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Biomedical subjects

C G Sahasrabuddhe

Publications and source records attributed to C G Sahasrabuddhe.

30 records · Page 2Linked to original sources
PubMed1982

Localization of mitotic factors on metaphase chromosomes.

The objective of this study was to determine whether the mitotic factors of HeLa cells, which induce meiotic maturation, i.e. germinal vesicle breakdown (GVBD) and chromosome condensation, when injected into fully grown Xenopus laevis oocytes, were localized in the cytoplasm or associated with the metaphase chromosomes. Cytoplasmic extracts were prepared by lysing mitotic HeLa cells in low-salt hypotonic buffer and separating the chromosomes by centrifugation. Th mitotic factors bound to chromosomes were extracted with high-salt (0.2 M-NaCl) buffer. Both the cytoplasmic and chromosomal protein fractions were evaluated for their maturation-promoting activity (MPA) in the Xenopus oocytes. The results of this study indicate that both the cytoplasmic and chromosomal fractions are identical in many respects, including their ability to induce GVBD, but the specific activity of the chromosomal fraction was at least threefold greater than that of the cytoplasmic fraction. These data suggest that a major portion of the mitotic factors is localized on the metaphase chromosomes. This association does not appear to be due to adventitious binding of mitotic proteins to chromosomes during the extraction procedures. Furthermore, when extracts were prepared in a similar way from early- and mid-G2-phase HeLa cells, only the nuclear extracts had MPA and no activity was found in the cytoplasmic fraction. Both the cytoplasmic and nuclear extracts of late-G2 cells exhibited MPA. These data support the conclusion that the mitotic factors become preferentially bound to chromatin as soon as they are synthesized, and as the cell synthesizes more of these factors in preparation for mitosis, increasing amounts of them are retained in the cytoplasm.

Animals↗
PubMed1980

Use of RNA polymerase as an enzymatic probe of nucleosomal structure.

Nucleosomes prepared from human placental nuclei and Escherichia coli DNA-dependent RNA polymerase (nucleoside triphosphate: RNA nucleotidyl transferase EC.2.7.7.6) form stable initiation complexes. This property is utilized as a probe of nucleosome structure. RNA polymerase initiation has been studied on purified nucleosomes, nucleosome cores, and nucleosomal DNA. The affinity of E. coli RNA polymerase for both nucleosome cores and monomers was 5-6 fold less than found for nucleosomal DNA. No difference in apparent initiation Km was found between cores and mononucleosomes. This suggests that initiation does not preferentially occur on the DNA tails of nucleosomes. Once initiated and allowed to form nascent RNA, these complexes are very stable to ionic strength changes. Under conditions in which free enzyme is inactivated with rifampicin, the enzyme in the complex retains activity as demonstrated by its ability to transcribe and reinitiate on both nucleosomes and free DNA. These complexes can be well resolved from free nucleosomes on preparative polyacrylamide gels and both can be eluted from gels for analysis of proteins and DNA sequence complexity. Studies using (125I) labelled nucleosomes show that histones are retained in the initiation complex, and are not dissociated by the enzyme during initiation.

Cell Nucleus↗
PubMed1978

Responses of mammalian metaphase chromosomes to endonuclease digestion.

Digestion of fixed metaphase chromosomes by endonucleases (micrococcal nuclease and DNase II) under optimal digestion conditions followed by Giemsa staining produces sharp banding patterns identical to G-bands. In 3H-thymidine labeled, synchronized metaphase cells of the chinese hamster (CHO line), the band induction is accompanied by the removal of DNA. The single strand specific nuclease S1 and DNase I do not produce such banding patterns.

Animals↗
PubMed1978

Transcription of nucleosomes from human chromatin.

Nucleosomes (chromatin subunits) prepared by micrococcal nuclease digestion of human nuclei are similar in histone content but substantially reduced in non-histone proteins as compared to undigested chromatin. Chromatin transcription experiments indicate that the DNA in the nucleosomes is accessible to DNA-dependent RNA polymerase in vitro. The template capacities of chromatin and nucleosomes are 1.5 and 10%, respectively, relative to high molecular weight DNA, with intermediate values for oligonucleosomes. Three distinct sizes of transcripts, 150, 120 and 95 nucleotides in length, are obtained when nucleosomes are used as templates. However, when nucleosomal DNA is used as a template, the predominant size of transcripts is 150 nucleotides. When oligonucleosomes are used as templates longer transcripts are obtained. This indicates that RNA polymerase can transcribe the DNA contained in the nucleosomes.

Binding Sites↗
PubMed1977

Salt-induced structural changes in nucleosomes.

Nucleosomes and oligonucleosomes were prepared by digestion of human placental nuclei with staphlococcal nuclease and fractionated by gel filtration chromatography. The effect of increasing salt on the structure of nucleosomes was examined in the presence and absence of 10 mM MgCl2. Nucleosomes and oligonucleosomes are insoluble over a broad range of salt concentration. Nucleosomes are insoluble in larger than or equal to 120 mM (NH4)2SO4 containing 10 mM MgCl2 allowing analyses of changes in nucleosomal DNA by C.D. spectroscopy. Nucleosomes are insoluble in less than or equal to 120 mM (NH4)2SO4 containing 10 mM MgCl2 as demonstrated by turbidity measurements. We conclude that the insolubility of nucleosomes accompanies salt-induced structural changes possibly due to individual particle condensation. As the salt concentration is increased the nucleosomes condense and then relax at higher salt concentrations.

Ammonium Sulfate↗
PubMed1976

Presence of messenger specifying sequences in the DNA of chromatin subunits.

Messenger-specifying sequences in subunits of human lymphocyte chromatin were detected by hybridization of DNA complementary to cytoplasmic polyadenylylated RNA with DNA isolated from the subunits. Comparison of the kinetics and extents of hybridization of complementary DNA with chromatin subunit DNA and with nuclear DNA showed that most of the repetitive sequences and single copy sequences in mRNA are present in chromatin subunits. This result indicates that inclusion of a DNA sequence into the subunit does not prevent its transcription in vivo.

Chromatin↗
PubMed1974

A model for particulate structure in chromatin.

A model is proposed for the structure of nuclease-resistant chromatin particles. The model is novel in that it proposes that the DNA in such a particle is wound about a protein core, made up of the hydrophobic regions of histone molecules.

Animals↗
PubMedDate not supplied

Cytogenetic characterization of eight human lymphoblastoid cell lines.

Cytogenetic analyses of eight human lymphoblastoid cell lines that were established by a new procedure using B-cell growth factor (BCGF) and interleukin-4 (IL-4) revealed that three cell lines (37.5%) showed structural anomalies in more than 97% of their metaphases, whereas others were predominantly normal diploid. Our results indicate that a) the structural anomalies seen in three cell lines were not induced by culture technique but were constitutional defects present in donors' peripheral blood samples; b) one donor, whose blood gave rise to cell line BCDI, is a constitutional mosaic because both normal diploid and altered metaphases were present; and c) genetic instability should be monitored in regular blood donors because some of them may harbor blood-mediated clastogen or chromosome breakage factor(s) that could induce higher rates of recombinations in somatic cells of some recipients and thus may potentiate such individuals to develop certain types of malignancies.

B-Lymphocytes↗