PubMed Health⌕ Search

Biomedical subjects

W D Rupp

Publications and source records attributed to W D Rupp.

At least 37 records · Page 2Linked to original sources
PubMed1983

Use of an Escherichia coli mutant strain permits measurement of single-stranded apurinic-apyrimidinic endonuclease in crude extracts: studies with untransformed cells and cells transformed with plasmids containing the uvrC gene.

We have constructed a strain of Escherichia coli that is defective in exonuclease VII and uracil-DNA glycosylase activities. This strain (xse ung) facilitates the quantitation of single-stranded apurinic-apyrimidinic endonuclease activity in crude extracts. Quantitative comparisons of single-stranded apurinic-apyrimidinic endonuclease activity under conditions in which uvrC protein is overexpressed showed no differences, suggesting that single-stranded apurinic-apyrimidinic endonuclease and uvrC protein are probably distinct.

Bacterial Proteins↗
PubMed1982

The uvrB gene of Escherichia coli has both lexA-repressed and lexA-independent promoters.

We have found that the uvrB gene of Escherichia coli is transcribed from at least two promoters, which we call P1 and P2. Transcription from P1 begins with an ATP at +1, and transcription from P2 begins primarily with a GTP at -31. A binding site for the lexA protein (LEXA), located between the -35 sequence and Pribnow box of P2, regulates transcription from this promoter. In vitro, LEXA inhibits transcription from P2 but has no detectable effect on transcription from P1. A third promoter, P3, was also detected at -341; transcription from P3 is toward uvrB but terminates in vitro in the region of the LEXA binding site. The binding of LEXA to P2 inhibits transcription from the P3 promoter even though several hundred nucleotides separate the two promoters. The data suggest that a transcribing RNA polymerase stalls when it reaches the repressor-operator complex but remains bound to the DNA, causing a jamming of RNA polymerases between P3 and the repressor-operator complex at P2. The physiological significance of P3 is unknown.

Bacterial Proteins↗
PubMed1981

A general approach for purifying proteins encoded by cloned genes without using a functional assay: isolation of the uvrA gene product from radiolabeled maxicells.

The uvrA protein (UVRA) of E. coli has been extensively purified from a strain in which UVRA is overproduced and specifically labeled with 35S-methionine. This approximately 100-fold overproduction relative to normal strains is a result of having the uvrA gene present on a multicopy plasmid in a spr recA cell that makes defective lexA protein, the normal repressor of the uvrA gene, while the specific labeling of UVRA is done with maxicells. This approach facilitates the preparation of the protein since enzyme assays do not have to be carried out during the intermediate stages of purification. The purified UVRA binds to DNA and has ATPase activity but does not have intrinsic endonuclease activity. When added to extracts of uvrA- cells, the purified UVRA does promote the specific cutting of UV-irradiated DNA. Since this approach for working out rapid purification procedures by specifically labeling the proteins encoded by cloned genes does not require the use of a functional assay, it is a general one that can be applied to a wide variety of other gene products in addition to UVRA.

Adenosine Triphosphatases↗
PubMed1981

Sequences of the ssb gene and protein.

We have determined the sequences of the ssb gene and protein of Escherichia coli. The coding region of ssb is 534 base pairs and is preceeded and followed by dyad symmetries of 39 base pairs and 27 base pairs, respectively. The promoter for ssb is close to that for uvrA and these two genes are transcribed in opposite directions: ssb clockwise and uvrA counterclockwise on the standard E. coli genetic map. The DNA helix-destabilizing protein encoded by the ssb gene (single-strand binding protein) contains 177 amino acids and has a calculated molecular weight of 18,873. Although there is no extensive sequence homology among the three helix-destabilizing proteins whose sequences are now known, both the E. coli and bacteriophage T4 DNA helix-destabilizing proteins do contain an acidic, alpha-helical region at their carboxy termini that may be functionally homologous. The remainder of the E. coli helix-destabilizing protein can be divided into two apparent domains on the basis of its amino acid sequence. The amino-terminal region (residues 1-105) contains 79% of the charged residues (27 out of 34 total) in the protein and is predicted to have a high degree of secondary structure (alpha helix and beta pleated sheet). In contrast, the region including residues 106-165 contains only two charged amino acids and is devoid of alpha helix or beta pleated sheet.

Amino Acid Sequence↗
PubMed1981

Identification of the uvrC gene product.

We have constructed a multicopy plasmid that carries the uvrC gene of Escherichia coli. By inserting the transposon Tn1000 (previously designated gamma delta) into this plasmid, we obtained many derivatives that fail to complement uvrC34. The proteins synthesized by the original plasmid and the uvrC::Tn1000 derivatives were labeled in maxicells and analyzed on gels, demonstrating that a protein of Mr 70,000 encoded by the original uvrC+ plasmid was absent from the mutated noncomplementing derivatives; this protein is presumed to be the uvrC gene product. We found that this protein of Mr 70,000 binds to DNA and have partially purified the uvrC protein by DNA-cellulose chromatography. Because some of the uvrC::Tn1000 derivatives produce truncated polypeptides, the orientation of expression and the location of the promoter were determined by correlating the sizes of the truncated polypeptides with the sites of insertion of Tn1000.

Bacterial Proteins↗
PubMed1980

Amplification of single-strand DNA binding protein in Escherichia coli.

An E. coli strain containing a recombinant plasmid carrying the E. coli ssbA+ gene has been shown to produce 12 to 15 fold increased amounts of single-strand DNA binding-protein relative to wild-type strains. In addition, a gamma transducing phage carrying the E. coli uvrA+ gene has been shown to also carry the ssbA+ gene and to be capable of producing increased amounts of binding protein.

Bacteriophage lambda↗
PubMed1980

Sequences of the recA gene and protein.

We have determined the nucleotide sequence of the recA gene of Escherichia coli; this permits the formulation of the primary structure for the recA protein. This structure is consistent with the amino acid composition of the tryptic peptides obtained from the recA protein. The coding region of the recA gene has 1059 base pairs, which specify 352 amino acids. The recA protein has alanine and phenylalanine as its NH2- and COOH-terminal amino acids, respectively, and has the following amino acid composition: Cys3 Asp20 Asn15 Met9 Thr17 Ser20 Glu30 Gln13 Pro10 Gly35 Ala38 Val22 Ile27 Leu31 Tyr7 Phe10 His2Lys27 Trp2 Arg14. Of the three cysteine residues, only two can be alkylated under reducing and denaturing conditions. The molecular weight of the recA polypeptide is 37,842.

Amino Acid Sequence↗
PubMed1980

Impaired incision of ultraviolet-irradiated deoxyribonucleic acid in uvrC mutants of Escherichia coli.

The production of single-strand breaks in the deoxyribonucleic acid of irradiated uvrC mutants of Escherichia coli K-12 was studied both in vivo and in vitro. In vivo, uvrC mutants displayed a slow accumulation of breaks after irradiation, and in this respect appeared different from uvrA mutants, in which very few breaks could be detected. The breakage observed in uvrC mutants differed from that observed in wild-type strains in both the slow rate of break accumulation and the very limited dose response. The behavior of the uvrC lig-7(Ts) double mutant was shown not to be consistent with the suggestion of ligase reversal as the explanation for the lower rate and limited dose response of break formation observed in ultraviolet-irradiated uvrC mutants in vivo. Rather, there appeared to be a real defect in incision. In toluene-treated cells, we studied the effect of the ligase inhibitor nicotinamide mononucleotide on strand incision. Whereas uvrC mutants displayed more strand breakage in the presence of this inhibitor, the same amount of breakage was seen in uvrA mutants, and as such the breakage could be judged as not due to the main excision repair pathway. Experiments using a cell-free system comprising the partially purified uvr+ gene products demonstrated clearly that there is a requirement for the uvrC+ gene product for strand incision. We suggest that in vivo in the absence of the uvrC+ gene product, a partial analog of this protein may allow some abnormal incision.

DNA Repair↗
PubMed1980

Effect of excision repair on the mutagenesis of misonidazole.

The mutagenic and toxic effects of the nitroimidazole hypoxic cell sensitizer misonidazole (Ro-07-0582) have been studied in two strains of E. coli that differ in their abilities to repair DNA. It was found that even though the strain defective in excision repair was only slightly more sensitive than the original wild-type strain to killing by misonidazole, a large difference in the mutagenesis of the two strains was observed. While the wild-type strain was not mutated by misonidazole at any of the concentrations tested, the uvr strain was strongly mutated by the drug. This demonstrates the presence of a mutagenic lesion in DNA that is removed by excision repair.

DNA Repair↗
PubMed1979

Potential radiosensitizing agents. Dinitroimidazoles.

New compounds of the nitroimidazole series have been synthesized as radiosensitizers which selectively sensitize hypoxic cells to the lethal effect of radiation. The reaction of 2,4(5)-dinitroimidazole (2) with chloroethanol or hydrochloric acid yielded 4(5)-nitro-5(4)-chloroimidazole (3), which upon reaction with ethylene oxide yielded the 4-nitro-5-chloroimidazole-1-ethanol (6). Reaction of 2 with ethylene oxide resulted in a mixture of two compounds, the 2,4-dinitroimidazole-1-ethanol (4) and 2,3-dihydro-5-nitroimidazo[2,1-b]oxazole (5). The structure of the new heterocyclic compound 5 was confirmed by 1H NMR, mass spectrum, and X-ray crystallography. These agents were tested for their ability to sensitize hypoxic Escherichia coli cells to killing by ionizing radiation. Compound 4 was found to be the most active agent of this series of compounds.

Chemical Phenomena↗
PubMed1979

Physical map of the recA gene.

We have cloned the recA gene of Echerichia coli K12 and some of its restriction fragments on the plasmid cloning vehicle pBR322. The recA gene was mapped with regard to the restriction sites of EcoRI, BamHI, Pst I, Hha I, Hae III, HinfI, and Taq I restriction endonucleases. The recA promoter was localized by the binding of RNA polymerase to restriction fragments. The initiation point of transcription of recA mRNA and the direction of transcription were determined from in vitro transcription of recA gene fragments and from analysis of the polypeptides made in maxicells that contain plasmids carrying only part of the recA gene.

Bacterial Proteins↗