Somatic Cell and Molecular Genetics最新文献

A method of measurement of exposed DNA (i.e. hypersensitive to DNase I hydrolysis) as opposed to sequestered (hydrolysis resistant) DNA in isolated nuclei of mammalian cells is described. While cell cultures exhibit some differences in behavior from day to day, the general pattern of exposed and sequestered DNA is satisfactorily reproducible and agrees with results previously obtained by other methods. The general pattern of DNA hydrolysis exhibited by all cells tested consists of a curve which at first rises sharply with increasing DNase I, and then becomes almost horizontal, indicating that roughly about half of the nuclear DNA is highly sequestered. In 4 cases where transformed cells (Raszip6, CHO, HL60 and PC12) were compared, each with its more normal homolog (3T3, and the reverse transformed versions of CHO, HL60 and PC12, achieved by dibutyryl cyclic AMP [DBcAMP], retinoic acid, and nerve growth factor [NGF] respectively), the transformed form displayed less genome exposure than the nontransformed form at every DNase I dose tested. When Ca++ was excluded from the hydrolysis medium in both the Raszip6-3T3 and the CHO-DBcAMP systems, the normal cell forms lost their increased exposure reverting to that of the transformed forms. Therefore Ca++ appears necessary for maintenance of the DNA in the more highly exposed state characteristic of the nontransformed phenotype. LiCl increases the DNA exposure of all transformed cells tested. Dextran sulfate and heparin each can increase the DNA exposure of several different cancers. Colcemid prevents the increase of exposure of CHO by DBcAMP but it must be administered before or simultaneously with the latter compound. Measurements on mouse biopsies reveal large differences in exposure in different normal tissues. Thus, the exposure from adult liver cells was greater than that of adult brain, but both fetal liver and fetal brain had significantly greater exposure than their adult counterparts. Exposure in normal human fibroblasts as revealed by in situ nick translation reveals a nuclear distribution pattern around the periphery, around the nucleoli and in punctate positions in the nuclear interior in parts of both S and G1 phases of the cell cycle. The same exposure pattern is duplicated by the pattern of DNA synthesis in S cells. It would appear that these nuclear regions represent positions of special activity. The previously proposed theory of genome regulation in mammalian cells is supported by these findings. The theory proposes that: a) gene activity requires exposure of the given locus followed by action of transcription factors on the exposed genes; b) the fiber system of the cell (cytoskeleton, nuclear fibers, and extracellular fibers) are required for normal exposure; c) active sites for gene expression and replication consist of the nuclear periphery where differentiation genes particularly are exposed; the nucleoli where at least some housekeeping genes are exposed; and possibly also p

We present the first documented NOR suppression in a hybridoma other than man-mouse for the hamster-chimpanzee hybrid cell line R48-26. Alu PCR and chromosome painting showed that in this cell line chimpanzee chromosomes 13-15-23 are maintained. NORs on chimpanzee chromosomes 15-23, whose presence was directly verified by FISH with H 28s rDNA, resulted inactive while telomeric rDNA on hamster chromosomes resulted active even if hamster chromosomes presented extensive rearrangements. We observed an all or nothing model in accordance with a model of regulation by selective transcriptional factors. The rearrangements of hamster chromosomes have not involved the location of NORs because they maintain a telomeric position.

Down regulation of Tropomyosins (TMs) is a consistent biochemical change observed in many transformed cells. Our previous work has demonstrated that Tropomyosin-1 is an antioncogene and it is a class II tumor suppressor. Using ras-transformed murine fibroblasts (DT cells), we have examined the effects of co-expression of two isoforms of TM on cell morphology, cytoskeleton and tumorigenecity. Enhanced expression of TM1, a suppressor of transformation, along with TM2 which is not a tumor suppressor results in the formation of well-organized microfilaments, a morphology that resembles normal fibroblasts, and suppression of tumorigenecity. Tumor formation in vivo was compatible with the persistence of high-level of TM2, but not TM1. Homodimers of TM1 and TM2 were observed in these cells. Thus, restoration of expression of TM1 and TM2 protein in ras-transformed cells suppresses the transformed phenotype with dramatic re-organization of microfilaments. These data show that TM2 cooperates with TM1 in the reorganization of microfilaments, while TM1 is a suppressor of the transformed phenotype.

A major problem with standard treatments of solid tumors such as chemotherapy is that the effects are not localized to the tumor. As a result, normal tissue function is often severely impaired. Here we show that myoblasts from skeletal muscle that have been engineered with retroviral vectors to express Fas ligand (FasL) have potential as site-specific anti-tumor agents. FasL-expression by myoblasts was previously shown to lead to neutrophil-mediated immunodestruction, both of the cells and the surrounding tissue. Moreover, myoblasts expressing FasL induced apoptosis in Fas-expressing human tumor cells in vitro. These findings led us to investigate the possibility that myoblasts expressing FasL could serve as anti-tumor agents acting by both apoptotic and immunological mechanisms. The C57BL/6 lpr/lpr mouse primary myoblasts either expressing or not expressing murine FasL were co-injected with Fas-positive or Fas-negative human rhabdomyosarcoma cells into the tibialis anterior of immunodeficient mice. After 19-31 days, FasL-expressing myoblasts resulted in a marked accumulation of neutrophils and inhibited tumor growth in every case. By contrast, control myoblasts did not prevent significant tumor growth. The status of Fas expression by the tumor tissue in vivo was confirmed by immunostaining tumor sections with antibodies against Fas. Tumor inhibition was observed regardless of the presence or absence of Fas on the tumor cells, suggesting that in vivo, the induction of a neutrophil response is remarkably potent and sufficient to inhibit tumors.

To construct the PAC and cosmid contig map spanning the HOXA cluster on human chromosome 7, we used 9 DNA markers (D7S2243, D7S3010, HOXA1, EVX1, 750, pBH8, p60, p8.0, and HOXA11), among which the final 4 were generated in this study by shotgun cloning strategy. From the libraries, 5 PAC and 35 cosmid clones were screened and as a result, an overlapping continuous array of cosmid and PAC clones covering the genomic region (about 200 kb) spanning the entire cluster were constructed. The isolated cosmids contained several consecutive HOX genes of regional group, probably sharing the regulatory processes such as alternative splicing or polyadenylation, and thus could be used as useful materials for elucidating the molecular mechanism of HOX gene expression in the future.

Although cationic lipids are successfully used for gene transfer in vitro, primary cells such as neonatal cardiomyocytes frequently resist efficient transfection. We show here that the polycationic lipid DOSPER in combination with histone H1 was much more efficient in transfection of neonatal cardiomyocytes than DOSPER alone or other cationic lipids. This has been shown for transfection with the reporter plasmids pSV beta-gal and pCMV luc. If viral transfections are not possible, this mild method is an alternative to transfect cardiomyocytes.

We have examined the expression of cloned genes following their stable integration into the genome of pluripotent embryonal carcinoma stem cells. Transfected genes integrate into the genome as tandem arrays. Expression of reporter genes from these tandem arrays in embryonal carcinoma cells is inefficient probably because genes are subject to repeat-induced gene silencing. We found that expression of reporter genes was significantly enhanced if co-transfected with cloned fragments derived from the murine Pgk-1 gene. The enhanced expression required (a) that the Pgk-1 fragment carries an active promoter, (b) that the promoter drives transcription through a region of more than 12 kbp, and (c) that this transcribed region contains both introns and exons. Reporter gene activity did not require specific Pgk-1 DNA sequences suggesting that the coupled processes of transcription and RNA processing conferred activity on neighboring genes probably by influencing local chromatin structure. Consistent with this idea, the effect of the Pgk-1 gene could be mimicked by exposing cells to butyrate or trichostatin A, inhibitors of histone deacetylase. Thus, the effect of the co-transfected Pgk-1 gene is to inhibit the process of gene inactivation possibly by functioning like an insulator or boundary element in the chromatin.

Huntington's disease (HD) is caused by an expanded CAG trinucleotide repeat encoding a tract of consecutive glutamines near the amino terminus of huntingtin, a large protein of unknown function. It has been proposed that the expanded polyglutamine stretch confers a new property on huntingtin and thereby causes cell and region-specific neurodegeneration. Genotype-phenotype correlations predict that this novel property appears above a threshold length (approximately 38 glutamines), becomes progressively more evident with increasing polyglutamine length, is completely dominant over normal huntingtin and is not appreciably worsened by a double genetic dose in HD homozygotes. Recently, an amino terminal fragment of mutant huntingtin has been found to form self-initiated fibrillar aggregates in vitro. We have tested the capacity for aggregation to assess whether this property matches the criteria expected for a fundamental role in HD pathogenesis. We find that that in vitro aggregation displays a threshold and progressivity for polyglutamine length remarkably similar to the HD disease process. Moreover, the mutant huntingtin amino terminus is capable of recruiting into aggregates normal glutamine tract proteins, such as the amino terminal segments of both normal huntingtin and of TATA-binding protein (TBP). Our examination of in vivo aggregates from HD post-mortem brains indicates that they contain an amino terminal segment of huntingtin of between 179 and 595 residues. They also contain non-huntingtin protein, as evidenced by immunostaining for TBP. Interestingly, like the in vitro aggregates, aggregates from HD brain display Congo red staining with green birefringence characteristic of amyloid. Our data support the view that the expanded polyglutamine segment confers on huntingtin a new property that plays a determining role in HD pathogenesis and could be a target for treatment. Moreover, the new property might have its toxic consequences by interaction with one or more normal polyglutamine-containing proteins essential for the survival of target neurons.

The G258 mutant cell line, isolated from the FM3A mouse mammary carcinoma cell line, is temperature-sensitive for both cell growth and asparagine-linked glycosylation due to mutation at a single location. The biochemical defect in the G258 mutant resides in the formation of lipid-linked oligosaccharide, presumably in one of the steps of GDP-mannose-dependent mannosylation (Y. Nishikawa, J. Cell. Physiol. 119, 260-266, 1984; Y. Nishikawa, Biochim. Biophys. Acta 1091, 135-140, 1991). In the present study, we transfected human genomic DNA fragments into the G258 mutant by the radiation hybrid method and isolated transformants (KK-1, -3 and -4) which showed recovery from both temperature-sensitive cell growth and asparagine-linked glycosylation. These transformants contained a common Alu-containing human DNA fragment (1.3 kb) which will be used as a marker for isolating the gene that complements the defect of lipid-liked oligosaccharide synthesis in the G258 mutant.

Electroporation was used to introduce a mixture of two plasmid-cloned genes into Chinese hamster ovary (CHO) cells, and the location of the two genes was subsequently determined by fluorescence in situ hybridization (FISH). The 25 kb Chinese hamster gene for dihydrofolate reductase (dhfr) in the form of a cosmid-derived 40 kb BglI fragment and the SV40 promoter-driven E. coli gene for guanine phosphoribosyltransferase (gpt) were co-electroporated and gpt + transfectants selected. Clones that had also integrated a single copy of the dhfr gene were studied by 2-color fluorescence in situ hybridization (FISH) to localize the integration site(s) of the exogenous DNA in metaphase chromosomes. All 9 clones examined showed co-localization of the two transgenes. The chromosomal site of integration was different in each clone. Co-integration was confirmed by co-amplification experiments. We conclude that, even when provided at low concentrations, separate soluble DNA molecules become linked upon gene transfer by electroporation, either by intracellular ligation prior to integration, or by co-integration at a common site in a given recipient cell.