15 results on '"Sandya Narayanswami"'
Search Results
2. Differential protection in two transgenic lines of NOD/Lt mice hyperexpressing the autoantigen GAD65 in pancreatic beta-cells
- Author
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Vijayakumar K. Ramiya, Margot Bridgett, Robert W. O'Rourke, Marina Cetkovic-Cvrlje, Steinunn Baekkeskov, Jeremy Lambert, Edward H. Leiter, Yuguang Shi, and Sandya Narayanswami
- Subjects
Male ,medicine.medical_specialty ,Adoptive cell transfer ,Ratón ,Insulin Antibodies ,Endocrinology, Diabetes and Metabolism ,Transgene ,Gene Dosage ,Congenic ,Gene Expression ,Mice, Transgenic ,Mice, SCID ,Weaning ,Nod ,Biology ,Autoantigens ,Islets of Langerhans ,Mice ,Mice, Inbred NOD ,Internal medicine ,Internal Medicine ,medicine ,Animals ,Transgenes ,Autoantibodies ,NOD mice ,geography ,Binding Sites ,geography.geographical_feature_category ,Glutamate Decarboxylase ,Reverse Transcriptase Polymerase Chain Reaction ,Incidence ,Pancreatic Diseases ,medicine.disease ,Islet ,Adoptive Transfer ,Diabetes Mellitus, Type 1 ,Endocrinology ,Genes ,Cytokines ,Female ,Insulitis - Abstract
Although expressed at very low levels in islets of NOD mice, GAD65 is a candidate islet autoantigen. Two transgenic lines of NOD/Lt mice expressing high levels of human GAD65 from a rat insulin promoter were generated. Transgenes were integrated on proximal chromosome 15 of the A line and on the Y chromosome of the Y line. Transgenic A-line mice were obligate hemizygotes, since homozygous expression resulted in developmental lethality. A twofold higher level of hGAD65 transcripts in A-line islets from young donors was associated with higher GAD protein and enzyme activity levels. Y-line males developed diabetes at a similar rate and incidence as standard NOD/Lt males. In contrast, A-line mice of both sexes exhibited a markedly lowered incidence of diabetes. Insulitis, present in both transgenic lines, developed more slowly in A-line mice and correlated with a reduction in the ratio of gamma-interferon to interleukin-10 transcripts. Splenic leukocytes from young A-line donors transferred diabetes into NOD-scid recipients at a retarded rate compared with those from nontransgenic donors. Further, nontransgenic NOD T-cells transferred diabetes more slowly in NOD-scid recipients that were congenic for A-line transgenes as compared with standard NOD-scid recipients. Primed T-cell responses and spontaneous humoral reactivity to GAD65 failed to distinguish transgenic from nontransgenic mice. Quantitative differences in expression level or insertional mutagenesis are possible mechanisms of protection in the A line.
- Published
- 1998
3. DNA sequence mapping using electron microscopy
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Barbara A. Hamkalo and Sandya Narayanswami
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Biotin ,Molecular Probe Techniques ,High resolution ,Biology ,Acetoxyacetylaminofluorene ,Applied Microbiology and Biotechnology ,DNA sequencing ,law.invention ,law ,Genetics ,Animals ,Humans ,Microscopy, Immunoelectron ,Repetitive Sequences, Nucleic Acid ,Whole mount ,Base Sequence ,Chromosome Mapping ,Nucleic Acid Hybridization ,DNA ,Immunohistochemistry ,Molecular biology ,Colloidal gold ,Biophysics ,Electron microscope ,Digoxigenin ,Dinitrophenols - Abstract
DNA sequences can be mapped on chromosomes at high resolution in the electron microscope after hybridization with a nonisotopically labeled probe followed by detection with a two-step antibody reaction employing a colloidal gold tag. Hybridization probes can be modified with biotin-dUTP, digoxigenin-dUTP, dinitrophenyl-dUTP, or N-acetoxy-2-acetylaminofluorene (AAF). The availability of different sizes of colloidal gold particles permits the simultaneous detection of several sequences. In addition, low signals can be amplified either with an antibody sandwich scheme or by silver intensification. This technology is applicable both to TEM and SEM preparations of chromosomes, and we have used it to map a number of highly and moderately repeated sequences on whole mount meta-phase chromosomes.
- Published
- 1991
4. An expanded collection of mouse Y Chromosome RDA clones
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Sandya Narayanswami, Janet K. Bayleran, Hailing Yan, David E. Bergstrom, Manjula M. Sonti, and Elizabeth M. Simpson
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Male ,Molecular Sequence Data ,Biology ,Y chromosome ,Polymerase Chain Reaction ,Article ,law.invention ,Mice ,chemistry.chemical_compound ,symbols.namesake ,law ,Sequence Homology, Nucleic Acid ,Y Chromosome ,Genetics ,Animals ,Cloning, Molecular ,In Situ Hybridization ,In Situ Hybridization, Fluorescence ,Polymerase chain reaction ,DNA Primers ,Cloning ,Deoxyribonuclease BamHI ,Chromosome Mapping ,Human genetics ,Mice, Inbred C57BL ,chemistry ,Mendelian inheritance ,symbols ,Female ,Representational difference analysis ,DNA ,Recombination - Abstract
If we are to understand the structure and function of the mammalian genome and its transmission to subsequent generations, we cannot neglect the Y Chromosome (Chr). However, certain properties of the Y have made it refractory to analysis. The bulk of the Y Chr does not recombine, and thus genetic maps based on standard Mendelian recombination are not feasible. For this reason, investigators have relied on physical approaches to map the human Y (Foote et al. 1992; Vollrath et al. 1992). We are using physical approaches to further our understanding of the mouse Y Chr by developing chromosome-specific DNA-based probes.
- Published
- 1997
5. Electron Microscopic Localization of in Situ Hybrids
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Sandya Narayanswami and Barbara A. Hamkalo
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In situ ,Biology ,Mitosis ,Electron microscopic ,Molecular Probe Techniques ,Hybrid ,Cell biology - Published
- 2003
6. The mouse Y chromosome: enrichment, sizing, and cloning by bivariate flow cytometry
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John J. Fawcett, C. Bell-Prince, Elizabeth M. Simpson, David E. Bergstrom, Manjula M. Sonti, Cram Ls, Donald A. Grieco, and Sandya Narayanswami
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medicine.medical_specialty ,Pseudoautosomal region ,Molecular Sequence Data ,Biology ,Molecular cloning ,Y chromosome ,Polymerase Chain Reaction ,Homology (biology) ,law.invention ,Mice ,Chromosome 16 ,law ,Sequence Homology, Nucleic Acid ,Y Chromosome ,Genetics ,medicine ,Animals ,Humans ,Genomic library ,Cloning, Molecular ,Polymerase chain reaction ,In Situ Hybridization, Fluorescence ,Gene Library ,Repetitive Sequences, Nucleic Acid ,Cytogenetics ,DNA ,Flow Cytometry ,Molecular biology ,Mice, Inbred C57BL ,Interleukin-2 ,Chromosomes, Human, Pair 16 ,Spleen - Abstract
In this report, we demonstrate the utility of interleukin-2 (IL-2) stimulation of spleen cell cultures and bivariate flow cytometry in the analysis and purification of the C57BL/6J mouse Y Chromosome. We determined that the DNA content of the C57BL/6J Y Chromosome is approximately 94.7 Mb, making it similar in size to human Chromosome 16 and significantly larger than previous estimates. In addition, we describe the bulk isolation of mouse Y Chromosomes and demonstrate enrichment of the isolated material using a fluorescencein situhybridization strategy. We detail the construction of two small insert Y Chromosome-specific libraries, ideal for sampling Y Chromosome sequences. From these libraries 1566 clones were analyzed. We provide a detailed characterization of 103 clones, generating nearly 50 kb of sequence. For 30 of these clones, we identify regions of homology to known Y chromosomal sequences, confirming the enrichment of the sorted DNA. From the remaining characterized clones, we describe the development of 15 male-specific PCR assays and 19 male–female PCR assays potentially originating from the pseudoautosomal region or other areas of X–Y or autosome–Y homology.
- Published
- 1998
7. Mouse Y-specific repeats isolated by whole chromosome representational difference analysis
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Donald A. Grieco, Ann Navin, Sandya Narayanswami, Elizabeth M. Simpson, Rytis Prekeris, Eric S. Lander, Manjula M. Sonti, and Nikolai A. Lisitsyn
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Genetics ,Male ,medicine.diagnostic_test ,Base Sequence ,Hybridization probe ,DNA–DNA hybridization ,Interspersed repeat ,Molecular Sequence Data ,Biology ,Y chromosome ,Polymerase Chain Reaction ,Sequence-tagged site ,Mice, Inbred C57BL ,Blotting, Southern ,Mice ,Y Chromosome ,medicine ,Animals ,Female ,Representational difference analysis ,DNA Probes ,In Situ Hybridization, Fluorescence ,Fluorescence in situ hybridization ,Southern blot - Abstract
Representational difference analysis (RDA) was used to generate Y-specific probes by enriching for and cloning the differences between the male (XY) and the female (XX) C57BL/6J mouse genomes. Characterization of 35 clones revealed 12 families related by sequence similarity. One clone from each family was chosen for detailed analysis by Southern blot hybridization, polymerase chain reaction (PCR) on normal and aberrant genomes (Sxr), and fluorescence in situ hybridization. From one difference product we have characterized 12 Y-specific probes for hybridization, created seven male-specific PCR assays, mapped all repeat families, and identified one repeat with a distinct XY homology. We report the first cloning of a Y-specific long interspersed repeat element (LINE) fragment. In total, RDA has identified six novel Y Chromosome repeat families and allowed us to extend the characterization of six known Y repeats. We conclude that this novel use of RDA for whole chromosome subtraction successfully enriches chromosome-specific sequences and is suitable for the rapid generation of new Y Chromosome-specific probes.
- Published
- 1996
8. Cytological and molecular characterization of centromeres in Mus domesticus and Mus spretus
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Barbara A. Hamkalo, Sandya Narayanswami, Norman A. Doggett, Heinz-Ulrich Weier, Lynn M. Clark, and C.E. Hildebrand
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Genetics ,biology ,Mus spretus ,animal diseases ,Centromere ,Chromosome ,Nucleic Acid Hybridization ,biology.organism_classification ,Genome ,Telomere ,Cell Line ,Electrophoresis, Gel, Pulsed-Field ,Muridae ,Nucleic acid thermodynamics ,Blotting, Southern ,Mice ,Microscopy, Electron ,Animals ,Satellite (biology) ,Ploidy - Abstract
We have applied EM in situ hybridization (EMISH) and pulsed field gel electrophoresis (PFGE) to samples from diploid primary cell cultures and an established cell line to examine in detail the relative organization of the major and minor satellite DNAs and telomere sequences in the genomes of Mus domesticus and Mus spretus. EMISH localizes the Mus domesticus minor satellite to a single site at the centromere-proximal end of each chromosome. Double label hybridizations with both minor satellite and telomere probes show that they are in close proximity and possibly are linked. In fact, PFGE of M. domesticus DNA digested with Sal I and Sfi I reveals the presence of fragments which hybridize to both probes and is consistent with the physical linkage of these two sequences. The M. domesticus minor satellite is the more abundant satellite in Mus spretus. Its distribution in M. spretus is characterized by diffuse labeling with no obvious concentration near chromosome ends. In addition to this repeat the M. spretus genome contains a small amount of DNA that hybridizes to a M. domesticus major satellite probe. Unlike the M. domesticus minor satellite, it is not telomere proximal but is confined to a domain at the border of the centromere and the long arm. Thus, although both species possess all three sequences, except for the telomeres, their distribution relative to one another is not conserved. Based on the results presented, we propose preliminary molecular maps of the centromere regions of Mus domesticus and Mus spretus.
- Published
- 1992
9. Chapter 5 Nucleic Acid Sequence Localization by Electron Microscopic in Situ Hybridization
- Author
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Nadja Dvorkin, Sandya Narayanswami, and Barbara A. Hamkalo
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In situ ,chemistry.chemical_compound ,Biochemistry ,chemistry ,Gene mapping ,Oligonucleotide ,Nucleic acid sequence ,Immunogold labelling ,In situ hybridization ,Biology ,Molecular biology ,Primer extension ,DNA - Abstract
Publisher Summary This chapter discusses the electron microscopic in situ hybridization (EMISH) technique for nucleic acid sequence localization. In situ hybridization is a pivotal genome mapping technique that provides the cytological location of a cloned sequence. The development of equivalent mapping techniques at the electron microscope (EM) level present the opportunity to determine the relative map positions of sequences. The EM localization is well suited for mapping sequences on small chromosomal structures and for subnuclear localization in small nuclei, such as yeast. It uses biotin-substituted probes and immunogold tagging of hybrid sites. EMISH is applied successfully to the localization of DNA and RNA sequences in both whole-mount metaphase chromosomes and nuclei in organisms from yeast to man. The methodology is analogous to that used at the light microscope (LM) level with a few modifications. The main source of background in EMISH appears to be the nonspecific binding of antibodies to grid films. In addition to modifying EMISH, the use of primer extension in the presence of biodUTP after hybridization of an oligonucleotide to chromosome preparations is being investigated.
- Published
- 1991
10. High resolution mapping of Xenopus laevis 5S and ribosomal RNA genes by EM in situ hybridization
- Author
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Barbara A. Hamkalo and Sandya Narayanswami
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Nucleolus ,Biophysics ,Xenopus ,Mitosis ,In situ hybridization ,Translocation, Genetic ,Pathology and Forensic Medicine ,Xenopus laevis ,Endocrinology ,Animals ,Ribosomal DNA ,Interphase ,Cells, Cultured ,biology ,Hybridization probe ,RNA, Ribosomal, 5S ,RNA ,Chromosome Mapping ,Nucleic Acid Hybridization ,Cell Biology ,Hematology ,RNA Probes ,Ribosomal RNA ,biology.organism_classification ,Molecular biology ,Immunohistochemistry ,Microscopy, Electron ,RNA, Ribosomal ,Oocytes ,Nucleolus organizer region ,DNA Probes - Abstract
We have developed a modification of in situ hybridization at the electron microscope level that permits simultaneous detection of at least two sequences. Probes are labelled with either biotin or AAF and detected with two distinct sizes of colloidal gold. This protocol has been applied to map the positions of Xenopus laevis oocyte-type 5S genes relative to ribosomal precursor genes in several independently derived cell lines. The results for the line TRXO, which expresses some oocyte 5S RNA, indicate that this inappropriate expression is not due to translocation from telomeric sites into the nucleolus organizer, as previously hypothesized (7). In addition we found that four other Xenopus cell lines, none of which express these genes, also contain distinct, 5S oocyte translocations. These results suggest that an alteration in chromosome position is insufficient to result in gene activation and that sequences which are telomeric-proximal are exceptionally prone to translocation.
- Published
- 1990
11. Large scale isolation of expression vector cassette by magnetic triple helix affinity capture
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Albert P. Kausch, Arijit Bose, Matthew C. Griffor, Charles R. Cantor, Sandya Narayanswami, Takeshi Sano, and Srinivas V. Sonti
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Scale (ratio) ,Genetic Vectors ,Oligonucleotides ,Biology ,Microsphere ,Magnetics ,chemistry.chemical_compound ,Genetics ,Binding site ,Base Composition ,Binding Sites ,Expression vector ,Oligonucleotide ,Hydrogen Bonding ,DNA ,Hydrogen-Ion Concentration ,Plants ,Molecular biology ,Microspheres ,DNA metabolism ,Pyrimidines ,chemistry ,Biophysics ,Nucleic Acid Conformation ,Plasmids ,Triple helix - Published
- 1995
12. Eukaryotic genome organization analyzed by electron microscope in situ hybridization
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Sandya Narayanswami, Barbara A. Hamkalo, and Nadja Dvorkin
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Chemistry ,law ,Eukaryotic genome ,General Medicine ,In situ hybridization ,Electron microscope ,law.invention ,Cell biology - Abstract
In situ hybridization is a powerful tool for the localization of DNA/RNA sequences in nuclei and chromosomes. The introduction of nonisotopic labelling methodologies in conjunction with fluorescent or enzyme-linked detection have resulted in a dramatic increase in the application of this technique at the light microscope (LM) level and has placed it in a pivotal role in cell biology, development and genetics. Development of equivalent mapping protocols at the EM level offers increased spatial resolution. We have combined the use of nonisotopic probes with invmunogold labelling to investigate eukaryotic genome organization at high resolution.Metaphase chromosomes released from mitotically-arrested cells are deposited on gold EM grids by centrifugation through a sucrose cushion. After fixation (0.1% glutaraldehyde, 20 min) and DNA denaturation, chromosomes are hybridized to cloned probes enzymatically labelled with biotin-dUTP, digoxigenin-dUTP, dinitrophenyl-dUTP or covalently coupled to N-acetoxyacetoaminofluorene. Hybrid sites typically are detected by a two-step antibody incubation and 1-30 nm colloidal gold particles.
- Published
- 1991
13. Localization of nucleic acid sequences by EM in situ hybridization using colloidal gold labels
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Barbara A. Hamkalo, Sandya Narayanswami, and Karen Lundgren
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In situ ,Base Sequence ,Chromosome Mapping ,Nucleic Acid Hybridization ,Chromosome Organization ,DNA ,In situ hybridization ,Computational biology ,Immunogold labelling ,Biology ,Molecular biology ,Mice ,Microscopy, Electron ,Xenopus laevis ,Nucleic acid thermodynamics ,Colloidal gold ,Gold particles ,Nucleic acid ,Animals ,RNA ,Gold ,Anatomy - Abstract
It is possible to combine hybridization to specimens on electron-microscope grids of nucleic-acid probes labelled nonisotopically with immunogold detection of hybrid sites to map the position of target sequences rapidly and precisely. The basic technique is described, and examples are provided to illustrate the types of questions which can be approached in the general area of higher-order chromosome organization and function. A combination of two differentially labelled probes and two different-sized gold particles permits the simultaneous detection of closely linked or interspersed sequences.
- Published
- 1989
14. Chromosomal location of a major tRNA gene cluster of Xenopus laevis
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Stuart G. Clarkson, Barbara A. Hamkalo, Sandya Narayanswami, Mary Lou Pardue, and Jennifer M. Fostel
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Xenopus ,Biology ,DNA, Ribosomal ,Xenopus laevis ,Nucleic acid thermodynamics ,chemistry.chemical_compound ,RNA, Transfer ,Gene cluster ,Genetics ,Animals ,Gene ,Genetics (clinical) ,Repetitive Sequences, Nucleic Acid ,Chromosome Mapping ,Nucleic Acid Hybridization ,Chromosome ,RNA ,biology.organism_classification ,Molecular biology ,chemistry ,Organ Specificity ,Transfer RNA ,Oocytes ,Female ,DNA - Abstract
In Xenopus laevis, genes encoding tRNAPhe, tRNATyr, tRNA 1 Met , tRNAAsn, tRNAAla, tRNALeu, and tRNALys are clustered within a 3.18-kb (kilobase) fragment of DNA. This fragment is tandemly repeated some 150 times in the haploid genome and its components are found outside the repeat only to a limited extent. The fragment hybridizes in situ to a single site very near the telomere on the long arm of one of the acrocentric chromosomes of the group comprising chromosomes 13–18. All the chromosomes of this group also hybridize with DNA coding for oocyte-specific 5S RNA. The tRNA gene cluster is slightly proximal to the cluster of 5S RNA genes.
- Published
- 1984
15. Mouse satellite DNA, centromere structure, and sister chromatid pairing
- Author
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Barbara A. Hamkalo, L. M. Lica, and Sandya Narayanswami
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Mice, Inbred BALB C ,Cohesin ,Kinetochore ,Satellite DNA ,Centromere ,Mitosis ,DNA Restriction Enzymes ,Articles ,Cell Biology ,Chromatids ,DNA, Satellite ,Fibroblasts ,Biology ,Molecular biology ,Chromosomes ,Cell Line ,Establishment of sister chromatid cohesion ,Mice ,L Cells ,Bisbenzimidazole ,Animals ,Sister chromatids ,Chromatid ,Metaphase - Abstract
The experiments described were directed toward understanding relationships between mouse satellite DNA, sister chromatid pairing, and centromere function. Electron microscopy of a large mouse L929 marker chromosome shows that each of its multiple constrictions is coincident with a site of sister chromatid contact and the presence of mouse satellite DNA. However, only one of these sites, the central one, possesses kinetochores. This observation suggests either that satellite DNA alone is not sufficient for kinetochore formation or that when one kinetochore forms, other potential sites are suppressed. In the second set of experiments, we show that highly extended chromosomes from Hoechst 33258-treated cells (Hilwig, I., and A. Gropp, 1973, Exp. Cell Res., 81:474-477) lack kinetochores. Kinetochores are not seen in Miller spreads of these chromosomes, and at least one kinetochore antigen is not associated with these chromosomes when they were subjected to immunofluorescent analysis using anti-kinetochore scleroderma serum. These data suggest that kinetochore formation at centromeric heterochromatin may require a higher order chromatin structure which is altered by Hoechst binding. Finally, when metaphase chromosomes are subjected to digestion by restriction enzymes that degrade the bulk of mouse satellite DNA, contact between sister chromatids appears to be disrupted. Electron microscopy of digested chromosomes shows that there is a significant loss of heterochromatin between the sister chromatids at paired sites. In addition, fluorescence microscopy using anti-kinetochore serum reveals a greater inter-kinetochore distance than in controls or chromosomes digested with enzymes that spare satellite. We conclude that the presence of mouse satellite DNA in these regions is necessary for maintenance of contact between the sister chromatids of mouse mitotic chromosomes.
- Published
- 1986
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