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R Kothary

Publications and source records attributed to R Kothary.

At least 37 records · Page 2Linked to original sources
PubMed1996

The beta-globin locus control region enhances transcription of but does not confer position-independent expression onto the lacZ gene in transgenic mice.

The beta-globin locus control region (LCR) confers high levels of position-independent, copy number-dependent expression onto globin transgenes. Here > 40 independent transgenic mouse lines and founders that carried the LCR in cis with the beta-globin gene promoter driving a lacZ reporter gene were studied. Expression of the lacZ transgene was assayed by measuring beta-galactosidase enzyme activity in fetal liver extracts, the levels of which correlated with the quantity of lacZ mRNA determined using RNase protection assays. Unexpectedly, expression of the lacZ transgene was found to show strong position effects, varying as much as 700-fold per transgene copy. These position effects occurred even if the whole beta-globin gene was incorporated as part of the lacZ reporter gene. Moreover, DNase I-hypersensitive sites appeared in the transgene LCR in high expressing but not in low expressing lines, suggesting that the LCR itself was position dependent. In contrast, MEL cell clones, in which transcriptionally active integration sites were selected for, gave < 13-fold variation in expression per copy of an LCR-lacZ construct. These results show that the lacZ reporter affects the ability of the LCR to activate chromatin in mice and that culture cells are not an adequate model for position-independent gene expression studies.

Animals↗
PubMed1996

Lung tumors in mice expressing an antisense RARbeta2 transgene.

Retinoic acid has been shown to be an anticancer agent, and a growing literature suggests that it is the nuclear retinoic acid receptor beta2 (RARbeta2) that is primarily responsible for mediating this effect, at least in some systems. To determine whether partial inactivation of RARbeta2 would predispose to lung cancer in mice, we generated three transgenic lines expressing antisense sequences. When killed at 13-3/4-18 months of age, 21/36 animals had a total of 43 pulmonary tumors superficially visible upon necropsy, whereas among 23 nontransgenic mice, only 1 had a single visible lung tumor. A twofold higher incidence of lung tumors was seen in homozygous vs. hemizygous antisense mice. The endogenous RARbeta2 message level was reduced in transgenic lung tissue and further reduced in the tumors. RARbeta4, a truncated isoform derived from the same transcript as RARbeta2, does not carry the sequence identified by the antisense construct and its message was not as strongly affected. Immunofluorescence studies showed that RARbeta was virtually undetectable in the tumors, but present in normal tissue. We conclude that RARbeta2, but probably not RARbeta4, plays an important role in suppression of murine lung tumorigenesis.

Animals↗
PubMed1995

The cytosolic chaperonin subunit TRiC-P5 begins to be expressed at the two-cell stage in mouse embryos.

The cytosolic chaperonin TRiC is a large protein complex involved in the folding of newly synthesized actin and tubulin. The fertilization of the mouse oocyte is followed by a remodelling of the actin and tubulin filaments. The TRiC subunit TCP1 is expressed only from the 4-cell stage on, even though actin and tubulin are synthesized in the previous stages. We investigated the onset of synthesis of another subunit, TRiC-P5, during early mouse embryogenesis. We report that TRiC-P5 is synthesized at the 2-cell stage in an alpha-amanitin sensitive manner. Thus, it is expressed before TCP1 and is one of the first proteins to be synthesized after zygotic genome activation.

Amanitins↗
PubMed1995

Cloning and characterization of the neural isoforms of human dystonin.

Dystonia musculorum (dt) is a hereditary neurodegenerative disease in mice that leads to a sensory ataxia. We have identified and cloned a gene encoded at the dt locus. The product of the dt gene, dystonin, is a neural isoform of a hemidesmosomal protein bullous pemphigoid antigen 1 (bpag1). To investigate the potential role of dystonin in human neuropathies, we have cloned the neural-specific 5' exons of the human DT gene that together with the previously cloned BPAG1 sequences comprise human dystonin. The mouse and human dystonin genes demonstrate the same spectrum of alternatively spliced products, and the amino acid sequences of the neural-specific exons in the mouse and human genes are over 96% identical.

Amino Acid Sequence↗
PubMed1995

Dystonin expression in the developing nervous system predominates in the neurons that degenerate in dystonia musculorum mutant mice.

Dystonia musculorum (dt) is an inherited neurodegenerative disorder in mice. The dt gene product, dystonin, contains the bullous pemphigoid antigen 1 coding region at its C-terminus and an actin binding domain at its N-terminus. We demonstrate that dystonin expression throughout mouse development predominates in neurons of the cranial and spinal sensory ganglia. These structures are the most severely affected in dystonic mice which could explain their severe sensory ataxia. Since we show expression in sensory neurons with small and large axoplasmic volumes, but degeneration is restricted primarily to the latter type, we suggest that caliber and size of the axon is an important factor in the disease process. Dystonin is also expressed in the extrapyramidal motor system and in the cerebellum. Functional defects in these cell types could account for the dystonic symptoms of dt mice not explained by simple sensory denervation. We also detect dystonin expression in motor neurons most of which are unaffected by the degenerative process in dt mice.

Animals↗
PubMed1995

The mouse dystonia musculorum gene is a neural isoform of bullous pemphigoid antigen 1.

Dystonia musculorum (dt) is a hereditary neurodegenerative disease in mice that leads to a sensory ataxia. We describe cloning of a candidate dt gene, dystonin, that is predominantly expressed in the dorsal root ganglia and other sites of neurodegeneration in dt mice. Dystonin encodes an N-terminal actin binding domain and a C-terminal portion comprised of the hemidesmosomal protein, bullous pemphigoid antigen 1 (bpag1). dt and bpag1 are part of the same transcription unit which is partially deleted in a transgenic strain of mice, Tg4, that harbours an insertional mutation at the dt locus, and in mice that carry a spontaneous dt mutation, dtAlb. We also demonstrate abnormal dystonin transcripts in a second dt mutant, dt24J. We conclude that mutations in the dystonin gene are the primary genetic lesion in dt mice.

Amino Acid Sequence↗
PubMed1994

Hyperplasia and tumours in lung, breast and other tissues in mice carrying a RAR beta 4-like transgene.

Transgenic mice were generated which express a truncated nuclear retinoic acid receptor beta (RAR beta), closely resembling the natural isoform RAR beta 4, under the control of the MMTV promoter. The transgene was expressed in salivary gland, testis, lung and mammary tissue in two different lines. At approximately 11-14 months virtually all the transgenic mice showed hyperplasia of the lung alveolar epithelium with an excess of type II pneumocytes. Hyperplasia of the mammary alveoli and terminal ducts was also seen in some females. Salivary glands and some sebaceous glands were hyperplastic in most male transgenic mice, but only rarely in females or in non-transgenics. Primary benign and malignant tumours were more numerous in transgenic mice than in controls, with a total of 23 in 43 mice versus two in 33 non-transgenic animals. Treatment with dexamethasone to increase transgene expression resulted in exaggerated versions of the above phenotypes. Overexpression of RAR beta 4 therefore appears to predispose various tissues to hyperplasia and neoplasia, and this by contrast to the RAR beta 2 isoform, which has tumour suppressor activity. A survey of ratios of RAR beta 4:RAR beta 2 expression in human lung tumour cell lines showed an increase compared with normal lung tissue, suggesting that RAR beta 4 may play a similar role in human tumorigenesis.

Animals↗
PubMed1994

The genomic structure of an insertional mutation in the dystonia musculorum locus.

We have previously identified a line of transgenic mice, Tg4, in which an hsp68-lacZ hybrid gene has inserted into the dystonia musculorum (dt) locus on chromosome 1. We have confirmed the localization of the Tg4 integration site to the proximal region of mouse chromosome 1 by interspecific backcross analysis. One end of the integration complex has been cloned and we have used single-copy probes from the flanking region to screen a mouse genomic library. Several overlapping lambda phage clones have been isolated and arranged into a contig spanning 75 kb of genomic DNA. Probes from the genomic contig have enabled us to characterize the wildtype and Tg4 loci. We report that the integration of the transgene was accompanied by a deletion of 45 kb of host genomic sequences with no other detectable rearrangement in the Tg4 genome.

Animals↗
PubMed1994

Human homolog of a mouse sequence from the dystonia musculorum locus is on chromosome 6p12.

Dystonia musculorum is a hereditary neurodegenerative disease in mice that affects sensory neurons. In an effort to clone the gene responsible for this disorder, we have assembled a genomic contig spanning 75 kb of the dystonia musculorum (dt) locus. Within this genomic contig, we have identified a small restriction fragment that shows evolutionary conservation to rat, hamster, rabbit, and human genomic DNA. Using this mouse sequence, we have cloned the conserved human genomic fragment. Sequence analysis of the mouse and human genomic fragments revealed that they share a sequence similarity of 82% over 175 bp. A panel of human/rodent somatic cell hybrids was used to map the human genomic sequence to Chromosome (Chr) 6, and high-resolution in situ hybridization (FISH) allowed it to be sublocalized to 6p12. The human homolog of the mouse Bpag1 gene, a gene tightly linked to the mouse dt gene, also maps to Chr 6. Thus, this comparative mapping reveals a new region of conserved synteny between the chromosomes of mouse and human. Mapping the human homolog of the mouse dt gene enables us to initiate linkage studies to identify neurodegenerative disorders that may be caused by mutations in this gene.

Animals↗
PubMed1993

Characterization of the neural crest defect in Splotch (Sp1H) mutant mice using a lacZ transgene.

We have reinvestigated the neural crest defect of Splotch (Sp1H) mutant embryos using the tissue specific expression of lacZ by the HCMV-IEP-lacZ (CMZ) transgene as a marker. The CMZ transgene was backcrossed onto the Sp1H mutant background, which has been shown to carry mutations in the Pax-3 gene. The CMZ transgene has previously been shown to be expressed in some neural crest-derived neural tissues of midgestation embryos. The pattern of CMZ expression in Splotch mutants is not caused by alterations of transgene transcription, but demonstrates morphological deviations of neural crest development. The gradual size reduction of spinal ganglia along a rostrocaudal gradient is shown to occur concomitantly with a size reduction of the sympathetic ganglia. CMZ expression also reveals the total absence of sympathetic ganglion cells in thoracic and lumbar segments of Sp1H homozygotes, which is confirmed in serial sections. Observations in whole mounts of CMZ transgenic homozygotes suggest that cranial nerve ganglia develop normally in these embryos. CMZ is expressed in epithelial cells around the neural tube defect in Splotch mutants at the epidermal/neuroepithelial boundary. It is proposed that this expression represents premigratory neural crest cells that remain within the epithelial layer around the neural tube defect. These observations are discussed with reference to the normal pattern of Pax-3 expression.

Animals↗
PubMed1993

The Splotch mutation interferes with muscle development in the limbs.

Homozygosity for the Splotch mutation causes neural tube and neural crest defects in mice. It has been demonstrated that Splotch mutant mice carry mutations in the homeodomain of the Pax-3 gene. Pax-3 is expressed in the neural tube, some neural crest derivatives, the mesenchyme of the limb bud and the somites. We have examined the development of the somite-derived skeletal muscles in homozygotes carrying the Splotch (Sp1H) mutation. Our results suggest that the Splotch mutation affects the development of skeletal muscles in a region-specific way: 1. The expression of the CMZ transgene in homozygotes reveals a disorganisation of the dermomyotome in whole stained embryos. 2. The axial musculature is reduced in size along a rostro-caudal gradient. 3. The muscle anlagen in the limbs develop much more slowly. Muscles of the head and the ventral body wall are normally developed in the mutant on day 13.5 of gestation. Recently, it has been shown that the myogenic precursors of the limbs are derived from the lateral half of the somite. The specific disturbance of muscle development in the limbs of Splotch mutants thus suggests a role for Pax-3 in the organisation of the somite, the production of trophic factors in the limb mesenchyme or an alteration of myogenic and mesenchymal cells.

Animals↗
PubMed1992

Parthenogenetic stem cells in postnatal mouse chimeras.

The ability of parthenogenetic (pg) cells to contribute to proliferating stem cell populations of postnatal aggregation chimeras was investigated. Using DNA in situ analysis, pg participation was observed in highly regenerative epithelia of various regions of the gastrointestinal tract, e.g., stomach, duodenum and colon, in the epithelia of tongue and uterus and in the epidermis. Pg cells also contributed to the epithelium of the urinary bladder, which is characterized by a relatively slow cellular turnover. Using a sensitive proliferation marker to determine division rate of pg and normal (wt) cells in tissues of a 24-day-old chimera, no significant differences between pg and fertilized cells were observed. However, in colon and uterus of a pg <==> wt chimera aged 101 days, a significant loss of proliferative capacity of pg cells was found. In the colon, this loss of proliferative potential was accompanied by an altered morphology of pg crypts. In general, they were situated at the periphery of the epithelium and lacked access to the lumen, with consequent cystic enlargement and flattened epithelium. No obvious morphological changes were observed in the pg-derived areas of the uterine epithelium of this chimera. Our results provide evidence that pg cells can persist as proliferating stem cells in various tissues of early postnatal chimeras. They suggest that pg-derived stem cells may cease to proliferate in restricted areas of the gastrointestinal tract and in the uterine epithelium of pg <==> wt chimeras of advanced age.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1991

A sequence motif found in a Drosophila heterochromatin protein is conserved in animals and plants.

Modifiers of position-effect-variegation in Drosophila encode proteins that are thought to modify chromatin, rendering it heritably changed in its expressibility. In an attempt to identify similar modifier genes in other species we have utilized a known sequence homology, termed chromo box, between a suppressor of position-effect-variegation, Heterochromatin protein 1 (HP1), and a repressor of homeotic genes, Polycomb (Pc). A PCR generated probe encompassing the HP1 chromo box was used to clone full-length murine cDNAs that contain conserved chromo box motifs. Sequence comparisons, in situ hybridization experiments, and RNA Northern blot analysis suggest that the murine and human sequences presented in this report are homologues of the Drosophila HP1 gene. Chromo box sequences can also be detected in other animal species, and in plants, predicting a strongly conserved structural role for the peptide encoded by this sequence. We propose that epigenetic (yet heritable) changes in gene expressibility, characteristic of chromosomal imprinting phenomena, can largely be explained by the action of such modifier genes. The evolutionary conservation of the chromo box motif now enables the isolation and study of putative modifier genes in those animal and plant species where chromosomal imprinting has been described.

Amino Acid Sequence↗
PubMed1991

Unusual cell specific expression of a major human cytomegalovirus immediate early gene promoter-lacZ hybrid gene in transgenic mouse embryos.

Transgenic mice carrying the human cytomegalovirus immediate early gene promoter driving the E. coli lacZ gene displayed an unusual cell specific expression of beta-galactosidase during development. LacZ expression was first detected in cells lining the apex of the neural fold of day 8.5 embryos. By day 10 of gestation, expression was prominent in the spinal ganglia, the ganglia of cranial nerves V, VII, VIII, IX, and X, in a line of cells marking the ventrolateral pathway adjacent to the dermamyotome, and in a column of differentiated cells in the entire ventrolateral neural tube posterior to the mesencephalon. Expression was also found in the myotomes. Neural tube explants from day 8.5 embryos cultured in vitro showed lacZ expression in cells migrating away from the explant. We conclude that the HCMV-IEP-lacZ transgene is expressed in a subpopulation of neural crest cells and its early derivatives.

Animals↗
PubMed1991

Developmental potential of parthenogenetic cells: role of genotype-specific modifiers.

The developmental potential of parthenogenetic cells derived from different mouse strains was investigated by examining their distribution in various tissues of adult aggregation chimeras. Using GPI-1 allozymes as marker, no striking differences were observed between chimeras whose parthenogenetic cells were derived from activated oocytes isolated from females of different genetic backgrounds, (C57BL/6 x CBA/J) F1, CFLP, 129, and SWR. In all the combinations tested, parthenogenetic cells were consistently absent from skeletal muscle, but there were varying contributions to most other tissues. These results suggest that the maternal duplication of chromosomes containing imprinted gene(s) responsible for the systematic elimination of parthenogenetic cells from skeletal muscle, are not subject to a pronounced influence of genotype-specific modifiers. However, the contribution of parthenogenetic cells to the brain does appear to be influenced by strain background, since a marked improvement in the survival of CFLP, 129 and perhaps SWR parthenogenetic cells in chimeric brains was observed compared with F2 cells.

Animals↗
PubMed1990

Genome imprinting and development in the mouse.

Development in mammals is influenced by genome imprinting which results in differences in the expression of some homologous maternal and paternal alleles. This process, initiated in the germline, can continue following fertilization with interactions between oocyte cytoplasmic factors and the parental genomes involving modifier genes. Further epigenetic modifications may follow to render the 'imprints' heritable through subsequent cell divisions during development. Imprinting of genes can be critical for their dosage affecting embryonic growth, cell proliferation and differentiation. The cumulative effects of all the imprinted genes are observed in androgenones (AG) and parthenogenones (PG), which reveal complementary phenotypes with respect to embryonic and extraembryonic tissues. The presence of PG cells in chimeras causes growth retardation, while that of AG cells enhanced growth. AG cells apparently have a higher cell proliferation rate and, unlike PG cells, are less prone to selective elimination. However, the PG germ cells are exempt from cell selection. In chimeras, PG cells are more likely to be found in ectodermal derivatives such as epidermis and brain in contrast to AG cells which make pronounced contributions to many mesodermal derivatives such as muscle, kidney, dermis and skeleton. The presence of androgenetic cells in chimeras also results in the disproportionate elongation of the anterior-posterior axis and sometimes in the abnormal development of skeletal elements along the axis. Genetic studies high-light the influence of subsets of imprinted genes, and identify those that are critical for development.

Animals↗
PubMed1989

Inducible expression of an hsp68-lacZ hybrid gene in transgenic mice.

Transgenic mice have been generated that express the E. coli beta-galactosidase gene under the control of the promoter from the mouse heat-shock gene, hsp68. Sequences from -664 to +113 relative to the start of transcription of the hsp68 gene were sufficient to direct stress-induced expression of the beta-galactosidase gene in adult tail tissue and various tissues of fetal stages of development. Expression was detected in situ by staining with the chromogenic substrate, X-gal. The hybrid gene was refractory to induction in preimplantation embryos until the blastocyst stage of development, as reported for the endogenous hsp68 gene. No constitutive expression was observed by in situ staining or Northern analysis at any stage of development, even in tissues that constitutively express the endogenous hsp68 gene. We conclude that the hsp68 promoter region included in the construct contains sufficient sequence information for heat and arsenite inducibility, but it does not contain sequences controlling tissue-specific expression during development. This tightly regulated inducible promoter may provide a useful tool for short-term inducible gene expression in transgenic mice.

Animals↗