Persistence of pyrimidine dimers during post-replication repair in ultraviolet light-irradiated Escherichia coli K12

We have used a new assay for pyrimidine dimers to obtain evidence regarding the mechanism of post-replication repair of ultraviolet light-induced damage in excision-deficient (uvr) mutants of Escherichia coli. Our data indicate that dimers are gradually removed from the irradiated DNA under conditions permitting post-replication repair. Concomitantly, dimers appear in daughter strands synthesized after irradiation. The daughter strands initially contain gaps. During post-replication repair the gaps are filled and the originally discontinuous DNA is joined into long molecules resembling those observed in unirradiated control cells. Density transfer experiments reported by other investigators have provided evidence that the gap-filling involves exchanges between irradiated parental DNA and unirradiated daughter strands. The results of our experiments are in accord with this possibility and suggest that some dimers are included in the exchanged regions. Our data imply that intact, dimer-free DNA molecules are not necessarily generated by gap-filling and may not appear in uvr cells until several hours after u.v. irradiation. Instead, dimers may be gradually diluted among successive generations of DNA molecules synthesized after irradiation.     

The genome of Bacillus subtilis phage SPP1: the arrangement of restriction endonuclease generated fragments.

Summary SPP1 DNA was cleaved by the restriction endonucleases, BglI, BglII, EcoRI, KpnI, SmaI, and SalI. The molecular weights of the DNA fragments obtained by single enzyme digestion or by consecutive digestion with two enzymes were determined by electron microscopic measurements of contour length and by gel electrophoresis. The major fragments from the six digests could be ordered to give a consistent restriction map of SPP1. The electropherograms of several digests indicated that certain fragments occurred in less than stoichiometric amounts or were heterogeneous in size. Such bands carried a major part of radioactivity, when SPP1 DNA was terminally labelled with P³² prior to degradation by restriction enzymes. These results, and studies of the effect of exonuclease III treatment on restriction enzyme patterns define the terminal restriction fragments. All data obaained support the conclusion drawn in the preceding paper (Morelli et al., 1978b) that the SPP1 genome is terminally redundant and partially circularly permuted.Part of this work is from the doctoral dissertations to be submitted to Stanford University¹ and the Freie Universität Berlin²     

DNA synthesis in vivo in Bacillus subtilis

DNA polymerase III is the enzyme responsible for deoxynucleotide addition to nascent DNA fragments in Bacillus subtilis protoplasts. Nascent single-stranded fragments separated from bulk DNA by hydroxyapatite chromatography cannot self-anneal. Partial inhibition of DNA polymerase III by 6-(hydroxyphenylazo)-uracil, a specific inhibitor, slows the rate of nascent fragment synthesis but has no effect on final size. Neither DNA polymerase I nor II can elongate nascent fragments in protoplasts when DNA polymerase III is completely inhibited.     

DNA replication in a polymerase I deficient mutant and the identification of DNA polymerases II and 3 in Bacillus subtilis

The partial amino acid sequences at the amino terminal of prothrombin and the intermediates of activation have been determined. These data indicate that the products of the first step of activation, whether derived from the action of factor Xa or thrombin, are identical. The data also show that the activation of prothrombin proceeds by the sequential cleavage of the amino terminal region of prothrombin and the intermediates, and confirm the mechanism of prothrombin activation as: NH₂-Prothrombin-COOH $\text{Xa or thrombin}$ NH₂-Intermediate 3 + Intermediate 1-COOH; NH₂-Intermediate 1-COOH $\text{Xa}$ NH₂-Intermediate 4 + Intermediate 2-COOH; NH₂-Intermediate 2-COOH $\text{Xa}$ NH₂-A chain α-thrombin -S-S-B chain α-thrombin-COOH.Previous reports from this laboratory have demonstrated that the activation of prothrombin proceeds through several single-chain intermediates prior to the appearance of thrombin activity. (1) Subsequent studies have sequence of the prothrombin molecule can be deduced from the sequences of its activation intermediates and we are continuing our studies toward this goal.     

Transfer and incorporation of genes controlling beta-D-galactosidase synthesis from Hfr and F' donors of Escherichia coli

Ganesan, Ann K. (Syntex Institute of Molecular Biology, Palo Alto, Calif.), and Boris Rotman. Transfer and incorporation of genes controlling beta-d-galactosidase synthesis from Hfr and F' donors of Escherichia coli. J. Bacteriol. 92:1378-1382. 1966.-Comparisons were made between Hfr(1) and F(13) donors with respect to the frequency of transfer and incorporation of genes controlling beta-d-galactosidase synthesis. The Hfr(1) donor transfers these genes as part of the chromosome, and the F(13) donor transfers them by F-duction. The criterion used for gene transfer was the acquisition by recipient cells of the ability to synthesize the enzyme, beta-d-galactosidase, measured by fluorogenic assays at the single-cell level. The criterion for incorporation was the formation of lac(+) recombinant colonies. It was found that the two types of donor showed the same frequency of gene transfer, but the probability of incorporation was 10-fold higher in F(13) matings than in Hfr(1) matings. In the former, between 46 and 97% of the merozygotes produced recombinant colonies; in the latter, 2 to 6% did so.     

Biological assay of prokaryotic genes in mouse cells following DNA mediated transformation

Summary Genes coding for leucine biosynthesis in Bacillus subtilis were introduced into mouse LTK^- cells by co-transformation with thymidine kinase⁺ (tk) DNA. Genomic DNA from the tk⁺ transformants was used to transform competent cultures of different B. subtilis leucine auxotrophs. Each auxotroph was transformed to prototrophy at a similar frequency and the number of leucine gene sequences per transformant genome as deduced by the B. subtilis bioassay strongly correlated with the number estimated by hybridization methods. Tk^- subclones were obtained by plating the transformants in 5-bromodeoxyuridine. One subclone still contained the non-selected leucine gene sequences and could transform auxotrophs of B. subtilis. No deletions or rearrangements in the linkage relationships of the leucine genes occurred in the LTK^- cells that inhibited transformation of B. subtilis.     

Induction of secretory acid proteinase in Candida albicans.

Candida albicans and some other pathogenic Candida species, when grown in a medium containing a protein as a sole source of nitrogen, secrete an acid proteinase. Culture supernatants were assayed for proteinase activity, and were also analysed by Western blotting with antibodies raised and affinity-purified against proteinase of C. albicans. Proteinases secreted by C. tropicalis and C. parapsilosis were antigenically related to that of C. albicans, but had different molecular masses. The proteinases secreted by C. lipolytica, C. rugosa and C. lusitaniae were not antigenically related. The kinetics of proteinase secretion by C. albicans were monitored by activity and by Western blotting. With BSA as the nitrogen source, proteinase secretion increased exponentially until about 16 h. Culture supernatants of BSA-grown cultures accumulated proteinase to about a 1000-fold higher level than those of ammonium-sulphate-grown cultures. In vivo labelling experiments showed that proteinase was not detectably accumulated in the cells, but was secreted immediately after synthesis. Immunoprecipitation of in vitro translated poly(A)-containing RNA identified a putative pre-protein of about 54 kDa. As well as BSA, other proteins (haemoglobin, ovalbumin, histone), peptone and tryptone, when used as nitrogen sources, could induce proteinase, but to different levels. When Casamino acids or an amino acid mixture (equivalent to the composition of BSA) was used as nitrogen source, no induction was observed. Ammonium sulphate, or any other ammonium salt, repressed secretion of proteinase.     

Pseudomonas aeruginosa exoenzyme S, a double ADP-ribosyltransferase, resembles vertebrate mono-ADP-ribosyltransferases

Previous data indicated that Pseudomonas aeruginosa exoenzyme S (ExoS) ADP-ribosylated Ras at multiple sites. One site appeared to be Arg41, but the second site could not be localized. In this study, the sites of ADP-ribosylation of c-Ha-Ras by ExoS were directly determined. Under saturating conditions, ExoS ADP-ribosylated Ras to a stoichiometry of 2 mol of ADP-ribose incorporated per mol of Ras. Nucleotide occupancy did not influence the stoichiometry or velocity of ADP-ribosylation of Ras by ExoS. Edman degradation and mass spectrometry of V8 protease generated peptides of ADP-ribosylated Ras identified the sites of ADP-ribosylation to be Arg41 and Arg128. ExoS ADP-ribosylated the double mutant, RasR41K,R128K, to a stoichiometry of 1 mol of ADP-ribose incorporated per mol of Ras, which indicated that Ras possessed an alternative site of ADP-ribosylation. The alternative site of ADP-ribosylation on Ras was identified as Arg135, which was on the same alpha-helix as Arg128. Arg41 and Arg128 are located within two different secondary structure motifs, beta-sheet and alpha-helix, respectively, and are spatially separated within the three-dimensional structure of Ras. The fact that ExoS could ADP-ribosylate a target protein at multiple sites, along with earlier observations that ExoS could ADP-ribosylate numerous target proteins, were properties that have been attributed to several vertebrate ADP-ribosyltransferases. This prompted a detailed alignment study which showed that the catalytic domain of ExoS possessed considerably more primary amino acid homology with the vertebrate mono-ADP-ribosyltransferases than the bacterial ADP-ribosyltransferases. These data are consistent with the hypothesis that ExoS may represent an evolutionary link between bacterial and vertebrate mono-ADP-ribosyltransferases.     

Effect of tsl (thermosensitive suppressor of lex) mutation on postreplication repair in Escherichia coli K-12.

Cells of Escherichia coli K-12 carrying lexA or recA mutations are more sensitive to UV radiation than corresponding wild-type cells and are defective in postreplication repair. Supressor mutations (tsl) have been described previously which increase the UV resistance of lexA uvr+, lexA uvrA, and recAI uvr+ strains, but not the resistance of recA1 uvrA strains. We have studied the effect of the tsl-1 mutation on postreplication repair and find that the enhanced survival conferred by this mutation is correlated with an increased capacity for postreplication repair.