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PubMed1976

DNA-dependent DNA polymerase from yeast mitochondria. Dependence of enzyme activity on conditions of cell growth, and properties of the highly purified polymerase.

The activity of DNA polymerase was determined in gradient-purified mitochondria from yeast cells grown under a variety of conditions. The specific enzyme activity was found to be dependent on the degree of aeration of the cells, and on the carbon source used for the medium. It was sensitive to glucose repression, and was enhanced about two-fold by the growth of yeast cells in the presence of ethidium bromide. Mitochondria DNA polymerase was highly purified and several properties were determined. Sucrose density gradient centrifugation, and dodecylsulfate-polyacylamide gel electrophoresis revealed the following structure: a monomer of molecular weight around 60 000 aggregated under relatively high salt concentration (0.2 M phosphate buffer) to a dimer of about 120 000 which under low salt concentration (0.2 M Tris-HCl buffer) formed higher aggregates. For optimal activity an Mg2+ ion concentration of 50 mM was found necessary, Mn ions did not promote activity at any concentration tested (0.5--50 mM). Indeed, if added to Mg2+-containing assays, Mn2+ strongly inhibited enzyme activity at low concentrations. This might be an explanation for the inducation of mitochondrial mutants in yeast cells grown in the presence of Mn2+ ions. Mitochondrial DNA polymerase activity was strongly inhibited by low concentrations of the -SH reagent p-chloromercuribenzoate, the nucleotide analogue cytosine arabinoside triphosphate also exerted an inhibitory effect. An about 50% decrease of activity was observed in the presence of 1 mM o-phenanthroline in assay mixture containing DNA at about the Km concentration. The enzyme preferred a gapped template primer, poly(dA) - (dT)10, over nicked DNA and was unable to use a polyribonucleotide template, poly(rA) - (dT)10. In the purest preparations no exonuclease activity could be detected.

Cell Division↗
PubMed1982

Regulatory properties of citrate synthase from Rickettsia prowazekii.

Citrate synthase [citrate (si)-synthase] (EC 4.1.3.7) was partially purified from extracts of highly purified typhus rickettsiae (Rickettsia prowazekii). Molecular exclusion and affinity column chromatography were used to prepare 200-fold-purified citrate synthase that contained no detectable malate dehydrogenase (EC 1.1.1.37) activity. Rickettsial malate dehydrogenase also was partially purified (200-fold) via this purification procedure. Catalytically active citrate synthase exhibited a relative molecular weight of approximately 62,000 after elution from a calibrated Sephacryl S-200 column. Acetyl coenzyme A saturation of partially purified enzyme was sensitive to strong competitive inhibition with adenylates (ATP greater than ADP much greater than AMP). [beta,gamma-methylene]ATP, dATP, and dADP also caused strong inhibition, but guanosine and cytosine nucleotides were significantly less inhibitory. Adenylates had no effect on oxalacetate saturation kinetics when acetyl coenzyme A was present in high concentration (greater than or equal to 50 microM). Neither NADH nor alpha-ketoglutarate affected the saturation kinetics of rickettsial citrate synthase. Thus, citrate synthase from R. prowazekii exhibits greater similarity to the eucaryotic and gram-positive procaryotic enzymes than to citrate synthase from free-living gram-negative bacteria. These results represent the first characterization of a highly purified key regulatory enzyme from these obligate intracellular parasitic bacteria.

Acetyl Coenzyme A↗
PubMed1994

De novo synthesis of thymidylate via deoxycytidine in dcd (dCTP deaminase) mutants of Escherichia coli.

dcd (dCTP deaminase) mutants of Escherichia coli were reported not to require thymidine for growth even though most of the thymidylate that is synthesized de novo arises from cytosine nucleotides through a pathway involving dCTP deaminase. We found, however, that the fresh introduction of dcd mutations into many strains of E. coli produced a requirement for thymidine for optimum aerobic growth, but the mutants readily reverted to prototrophy via mutations in other genes. One such mutation was in deoA, the gene for deoxyuridine phosphorylase. However, a dcd deo mutant became thymidine dependent once again if a cdd mutation (affecting deoxycytidine deaminase) were introduced. The results indicate that dcd mutants utilize an alternative pathway of TMP synthesis in which deoxycytidine and deoxyuridine are intermediates. A cdd mutation blocks the pathway by preventing the conversion of deoxycytidine to deoxyuridine, whereas a deoA mutation enhances it by sparing deoxyuridine from catabolism. The deoxycytidine must arise from dCTP or dCDP via unknown steps. It is not known to what extent this pathway is utilized in wild-type cells, which, unlike the dcd mutants, do not accumulate dCTP.

Anaerobiosis↗
PubMed2002

Dichotomy of glycoprotein g gene in herpes simplex virus type 1 isolates.

Herpes simplex virus type 1 (HSV-1) encodes 11 envelope glycoproteins, of which glycoprotein G-1 (gG-1) induces a type-specific antibody response. Variability of the gG-1 gene among wild-type strains may be a factor of importance for a reliable serodiagnosis and typing of HSV-1 isolates. Here, we used a gG-1 type-specific monoclonal antibody (MAb) to screen for mutations in the immunodominant region of this protein in 108 clinical HSV-1 isolates. Of these, 42 isolates showed no reactivity to the anti-gG-1 MAb. One hundred five strains were further examined by DNA sequencing of the middle part of the gG-1 gene, encompassing 106 amino acids including the immunodominant region and epitope of the anti-gG-1 MAb. By phylogenetic comparisons based on the sequence data, we observed two (main) genetic variants of the gG-1 gene among the clinical isolates corresponding to reactivity or nonreactivity to the anti-gG-1 MAb. Furthermore, four strains appeared to be recombinants of the two gG-1 variants. In addition, one strain displayed a gG-1-negative phenotype due to a frameshift mutation, in the form of insertion of a cytosine nucleotide. When immunoglobulin G reactivity to HSV-1 in sera from patients infected with either of the two variants was investigated, no significant differences were found between the two groups, either in a type-common enzyme-linked immunosorbent assay (ELISA) or in a type-specific gG-1 antigen-based ELISA. Despite the here-documented existence of two variants of the gG-1 gene affecting the immunodominant region of the protein, other circumstances, such as early phase of infection, might be sought for explaining the seronegativity to gG-1 commonly found in a proportion of the HSV-1-infected patients.

Amino Acid Sequence↗
PubMed2003

DNA methylation markers in patients with gastrointestinal cancers. Current understanding, potential applications for disease management and development of diagnostic tools.

DNA methylation, the modification of a cytosine nucleotide immediately preceding a guanine base in a stretch of DNA, is rapidly gaining strength in the diagnostic field as a powerful tool to be utilized for the discrimination of neoplastic tissue from its healthy counterpart. This epigenetic modification occurs often in the promoter region of genes and is associated with transcriptional silencing of tumor suppressors or other genes important for normal cellular function. These changes have been found to occur at very early stages in the progression of healthy to malignant phenotype in many cancer types. We are taking a targeted approach to finding methylation-based markers that can be used not only for the early detection of cancer but also for determining risk, monitoring patient response to therapy and even determining the degree of aggressiveness of a tumor. In this paper, we review the progress in our understanding of methylation in gastrointestinal tumors, the potential clinical applications of methylation-based markers and our process for the discovery and validation of highly specific and sensitive markers for the use in these applications.

Biomarkers, Tumor↗
PubMed2002

Mutational analysis of the GNAS1 exons encoding the stimulatory G protein in five patients with pseudohypoparathyroidism type 1a.

We analyzed the GNAS1 gene in five patients with pseudohypoparathyroidism type 1a (PHP1a) by performing polymerase chain reaction, followed by sequencing all 13 exons of the gene, single-stranded conformational polymorphism (SSCP) or heteroduplex analysis (HD). Three novel mutations were discovered: (1) a de novo 3 bp insertion of CTG in codon 47 of exon 1; (2) a missense mutation 1103T in exon 4; and (3) a de novo mutation of Arg280Gly in exon 10. Two other mutations, previously described in the literature, include: (1) a de novo 4 bp deletion (deltaGACT) involving codons 189 and 190 in exon 7, and (2) a deletion of a cytosine nucleotide at codon 115 in exon 5. We conclude that mutational analysis of the GNAS1 gene is a strong supportive tool for the diagnosis of PHP1a, and is a useful adjunct to the synthetic parathyroid hormone infusion test for PTH resistance.

Adolescent↗
PubMed2003

Epigenetic regulation of proprotein convertase PACE4 gene expression in human ovarian cancer cells.

Proprotein convertases (PC) are a family of serine endoproteases that play important roles in regulating cell function by converting proproteins to biologically active molecules. Several lines of evidence suggest that overexpression of PCs contributes to tumor formation and progression in various types of cancer. In this study, we examined PC expression in six normal ovarian surface epithelium (OSE) cultures, nine primary ovarian cancer (OC) cultures, and three established OC cell lines (Hey, HeyC2, and OCC-1). Our results show that furin and PC7 expression in OC cells was comparable to that in normal OSE. However, PACE4 expression was greatly reduced in all OC samples studied. PACE4 promoter activity was measured in HeyC2 and OCC-1 cells using transiently transfected luciferase reporter plasmids. Both cell lines supported PACE4 promoter activity, showing that the transcription factors critical for PACE4 expression are present in OC cells. The observation that established OC cell lines have reduced PACE4 expression, but maintained the ability to support PACE4 promoter activity, led to the hypothesis that reduced expression may be due to epigenetic modification of the PACE4 gene, such as DNA methylation and histone deacetylation. Methylation analysis of 79 CpG dinucleotides within the PACE4 promoter and exon I (-196/+340) revealed that the percentage of methylated cytosine nucleotides was 8-9% in normal OSE, but 58-93% in OC cells. Treatment with the demethylating agent 5-aza-2'-deoxycytidine and/or the histone deacetylase inhibitor trichostatin A greatly increased PACE4 expression in OC cells. These data suggest that the reduction of PACE4 expression in OC cells is caused, in part, by DNA hypermethylation and histone deacetylation.

DNA Methylation↗
PubMed1979

Synergistic action of 5-aza-2'-deoxycytidine and 3-deazauridine on L1210 leukemic cells and EMT6 tumor cells.

The biochemical and biological effects of the combination of 5-aza-2'-deoxycytidine (5-aza-dCyd) and 3-deazauridine (3-DU) on L1210 leukemic cells and EMT6 tumor cells were investigated. The cytotoxic action of 5-aza-dCyd and 3-DU on both L1210 and EMT6 cells in vitro was synergistic when these agents were used in combination. The combination of 5-aza-dCyd and 3-DU produced a greater inhibition of in vitro growth of L1210 and EMT6 cells than did either agent alone. The in vivo antineoplastic activity of this combination was synergistic with respect to the increased survival time of BALB/c x DBA/2 F1 mice with L1210 leukemia. 3-DU, an agent that reduces the intracellular pool size of cytosine nucleotides, stimulated the incorporation of [3H]-5-aza-dCyd into DNA of both L1210 and EMT6 cells, suggesting that the synergistic action of this combination is related to the increased incorporation of 5-aza-dCyd in the presence of 3-DU.

3-Deazauridine↗