodies directed against both soluble and insoluble glucan-synthesizing GTFs. The enzyme present in crude Escherichia coli extracts synthesized both soluble and insoluble glucans. The enzyme was partially purified from lysates of the lambda DS-76 clone and synthesized both types of glucans in a primer-independent fashion. In addition, the purified enzyme exhibited a pI of approximately 5.0. Southern blot analysis indicated that the cloned GTF gene represented a contiguous nucleotide sequence on the strain GS-5 chromosome. Furthermore, evidence for the existence of a distinct gene sharing partial homology with gtfB was also obtained. The gtfB gene was subcloned into plasmid pACYC184 into E. coli and exhibited GTF activity when carried on GS-5 inserts as small as 5 kilobases. The approximate location of the GTF promoter and the direction of gene transcription were also determined. The cloned enzyme was not secreted through the cytoplasmic membrane of E. coli, since most of the activity was found in the cytoplasm and, in lesser amounts, associated with the cytoplasmic membrane. The gtfB gene was insertionally inactivated by introducing a gene fragment coding for erythromycin resistance into the GTF coding region. After transformation of strain GS-5 with the altered gene, transformants defective in insoluble glucan synthesis were identified. These results indicate that the gtfB gene codes for a GTF involved in insoluble glucan synthesis in strain GS-5. MH - Base Sequence ; *Cloning, Molecular ; DNA Insertion Elements ; Electrophoresis, Polyacrylamide Gel ; Escherichia Coli/ENZYMOLOGY ; *Genes, Bacterial ; Glucans/*BIOSYNTHESIS ; Glucosyltransferases/ *GENETICS/ISOLATION & PURIFICATION ; Streptococcus mutans/ENZYMOLOGY/ *GENETICS ; Support, Non-U.S. Gov't ; Support, U.S. Gov't, P.H.S. SO - Infect Immun 1986 Sep;53(3):587-94 23 UI - 86301872 AU - Armengod ME ; Lamb:ies E TI - Overlapping arrangement of the recF and dnaN operons of Escherichia coli; positive and negative control sequences. AB - The recF gene of Escherichia coli controls one of the recombination pathways and UV sensitivity, but its precise function and expression pattern are still largely unknown. We have characterized the promoter region of the recF gene by mapping for E. coli RNA polymerase binding sites, in vitro transcription experiments, cloning, and S1 mapping of in vivo mRNAs. It contains three overlapping promoters, two initiating transcription towards recF and one in the opposite direction. The recF promoter region is located about 600 bp upstream from the start codon of the recF structural gene and resides entirely within the translated region of the preceding gene, dnaN, which encodes for the beta subunit of DNA polymerase III. This unusual arrangement might provide discoordinate regulation of the recF and dnaN genes, thus controlling the level of DNA polymerase III holoenzyme. Expression of recF is also negatively controlled by sequences located upstream as well as inside the recF coding frame. Such negative regulation may serve to prevent toxic effects due to accumulation of an excessive number of copies of the recF gene product. MH - Chromosome Mapping ; DNA Polymerase III/*GENETICS ; DNA Polymerases/ *GENETICS ; DNA, Bacterial/GENETICS ; Escherichia Coli/*GENETICS ; Gene Expression Regulation ; *Genes, Bacterial ; Genetic Complementation Test ; Operon ; Promoter Regions (Genetics) ; *Recombination, Genetic ; RNA Polymerases/METABOLISM ; Support, Non-U.S. Gov't ; Transcription, Genetic ; Ultraviolet Rays SO - Gene 1986;43(3):183-96 24 UI - 86284313 AU - Johnson LP ; L'Italien JJ ; Schlievert PM TI - Streptococcal pyrogenic exotoxin type A (scarlet fever toxin) is related to Staphylococcus aureus enterotoxin B. AB - The nucleotide sequence of the gene encoding group A streptococcal pyrogenic exotoxin type A (SPE A) was determined by the dideoxy chain termination method. The first 30 residues of the translation product represented a hydrophobic signal peptide. The mature protein was 220 amino acids in length and had a molecular weight of 25,805. It has significant protein sequence homology with Staphylococcus aureus enterotoxin B but not with other proteins in the Dayhoff library. MH - Amino Acid Sequence ; Enterotoxins/*GENETICS ; Exotoxins/*GENETICS ; *Genes, Bacterial ; *Genes, Structural ; Sequence Homology, Nucleic Acid ; Staphylococcus aureus/*GENETICS ; Streptococcus Pyogenes/*GENETICS ; Support, Non-U.S. Gov't SO - MGG 1986 May;203(2):354-6 25 UI - 86259068 AU - Blanco L ; Guti:errez J ; L:azaro JM ; Bernad A ; Salas M TI - Replication of phage phi 29 DNA in vitro: role of the viral protein p6 in initiation and elongation. AB - The phi 29 protein p6 stimulates the formation of the protein p3-dAMP initiation complex when added to a minimal system containing the terminal protein p3, the phi 29 DNA polymerase p2 and phi 29 DNA-protein p3 complex, by decreasing about 5 fold the Km value for dATP. In addition, protein p6 stimulates elongation of the p3-dAMP initiation complex. Whereas the effect of protein p6 on initiation is similar with protein p3-containing fragments from the right or left phi 29 DNA ends, the stimulation of elongation is higher with the right than with the left phi 29 DNA terminal fragment, suggesting DNA sequence specificity. The stimulation by protein p6 of the initiation and elongation steps of phi 29 DNA replication does not require the presence of the parental protein p3 at the phi 29 DNA ends. No effect of protein p6 was obtained on the elongation of the template-primer poly(dT)-(dA) 12-18 by the phi 29 DNA polymerase. MH - Bacillus Subtilis/*GENETICS ; Bacteriophages/*GENETICS ; Base Sequence ; Deoxyadenine Nucleotides/METABOLISM ; DNA Polymerases/METABOLISM ; *DNA Replication ; DNA, Viral/GENETICS ; Support, Non-U.S. Gov't ; Support, U.S. Gov't, P.H.S. ; Templates ; Viral Proteins/GENETICS SO - Nucleic Acids Res 1986 Jun 25;14(12):4923-37 26 UI - 86250622 AU - Rowe DB ; Iismaa TP ; Wake RG TI - Nonrandom cosmid cloning and prophage SP beta homology near the replication terminus of the Bacillus subtilis chromosome. AB - From a library of Bacillus subtilis DNA cloned with the Escherichia coli cosmid vector pHC79, 85 recombinant cosmids containing DNA from near the replication terminus, terC, were identified. The DNA inserts of these cosmids were confined to three regions of a 350-kilobase segment of the chromosome extending from the left end of the SP beta prophage to approximately 75 kilobases on the right of terC. All B. subtilis genes known to reside in this segment, as well as the portion of the SP beta prophage that is expressed early in the lytic cycle of the phage, appeared to be absent from the library. A region of SP beta homology distinct from the prophage and just to the left of terC was identified. MH - Bacillus Subtilis/*GENETICS ; Bacteriophages/*GENETICS ; Chromosomes, Bacterial ; *Cloning, Molecular ; *DNA Replication ; *Genes, Bacterial ; Genes, Viral ; Nucleic Acid Hybridization ; Plasmids ; Sequence Homology, Nucleic Acid ; Support, Non-U.S. Gov't SO - J Bacteriol 1986 Jul;167(1):379-82 27 UI - 86250621 AU - Hamood AN ; Pettis GS ; Parker CD ; McIntosh MA TI - Isolation and characterization of the Vibrio cholerae recA gene. AB - A 3.6-kilobase PstI fragment was isolated from a Vibrio cholerae chromosomal DNA library and shown to encode RecA-like activity in complementation studies with Escherichia coli recA mutants. Although DNA hybridization experiments failed to detect any homology between the E. coli and V. cholerae recA genes, hyperimmune antiserum produced against purified E. coli RecA protein recognized epitopes shared by the V. cholerae protein. The V. cholerae chromosomal fragments, when cloned and transferred to E. coli, provided the missing recA functions, including resistance to the alkylating agent methyl methanesulfonate, resistance to UV irradiation, and promotion of homologous recombination in Hfr mating experiments. MH - Antigens, Bacterial/IMMUNOLOGY ; DNA, Bacterial/GENETICS ; DNA, Recombinant ; Escherichia Coli/GENETICS ; *Genes, Bacterial ; Genetic Complementation Test ; Methyl Methanesulfonate/PHARMACOLOGY ; Nucleic Acid Hybridization ; Rec A Protein/*GENETICS/IMMUNOLOGY ; Recombination, Genetic ; Sequence Homology, Nucleic Acid ; Support, U.S. Gov't, P.H.S. ; Ultraviolet Rays ; Vibrio cholerae/DRUG EFFECTS/*GENETICS/RADIATION EFFECTS SO - J Bacteriol 1986 Jul;167(1):375-8 28 UI - 86250582 AU - Fowler RG ; Schaaper RM ; Glickman BW TI - Characterization of mutational specificity within the lacI gene for a mutD5 mutator strain of Escherichia coli defective in 3'----5' exonuclease (proofreading) activity. AB - The mutD (dnaQ) gene of Escherichia coli codes for the epsilon subunit of the DNA polymerase III holoenzyme which is involved in 3'----5' exonuclease proofreading activity. We determined the mutational specificity of the mutator allele, mutD5, in the lacI gene of E. coli. The mutD5 mutation preferentially produces single base substitutions as judged from the enhanced fraction of lacI nonsense mutations and the spectrum of sequenced dominant lacI (lacId) and constitutive lacO (lacOc) mutations which were predominantly (69/71) single nucleotide substitutions. The distribution of amber lacI and sequenced lacId mutations revealed that transitions occur more frequently than transversions. A . T----G . C and G . C----A . T transitions were equally frequent and, with one major exception, evenly distributed among numerous sites. Among the transversions, A . T----T . A events were the most common, A . T----C . G substitutions were rare, and G . C----C . G changes were not detected. Transversions were unequally distributed among a limited number of sites with obvious hotspots. All 11 sequenced transversions had a consensus neighboring sequence of 5'-C-C-(mutated G or A)-C-3'. Although no large deletions or complex mutational events were recovered, sequencing revealed that mutD5 induced single nucleotide deletions within consecutive G X C sequences. An extraordinary A . T----G . C transition hotspot occurred at nucleotide position +6 in the lac operator region; the mutD5 mutation frequency of this single base pair was calculated to be 1.2 X 10(-3). MH - Base Sequence ; DNA Polymerase III/*GENETICS/METABOLISM ; DNA Polymerases/ *GENETICS ; DNA Replication ; DNA, Bacterial/BIOSYNTHESIS ; Escherichia Coli/ENZYMOLOGY/*GENETICS ; Genes, Bacterial ; *Mutation ; Operator Regions (Genetics) SO - J Bacteriol 1986 Jul;167(1):130-7 29 UI - 86223771 AU - Peet RC ; Lindgren PB ; Willis DK ; Panopoulos NJ TI - Identification and cloning of genes involved in phaseolotoxin production by Pseudomonas syringae pv. "phaseolicola:. AB - Genes involved in the production of phaseolotoxin by Pseudomonas syringae pv. "phaseolicola: NPS3121 were identified by Tn5 mutagenesis and cosmid cloning. A total of 5,180 kanamycin-resistant colonies were screened for the loss of phaseolotoxin production by a microbiological assay. Six independent, prototrophic, Tox- mutants were isolated that had Tn5 insertions in five different EcoRI fragments. All six mutants had Tn5 inserted in the same KpnI fragment, which had a length of ca. 28 kilobases including Tn5. The mutants produced residual toxin in vitro. An EcoRI fragment containing Tn5 and flanking sequences from mutant NPS4336 was cloned and used to probe a wild-type genomic library by colony hybridization. Seven recombinant plasmids showing homology to this probe were identified. Each Tox- mutant was restored in OCTase-specific toxin production by two or more of the recombinant plasmids. The data suggest that at least some of the genes involved in phaseolotoxin production were clustered in a large KpnI fragment. No homology was detected between the Tn5 target fragment cloned from mutant NPS4336 and the total genomic DNA from closely or distantly related bacteria that do not produce phaseolotoxin. MH - *Cloning, Molecular ; Drug Resistance, Microbial ; DNA Insertion Elements ; DNA Restriction Enzymes/METABOLISM ; DNA, Bacterial/ANALYSIS ; Exotoxins/*BIOSYNTHESIS ; Kanamycin/PHARMACOLOGY ; Molecular Weight ; Plasmids ; Pseudomonas/*GENETICS ; Support, U.S. Gov't, Non-P.H.S. SO - J Bacteriol 1986 Jun;166(3):1096-105 30 UI - 86230114 AU - Gram H ; R:uger W TI - The alpha-glucosyltransferases of bacteriophages T2, T4 and T6. A comparison of their primary structures. AB - With the aim of comparing the primary structures of gene products coded for by T-even bacteriophages we constructed clone libraries of the DNAs of bacteriophages T2 and T6. Using hybrid M13 phages carrying the gene for the T4-coded alpha-glucosyl transferase (alpha gt) we isolated corresponding T2 and T6 clones. The nucleotide sequences of the three alpha gt genes and the amino acid sequences derived were compared. The differences between the genes and their products are discussed in terms of structure, function and evolutionary aspects. MH - Amino Acid Sequence ; Base Sequence ; Cloning, Molecular ; Comparative Study ; DNA/ANALYSIS ; Escherichia Coli/ENZYMOLOGY/*GENETICS ; Glucosyltransferases/*GENETICS ; Support, Non-U.S. Gov't ; T-Phages/ ENZYMOLOGY/*GENETICS SO - MGG 1986 Mar;202(3):467-70 31 UI - 86139859 AU - Liu J ; Burns DM ; Beacham IR TI - Isolation and sequence analysis of the gene (cpdB) encoding periplasmic 2',3'-cyclic phosphodiesterase. AB - The cpdB gene encodes a periplasmic 2',3'-cyclic phosphodiesterase (3'-nucleotidase). This enzyme has been purified previously and the gene is located at 96 min on the Escherichia coli chromosome. In this study the cpdB gene was cloned from ClaI-cleaved DNA, and the gene product was identified. DNA blotting experiments showed that the recombinant plasmid contains a deletion with respect to the expected genomic fragment of approximately 4 kilobases, which extends into the vector. Furthermore, the gene was absent from three other recombinant libraries. Together, these findings suggest the presence in the genome of an adjacent gene whose product is lethal when it is present on a multicopy plasmid. The nucleotide sequence of the cpdB gene was also determined. The 5' and 3' untranslated sequences contain characteristic sequences that are involved in the initiation and termination of transcription, including two possible promoters, one of which may contain two overlapping -10 sequences. A strong Shine-Dalgarno sequence is followed by an open reading frame which corresponds to a protein having a molecular weight of 70,954. The first 19 amino acid residues have the characteristics of a signal peptide. The 3' untranslated sequence contains two putative rho-independent transcription terminators having low thermodynamic stability. MH - Amino Acid Sequence ; Base Sequence ; Cloning, Molecular ; DNA, Bacterial ; Escherichia Coli/ENZYMOLOGY/*GENETICS ; Genes, Bacterial ; Genes, Structural ; Plasmids ; Promoter Regions (Genetics) ; Support, Non-U.S. Gov't ; Terminator Regions (Genetics) ; Transcription, Genetic ; 2',3'-Cyclic Nucleotide Phosphodiesterases/*GENETICS SO - J Bacteriol 1986 Mar;165(3):1002-10 32 UI - 86205238 AU - Charette MF ; Weaver DT ; DePamphilis ML TI - Persistence of DNA synthesis arrest sites in the presence of T4 DNA polymerase and T4 gene 32, 44, 45 and 62 DNA polymerase accessory proteins. AB - DNA synthesis by phage T4 DNA polymerase is arrested at specific sequences in single-stranded DNA templates. To determine whether or not T4 DNA polymerase accessory proteins 32, 44, 45 and 62 eliminated recognition of these arrest sites, unique primer-templates were constructed in which DNA synthesis began at a DNA primer located at different distances from palindromic and nonpalindromic arrest sites. Nucleotide positions that caused polymerase to pause or leave the template were identified by sequence analysis of 5'-end labeled nascent DNA chains. Stable hairpin structures at palindromic sequences were confirmed by acetylation of single-stranded sequences with bromoacetaldehyde. Our results confirmed that these T4 DNA polymerase accessory proteins stimulated T4 DNA polymerase activity and processivity on natural as well as homopolymer primer-templates. However, they did not alter recognition of DNA synthesis arrest sites by T4 DNA polymerase. Extensive DNA synthesis resulted from an increased rate of translocation and/or processivity to the same extent over all DNA sequences. MH - DNA Polymerases/GENETICS/*METABOLISM ; *DNA Replication ; DNA, Viral/ ISOLATION & PURIFICATION ; Escherichia Coli/ENZYMOLOGY/*GENETICS ; *Genes, Structural ; *Genes, Viral ; Kinetics ; Molecular Weight ; Support, Non-U.S. Gov't ; Support, U.S. Gov't, P.H.S. ; T-Phages/ ENZYMOLOGY/*GENETICS ; Templates ; Viral Proteins/*GENETICS/METABOLISM SO - Nucleic Acids Res 1986 Apr 25;14(8):3343-62 33 UI - 86196066 AU - Cudny H ; Lupski JR ; Godson GN ; Deutscher MP TI - Cloning, sequencing, and species relatedness of the Escherichia coli cca gene encoding the enzyme tRNA nucleotidyltransferase. AB - The Escherichia coli cca gene which encodes the enzyme tRNA nucleotidyltransferase has been cloned by taking advantage of its proximity to the previously cloned dnaG locus. A series of recombinant bacteriophages, spanning the chromosomal region between the dnaG and cca genes at 66 min on the E. coli linkage map, were isolated from a lambda Charon 28 partial Sau3A E. coli DNA library using recombinant plasmids containing regions between dnaG and cca as probes. Two of the recombinant phage isolates, lambda c1 and lambda c4, contained the cca gene. A BamHI fragment from lambda c1 was subcloned into pBR328, and cells containing this recombinant plasmid, pRH9, expressed tRNA nucleotidyltransferase activity at about 10-fold higher level than the wild type control. The cca gene was further localized to a 1.4-kilobase stretch of DNA by Bal31 deletion analysis. The nucleotide sequence of the cca gene was determined by the dideoxy method, and revealed an open reading frame extending for a total of 412 codons from an initiator GTG codon that would encode a protein of about 47,000 daltons. Southern analysis using genomic blots demonstrated that the cca gene is present as a single copy on the E. coli chromosome and that there is no homology on the DNA level between the E. coli cca gene, and the corresponding gene in the Bacillus subtilis, Saccharomyces cerevisiae, Petunia hybrida, or Homo sapiens genomes. Homology was found only with DNA from the closely related species, Salmonella typhimurium. These studies have also allowed exact placement of the cca gene on the E. coli genetic map, and have shown that it is transcribed in a clockwise direction. MH - Amino Acid Sequence ; Base Sequence ; Chromosome Mapping ; *Cloning, Molecular ; DNA Restriction Enzymes/METABOLISM ; DNA, Bacterial/ METABOLISM ; Escherichia Coli/*GENETICS ; RNA Nucleotidyltransferases/ *GENETICS ; Support, U.S. Gov't, P.H.S. SO - J Biol Chem 1986 May 15;261(14):6444-9 34 UI - 86177565 AU - Goddard JM ; Weiland JJ ; Capecchi MR TI - Isolation and characterization of Caenorhabditis elegans DNA sequences homologous to the v-abl oncogene. AB - DNA sequences homologous to the v-abl oncogene were isolated from a Caenorhabditis elegans genomic library by their ability to hybridize with a v-src probe. The DNA sequence of 2465 nucleotides of one clone was determined. This region corresponds to the 5' protein kinase domain of v-abl plus approximately equal to 375 base pairs toward the 3' end. Four potential introns were identified. The homology between the deduced amino acid sequence of the C. elegans clone and that of the 1.2-kilobase-pair protein kinase region of v-abl is 62%. The tyrosine residue corresponding to the tyrosine that is phosphorylated in the v-src protein is conserved in the C. elegans sequence. When 95 amino acids around this tyrosine were compared with the corresponding sequences of Drosophila c-abl, v-abl, and v-src, the identities were 83%, 79%, and 56%, respectively. Hybridization of the cloned DNA with C. elegans poly(A)+ RNA revealed a major transcript of 4.4 kilobases. MH - Amino Acid Sequence ; Animal ; Base Sequence ; Caenorhabditis/*GENETICS ; Gene Expression Regulation ; Nucleic Acid Hybridization ; *Oncogenes ; Protein-Tyrosine Kinase/GENETICS ; *Proto-Oncogenes ; Sequence Homology, Nucleic Acid ; Support, U.S. Gov't, P.H.S. ; Transcription, Genetic SO - Proc Natl Acad Sci USA 1986 Apr;83(7):2172-6 35 UI - 86174349 AU - Piechocki R ; Kupper D ; Qui:nones A ; Langhammer R TI - Mutational specificity of a proof-reading defective Escherichia coli dnaQ49 mutator. AB - The dnaQ (mutD) gene product which encodes the epsilon-subunit of the DNA polymerase III holoenzyme has a central role in controlling the fidelity of DNA replication because both mutD5 and dnaQ49 mutations severely decrease the 3'-5' exonucleolytic editing capacity. It is shown in this paper that more than 95% of all dnaQ49-induced base pair substitutions are transversions of the types G:C-T:A and A:T-T:A. Not only is this unusual mutational specificity precisely that observed recently for a number of potent carcinogens such as benzo(a) pyrene diolepoxide (BPDE) and aflatoxin B1 (AFB1), which are dependent on the SOS system to mutagenize bacteria, but it is also seen for the constitutively expressed SOS mutator activity in E. coli tif-1 strains as well as for the SOS mutator activity mediated gap filling of apurinic sites. Because the G:C-T:A and A:T-T:A transversions can either result from the insertion of an adenine across from apurinic sites or arise due to the incorporation of syn-adenine opposite a purine base, we postulate that the DNA polymerase III holoenzyme also has a reduced discrimination ability in a dnaQ49 background. The introduction of a lexA (Ind-) allele, which prevents the expression of SOS functions, led to a significant reduction in the dnaQ49-caused mutator effect. Both, the mutational specificity observed and the partial lexA+ dependence of the mutator effect provoke a reanalysis of the hypothesis that the DNA polymerase III holoenzyme can be converted into the postulated but until now unidentified SOS polymerase. MH - Anticodon ; Base Sequence ; Codon ; DNA Polymerase III/*GENETICS ; DNA Polymerases/*GENETICS ; Escherichia Coli/*GENETICS ; F Factor ; *Genes, Bacterial ; *Genes, Structural ; Genotype ; Macromolecular Systems ; *Mutation ; Phenotype ; Support, Non-U.S. Gov't ; Suppression, Genetic SO - MGG 1986 Jan;202(1):162-8 36 UI - 86168006 AU - Naito S ; Uchida H TI - RNase H and replication of ColE1 DNA in Escherichia coli. AB - Amber mutations within the rnh (RNase H) gene of Escherichia coli K-12 were isolated by selecting for bacteria capable of replicating in a sup+ background replication-defective cer-6 mutant of the ColE1 replicon. The cer-6 mutation is an alteration of one base pair located 160 nucleotides upstream of the unique replication origin of this plasmid. Subsequently, we determined the DNA alterations present within these mutants. ColE1 DNA replicated in rnh(Am) recA cells, indicating that (i) RNase H, which has been shown to be absolutely required for in vitro initiation of ColE1 DNA replication, is dispensable in vivo, and (ii) ColE1 replication in the absence of RNase H is not dependent on "stable DNA replication,: which has been reported to be an alternative mode of chromosomal DNA replication. Another class of bacterial mutations was also isolated. These mutations, named herB, suppressed cer-6 replication in rnh+ bacteria. herB mutations mapped close to the polA gene on the E. coli chromosome and increased the activity of DNA polymerase I. These findings suggest that when the DNA polymerase I has an opportunity to initiate DNA synthesis before RNase H acts, the replication-defective cer-6 mutant or the wild-type ColE1 replicates in E. coli. MH - DNA Polymerase I/ANALYSIS ; *DNA Replication ; Endoribonucleases/ *GENETICS/PHYSIOLOGY ; Escherichia Coli/*GENETICS ; Mutation ; *Plasmids ; Support, Non-U.S. Gov't ; Suppression, Genetic SO - J Bacteriol 1986 Apr;166(1):143-7 37 UI - 86168005 AU - Spring KJ ; Jerlstr:om PG ; Burns DM ; Beacham IR TI - L-asparaginase genes in Escherichia coli: isolation of mutants and characterization of the ansA gene and its protein product. AB - Mutants of Escherichia coli have been isolated which are resistant to beta-aspartyl hydroxamate, a lethal substrate of asparaginase II in fungi and a substrate for asparaginase II in E. coli. Among the many phenotypic classes observed, a single mutant (designated GU16) was found with multiple defects affecting asparaginases I and II and aspartase. Other asparaginase II-deficient mutants have also been derived from an asparaginase I-deficient mutant. The mutant strain, GU16, was unable to utilize asparagine and grew poorly on aspartate as the sole source of carbon; transformation of this strain with an E. coli recombinant plasmid library resulted in a large recombinant plasmid which complemented both these defects. Two subclones were isolated, designated pDK1 and pDK2; the former complemented the partial defect in the utilization of aspartate, although its exact function was not established. pDK2 encoded the asparaginase I gene (ansA), the coding region of which was further defined within a 1.7-kilobase fragment. The ansA gene specified a polypeptide, identified in maxicells, with a molecular weight of 43,000. Strains carrying recombinant plasmids encoding the ansA gene overproduced asparaginase I approximately 130-fold, suggesting that the ansA gene might normally be under negative regulation. Extracts from strains overproducing asparaginase I were electrophoresed, blotted, and probed with asparaginase II-specific antisera; no cross-reaction of the antisera with asparaginase I was observed, indicating that asparaginases I and II are not appreciably related immunologically. When a DNA fragment containing the ansA gene was used to probe Southern blots of restriction endonuclease-digested E. coli chromosomal DNA, no homologous sequences were revealed other than the expected ansA-containing fragments.(ABSTRACT TRUNCATED AT 250 WORDS) MH - Asparaginase/ANALYSIS/BIOSYNTHESIS/*GENETICS ; Asparagine/ANALOGS & DERIVATIVES/PHARMACOLOGY ; Cross Reactions ; DNA, Bacterial/ANALYSIS ; DNA, Recombinant/ISOLATION & PURIFICATION ; Escherichia Coli/ENZYMOLOGY/ *GENETICS ; *Genes, Bacterial ; Mutation ; Plasmids ; Recombination, Genetic ; Sequence Homology, Nucleic Acid ; Support, Non-U.S. Gov't SO - J Bacteriol 1986 Apr;166(1):135-42 38 UI - 86148514 AU - Argara:na CE ; Kuntz ID ; Birken S ; Axel R ; Cantor CR TI - Molecular cloning and nucleotide sequence of the streptavidin gene. AB - Using synthetic oligonucleotides as probes we have cloned the streptavidin gene from a genomic library of Streptomyces avidinii. Nucleotide sequence analysis indicated that a 2 Kb DNA-fragment contained the entire coding region, a signal peptide region and the 3' and 5' flanking regions of the gene. The deduced amino acid sequence shows several interrupted blocks of homology with the amino acid sequence of chicken egg-white avidin. Analysis of the secondary structure suggests a high content of beta-structure in both proteins and considerable overall structural similarity between them. MH - Amino Acid Sequence ; Avidin ; Bacterial Proteins/*GENETICS ; Base Sequence ; Cloning, Molecular ; Comparative Study ; DNA, Bacterial/ GENETICS ; Genes, Bacterial ; Protein Conformation ; Streptomyces/ *GENETICS ; Support, Non-U.S. Gov't ; Support, U.S. Gov't, P.H.S. SO - Nucleic Acids Res 1986 Feb 25;14(4):1871-82 39 UI - 86139912 AU - Ott RW ; Barnes MH ; Brown NC ; Ganesan AT TI - Cloning and characterization of the polC region of Bacillus subtilis. AB - The polC gene of Bacillus subtilis is defined by five temperature-sensitive mutations and the 6-(p-hydroxyphenylazo)-uracil (HPUra) resistance mutation azp-12. Biochemical evidence suggests that polC codes for the 160-kilodalton DNA polymerase III. A recombinant plasmid, p154t, was isolated and found to contain the azp-12 marker and one end of the polC gene (N. C. Brown and M. H. Barnes, J. Cell. Biochem. 78 [Suppl.]: 116, 1983). The azp-12 marker was localized to a 1-kilobase DNA segment which was used as a probe to isolate recombinant lambda phages containing polC region sequences. A complete polC gene was constructed by in vitro ligation of DNA segments derived from two of the recombinant phages. The resulting plasmid, pRO10, directed the synthesis of four proteins of 160, 76, 39, and 32 kilodaltons in Escherichia coli maxicells. Recombination-deficient (recE) B. subtilis PSL1 containing pRO10 produced an HPUra-resistant polymerase III activity which was lost when the strain was cured of pRO10. In vivo, the HPUra resistance of the plasmid-encoded polymerase III appeared to be recessive to the resident HPUra-sensitive polymerase III enzyme. MH - Bacillus Subtilis/ENZYMOLOGY/*GENETICS ; Chromosome Mapping ; Chromosomes, Bacterial ; *Cloning, Molecular ; DNA Polymerase III/ *GENETICS/METABOLISM ; DNA Polymerases/*GENETICS ; DNA, Recombinant ; Escherichia Coli/GENETICS ; Genes, Bacterial ; Genetic Marker ; Hydroxyphenylazouracil/PHARMACOLOGY ; Phage Lambda/GENETICS ; Plasmids ; Recombination, Genetic ; Support, Non-U.S. Gov't ; Support, U.S. Gov't, P.H.S. ; Transduction, Genetic SO - J Bacteriol 1986 Mar;165(3):951-7 40 UI - 86139876 AU - Gutterson NI ; Layton TJ ; Ziegle JS ; Warren GJ TI - Molecular cloning of genetic determinants for inhibition of fungal growth by a fluorescent pseudomonad. AB - Pseudomonas fluorescens HV37a inhibits growth of the fungus Pythium ultimum in vitro. Optimal inhibition is observed on potato dextrose agar, a rich medium. Mutations eliminating fungal inhibition were obtained after mutagenesis with N-methyl-N'-nitro-N-nitrosoguanidine. Mutants were classified by cosynthesis and three groups were distinguished, indicating that a minimum of three genes are required for fungal inhibition. Cosmids that contain wild-type alleles of the genes were identified in an HV37a genomic library by complementation of the respective mutants. This analysis indicated that three distinct genomic regions were required for fungal inhibition. The cosmids containing these loci were mapped by transposon insertion mutagenesis. Two of the cosmids were found to contain at least two genes each. Therefore, at least five genes in HV37a function as determinants of fungal inhibition. MH - Alleles ; Antibiosis ; *Antifungal Agents/METABOLISM ; *Cloning, Molecular ; Cosmids ; DNA Insertion Elements ; *Genes, Bacterial ; Genetic Complementation Test ; Mastigomycotina/*GROWTH & DEVELOPMENT ; Mutation ; Pseudomonas Fluorescens/*GENETICS/METABOLISM ; Pythium/*GROWTH & DEVELOPMENT SO - J Bacteriol 1986 Mar;165(3):696-703 41 UI - 86085707 AU - Donald RG ; Nees DW ; Raymond CK ; Loroch AI ; Ludwig RA TI - Characterization of three genomic loci encoding Rhizobium sp. strain ORS571 N2 fixation genes. AB - Sixty-five independent, N2 fixation-defective (Nif-) vector insertion (Vi) mutants were selected, cloned, and mapped to the ORS571 genome. The recombinant Nif::Vi plasmids obtained in this way were used as DNA hybridization probes to isolate homologous phages from a genomic library of ORS571 constructed in lambda EMBL3. Genomic maps were drawn for three ORS571 Nif gene loci. Forty-five Nif::Vi mutants in genomic Nif locus 1 defined two gene clusters separated by 8 kilobase pairs (kb) of DNA. In the first cluster, 36 Nif::Vi mutants mapped to a 7-kb DNA segment that showed DNA homology with Klebsiella pneumoniae nifHDKE and encoded at least two Nif operons. In the other cluster, nine Nif::Vi mutants mapped to a 1.5-kb DNA segment that showed homology with K. pneumoniae and Rhizobium meliloti nifA; this DNA segment encoded a separate Nif operon. Fifteen Nif::Vi mutants mapped to a 3.5-kb DNA segment defined as Nif locus 2 and showed DNA homology with the R. meliloti P2 fixABC operon. Nif locus 2 carries a second nifH (nifH2) gene. Four Nif::Vi mutants mapped to a 2-kb DNA segment defined as Nif locus 3 and showed DNA homology with K. pneumoniae nifB. DNA from lambda Nif phages comprising all three genomic Nif loci was subcloned in plasmid vectors able to stably replicate in ORS571. These plasmid subclones were introduced into ORS571 strains carrying physically mapped Nif::Vi insertions, and genetic complementations were conducted. With the exception of certain mutants mapping to the nifDK genes, all mutants could be complemented to Nif+ when they carried plasmid subclones of defined genomic DNA regions. Conversely, most nifDK mutants behaved as pseudodominant alleles. MH - Alleles ; Chromosome Mapping ; Cloning, Molecular ; DNA Insertion Elements ; DNA, Bacterial/ANALYSIS ; *Genes, Bacterial ; Genetic Complementation Test ; Mutation ; *Nitrogen Fixation ; Nucleic Acid Hybridization ; Rhizobium/*GENETICS/METABOLISM ; Sequence Homology, Nucleic Acid ; Support, U.S. Gov't, Non-P.H.S. SO - J Bacteriol 1986 Jan;165(1):72-81 42 UI - 86085659 AU - Gianni M ; Galizzi A TI - Isolation of genes preferentially expressed during Bacillis subtilis spore outgrowth. AB - From the Charon 4A library of Ferrari et al (J. Bacteriol. 146:430-432, 1981) we isolated three genes involved in Bacillus subtilis spore outgrowth by screening the library by hybridization with labeled RNA from outgrowing spores in the presence of an excess of unlabeled vegetative RNA. Hybridization competition experiments with purified clones showed that the clones contained sequences that were transcribed only during spore outgrowth or preferentially during spore outgrowth. Fragments of the cloned DNAs were subcloned in plasmid pJH101, and by using plasmid integration and PBS1 transduction the chromosomal loci were mapped. The three loci which we mapped are outG and outH, which are located between polC and dnaA, and outI, which is located near pycA. Using the cloned DNAs and derived plasmids in dot hybridization experiments with RNA extracted from cells at different developmental stages, we defined for two clones a region that is transcribed only during the outgrowth phase. MH - Bacillus Subtilis/GROWTH & DEVELOPMENT/*GENETICS ; Base Sequence ; Chromosome Mapping ; Cloning, Molecular ; DNA, Bacterial/ANALYSIS ; *Genes, Bacterial ; Nucleic Acid Hybridization ; Phage Lambda/GENETICS ; Plasmids ; Spores, Bacterial/GROWTH & DEVELOPMENT ; Support, Non-U.S. Gov't SO - J Bacteriol 1986 Jan;165(1):123-32 43 UI - 86082357 AU - Toh H TI - T7 and E. coli share homology for replication-related gene products. AB - Recently, the complete nucleotide sequence of the bacteriophage T7 genome was determined and 50 genes were identified on the genome. We compared amino acid sequences of all the gene products of T7 and replication-related gene products of E. coli. As a result, we found that T7 and E. coli share homology for each pair of exonuclease, DNA primase and helix-destabilizing protein. For E. coli, these gene products are known to be involved in the process of discontinuous DNA replication. These observations suggest that T7 and E. coli have a common origin for a part of their replication systems. MH - Amino Acid Sequence ; *Base Sequence ; Comparative Study ; DNA Polymerase I/METABOLISM ; DNA Replication ; Escherichia Coli/ENZYMOLOGY/*GENETICS ; *Genes, Bacterial ; *Genes, Viral ; RNA Nucleotidyltransferases/ METABOLISM ; *Sequence Homology, Nucleic Acid ; T-Phages/ENZYMOLOGY/ *GENETICS SO - FEBS Lett 1986 Jan 6;194(2):245-8