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D Bhaumik

Publications and source records attributed to D Bhaumik.

14 recordsLinked to original sources
PubMed1999

Mutator phenotype induced by aberrant replication.

We have identified thermosensitive mutants of five Schizosaccharomyces pombe replication proteins that have a mutator phenotype at their semipermissive temperatures. Allele-specific mutants of DNA polymerase delta (poldelta) and mutants of Polalpha, two Poldelta subunits, and ligase exhibited increased rates of deletion of sequences flanked by short direct repeats. Deletion of rad2(+), which encodes a nuclease involved in processing Okazaki fragments, caused an increased rate of duplication of sequences flanked by short direct repeats. The deletion mutation rates of all the thermosensitive replication mutators decreased in a rad2Delta background, suggesting that deletion formation requires Rad2 function. The duplication mutation rate of rad2Delta was also reduced in a thermosensitive polymerase background, but not in a ligase mutator background, which suggests that formation of duplication mutations requires normal DNA polymerization. Thus, although the deletion and duplication mutator phenotypes are distinct, their mutational mechanisms are interdependent. The deletion and duplication replication mutators all exhibited decreased viability in combination with deletion of a checkpoint Rad protein, Rad26. Interestingly, deletion of Cds1, a protein kinase functioning in a checkpoint Rad-mediated reversible S-phase arrest pathway, decreased the viability and exacerbated the mutation rate only in the thermosensitive deletion replication mutators but had no effect on rad2Delta. These findings suggest that aberrant replication caused by allele-specific mutations of these replication proteins can accumulate potentially mutagenic DNA structures. The checkpoint Rad-mediated pathways monitor and signal the aberrant replication in both the deletion and duplication mutators, while Cds1 mediates recovery from aberrant replication and prevents formation of deletion mutations specifically in the thermosensitive deletion replication mutators.

Alleles↗
PubMed1998

Mutational effect of fission yeast polalpha on cell cycle events.

Polalpha is the principal DNA polymerase for initiation of DNA replication and also functions in postinitiation DNA synthesis. In this study, we investigated the cell cycle responses induced by mutations in polalpha+. Germinating spores carrying either a deletion of polalpha+ (polalphaDelta) or a structurally intact but catalytically dead polalpha mutation proceed to inappropriate mitosis with no DNA synthesis. This suggests that the catalytic function, and not the physical presence of Polalpha, is required to generate the signal that prevents the cells from entering mitosis prematurely. Cells with a polalphats allele arrest the cell cycle near the hydroxyurea arrest point, but, surprisingly, polalphats in cdc20 (polepsilon mutant) background arrested with a cdc phenoytpe, not a polalphats-like phenotype. At 25 degrees C, replication perturbation caused by polalphats alleles induces Cds1 kinase activity and requires the checkpoint Rads, Cds1, and Rqh1, but not Chk1, to maintain cell viability. At 36 degrees C, replication disruption caused by polalphats alleles induces the phosphorylation of Chk1; however, mutant cells arrest with heterogeneous cell sizes with a population of the cells entering aberrant mitosis. Together, our results indicate that the initiation DNA structure synthesized by Polalpha is required to bring about the S phase to mitosis checkpoint, whereas replication defects of different severity caused by polalphats mutations induce differential downstream kinase responses.

Amino Acid Sequence↗
PubMed1994

Initiation of transcription at the human terminal deoxynucleotidyl transferase gene promoter: a novel role for the TATA binding protein.

Control of initiation of transcription of the human terminal deoxynucleotidyl transferase (TdT) gene was investigated by using an in vitro transcription assay. The precise contribution of discrete basal promoter elements to transcription initiation was determined by testing deletion and substitution mutations. The primary element, contained within the region spanning -34 to -14 bp relative to the transcription start site, accounted for 80% of basal promoter activity. TdT promoter activity required the sequence ACCCT at -24 to -20 bp since a dramatic decrease in transcription initiation was observed after mutation of this sequence, whereas mutation of the adjacent sequence from -32 to -25 bp did not alter promoter activity. The secondary element contained sequences surrounding the transcription start site and had 20% of promoter activity. Deletion of both elements completely abolished transcription initiation. Initiator characteristics of the secondary element were revealed by using the in vitro assay: promoter sequences at the transcription start site were sufficient to direct accurate initiation at a single site. Mutation of the sequence GGGTG spanning the transcription start site resulted in loss of transcription initiation. Both the primary and secondary elements were nonhomologous to corresponding regions from the mouse TdT gene promoter. While the human basal promoter functioned in the absence of TATA consensus sequences or GC-rich SP1 binding sites, it was dependent on active TFIID. In contrast to other TATA-less promoters, purified TATA binding protein substituted for the TFIID complex and restored promoter activity to TFIID-inactivated nuclear extracts.

Base Sequence↗
PubMed1994

Identification of a tripartite basal promoter which regulates human terminal deoxynucleotidyl transferase gene expression.

In order to locate the promoter region of the human terminal deoxynucleotidyl transferase gene, serially truncated segments of the 5'-flanking region of the gene were cloned into a chloramphenicol acetyltransferase reporter vector. Transient transfection analyses of the terminal transferase-reporter gene constructs identified the basal promoter region within -34 to +40 base pairs relative to the transcription start site. Three promoter elements were defined in this region. The primary element is within 34 base pairs upstream of the transcription start site. The CAP site is 62 base pairs upstream of the translation start site. The secondary element involves sequences around the transcription start site. The third is located 25 base pairs downstream from the initiation site (+25 to +40). This tripartite basal promoter was not tissue specific; similar patterns of promoter activity were observed in terminal transferase expressing and non-expressing cells. Transfection analyses also indicated the presence of negative regulatory elements upstream of the basal promoter region, and these elements were preferentially active in cells expressing terminal transferase.

Base Sequence↗
PubMed1993

Mutational analysis of active site residues of human adenosine deaminase.

Adenosine deaminase was overexpressed in a baculovirus system. The pure recombinant and native enzymes were identical in size, Zn2+ content, and activity. Five amino acids, in proximity to the active site, were replaced by mutagenesis. The altered enzymes were purified to homogeneity and compared to wild-type adenosine deaminase with respect to zinc content, enzymatic activity, and kinetic parameters. All but one of the alterations produced significant activity perturbations. Replacement of Cys262 produced a protein that retained at least 30-40% of wild-type activity. In contrast, replacements of His17, His214, His238, and Glu217 resulted in dramatic losses of enzyme activity. None of these mutants exhibited large variations in Km. The proteins produced from alterations of amino acids implicated in metal coordination were slightly activated by inclusion of Zn2+ throughout purification. These experiments confirm that in the active enzyme Zn2+ plays a critical role in catalysis, that a histidine or glutamate residue plays a mechanistic role in the hydrolytic deamination step, and that cysteine is not involved in the catalytic mechanism of adenosine deaminase. These data support the roles for these amino acid residues suggested from the x-ray structure of murine adenosine deaminase (Wilson, D. K., Rudolf, F. B., and Quicho, F. A. (1991) Science 252, 1278-1284).

Adenosine Deaminase↗
PubMed1992

Active site thiol(s) in Leishmania donovani adenosine kinase: comparison with hamster enzyme and evidence for the absence of regulatory adenosine binding site.

Adenosine kinase (ATP, adenosine 5'-phosphotransferase, E.C. 2.7.1.20) from Leishmania donovani, unlike adenosine kinase from other known eukaryotic sources, does not elicit an inhibitory response at high concentrations of adenosine. The mechanistic basis for this unique catalytic behavior of the parasite enzyme has been probed with the help of chemical modification and enzyme inhibition kinetics experiments. The use of cysteine-directed reagents has shown that chemical integrity of cysteinyl residues is essential for the expression of functional activity of the enzyme. Thiol group titration revealed that the enzyme contains 3 cysteine residues. However, in contrast to adenosine kinase from other sources, inactivation of the parasite enzyme could be correlated with alkylation of 2 cysteinyl residues. Adenosine, but not ATP, protected 2 thiols against -SH blocker-mediated inactivation of the enzyme. The thiol groups were shown to map at positions corresponding to approximately 16, 22, and 36 kDa sites from the protein's N-terminal end. The functions of 2 thiols at the catalytic site were functional thiol groups yielded a 'protection constant' (KpAd) of 3.4 microM, while the dissociation constant (KsAD) of the enzyme-substrate complex was 2.7 microM, hence supporting involvement of the same in both processes, namely catalysis and protection. The overall results were therefore interpreted as showing that (a) the leishmanial enzyme, in contrast to adenosine kinase from other sources, contains 2 functional thiol groups at the catalytic site; and (b) the enzyme binds adenosine exclusively through the catalytic site and as a consequence is not amenable to inhibition at high adenosine concentrations.(ABSTRACT TRUNCATED AT 250 WORDS)

Adenosine↗
PubMed1989

Immunochemical and catalytic characteristics of adenosine kinase from Leishmania donovani.

Polyclonal antibodies to homogeneous preparation of adenosine kinase from Leishmania donovani were raised in rabbit. The antiserum was inhibitory and precipitated enzyme activity from both homogeneous and partially purified adenosine kinase from the parasite. However, the antiserum did not immunoprecipitate adenosine kinase of other higher eukaryotic sources tested so far. Immunoblot analysis of extracts from L. donovani and other sources revealed specific reaction of the antiserum with only the parasite enzyme. Under similar conditions, the enzyme monophosphorylated adenosine and 7-amino-3[beta-D-ribofuranosyl]-1H-pyrazolo[4,3-d]pyrimidine (formycin A) with almost equal efficiency, exhibiting Km values of 16 and 24 microM, respectively. The turnover number (Kcat) of the enzyme with both adenosine and formycin A was 24 s-1, whereas Kcat/Km yielded values of 1.5 and 1.0 microM-1 s-1, respectively. Substrate competition experiments indicated strong inhibition of [3H]formycin A phosphorylation by adenosine. In contrast, [3H]adenosine phosphorylation was insensitive to formycin A except at very high concentrations. The inhibitions of [3H]formycin A and [3H]adenosine phosphorylation by adenosine and formycin A were noncompetitive with respect to each other. Of the two nucleosides, adenosine was found to be effective in eluting the enzyme from the 5'-AMP Sepharose 4B column. Phosphorylation of [3H]formycin A was strongly inhibited by N-ethylmaleimide at concentrations which exerted minimal effect on [3H]adenosine phosphorylation. Adenosine exclusively, but not formycin A, protected the enzyme from N-ethylmaleimide-mediated inactivation. Taken together the results suggest that (a) adenosine kinase from L. donovani is immunologically distinct and (b) the enzyme possibly has two discrete catalytically active nucleoside interacting sites.

Adenosine↗
PubMed1988

Reaction kinetics and inhibition of adenosine kinase from Leishmania donovani.

The reaction kinetics and the inhibitor specificity of adenosine kinase (ATP:adenosine 5'-phosphotransferase, EC 2.7.1.20) from Leishmania donovani, have been analysed using homogeneous preparation of the enzyme. The reaction proceeds with equimolar stoichiometry of each reactant. Double reciprocal plots of initial velocity studies in the absence of products yielded intersecting lines for both adenosine and Mg2+-ATP. AMP is a competitive inhibitor of the enzyme with respect to adenosine and noncompetitive inhibitor with respect to ATP. In contrast, ADP was a noncompetitive inhibitor with respect to both adenosine and ATP, with inhibition by ADP becoming uncompetitive at very high concentration of ATP. Parallel equilibrium dialysis experiments against [3H]adenosine and [gamma-32P]ATP resulted in binding of adenosine to fre enzyme. Tubercidin (7-deazaadenosine) and 6-methyl-mercaptopurine riboside acted as substrates for the enzyme and were found to inhibit adenosine phosphorylation competitively in vitro. 'Substrate efficiency (Vmax/Km)' and 'turnover numbers (Kcat)' of the enzyme with respect to specific analogs were determined. Taken together the results suggest that (a) the kinetic mechanism of adenosine kinase is sequential Bi-Bi, (b) AMP and ADP may regulate enzyme activity in vivo and (c) tubercidin and 6-methylmercaptopurine riboside are monophosphorylated by the parasite enzyme.

Adenosine↗
PubMed1987

Isolation and characterization of adenosine kinase from Leishmania donovani.

Adenosine kinase (ATP:adenosine 5'-phosphotransferase, EC 2.7.1.20) has been purified 3250-fold from Leishmania donovani promastigotes using ion-exchange, gel filtration, and affinity chromatography techniques. Both native and sodium dodecyl sulfate-gel electrophoresis of the enzyme revealed a single polypeptide of around 38,000 molecular weight. Biophysical and biochemical analyses of the enzyme reveal unique characteristics different from those of adenosine kinases from other eukaryotic sources. The isoelectric pH of the enzyme is 8.8. In native acrylamide gels the enzyme moves with an RF of about 0.62. The enzyme displays a maximum activity at pH between 7.5 and 8.5 and is dependent upon an optimum ATP/Mg2+ ratio. ATP at high concentration inhibits the reaction. Adenosine and Mg2+ are not inhibitory. EDTA completely knocks off the activity. Enzyme activity is dependent upon the presence of active thiol group(s) at or near the active center. Under a defined set of conditions the enzyme exhibited an apparent Km for adenosine and ATP of 33 and 50 microM, respectively. Of the nucleoside triphosphates tested ATP and GTP were the most effective phosphate donors. Marginal inhibition of activity was detected with other nucleosides as competitors. However, adenosine analogs, such as 7-deaza-adenosine (tubercidin) and 6-methylmercaptopurine riboside at very low concentrations, were found to be excellent inhibitors and substrates as well. S-Adenosylhomocysteine does not inhibit the reaction even at very high concentration.

Adenosine Kinase↗
PubMedDate not supplied

Assessment of the relationship of sperm morphology with seminal and other clinical conditions of semen donors.

The occurrence of abnormal forms of spermatozoa in human semen is quite common. According to WHO, semen is considered normal even if it contains 50% morphologically abnormal spermatozoa. This study assessed whether the sperm morphology maintains any relation with the relevant clinical conditions of the semen donor. One hundred samples representing normal and different types of male factor etiologies underwent semen and morphological analysis. Clinical information such as race, age, weight, profession, medication, medical history, and smoking habit of the semen contributors were recorded. The influence of seminal and clinical features on sperm morphology was evaluated with multiple regression analysis. Head abnormalities were more common than tail abnormalities. Acrosomal defects and coiled tails were the most prevalent head and tail abnormalities, respectively. Regression analysis failed to confirm any strong association between sperm morphology and other seminal parameters. Accessory gland-related seminal parameters such as viscosity, volume, pH, and liquefaction showed the least association with the morphological variability. Sperm morphology also showed poor correlation with race, age, weight, smoking habit, and work environment.

Acrosome↗