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Biomedical subjects

Y M Bhatnagar

Publications and source records attributed to Y M Bhatnagar.

At least 19 recordsLinked to original sources
PubMed2003

Analysis of detached human kinetochores.

A method has recently been established for inducing the physical detachment of kinetochores from chromosomes in human HeLa cells, and was used in the studies reported here to investigate the organization and function of dissociated HeLa kinetochores. Immunofluorescence labeling demonstrated that the detached HeLa kinetochores were relatively intact, with the number of detached kinetochores being only moderately more than the diploid number of chromosomes in HeLa cells. In addition, the detached kinetochores could be labeled with antibodies specific for the inner kinetochore plate, outer kinetochore, and subjacent centromeric heterochromatin. A functional assay demonstrated that detached kinetochores retained the capacity to activate the spindle checkpoint, leading to metaphase arrest. Analysis of kinetochore DNA indicated that it consisted primarily of DNA fragments of 130-160 kb in size, while the remainder of the chromosomes were sheared into much smaller fragments during the kinetochore detachment event. Further analysis of kinetochore DNA indicated that it was first cleaved into high molecular weight DNA (>200 kb) fragments during the initial stages of the kinetochore detachment process, and then underwent further maturation following nuclear envelope breakdown to give rise to the 130-160 kb fragment in detached kinetochores. Collectively, these data indicate that detached human kinetochores will be a useful system for investigating the organization, assembly, and function of human kinetochores.

Antibodies↗
PubMed1995

Increased accessibility of the N-terminus of testis-specific histone TH2B to antibodies in elongating spermatids.

Changes in chromatin structure during spermatogenesis were investigated using a monoclonal antibody that immunoreacts with the N-terminus of the testis-specific histone TH2B. This monoclonal antibody, which had been raised against rat tyrosine hydroxylase (TH), cross-reacted with TH2B because of sequence homology at the N-termini of TH and TH2B. The epitope was localized to the N-terminus of TH2B as trypsin-digested chromatin which lacked the N-terminal tail did not react with anti-TH and preincubating anti-TH with a synthetic peptide made from the homologous sequence between TH2B and TH inhibited its binding to TH and TH2B. In histological sections of rat testis, the primary spermatocytes and round spermatids immunoreacted weakly, whereas elongating spermatids at steps 10-12 immunoreacted intensely with anti-TH. Increased staining of elongating spermatids was also observed in mouse and hamster by immunohistochemistry. However, immunoblotting proteins extracted from separated rat testis cells showed no increase in the TH2B content of these late steps of spermatids. The apparent increase in the immunohistochemical staining corresponds to increased accessibility of the epitope in the elongating spermatids. This indicated that the N-terminus of TH2B is less tightly bound to DNA or to other proteins at this time in preparation for the removal of TH2B and other histones.

Amino Acid Sequence↗
PubMed1995

Isodicentric Y chromosome: cytogenetic, molecular and clinical studies and review of the literature.

Dicentrics are among the most common structural abnormalities of the human Y chromosome. Predicting the phenotypic consequences of different duplications and deletions of dicentric Y chromosomes is usually complicated by varying degrees of mosaicism (45,X cell lines), which may, in some cases, remain undetected. Molecular studies in patients with dicentric Y chromosomes have been few, and only two studies have attempted to determine the presence of SRY (the putative testis-determining factor gene). We report an 18-year-old female with short stature, amenorrhea, hirsutism, hypoplastic labia minora, and clitoromegaly who has a 45,X/46,X,idic(Y)(p11.32)/47,X,idic(Y)(p11.32),idic(Y) (p11.32) karyotype. Southern analysis using Y-specific probes (Y97, 2D6, 1F5, pY3.4) and polymerase chain reaction (PCR) analysis using primers for ZFY and SRY were positive for all loci tested, indicating that almost all of the Y chromosome was present. Our findings and an extensive review of the literature emphasize the importance of molecular analyses of abnormal Y chromosomes before any general conclusions can be reached concerning the relative effects of the Y-chromosome abnormality and mosaicism on sexual differentiation.

Adolescent↗
PubMed1995

Telomeres of higher primates.

The telomeres of gorilla, chimpanzee and human peripheral blood cells have been examined by hybridization to an oligonucleotide probe, (TTAGGG)4, following conventional and pulsed-field electrophoresis procedures. The MspI site present near the chromosome terminus undergoes methylation in gorilla, chimpanzee and human genome as shown by the HpaII digestion. Minor (TTAGGG)4-hybridizing sequences have been also detected in the chimpanzee HindIII and MspI digests.

Animals↗
PubMed1992

Highly acetylated H4 is associated with histone displacement in rat spermatids.

The presence of highly acetylated histone H4 during spermatogenesis was studied to evaluate its correlation with the events of gene transcription, histone deposition, and histone displacement. We utilized an antibody raised to a pentaacetylated synthetic peptide that preferentially recognizes highly (tetra- and tri-) acetylated forms of rat testis H4. Electrophoretic separation of histones from enriched fractions of spermatogenic cells followed by detection of these forms by staining and by immunoblotting using this antibody showed that the highly acetylated forms were limited almost exclusively to spermatids beginning at step 11 of development. Immunoflurescence also revealed a striking polarity in the progression of histone from the spermatid nucleus. Highly acetylated H4 was displaced from the anterior to the caudal portion of the spermatid nucleus during steps 11 and 12, along with other histones, prior to their displacement by transition proteins. Thus, while monoacetylated and low levels of diacetylated forms of H4 were associated with stages at which histone deposition and transcription occur, the more highly acetylated forms appeared in high levels only at the stage at which histone displacement occurs.

Acetylation↗
PubMed1991

Evolution and sequence analysis of a human Y-chromosomal DNA fragment.

A Y-chromosomal DNA fragment has been isolated from a human Y-Charon 21A recombinant library. Evolutionary analysis of 1F5 indicates that the size and sequence of this fragment have been conserved in higher primates. Deletion mapping and in situ hybridization analysis have localized 1F5 to the middle euchromatic portion of the long arm of the human Y chromosome at Yq11.2. Sequence analysis revealed the presence of an atypical Alu element and two regions rich in polypyrimidine-polypurine residues.

Base Sequence↗
PubMed1991

A Y-chromosomal DNA fragment is conserved in human and chimpanzee.

A human male-specific Y-chromosomal DNA fragment (lambda YH2D6) has been isolated. By deletion-mapping analysis, 2D6 has been localized to the euchromatic portion of the long arm (Yq11) of the human Y chromosome. Among great apes, this fragment was found to be conserved in male chimpanzee but was lacking in male gorilla and male orangutan. No homologous fragments were detected in females of orangutan, gorilla, chimpanzee, or human. Nucleotide sequence analysis indicated the presence of partial-Alu-elements and of sequences similar to the GATA repeats of the snake Bkm sequence.

Animals↗
PubMed1990

Physical mapping of mouse histone gene clusters.

A chicken histone H2A probe has been used to isolate five different clones from a mouse genomic library in lambda Charon 4A. The size of the inserts in these clones ranged from 11 to 16 kb. The full complement of histone genes was not present in any of the five isolated clones. A physical map, for each of the five clones, has been constructed using partial- and double-restriction digests.

Animals↗
PubMed1989

Biochemical and immunocytochemical analysis of a histone H1 variant from the mouse testis.

An H1 histone variant, H1a, has been isolated and purified from the mouse testis. Biochemical and amino acid analyses indicate its similarity with the rat testis H1a. Specific antibodies against the purified H1a have been generated in rabbits and used to study its tissue and species distribution using protein blotting procedures. We have also used the immunocytochemical technique to determine in situ distribution of H1a in spermatogenic cells and somatic tissues of the mouse. A non-random distribution of H1a has been noted in the nuclei of certain somatic cell types such as Sertoli cells, Leydig cells and neurons. By contrast, hepatocyte nuclei lacked detectable levels of H1a. In adult seminiferous tubules, the early primary spermatocyte nuclei displayed a greater level of immunoreactivity relative to other cell types. Developmental studies indicate its initial expression in the 7-day-old mouse testis concomitant with the appearance of intermediate and type B spermatogonia.

Animals↗
PubMed1987

A protease activity is associated with testicular chromatin of the mouse.

A protease activity associated with the micrococcal nuclease-solubilized chromatin from mouse seminiferous tubules has been characterized. Proteolysis of histone H1 and core histones is stimulated in the presence of 3 M urea. The pH optimum of this protease is between pH 8 and 9, and the activity is not inhibited by trypsin or chymotrypsin-active site inhibitors. Leupeptin is an effective inhibitor of the protease at low concentrations. Soluble chromatin from neonatal and prepubertal mice lacks this proteolytic activity until three to four weeks after birth. That the protease activity is localized in the dinucleosomes and higher oligomers but is lacking in mononucleosome populations suggests its association with the linker DNA. Rat testis-soluble chromatin apparently lacks such a protease activity. The developmental expression of this protease and its in situ localization are consistent with a role in histone displacement during mouse spermiogenesis.

Animals↗
PubMed1986

Histone messenger RNAs of the mouse testis.

A 6-12S RNA fraction has been isolated following sucrose gradient fractionation of mouse testis RNA, and further resolved into poly A+ and poly A- RNA fractions by oligo-(dt)-cellulose chromatography. Polyacrylamide gel electrophoresis of products formed in a reticulocyte lysate-dependent cell-free translation system has enabled identification of histone variants, H1t, H2S, H2A . X, an H4-like protein and a low Mr protein (presumably TP and/or protamine). Cell-free synthesis of a number of these histone variants appears to be directed by poly A+ mRNAs.

Animals↗
PubMed1985

A high performance liquid chromatography method to obtain rat epidermal filaggrin.

Sodium pyrophosphate-glycerol buffer, a non-denaturing buffer has been used to solubilize epidermal proteins. The extracted proteins are different in their electrophoretic profile in various mammalian species, 47 K in rat and guinea pig, 31 K and 60 K in mouse and 43 K and 60 K proteins in human epidermis were most predominant. Electrophoretic analyses show synthesis of new proteins as a function of age in rat tissue. Purification of the major rat protein fraction was achieved using a reverse phase high-performance liquid chromatography using a gradient of 5-50% acetonitrile. Based upon the molecular size, amino acid data and immunodiffusion analysis, we conclude the purified rat protein to be filaggrin.

Amino Acids↗
PubMed1985

Biochemical and immunological characterization of an H2A variant from the mouse testis.

A histone H2A variant, protein 'A', has been isolated and purified from the mouse testis. Amino acid composition analysis and electrophoretic properties indicate it to be apparently similar to H2A X X variant present in the mouse L1210 cells. Specific antibodies against protein 'A' have been generated in rabbits and used to study tissue and species distribution in mammals. Protein transfer experiments indicate the presence of antigenically similar proteins in somatic tissues of the mouse. Immunologically similar proteins were also detected in other mammalian testes. The data further indicate that protein A is antigenically distinct from the other members of the H2A family, H2A X 1, H2A X 2 and H2A X Z.

Amino Acids↗
PubMed1985

Biosynthesis of specific histones during meiotic prophase of mouse spermatogenesis.

A kinetics study has demonstrated histone synthesis occurring at two distinct phases during meiotic prophase of mouse spermatogenesis. These two periods have been delineated by quantifying the synthesis of DNA and basic nuclear proteins in spermatogenic cells at discrete intervals following the intratesticular injection of [3H] thymidine and [14C] arginine, respectively. One phase of histone synthesis occurs coincident with DNA synthesis in preleptotene spermatocytes. By contrast, a second phase of histone synthesis occurs during midprophase of meiosis, independent of semiconservative DNA synthesis. The [14C] arginine incorporated into the basic nuclear proteins of pachytene spermatocytes is conserved during spermiogenesis and then subsequently discarded within the residual bodies, which are formed during late spermiogenesis. Fluorographic analyses of isotopically labeled basic nuclear proteins in pachytene spermatocytes has shown that only the somatic complement of histones are synthesized during the preleptotene period, whereas the second phase involves the synthesis of proteins H1t, H2S, and "A". In addition, several nonhistone basic nuclear proteins are synthesized concomitant with the germ cell-specific histones. Thus, the data clearly demonstrate that pachytene spermatocytes actively synthesize a number of novel chromatin-associated polypeptides.

Animals↗
PubMed1985

Immunocytochemical localization of an H2A variant in the spermatogenic cells of the mouse.

Immunocytochemical localization of protein "A," an H2A variant, has been carried out in the adult, neonatal, and embryonic spermatogenic cells of the mouse using the peroxidase-antiperoxidase technique. The results indicate an apparent enrichment of this protein in the meiotic cells of the adult testis. In addition, T-prospermatogonia present in the neonatal mouse and 16-day-old embryos were found to be immunoreactive. By contrast, Sertoli cells and other somatic elements of the neonatal and embryonic gonads were only weakly immunoreactive. These data suggest potential usefulness of protein "A" as a nuclear marker of the embryonic spermatogenic cells.

Aging↗
PubMed1984

Immunocytochemical localization of a histone H2A variant in the mammalian nucleolar chromatin.

The distribution of protein "A", a minor variant of H2A present in the mouse testis, was studied in the liver and brain nuclei using peroxidase-antiperoxidase technique. The data presented here suggest that nucleolar-associated chromatin is highly enriched in protein "A". Microspectrophotometric measurements corroborate the immunocytochemical data. The regional differentiation in the eukaryotic chromatin, therefore, may involve qualitative changes in the histone composition.

Animals↗
PubMed1983

Biochemical and immunological characterization of a histone variant associated with spermatogenesis in the mouse.

A chromosomal histone, H2S, specific to the mouse testis has been purified. Amino acid analysis indicated lack of cysteine and a high basic amino acid content typical of histones. Specific antibodies against histones H2S have been generated in rabbits and partially purified using (NH4)2SO4 precipitation and ion-exchange chromatography. Protein transfer experiments indicate presence of antigenically similar histones in the rat and rabbit testes but not in the guinea pig and dog testes. In addition, histone complement of somatic tissues such as lung, kidney, liver and spleen lacked antigenically similar proteins. Immunocytochemical studies using peroxidase-antiperoxidase complex indicated presence of immunoreactive cells in the seminiferous epithelium which were lacking in the interstitium. These data demonstrate histone H2S to be a unique histone associated with spermatogenesis in the mouse.

Amino Acids↗