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T Conway

Publications and source records attributed to T Conway.

At least 73 records · Page 4Linked to original sources
PubMed1992

Molecular characterization of the Zymomonas mobilis enolase (eno) gene.

The Zymomonas mobilis gene encoding enolase was cloned by genetic complementation of an Escherichia coli eno mutant. An enzyme assay and sodium dodecyl sulfate-polyacrylamide gel electrophoresis confirmed the overexpression of enolase in E. coli clones carrying the Z. mobilis eno gene. The eno gene is present in a single copy of the Z. mobilis genome. Nucleotide sequence analysis of the eno region revealed an open reading frame of 1,293 bp that encodes a protein of 428 amino acids with a predicted molecular weight of 45,813. Comparison of the sequence of Z. mobilis enolase with primary amino acid sequences for other enolases indicates that the enzyme is highly conserved. Unlike all of the previously studied glycolytic genes from Z. mobilis that possess canonical ribosome binding sites, the eno gene is preceded by a modest Shine-Dalgarno sequence. The transcription initiation site was mapped by primer extension and found to be located within a 115-bp sequence that is 55.7% identical to a highly conserved consensus sequence found within the regulatory regions of highly expressed Z. mobilis genes. Northern RNA blot analysis revealed that eno is encoded on a 1.45-kb transcript. The half-life of the eno mRNA was determined to be 17.7 +/- 1.7 min, indicating that it is unusually stable. The abundance of the eno message is proposed to account for enolase being the most prevalent protein in Z. mobilis.

Amino Acid Sequence↗
PubMed1992

The Zymomonas mobilis glf, zwf, edd, and glk genes form an operon: localization of the promoter and identification of a conserved sequence in the regulatory region.

The Zymomonas mobilis genes that encode the glucose-facilitated diffusion transporter (glf), glucose-6-phosphate dehydrogenase (zwf), 6-phosphogluconate dehydratase (edd), and glucokinase (glk) are clustered on the genome. The data presented here firmly establish that the glf, zwf, edd, and glk genes form an operon, in that order. The four genes of the operon are cotranscribed on a 6.14-kb mRNA. The site of transcriptional initiation for the polycistronic message was mapped by primer extension and nuclease S1 protection analysis. The glf operon promoter region showed significant homology to other highly expressed Z. mobilis promoters, but not to consensus promoters from other bacteria. The highly expressed Z. mobilis promoter set contains two independent, overlapping, conserved sequences that extend from approximately bp -100 to +15 with respect to the transcriptional start sites. Expression of the glf operon was shown to be subject to carbon source-dependent regulation. The mRNA level was threefold higher in cells grown on fructose than in cells grown on glucose. This increase was not the result of differential mRNA processing when cells were grown on the different carbon sources, nor was it the result of differential transcript stability. Degradation of the 6.14-kb glf operon mRNA was biphasic, with initial half-lives of 11.5 min in fructose-grown cells and 12.0 min in glucose-grown cells. Thus, the higher level of glf operon mRNA in fructose-grown cells is the result of an increased rate of transcription. The importance of increasing glf expression in cells growing on fructose is discussed.

Base Sequence↗
PubMed1992

The polycistronic mRNA of the Zymomonas mobilis glf-zwf-edd-glk operon is subject to complex transcript processing.

The full-length 6.14-kb polycistronic glf-zwf-edd-glk mRNA from Zymomonas mobilis appears to be processed by endonucleolytic cleavage, resulting in the formation of several discrete transcripts. Northern analysis and transcript mapping revealed that the processed transcripts correspond to functional mono-, di-, or tricistronic messages. The relative abundance of the gene-specific, functional messages was measured. Expression of zwf and edd correlated well with functional message levels. Disproportionally high levels of the glk-specific mRNAs might compensate for the instability of glucokinase by allowing increased translation. The relative abundance of the discrete transcripts was shown to be a function of their respective decay rates. Northern analysis of the fate of the 6.14-kb transcript after inhibition of transcription by rifampin showed that the abundance of shorter, more stable transcripts increased at the expense of longer, less stable transcripts. This is suggestive of endonucleolytic mRNA processing. The most abundant 5' and 3' transcript ends were found to lie within secondary structures that probably impart stability to the most abundant mRNAs.

Base Sequence↗
PubMed1991

Familial breast-ovarian cancer locus on chromosome 17q12-q23.

Familial breast cancer has been linked to the D17S74 locus on chromosome 17q. To confirm this finding and to investigate whether ovarian cancer is also linked to this locus, five large families with a hereditary predisposition to cancer of the breast and ovary were investigated. Three families were positive for linkage. For the largest family the lod score was 2.72. These findings suggest that the chromosomal region 17q12-q23, previously shown to contain a gene for early-onset breast cancer, is also associated with a proportion of hereditary ovarian cancers.

Adult↗
PubMed1991

Characterization of a maize cDNA that complements an enolase-deficient mutant of Escherichia coli.

A cDNA encoding maize enolase (2-phospho-D-glycerate hydrolase) was purified by functional genetic complementation using an enolase deficient mutant of Escherichia coli, DF261. This cDNA, pZM245, was characterized by restriction mapping and DNA sequence analysis. The cDNA contained an open reading frame encoding a protein of 446 amino acids with a high degree of similarity to enolase sequences from other organisms (72% identity to yeast enolase and 82% identity to human enolase). The pZM245 contains a correctly positioned consensus prokaryotic translation initiation sequence. The specific activity of enolase in maize increases to about twice its initial level after 48 hours of anaerobiosis. Northern-blot analysis showed a five-fold anaerobic induction in enolase mRNA, while heat shock or cold shock increased enolase mRNA levels only slightly. Southern-blot analysis of maize genomic DNA indicated that there is one copy of the pZM245 hybridizing sequence per haploid genome in maize.

Amino Acid Sequence↗
PubMed1991

Pharmacokinetics of 11C-labelled BCNU and SarCNU in gliomas studied by PET.

This paper describes the study of the pharmacodynamics of two 11C-labelled nitrosoureas, 1,3-bis-(2-chloroethyl) nitrosourea (BNCU) and sarcosinamide chloroethylnitrosourea (SarCNU), both labelled in the carbonyl position. Distribution of the radioactivity as measured by positron emission tomography was compared to the distribution of radioactivity observed after injection of 68Ga-EDTA, this being used as an indicator of the blood-brain barrier integrity around the brain tumor. Data suggest that the new nitrosourea, SarCNU, most likely enters brain tissue by different mechanism(s) than BCNU, which enters by diffusion. Data also indicate that use of SarCNU may result in a better tumor to brain ratio than BCNU.

Antineoplastic Agents↗
PubMed1991

Hereditary ovarian cancer. Pedigree studies, Part II.

Hereditary ovarian carcinoma is heterogenous. There are at least three genetic variants, namely, hereditary site-specific ovarian carcinoma, hereditary breast/ovarian carcinoma syndrome, and Lynch syndrome II. Early age of onset characterizes these disorders. A crucial hallmark of these disorders is the integral association of extraovarian cancers, such as carcinoma of the endometrium and colon in Lynch syndrome II. We have described 24 pedigrees of ovarian cancer-prone families in order to depict the several differing heterogenous variants. Interest in hereditary ovarian cancer has increased remarkably, due in part to the fact that its surveillance has been wholly unsatisfactory, as have therapeutic measures. Prevention through prophylactic oophorectomy offers hope. However, there is a risk for extraovarian peritoneal serous papillary carcinoma, consonant with primary cancer of the ovary. This must be discussed with these at-risk patients. Until a biomarker of acceptable sensitivity and specificity is identified, the family history must remain the key to hereditary ovarian cancer diagnosis.

Adult↗
PubMed1991

PET studies of potential chemotherapeutic agents--X. Synthesis of "no-carrier-added" (11C)-HECNU: the hydroxyethyl analog of the chemotherapeutic agent BCNU.

Carbon-11-labeled HECNU [1-(2-chloroethyl)-1-nitroso-3-(2-hydroxyethyl) urea] a potential chemotherapeutic agent, has been prepared by the nitrosation of the corresponding carbon-11-labeled urea, HECU, [1-(2-chloroethyl)-3-(2-hydroxyethyl) urea]. The isometric byproduct of nitrosation, 1-(2-chloroethyl)-3-nitroso-3-(2-hydroxyethyl) urea can be efficiently removed by preparative scale HPLC on a Partisil column. (11C)-HECU was prepared by reacting ethanolamine with (11C)-2-chloroethyl-isocyanate which was itself prepared by reacting [11C)-phosgene with 2-chloroethylamine hydrochloride suspended in dioxane at 60-65 degrees C. This synthesis yielded (11C)-HECNU with an average radiochemical purity of 98% in an average radiochemical yield of 18% relative to the radioactivity measured at the end of the 11C-phosgene introduction.

Carbon Radioisotopes↗
PubMed1991

Cloning, characterization and expression of the Zymononas mobilis eda gene that encodes 2-keto-3-deoxy-6-phosphogluconate aldolase of the Entner-Doudoroff pathway.

The eda gene that encodes 2-keto-3-deoxy-6-phosphogluconate aldolase of the Entner-Doudoroff pathway was cloned from Zymomonas mobilis by genetic complementation of an Escherichia coli mutant. The gene is present in a single copy on the Z. mobilis genome and is not tightly linked to the edd gene. Nucleotide sequence analysis of the eda region revealed that the structural gene is 627 bp long and capable of encoding a protein of 208 amino acids with a deduced molecular weight of 21,505. The eda gene is monocistronic and is transcribed from a single promoter. The transcriptional initiation site was determined and an improved consensus promoter sequence for Z. mobilis was derived. High-level expression of the eda gene can be attributed to very efficient translational initiation caused by the high quality of the ribosome-binding site and stability of the mRNA, which has a decay rate of 7.6 min. A comparison of highly expressed Z. mobilis genes indicated that the relative quality of the ribosome-binding sites of these genes might play an important role in determining the level of enzyme synthesis. This possibility is discussed with regard to the role of gene expression in co-ordinating the enzyme levels of the Entner-Doudoroff glycolytic pathway.

Aldehyde-Lyases↗
PubMed1991

Nutritional complementation of oxidative glucose metabolism in Escherichia coli via pyrroloquinoline quinone-dependent glucose dehydrogenase and the Entner-Doudoroff pathway.

Two glucose-negative Escherichia coli mutants (ZSC113 and DF214) were unable to grow on glucose as the sole carbon source unless supplemented with pyrroloquinoline quinone (PQQ). PQQ is the cofactor for the periplasmic enzyme glucose dehydrogenase, which converts glucose to gluconate. Aerobically, E. coli ZSC113 grew on glucose plus PQQ with a generation time of 65 min, a generation time about the same as that for wild-type E. coli in a defined glucose-salts medium. Thus, for E. coli ZSC113 the Enter-Doudoroff pathway was fully able to replace the Embden-Meyerhof-Parnas pathway. In the presence of 5% sodium dodecyl sulfate, PQQ no longer acted as a growth factor. Sodium dodecyl sulfate inhibited the formation of gluconate from glucose but not gluconate metabolism. Adaptation to PQQ-dependent growth exhibited long lag periods, except under low-phosphate conditions, in which the PhoE porin would be expressed. We suggest that E. coli has maintained the apoenzyme for glucose dehydrogenase and the Entner-Doudoroff pathway as adaptations to an aerobic, low-phosphate, and low-detergent aquatic environment.

Aerobiosis↗
PubMed1991

Cloning, characterization, and nucleotide sequence analysis of a Zymomonas mobilis phosphoglucose isomerase gene that is subject to carbon source-dependent regulation.

The Zymomonas mobilis gene encoding phosphoglucose isomerase (pgi) was cloned by genetic complementation of an Escherichia coli pgi mutant. An enzyme assay and sodium dodecyl sulfate-polyacrylamide gel electrophoresis confirmed the presence of excess amounts of phosphoglucose isomerase in E. coli clones carrying the Z. mobilis pgi gene. The pgi gene is present in only one copy on the Z. mobilis genome. Nucleotide sequence analysis of the pgi region revealed an open reading frame of 1,524 bp preceded by a strong Shine-Dalgarno sequence. The pgi gene encodes a 507-amino-acid protein with a predicted molecular weight of 55,398. Z. mobilis phosphoglucose isomerase is between 38 and 43% identical to the enzyme from other species. Northern (RNA) blot analysis showed that the pgi transcript is 1.8 kb in length. The level of the pgi transcript was found to be influenced by the phase of growth and by the carbon and energy sources. Transcript levels increased with respect to total RNA during logarithmic growth and were threefold higher when grown on fructose than on glucose. These changes in transcript levels paralleled phosphoglucose isomerase activities in the cultures. Differential mRNA stability was not a factor, since the half-life of the pgi transcript was 6.3 min in glucose-grown cells and 6.0 min in fructose-grown cells. Thus, an increase in the rate of transcription appears to be at least partially responsible for the increased levels of phosphoglucose isomerase observed for Z. mobilis grown on fructose.

Amino Acid Sequence↗
PubMed1990

Sequence and genetic organization of a Zymomonas mobilis gene cluster that encodes several enzymes of glucose metabolism.

The Zymomonas mobilis genes that encode glucose-6-phosphate dehydrogenase (zwf), 6-phosphogluconate dehydratase (edd), and glucokinase (glk) were cloned independently by genetic complementation of specific defects in Escherichia coli metabolism. The identity of these cloned genes was confirmed by various biochemical means. Nucleotide sequence analysis established that these three genes are clustered on the genome and revealed an additional open reading frame in this region that has significant amino acid identity to the E. coli xylose-proton symporter and the human glucose transporter. On the basis of this evidence and structural analysis of the deduced primary amino acid sequence, this gene is believed to encode the Z. mobilis glucose-facilitated diffusion protein, glf. The four genes in the 6-kb cluster are organized in the order glf, zwf, edd, glk. The glf and zwf genes are separated by 146 bp. The zwf and edd genes overlap by 8 bp, and their expression may be translationally coupled. The edd and glk genes are separated by 203 bp. The glk gene is followed by tandem transcriptional terminators. The four genes appear to be organized in an operon. Such an arrangement of the genes that govern glucose uptake and the first three steps of the Entner-Doudoroff glycolytic pathway provides the organism with a mechanism for carefully regulating the levels of the enzymes that control carbon flux into the pathway.

Amino Acid Sequence↗
PubMed1989

Differential expression of gap and pgk genes within the gap operon of Zymomonas mobilis.

In Zymomonas mobilis, the genes encoding glyceraldehyde-3-phosphate dehydrogenase (GAP) and phosphoglycerate kinase (PGK) are encoded in an operon that is transcribed from tandem promoters. The promoter-proximal gap gene is expressed at six- to ninefold higher levels than the pgk gene from chromosomal genes and from multiple copies of plasmid-borne genes. Two dominant transcripts were identified. The smaller, most abundant transcript contained primarily the gap message, whereas the larger, less abundant message contained both genes. The ratio of message levels for gap and pgk was calculated to be 5:1 and is sufficient to account for the observed differences in levels of GAP and PGK. The differences in message abundance are proposed to result from either transcriptional attenuation or preferential degradation of the 3' region encoding pgk. Increases in gene dosage were accompanied by one-third the expected increase in enzymatic activity on the basis of estimates of copy number, consistent with the presence of a limiting, positive regulatory factor. However, GAP and PGK expressions were not reduced from the chromosome in recombinants that contained multiple copies of the gap operon with inactive genes.

Bacteria↗
PubMed1989

Cloning, sequencing, and characterization of the principal acid phosphatase, the phoC+ product, from Zymomonas mobilis.

The Zymomonas mobilis gene encoding acid phosphatase, phoC, has been cloned and sequenced. The gene spans 792 base pairs and encodes an Mr 28,988 polypeptide. This protein was identified as the principal acid phosphatase activity in Z. mobilis by using zymograms and was more active with magnesium ions than with zinc ions. Its promoter region was similar to the -35 "pho box" region of the Escherichia coli pho genes as well as the regulatory sequences for Saccharomyces cerevisiae acid phosphatase (PHO5). A comparison of the gene structure of phoC with that of highly expressed Z. mobilis genes revealed that promoters for all genes were similar in degree of conservation of spacing and identity with the proposed Z. mobilis consensus sequence in the -10 region. The phoC gene contained a 5' transcribed terminus which was AT rich, a weak ribosome-binding site, and less biased codon usage than the highly expressed Z. mobilis genes.

Acid Phosphatase↗
PubMed1989

Similarity of Escherichia coli propanediol oxidoreductase (fucO product) and an unusual alcohol dehydrogenase from Zymomonas mobilis and Saccharomyces cerevisiae.

The gene that encodes 1,2-propanediol oxidoreductase (fucO) from Escherichia coli was sequenced. The reading frame specified a protein of 383 amino acids (including the N-terminal methionine), with an aggregate molecular weight of 40,642. The induction of fucO transcription, which occurred in the presence of fucose, was confirmed by Northern blot analysis. In E. coli, the primary fucO transcript was approximately 2.1 kilobases in length. The 5' end of the transcript began more than 0.7 kilobase upstream of the fucO start codon within or beyond the fucA gene. Propanediol oxidoreductase exhibited 41.7% identity with the iron-containing alcohol dehydrogenase II from Zymomonas mobilis and 39.5% identity with ADH4 from Saccharomyces cerevisiae. These three proteins did not share homology with either short-chain or long-chain zinc-containing alcohol dehydrogenase enzymes. We propose that these three unusual alcohol dehydrogenases define a new family of enzymes.

Alcohol Oxidoreductases↗
PubMed1989

Expression of Zymomonas mobilis adhB (encoding alcohol dehydrogenase II) and adhB-lacZ operon fusions in recombinant Z. mobilis.

The Zymomonas mobilis alcohol dehydrogenase II gene (adhB) was overexpressed 7- to 14-fold on a recombinant plasmid, accompanied by a small decrease in growth rate. A fragment containing the truncated gene with promoter reduced expression from the chromosomal gene as measured immunologically and enzymatically, consistent with the presence of a trans-active regulatory factor and positive regulatory control. Both the complete gene and the promoter fragment increased pyruvate decarboxylase and glucokinase activities, with no effect on alcohol dehydrogenase I or eight glycolytic enzymes. Tandem promoters from adhB expressed beta-galactosidase at higher levels than did either promoter alone in operon fusions. Addition of 50 microM zinc sulfate in minimal medium reduced the expression of adhB and of the operon fusions. Abundant but inactive alcohol dehydrogenase II was produced in iron-limited cells. This inactive enzyme did not form intracellular aggregates, and no morphological changes were apparent by transmission electron microscopy.

Alcohol Dehydrogenase↗
PubMed1988

Tumorigenic and molecular characterization of novel phorbol ester-resistant and -sensitive lines of mice.

Two outbred lines of CD-1 mice were developed using males and females in an initiation (dimethylbenz[a]anthracene; DMBA), promotion (12-O-tetradecanoylphorbol-13-acetate; TPA) challenge, posttumorigenesis breeding protocol. Our results indicate that the phorbol ester-sensitive (PESTI) line developed tumors at a rate 4.1 times faster than the CD-1 parental line, while the phorbol ester-resistant (PERTI) line developed tumors at a rate 36 times slower than the CD-1 parents. The average number of tumors per mouse reached levels of 27.5 at 12 wk in the PESTI line, 0.1 at 16 wk in the PERTI line, and 6.7 at 16 wk in the CD-1 line. Biochemical tests showed that the PESTI line had both a high basal level and an enhanced epidermal ornithine decarboxylase (E.C. 4.1.1.17) response to TPA, the latter being nine times that of the PERTI line at their maximum dosages. An autoradiographic analysis of in vivo epidermal cell protein phosphorylation indicated marked differences in basal protein phosphorylation profiles (with high phosphate incorporation, PERTI, 112.7, 95.5, 64.4, 40.8, 18.6, 17.4, and 12.3 kDa; PESTI, 64.4, 40.8, 31.8, and 12.3 kDa) as well as TPA-dependent changes in these profiles (difference from basal levels, PERTI, 31.8 and 12.8 kDa; PESTI, 139.6, 126.3, 37.2, and 18.6 kDa). These heterogeneous profiles indicate strong genetic segregation of these protein kinase C target substrates.(ABSTRACT TRUNCATED AT 250 WORDS)

9,10-Dimethyl-1,2-benzanthracene↗