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K McEntee

Publications and source records attributed to K McEntee.

At least 73 records · Page 4Linked to original sources
PubMed1986

Transcriptional regulation of DNA damage responsive (DDR) genes in different rad mutant strains of Saccharomyces cerevisiae.

The roles of the RAD genes of Saccharomyces cerevisiae in the regulation of transcription of two DNA damage responsive (DDR) genes were investigated by examining the levels of the DDRA2 and DDR48 transcripts in different rad mutants after exposure to two different DNA damaging agents. Strains carrying mutations in either the RAD3, RAD6 or RAD52 genes were treated with increasing concentrations of 4-nitroquinoline-1-oxide (NQO) or N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) and the DDR transcript levels were determined by Northern hybridization analysis. Our results indicate that the RAD3 gene is required for DDRA2 transcript production following NQO or MNNG treatments. Strains carrying mutations in either the RAD6 or RAD52 genes show an increased level of DDRA2 transcript in undamaged cells. However, the rad6 and rad52 mutants show a normal dose-dependent increase in DDRA2 transcript levels after NQO or MNNG exposure. The DDR48 gene appears to be regulated differently from DDRA2 in that this gene is induced in rad3 cells after damaging treatment but transcript induction is severely reduced in both rad6 and rad52 mutant strains. Although the rad mutations influence the kinetics of transcript accumulation, these effects do not account for the altered dose responses of the DDRA2 and DDR48 genes. Our results also demonstrate that the regulation of DDRA2 and DDR48 transcript levels by heat shock treatment is affected less severely in the different rad strains, a result which suggests that the RAD genes play an indirect role in DDR gene control.(ABSTRACT TRUNCATED AT 250 WORDS)

4-Nitroquinoline-1-oxide↗
PubMed1986

Nucleotide binding by a 24-residue peptide from the RecA protein of Escherichia coli.

We have recently demonstrated that two ATP analog affinity labels, 8-azidoadenosine 5'-triphosphate (N3ATP) and 5'-p-fluorosulfonylbenzoyladenosine (5'FSBA), covalently modify RecA protein of Escherichia coli at a specific tyrosine residue (Tyr-264) located within a 24-residue tryptic peptide (T-31) spanning residues 257-280. Here we show that N3ATP efficiently modifies purified peptide T-31 and show that the interaction is specific by the following criteria: photolabeling of peptide T-31 is saturable with respect to the N3ATP concentration; photolabeling is competitive with ATP and adenosine but not with adenine, UTP, or TTP; and other peptides derived from RecA protein were poor substrates for photolabeling except for one fragment that showed a nonspecific interaction with the photoaffinity analog. Analysis of N3ATP-modified T-31 shows that the photolabel attaches to more than one site within the peptide. These data argue that peptide T-31 contains some sites of contact for adenine and ribose moieties of ATP when it is bound to RecA protein.

Adenosine Triphosphate↗
PubMed1986

DNA damage and heat shock dually regulate genes in Saccharomyces cerevisiae.

Two Saccharomyces cerevisiae genes isolated in a differential hybridization screening for DNA damage regulation (DDR genes) were also transcriptionally regulated by heat shock treatment. A 0.45-kilobase transcript homologous to the DDRA2 gene and a 1.25-kilobase transcript homologous to the DDR48 gene accumulated after exposure of cells to 4-nitroquinoline-1-oxide (NQO; 1 to 1.5 microgram/ml) or brief heat shock (20 min at 37 degrees C). The DDRA2 transcript, which was undetectable in untreated cells, was induced to high levels by these treatments, and the DDR48 transcript increased more than 10-fold as demonstrated by Northern hybridization analysis. Two findings argue that dual regulation of stress-responsive genes is not common in S. cerevisiae. First, two members of the heat shock-inducible hsp70 family of S. cerevisiae, YG100 and YG102, were not induced by exposure to NQO. Second, at least one other DNA-damage-inducible gene, DIN1, was not regulated by heat shock treatment. We examined the structure of the induced RNA homologous to DDRA2 after heat shock and NQO treatments by S1 nuclease protection experiments. Our results demonstrated that the DDRA2 transcript initiates equally frequently at two sites separated by 5 base pairs. Both transcriptional start sites were utilized when cells were exposed to either NQO or heat shock treatment. These results indicate that DDRA2 and DDR48 are members of a unique dually regulated stress-responsive family of genes in S. cerevisiae.

Cloning, Molecular↗
PubMed1986

Pathology of the canine and feline uterine tube.

Lesions of the uterine tube were described in 37 dogs and nine cats. Of the 52 lesions noted, 18 were developmental abnormalities; four paramesonephric remnants, and 14 mesonephric remnants. Uterine tube cysts were seen in 16 animals, and seven animals had salpingitis. One dog had hyperplastic changes of the infundibular mucosa, and one cat and one dog demonstrated adenomyosis. There were three primary benign uterine tube neoplasms, one adenocarcinoma, two metastatic neoplasms in the uterine tube, and two bursal lipomas, all in dogs. With the exception of neoplasia of the uterine tube, most lesions were unlikely to affect the reproductive performance of these animals and/or were incidental or secondary to disorders elsewhere in the reproductive tract.

Animals↗
PubMed1986

Pathophysiology of small testes in beef bulls: relationship between scrotal circumference, histopathologic features of testes and epididymides, seminal characteristics, and endocrine profiles.

Tissue sections from testes and epididymides obtained from 17 young beef bulls with scrotal circumference (SC) between 27 and 40.5 cm were studied to determine whether small testes were a manifestation of lesions or a result of less, but otherwise normal, seminiferous epithelium. The SC correlated negatively with the estimates of germinal epithelial loss and positively with seminiferous epithelial area. Four bulls with SC less than 30 cm had severe lesions in their testes. Hypoplastic tubules were characterized by Sertoli's cells only with no evidence of germinal cells. Loss of germinal cells, leaving vacuolated epithelium and atrophy, were observed in degenerated tubules. Hyperplasia of Leydig's cells was observed in the vicinity of Sertoli's cell-only tubules, resulting either from degeneration or hypoplasia, and atrophy of Leydig's cells was associated with tubules devoid of Sertoli's cells. These findings indicated that Sertoli's cells may produce a factor(s) required for maintenance and regulation of Leydig's cell function. Epididymal epithelium, especially in the head, had regressed in bulls with hypoplastic and degenerative changes in their testes. Decreased sperm concentration and motility and an increased frequency of morphologic defects were observed in the 4 bulls with testicular lesions and regressed epididymal epithelium. Blood plasma profiles of cortisol, follicle-stimulating hormone, luteinizing hormone, and testosterone were determined in the 4 bulls with SC less than 30 cm and 10 of the 13 bulls with SC greater than 30 cm. There were no statistically significant (P greater than 0.1) differences in the responses to exogenous gonadotropin-releasing hormone or base-line patterns of blood plasma follicle-stimulating hormone and luteinizing hormone between the 2 groups. However, in the bulls with SC less than 30 cm, the mean concentration of testosterone was lower, whether spontaneous (P less than 0.05) or exogenous gonadotropin-releasing hormone induced (P less than 0.1). The fact that these bulls were not deficient in gonadotropins indicated that Leydig's cell function was impaired by local factors, either the factors that caused the tubular damage or those consequent to the tubular damage.

Animals↗
PubMed1985

Affinity labeling of a tyrosine residue in the ATP binding site of the recA protein from Escherichia coli with 5'-p-fluorosulfonylbenzoyladenosine.

We have covalently modified the recA protein from Escherichia coli with the adenine nucleotide analog 5'-p-fluorosulfonylbenzoyladenosine (5'-FSBA). The rate at which the protein is modified shows a sigmoidal dependence on the concentration of 5'-FSBA suggesting that binding of the analog is characterized by positive cooperativity. Covalent modification of the protein results in irreversible inactivation of its single-stranded DNA-dependent ATPase activity such that 100% inactivation is achieved when 25% of the enzyme monomers have been modified. Attachment of 5'-FSBA is specific for the ATP-binding site of recA protein as judged by the following criteria: (i) attachment of the affinity label to the protein appears to saturate at 1 mol of 5'-FSBA/mol of protein; (ii) binding of 5'-FSBA to recA protein is inhibited by ATP and competitive inhibitors of its ATP hydrolytic activity, e.g. adenosine-5'-O-(thiotriphosphate), ADP, UTP, and GTP, but not by adenosine; (iii) attachment of 5'-FSBA to the protein occurs at a single site as determined by high pressure liquid chromatography peptide separation. Following trypsin digestion of recA protein that had been covalently modified with [3H]5'-FSBA we isolated a single labeled peptide (T31) containing the exclusive site of 5'-FSBA attachment. A secondary proteolytic digestion was performed on both 5'-FSBA modified T31 and unmodified T31 using Staphylococcus aureus V8 protease, and by comparison of the amino acid compositions of the resulting peptides we identified Tyr-264 as the exclusive site of 5'-FSBA attachment in recA protein.

Adenosine↗
PubMed1985

Tyrosine 264 in the recA protein from Escherichia coli is the site of modification by the photoaffinity label 8-azidoadenosine 5'-triphosphate.

The photoaffinity label 8-azidoadenosine 5'-triphosphate (N3-ATP) was used to covalently modify the recA protein from Escherichia coli within its ATP-binding site. We have previously demonstrated that N3-ATP modification of recA protein is specific for the ATP-binding site and have isolated a unique tryptic peptide (T31), spanning residues 257-280, that contains the exclusive site of attachment of this ATP analog (Knight, K. L., and McEntee, K. (1985) J. Biol. Chem. 260, 867-872). We performed a secondary proteolytic digestion of the [alpha-32P]N3-ATP-labeled T31 peptide using Staphylococcus aureus V8 protease and purified the resulting peptide fragments by high-pressure liquid chromatography (HPLC). Based on a comparison of the amino acid compositions of all purified fragments and sequence analysis of one labeled fragment we determined that Tyr-264 is the exclusive site of N3-ATP attachment in recA protein. Photoaffinity labeling of recA protein was also performed in the presence of single-stranded DNA. Following trypsin treatment and separation of peptides by HPLC we showed that tryptic peptide T31 contained the exclusive site of N3-ATP attachment. A secondary proteolytic digestion was performed on both [alpha-32P]N3ATP-modified T31 and unmodified T31 using alpha-chymotrypsin. Comparison of the HPLC profiles and amino acid compositions of the resulting fragments was consistent with Tyr-264 as the exclusive site of N3-ATP attachment to recA protein.

Adenosine Triphosphate↗
PubMed1985

Kinetics of DNA renaturation catalyzed by the RecA protein of Escherichia coli.

The recA enzyme of Escherichia coli catalyzes renaturation of DNA coupled to hydrolysis of ATP. The rate of enzymatic renaturation is linearly dependent on recA protein concentration and shows saturation kinetics with respect to DNA concentration. The kinetic analysis of the reaction indicates that the Km for DNA is 65 microM while the kcat is approximately 48 pmol of duplex formed (pmol of recA)-1 (20 min)-1. RecA protein catalyzed renaturation has been characterized with respect to salt sensitivity, Mg2+ ion and pH optima, requirements for nucleoside triphosphates, and inhibition by nonhydrolyzable nucleoside triphosphates and analogues. These results are consistent with a Michaelis-Menten mechanism for DNA renaturation catalyzed by recA protein. A model is described in which oligomers of recA protein bind rapidly to single-stranded DNA, and in the presence of ATP, these nucleoprotein intermediates aggregate to bring complementary sequences into close proximity for homologous pairing. As with other DNA pairing reactions catalyzed by recA protein, ongoing DNA hydrolysis is required for renaturation. However, unlike the strand assimilation or transfer reaction, renaturation is inhibited by E. coli helix-destabilizing protein.

Coliphages↗
PubMed1985

Covalent modification of the recA protein from Escherichia coli with the photoaffinity label 8-azidoadenosine 5'-triphosphate.

We have covalently modified the recA protein from Escherichia coli with the photoaffinity ATP analog 8-azido-[alpha-32P]ATP (N3-ATP). Covalent attachment of N3-ATP to recA protein is dependent on native protein conformation and is shown to be specific for the site of ATP hydrolysis by the following criteria. (i) Binding of the probe to recA protein is inhibited by ATP and competitive inhibitors of its ATP hydrolytic activity, e.g. adenosine 5'-O-(thiotriphosphate), ADP, and UTP, but not by adenosine; (ii) N3-ATP is efficiently hydrolyzed by recA protein in the presence of single-stranded DNA; (iii) labeling of recA protein occurs at a single site as judged by two-dimensional thin-layer peptide mapping and high-performance liquid chromatography peptide separation. We have purified and identified a tryptic fragment, spanning amino acid residues 257-280, which contains the primary site of attachment of N3-ATP. This peptide is likely to be contained within the ATP hydrolytic site of recA protein.

Adenosine Triphosphate↗
PubMed1985

Response of S. cerevisiae to N-methyl-N'-nitro-N-nitrosoguanidine: mutagenesis, survival and DDR gene expression.

We have examined the effects of low concentrations of the alkylating agent N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) on survival and mutagenesis of logarithmically growing Saccharomyces cerevisiae. Pretreatment of cells with nontoxic and submutagenic concentrations of MNNG for several generations does not reduce the cytotoxic and mutagenic effects of exposure to a high concentration of drug. This lack of 'adaptation' in S. cerevisiae was further investigated biochemically and cell extracts prepared from pretreated cells were shown to be deficient in the ability to remove O6-methyl guanine from alkylated DNA. Moreover, we could not detect transfer of the methyl group from the DNA to a protein acceptor in yeast cell extracts suggesting that the level of O6-methyl guanine transferase is below 200 molecules/cell following pretreatment. Exposure of S. cerevisiae to mutagenic concentrations of MNNG stimulates transcription of at least three DNA damage responsive genes that also respond to UV-irradiation and 4-nitroquinoline-1-oxide treatment. These results support the contention that in Saccharomyces alkylation damage is processed by repair pathways that operate on a variety of lesions, and that one or more of these pathways is inducible.

4-Nitroquinoline-1-oxide↗
PubMed1985

Feline ovarian neoplasms.

Primary ovarian neoplasms from 22 cats were described. A single cat had tumors of epithelial origin--bilateral cystadenomas. Seven animals had germ cell tumors--dysgerminomas or teratomas; two cats had bilateral tumors. Fourteen animals had neoplasms of sex cord-stromal origin--granulosa cell tumors and interstitial gland tumors. Four cats with granulosa cell tumors had clinical evidence of hormonal disturbance.

Animals↗
PubMed1984

Identification of the amino acid substitutions in two mutant forms of the recA protein from Escherichia coli: recA441 and recA629.

We have identified the amino acid substitutions in two mutant forms of the recA protein from Escherichia coli. The recA441 mutant, which shows constitutive expression of the recA-mediated SOS response at 42 degrees C, contains two amino acid substitutions, glutamic acid to lysine at residue 38 and isoleucine to valine at residue 298. The recA629 mutant is an unusual pseudorevertant of recA441 that is no longer capable of spontaneous expression of SOS functions at 42 degrees C. Purified recA629 protein is cold-labile for several of the wild-type enzymatic activities and is shown here to contain three amino acid substitutions, the two found in the recA441 protein at residues 38 and 298, as well as an aspartic acid-to-glycine change at residue 32. The mutation at residue 32 was verified by restriction digestion of the 5' region of the recA629 structural gene.

Alleles↗
PubMed1984

Homologous pairing of single-stranded circular DNAs catalyzed by recA protein.

RecA protein catalyzes annealing between pairs of circular single-stranded DNA molecules containing complementary sequences varying in length from 3550 nucleotides to 181 nucleotides. The reaction requires ATP and catalytic amounts of recA protein. Molecules containing large complementary inserts are annealed by recA protein to form large multimeric aggregates that migrate slowly in agarose gels. In contrast the products formed from circular molecules containing short complementary regions are principally dimeric structures. We have used electron microscopy, thermal denaturation and kinetic studies to analyze these reaction products. Our results indicate that recA protein catalyzes multiple nucleation events between complementary DNA sequences in the absence of a free end and when these sequences are flanked by extensive noncomplementary regions.

Base Sequence↗
PubMed1984

Cloning and characterization of recA genes froM Proteus vulgaris, Erwinia carotovora, Shigella flexneri, and Escherichia coli B/r.

The recA genes of Proteus vulgaris, Erwinia carotovora, Shigella flexneri and Escherichia coli B/r have been isolated and introduced into Escherichia coli K-12. All the heterologous genes restore resistance to killing by UV irradiation and the mutagen 4-nitroquinoline-1-oxide in RecA- E. coli K-12 hosts. Recombination proficiency is also restored as measured by formation of Lac+ recombinants from duplicated mutant lacZ genes and the ability to propagate phage lambda derivatives requiring host recombination functions for growth (Fec-). The cloned heterologous genes increase the spontaneous induction of lambda prophage in lysogens of a recA strain. Addition of mitomycin C stimulates phage production in cells carrying the E. coli B/r and S. flexneri recA genes, but little or no stimulation is seen in cells carrying the E. carotovora and P. vulgaris recA genes. After treatment with nalidixic acid, the heterologous RecA proteins are synthesized at elevated levels, a result consistent with their regulation by the E. coli K-12 LexA repressor. Southern hybridization and preliminary restriction analysis indicate divergence among the coding sequences, but antibodies prepared against the E. coli K-12 RecA protein cross-react with the heterologous enzymes, indicating structural conservation among these proteins.

Bacteria↗
PubMed1984

Specific transcripts are elevated in Saccharomyces cerevisiae in response to DNA damage.

Differential hybridization has been used to identify genes in Saccharomyces cerevisiae displaying increased transcript levels after treatment of cells with UV irradiation or with the mutagen/carcinogen 4-nitroquinoline-1-oxide (NQO). We describe the isolation and characterization of four DNA damage responsive genes obtained from screening ca. 9,000 yeast genomic clones. Two of these clones, lambda 78A and pBR178C, contain repetitive elements in the yeast genome as shown by Southern hybridization analysis. Although the genomic hybridization pattern is distinct for each of these two clones, both of these sequences hybridize to large polyadenylated transcripts ca. 5 kilobases in length. Two other DNA damage responsive sequences, pBRA2 and pBR3016B, are single-copy genes and hybridize to 0.5- and 3.2-kilobase transcripts, respectively. Kinetic analysis of the 0.5-kilobase transcript homologous to pBRA2 indicates that the level of this RNA increases more than 15-fold within 20 min after exposure to 4-nitroquinoline-1-oxide. Moreover, the level of this transcript is significantly elevated in cells containing the rad52-1 mutation which are deficient in DNA strand break repair and gene conversion. These results provide some of the first evidence that DNA damage stimulates transcription of specific genes in eucaryotic cells.

4-Nitroquinoline-1-oxide↗
PubMed1984

Feline cystic rete ovarii.

Cysts of the rete ovarii system from 20 cats are described. The cellular morphology of these cysts varies because the normal rete ovarii in the cat consists of three anatomically distinct parts. Cysts arise in the ovarian medulla or at the tubal extremity of the ovary, expand into ovarian stroma rather than the mesovarium , and are lined by ciliated columnar to flattened epithelium. Their functional significance is unknown.

Animals↗
PubMed1984

Hyperplastic endometrial polyps in the dog and cat.

Hyperplastic endometrial polyps from 17 dogs and 14 cats are described. These growths were pedunculated to broad-based and projected into the uterine lumen. They were lined by endometrial epithelium and contained cystic endometrial glands surrounded by a well-vascularized connective tissue stroma. The morphologic evidence suggests that endometrial polyps arise from focal areas of cystic endometrial hyperplasia.

Animals↗