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K McEntee

Publications and source records attributed to K McEntee.

At least 55 records · Page 3Linked to original sources
PubMed1990

Evidence for a heat shock transcription factor-independent mechanism for heat shock induction of transcription in Saccharomyces cerevisiae.

Transcription of the DNA damage-responsive gene, DDRA2, of Saccharomyces cerevisiae is activated by heat shock treatment as well as by mutagen/carcinogen exposure. Deletion analysis of upstream noncoding sequence indicated that sequences between approximately -190 and -140 base pairs were necessary for heat shock and DNA damage regulation of transcription. Fusion of this region to a CYC1-lacZ reporter gene demonstrated that the sequence between -202 and -165 base pairs was sufficient for basal level and heat shock-induced expression. This DNA sequence was unable to bind heat shock transcription factor as judged by binding competition experiments in vitro. These results indicate that yeast possesses a second, heat shock transcription factor-independent mechanism for activating transcription in response to thermal stress.

Amino Acid Sequence↗
PubMed1990

DNA polymerase II is encoded by the DNA damage-inducible dinA gene of Escherichia coli.

The structural gene for DNA polymerase II was cloned by using a synthetic inosine-containing oligonucleotide probe corresponding to 11 amino acids, which were determined by sequencing the amino terminus of the purified protein. The labeled oligonucleotide hybridized specifically to the lambda clone 7H9 from the Kohara collection as well as to plasmid pGW511 containing the SOS-regulated dinA gene. Approximately 1400 base pairs of dinA sequence were determined. The predicted amino-terminal sequence of dinA demonstrated that this gene encoded DNA polymerase II. Sequence analysis of the upstream region localized a LexA binding site overlapping the -35 region of the dinA promoter, and this promoter element was found to be only two nucleotides downstream from the 3' end of the araD gene. These results demonstrate that the gene order is thr-dinA (pol II)-ara-leu on the Escherichia coli chromosome and that the DNA polymerase II structural gene is transcribed in the same direction as the araBAD operon. Based on the analysis of the predicted protein, we have identified a sequence motif Asp-Xaa-Xaa-Ser-Leu-Tyr-Pro-Ser in DNA polymerase II that is highly conserved among a diverse group of DNA polymerases, which include those from humans, yeast, Herpes and vaccinia viruses, and phages T4 and PRD1. The demonstration that DNA polymerase II is a component of the SOS response in E. coli suggests that it plays an important role in DNA repair and/or mutagenesis.

Amino Acid Sequence↗
PubMed1990

The DNA damage-inducible gene DIN1 of Saccharomyces cerevisiae encodes a regulatory subunit of ribonucleotide reductase and is identical to RNR3.

The sequence of the DIN1 gene of Saccharomyces cerevisiae is identical to RNR3, a gene encoding a DNA damage-inducible regulatory subunit of ribonucleotide reductase. Two sequence elements located upstream of DIN1 (RNR3) are homologous to putative DNA damage regulatory elements in the promoter of the reductase catalytic subunit gene, RNR2. The transcript start sites for DIN1(RNR3) have been localized, and induction by different agents has been compared with other DNA damage-regulated genes.

Amino Acid Sequence↗
PubMed1990

Structure of the DNA damage-inducible gene DDR48 and evidence for its role in mutagenesis in Saccharomyces cerevisiae.

The DDR48 gene of Saccharomyces cerevisiae is a member of a set of genes that displays increased transcription in response to treatments that produce DNA lesions or to heat-shock stress. Other members of this group include the DDRA2 and UBI4 genes. DNA sequence analysis of the DDR48 gene demonstrates the presence of two overlapping open reading frames, each of which has the capacity to encode a protein with a molecular mass of approximately 45 kilodaltons. Fusions of the DDR48 coding sequences to lacZ demonstrates that only one of these frames is expressed in yeast cells. The protein predicted from this sequence is extremely hydrophilic and contains multiple repeats of the peptide sequence Ser-Asn-Asn-X-Asp-Ser-Tyr-Gly where X is either Asn or Asp. Additionally, closely related sequences are found throughout the primary sequence. Primer extension data indicate that, after 4-nitroquinoline-1-oxide and heat-shock treatments, there are three major and two minor transcriptional start sites which are utilized. The function of the DDR48 gene was investigated by disrupting this gene in diploid cells. Viable haploid cells containing the DDR48 gene disruption were isolated after tetrad analysis. Although the ddr48 mutant showed a slightly altered sensitivity to killing by 4-nitroquinoline-1-oxide and to heat shock compared with the DDR48 haploid, the spontaneous mutation rate of reversion of a his4 mutation was reduced 6- to 14-fold in the ddr48 strain. These results implicate the DDR48 gene in the production or recovery of mutations in S. cerevisiae.

4-Nitroquinoline-1-oxide↗
PubMed1989

Purification and characterization of a DNA-pairing and strand transfer activity from mitotic Saccharomyces cerevisiae.

An enzyme catalyzing homologous pairing of DNA chains has been extensively purified from mitotic yeast. The most highly purified fractions are enriched for a polypeptide with a molecular mass of approximately 120 kDa as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Protein-dependent pairing of single-stranded DNAs requires a divalent cation (Mg2+ or Ca2+) but proceeds rapidly in the absence of any nucleoside triphosphates. The kinetics of reassociation are extremely rapid, with more than 60% of the single-stranded DNA becoming resistant to S1 nuclease within 1 min at a ratio of 1 protein monomer/50 nucleotides. The results of enzyme titration and DNA challenge experiments suggest that this protein does not act catalytically during renaturation but is required stoichiometrically. The protein promotes formation of joint molecules between linear M13 replicative form DNA (form III) containing short single-stranded tails and homologous single-stranded M13 viral DNA. Removal of approximately 50 nucleotides from the ends of the linear duplex using either exonuclease III (5' ends) or T7 gene 6 exonuclease (3' ends) activates the duplex for extensive strand exchange. Electron microscopic analysis of product molecules suggests that the homologous circular DNA initially associates with the single-stranded tails of the duplexes, and the heteroduplex region is extended with displacement of the noncomplementary strand. The ability of this protein to pair and to promote strand transfer using either exonuclease III or T7 gene 6 exonuclease-treated duplex substrates suggests that this activity promotes heteroduplex extension in a nonpolar fashion. The biochemical properties of the transferase are consistent with a role for this protein in heteroduplex joint formation during mitotic recombination in Saccharomyces cerevisiae.

Cations, Divalent↗
PubMed1989

A rapid and efficient procedure for transformation of intact Saccharomyces cerevisiae by electroporation.

A rapid and efficient procedure is described for transforming Saccharomyces cerevisiae using electroporation to render intact cells permeable to DNA. The technique uses relatively low voltages and is particularly sensitive to low concentrations of plasmid DNA. At the highest voltage used (400 volts), the frequency of transformation increased with the amount of plasmid DNA between 25 ng and 100 ng. At higher concentrations of DNA (1-1.5 micrograms) electroporation yielded one-third to one-half the number of transformants obtained with a standard lithium acetate pretreatment. Because this method requires neither pretreatment of cells nor addition of polyethylene glycol (PEG), it has several advantages over currently used transformation procedures.

Electric Stimulation↗
PubMed1989

DNA damage activates transcription and transposition of yeast Ty retrotransposons.

A set of genes isolated from Saccharomyces cerevisiae showed increased transcript levels after yeast had been exposed to ultraviolet (UV) light or 4-nitroquinoline-1-oxide (4NQO). Included among these DNA damage responsive (DDR) genes were members of the Ty retrotransposon family of yeast. Northern hybridization analysis indicated that maximal levels of a 5.6 kb transcript encoded by the Ty elements accumulated in cells after 4 to 6 h of exposure to 4NQO. The induced levels of transcripts varied from two- to tenfold for different Ty probes although similar kinetics and dose responses were observed for transcripts hybridizing to the different Ty family members. Pulse labeling experiments suggested that the accumulation of Ty transcripts was due, in part, to an increased rate of Ty message synthesis. Transposition of Ty elements to two target loci encoding distinct alcohol dehydrogenase enzymes, ADH2 and ADH4, was examined in cells exposed to increasing doses of UV light or 4NQO. The frequency of Ty insertion into these genetic regions following DNA damaging treatments increased by as much as 17-fold compared with untreated cells. These results provide direct evidence that transposable elements can be activated by physical and chemical mutagens/carcinogens and that transpositional mutagenesis is induced by these agents in S. cerevisiae.

Blotting, Northern↗
PubMed1989

"Activated"-RecA protein affinity chromatography of LexA repressor and other SOS-regulated proteins.

We have developed an affinity column to study the interaction of LexA repressor and other substrates with the activated form of RecA protein. Nucleoprotein complexes of RecA protein, (dT)25-30, and adenosine 5'-[gamma-S]thio-triphosphate were formed in solution and bound to RecA protein-agarose columns. These "activated"-RecA nucleoprotein complexes were retained by strong hydrophobic interactions. Purified LexA protein bound tightly to these activated RecA columns, whereas the LexA protein bound poorly to RecA-agarose alone. Once bound, LexA protein underwent specific proteolysis, and the fragments were released from the complex. The mutant LexA protein, LexA-SA119, which cannot carry out self-cleavage or RecA-mediated cleavage in solution, bound efficiently to the activated RecA column but was not cleaved, indicating that these columns can be used to identify residues involved in RecA-LexA binding. As an example of this use, nucleoprotein complexes were prepared using the RecA430 protein. In vivo the recA430 mutation blocks induction of the SOS response. LexA protein was not efficiently retained on the immobilized RecA430 complexes, suggesting that Gly-204 is required for efficient repressor binding. These results show that activated RecA affinity columns can be used to investigate the binding and cleaving properties of mutationally altered RecA and LexA proteins. Additionally, these activated RecA columns have been used to investigate binding interactions of phage lambda repressor, as well as the UmuC protein, which is required for chemical mutagenesis.

Bacterial Proteins↗
PubMed1988

Affinity chromatography of RecA protein and RecA nucleoprotein complexes on RecA protein-agarose columns.

We have analyzed the nature of RecA protein-RecA protein interactions using an affinity column prepared by coupling RecA protein to an agarose support. When radiolabeled soluble proteins from Escherichia coli are applied to this column, only the labeled RecA protein from the extract was selectively retained and bound tightly to the affinity column. Efficient binding of purified 35S-labeled RecA protein required Mg2+, and high salt did not interfere with the binding of RecA protein to the column. Complete removal of the bound enzyme from the affinity column required treatment with guanidine HCl (5 M) or urea (8 M). These and other properties suggest that hydrophobic interactions contribute significantly to RecA protein subunit recognition in solution. Using a series of truncated RecA proteins synthesized in vitro, we have obtained evidence that at least some of the sequences involved in protein recognition are localized within the first 90 amino-terminal residues of the protein. Based on the observation that RecA proteins from three heterologous bacteria are specifically retained on the E. coli RecA affinity column, it is likely that this binding domain is highly conserved and is required for interaction and association of RecA protein monomers. Stable ternary complexes of RecA protein and single-stranded DNA were formed in the presence of the nonhydrolyzable ATP analog adenosine 5'-O-(thiotriphosphate) and applied to the affinity columns. Most of the complexes formed with M13 DNA could be eluted in high salt, whereas a substantial fraction of those formed with the oligonucleotide (dT)25-30 remained bound in high salt and were quantitatively eluted with guanidine HCl (5 M). The different binding properties of these RecA protein-DNA complexes likely reflect differences in the availability of a hydrophobic surface on RecA protein when it is bound to long polynucleotides compared to short oligonucleotides.

Chromatography, Affinity↗
PubMed1988

Purification and characterization of an inducible Escherichia coli DNA polymerase capable of insertion and bypass at abasic lesions in DNA.

We have investigated the ability of DNA polymerases from SOS-induced and uninduced Escherichia coli to incorporate nucleotides at a well-defined abasic (apurinic/apyrimidinic) DNA template site and to extend these chains from this unpaired 3' terminus. A DNA polymerase activity has been purified from E. coli, deleted for DNA polymerase I, that appears to be induced 7-fold in cells following treatment with nalidixic acid. Induction of this polymerase (designated DNA polymerase X) appears to be part of the SOS response of E. coli since it cannot be induced in strains containing a noncleavable form of the LexA repressor (Ind-). The enzyme is able to incorporate nucleotides efficiently opposite the abasic template lesion and to continue DNA synthesis. Although we observe an approximate 2-fold induction of DNA polymerase III in cells treated with nalidixic acid, several lines of evidence argue that DNA polymerase X is unrelated to DNA polymerase III (pol III). In contrast to pol X, pol III shows almost no detectable ability to incorporate at or extend beyond the abasic site; incorporation efficiency at the abasic lesion is at least 100-fold larger for pol X compared to pol III holoenzyme, pol III core, or pol III* (the polymerase III holoenzyme subassembly lacking the beta subunit). Pol X does not cross-react with polyclonal antibody directed against pol III holoenzyme complex or with monoclonal antibody prepared to the alpha subunit of pol III. Despite these structural and biochemical differences, pol X appears to interact specifically with the beta subunit of the pol III holoenzyme in the presence of single-stranded binding protein. Pol X has a molecular mass of 84 kDa. Our results indicate that this novel activity is likely to be identical to DNA polymerase II of E. coli.

Base Sequence↗
PubMed1988

Conservation of an ATP-binding domain among RecA proteins from Proteus vulgaris, Erwinia carotovora, Shigella flexneri, and Escherichia coli K-12 and B/r.

The purified RecA proteins encoded by the cloned genes from Proteus vulgaris, Erwinia carotovora, Shigella flexneri, and Escherichia coli B/r were compared with the RecA protein from E. coli K-12. Each of the proteins hydrolyzed ATP in the presence of single-stranded DNA, and each was covalently modified with the photoaffinity ATP analog 8-azidoadenosine 5'-triphosphate (8N3ATP). Two-dimensional tryptic maps of the four heterologous RecA proteins demonstrated considerable structural conservation among these bacterial genera. Moreover, when the [alpha-32P]8N3ATP-modified proteins were digested with trypsin and analyzed by high-performance liquid chromatography, a single peak of radioactivity was detected in each of the digests and these peptides eluted identically with the tryptic peptide T31 of the E. coli K-12 RecA protein, which was the unique site of 8N3ATP photolabeling. Each of the heterologous recA genes hybridized to oligonucleotide probes derived from the ATP-binding domain sequence of the E. coli K-12 gene. These last results demonstrate that the ATP-binding domain of the RecA protein has been strongly conserved for greater than 10(7) years.

Adenosine Triphosphate↗
PubMed1988

Expression of the yeast UB14 gene increases in response to DNA-damaging agents and in meiosis.

The polyubiquitin gene, UB14, of Saccharomyces cerevisiae is regulated by a variety of environmental stresses and physiological conditions. After exposure of rapidly growing yeast cells to DNA-damaging agents (4-nitroquinoline-1-oxide and N-methyl-N'-nitro-N-nitrosoguanidine), intracellular levels of UB14 transcript increased rapidly. Induction of UB14 transcripts occurred within 30 to 60 min of exposure to 4-nitroquinoline-1-oxide in RAD+, rad52, and rad6 repair-deficient yeast strains. In high-density RAD+ cultures, the effect of alkylating agents on UB14 transcript levels is attenuated, in part because of significant increases in the basal level of this message in untreated cells. We also observed that the levels of UB14 transcripts increased significantly when diploid cells were exposed to sporulation conditions. Maximal levels of UB14 transcripts were reached after 6 to 8 h in sporulation medium. Accumulation of UB14 transcripts occurred in a/alpha diploids that undergo meiosis but not in asporogenous alpha/alpha diploids exposed to the same nutritional conditions.

4-Nitroquinoline-1-oxide↗
PubMed1987

Primary structure of beta-galactoside alpha 2,6-sialyltransferase. Conversion of membrane-bound enzyme to soluble forms by cleavage of the NH2-terminal signal anchor.

This report describes the primary structure of a rat liver beta-galactoside alpha 2,6-sialyltransferase (EC 2.4.99.1), a Golgi apparatus enzyme involved in the terminal sialylation of N-linked carbohydrate groups of glycoproteins. The complete amino acid sequence was deduced from the nucleotide sequence of cDNA clones of the enzyme. The primary structure suggests that the topology of the enzyme in the Golgi apparatus consists of a short NH2-terminal cytoplasmic domain, a 17-residue hydrophobic sequence which serves as the membrane anchor and signal sequence, and a large lumenal, catalytic domain. NH2-terminal sequence analysis of a truncated form of the enzyme, obtained by purification from tissue homogenates, reveals that it is missing a 63-residue NH2-terminal peptide which includes the membrane binding domain. These and supporting results show that soluble forms of the sialyltransferase can be generated by proteolytic cleavage between the NH2-terminal signal-anchor and the catalytic domain.

Amino Acid Sequence↗
PubMed1987

Evidence for nucleotide-mediated changes in the domain structure of the recA protein of Escherichia coli.

We have used limited trypsin digestion as a means of investigating changes in the structural properties of recA protein accompanying the binding of different nucleoside triphosphates. The levels of four partial digestion products are greatly increased in digests of recA protein complexed with dTTP, dATP, ATP, or the ATP analogue adenosine 5'-O-(3-thiotriphosphate) (ATP gamma S). These bands (22, 19, and 17.5 kilodaltons) are absent or present at reduced levels in digests of recA protein alone. Unlike these nucleotides, all of which bind tightly to recA protein, nucleotides and analogues that bind poorly produce little or no change in the digestion pattern of recA protein. We have compared the rates of fragment accumulation in the presence of dTTP and show a saturable dependence on nucleotide concentration. Binding of single-stranded DNA to recA protein does not alter the pattern of digestion products compared to protein alone, and the digestion pattern of recA protein-DNA-ATP gamma S ternary complexes is similar to that of uncomplexed enzyme. We have used monoclonal antibody binding, high-performance liquid chromatography separation of peptides, and amino acid composition analyses to localize the regions of recA protein which are altered in their susceptibility to trypsin when nucleoside triphosphates are present. The results of these analyses indicate that the fragments arise from trypsin cutting at two or more sites near the middle of the primary sequence. These cleavage sites are more than 80-110 residues away from the site of photoaffinity labeling by 8-N3ATP (Tyr-264). Our results suggest that, in the presence of certain nucleotides, recA protein is organized into two stable structural domains.

Escherichia coli↗
PubMed1987

Equine testicular interstitial cell tumors.

Interstitial cell tumors from nine stallions were described. In all but one horse the tumors were found in undescended testes. Five animals had bilateral tumors. Two animals showed increased aggression. Tumors contained two cell types. The first type were large distinctly bordered eosinophilic cells interpreted to be hyperplastic and hypertrophic interstitial cells. They blended with pleomorphic often spindloid neoplastic cells which had fibrillar, vacuolated cytoplasm and indistinct cell borders. This latter cell population was arranged in nodules or broad sheets as endocrine-like packets or interweaving fascicles. Biologic behavior of the neoplasms could not be ascertained from histologic examination.

Animals↗
PubMed1987

Effects of zeranol on reproduction in beef bulls: scrotal circumference, serving ability, semen characteristics, and pathologic changes of the reproductive organs.

Effects of zeranol on scrotal circumference, serving ability, semen characteristics, and postmortem measurements of the genital organs were determined in beef bulls from 9 to 20 months of age. Group 1 (n = 5) served as a nonimplanted control group. Group 2A (n = 5) was implanted with 36 mg of zeranol at birth and at 3 and 6 months of age. Group 2B (n = 5) was implanted with 36 mg of zeranol every 3 months from birth through 18 months of age. Scrotal circumference was adversely affected by zeranol in groups 2A and 2B, but values approached those of group 1 with increasing age. Serving ability was also affected adversely but tended to recover with increasing age. Semen quality was low in groups 2A and 2B and did not improve with increasing age. There was no difference in testicular weight, vesicular gland weight, and penis length among groups when bulls were slaughtered at 20 months of age. Epididymal weight was greater in group-2B bulls and was most likely a consequence of epididymal lesions. Histologic examination of the genital organs revealed that zeranol induced adenomyosis and sperm granulomas in the caudae epididymidis and markedly altered the structure of the sexual accessory glands of bulls in groups 2A and 2B. Alterations in the vesicular glands were characterized by reduced alveolar development and an increase in connective tissue. Low epithelium associated with focal areas of squamous metaplasia were common in the prostate of groups 2A and 2B bulls. Lesions in the bulbourethral glands were characterized by low glandular epithelium, focal areas of squamous metaplasia, cystic collecting ducts, and an increase in connective tissue. Groups 2A and 2B had more abnormal seminiferous tubules than did group 1. Lesions in groups 2A and 2B may have been direct effects of zeranol or may have resulted from reduced testosterone secretion.

Animals↗
PubMed1987

Changes in basal lamina of seminiferous tubules associated with deranged spermatogenesis in the bull.

The basal lamina of seminiferous tubules of 16 beef bulls, with scrotal circumferences (SC) from 40.5 to 28 cm, was characterized. The mean thickness of basal laminae was from 0.55 to 0.94 micron in normospermic bulls with SC greater than 30 cm. Of 3 bulls that had a SC less than 30 cm, 1 had hypoplastic testes and was azoospermic, whereas 2 bulls had atrophic changes in their testes and were oligospermic. The latter 2 bulls had thick basal laminae, with a mean of 1.35 micron and 1.68 micron, respectively, whereas basal laminae of the bull with hypoplastic testes had a mean thickness of 0.96 micron, comparable with that of bulls with normal testes (SC, greater than 30 cm). Thickness of the basal lamina of seminiferous tubules might be a useful criterion in differentiating atrophy from hypoplasia.

Animals↗