Somatic Cell and Molecular Genetics最新文献

Neurofibromatosis type 1 (NF1) is a common genetic disorder caused by inactivation of neurofibromin, a protein capable of modulating signal transduction by activating Ras-GTPase activity. We have used cDNA cloning and Northern blot analysis to confirm the NF1 gene produces alternatively polyadenylated mRNAs with 3' untranslated regions (3' UTR) that show striking evolutionary conservation. Scanning of the 3'UTRs for genetic variation revealed three common sequence polymorphisms (> 30% heterozygosity), one less informative polymorphism (approximately 5% heterozygosity) and one rare variant (1/144 chromosomes). These differences were used to examine relative levels of expression of normal and mutant NF1 alleles in lymphoblast cell lines and in one case, autopsy tissue, from patients with NF1. Unequal allelic expression (up to 4-fold) was observed in a subset of both sporadic and familial NF1 cases. Where linkage phase could be determined, the allele segregating with the disorder displayed a relative reduction in expression. However, the magnitude of this effect was variable suggesting the operation of additional, non-genetic factors in determining the degree of relative expression of the mutant allele.

We have employed a direct cDNA selection methodology to isolate transcribed sequences encoded in the human chromosomal interval Xq26 that contains the gene for X-chromosome linked albinism deafness syndrome (ADFN). ADFN had been previously mapped to an 8 centi Morgan region on chromosome Xq26. We have constructed six cDNA libraries specific to six YACs mapping to a 1.5 mb span at the distal boundary of the ADFN locus. The YAC specific libraries were characterized for the presence of unique cDNAs. We have identified 15 transcribed sequences from the selected cDNA libraries. These cDNAs matched to three well characterized sequences corresponding to steroid 5-alpha reductase, ribosomal protein L28, and a short transcript that has been shown to be expressed in human brain cortex. Seven of the cDNAs matched to expressed sequence tags or other sequences of unknown function, and five cDNAs shared no homology with sequences in the public data bases. Each one of these sequences was represented as 3-10 clones in the set that was subjected to sequencing. Further characterization of these transcribed sequences may indicate potential candidates responsible for ADFN. We have discussed the utility of cDNA selection methodology in assembling transcript maps and identifying potential candidates for genetic deafness.

The DNA repair gene XRCC1 was the first cloned human DNA repair gene involved in resistance to ionizing radiation. Previous studies have shown that rodent and baboon homologs of XRCC1 are expressed in all tested tissues with significantly higher levels in testis. Furthermore, expression of murine XRCC1 is most abundant in pachytene spermatocytes and round spermatids. To begin to study regulation of XRCC1 expression, the 5' region of baboon XRCC1 was cloned and characterized. 400 bp of 5'-flanking region showed the greatest promoter activity, while -194 to -8 bp of the 5'-flanking region displayed core promoter activity in transient transfection assays. A comparison between baboon and human 5'-flanking sequences in the core promoter region revealed a potential CAAT-box, an imperfect CREB-binding site and two putative Sp1-binding sites. Results from transient transfection assays in which each putative binding site was individually mutated, indicated that the distal Sp1-binding site has a functional role in transcription. In comparison, both putative Sp1-binding sites bound protein(s) from HeLa cell nuclear extracts in vitro. In vitro binding was lost when mutated Sp1 sites were used in gel mobility shift assays. Finally, anti-Sp1 antibodies produced mobility supershifts, thereby indicating Sp1 or an Sp1-like protein bound to the DNA fragment in vitro.

We describe a series of retroviral vectors containing two internal ribosome entry sites (IRES) for the co-transcription of three genes. Transcription of the single-transcript tricistronic mRNA is under the control of a Harvey murine sarcoma virus long terminal repeat. The 5'-most open reading frame is under either cap-dependent or cap-independent translational control, while the two downstream open reading frames are translated in a cap-independent fashion using the initiation codons of their respective IRES elements. Both IRES elements are taken from the encephalomyocarditis virus. To characterize these vectors, we used the human multidrug resistance gene (MDR1) in the 5' position, the gene for green fluorescent protein (GFP) in the middle position, and neo in the 3' position. The vectors were either transfected directly into NIH3T3 mouse fibroblasts or packaged into retrovirus and then transduced into NIH3T3 cells. Gene transfer was followed by selection with colchicine, which selects for expression of the MDR1 gene, or with G418, which selects for expression of the neo gene. Thus, we could determine the function of the tricistronic vectors under conditions of selection for either the 5'-most or the 3'-most gene. In DNA-mediated transfections, we were able to achieve expression of all three open reading frames under either selection condition. We obtained higher expression of all three genes when colchicine was used to select for MDR1 expression than when G418 was used to select for neo expression. Expression of the non-selected GFP gene (the middle cistron) was unstable, most likely due to loss of integrated GFP DNA sequences during long-term culturing. We were able to achieve retrovirus-mediated transduction of all three genes, but this was an inefficient process.

Hypervariable minisatellite DNA repeats are found at tens of thousands of loci in the mammalian genome. These sequences stimulate homologous recombination in mammalian cells [Cell 60:95-103]. To test the hypothesis that protein-DNA interaction is required for hotspot function in vivo, we determined whether a second protein binding nearby could abolish hotspot activity. Intermolecular recombination between pairs of plasmid substrates was measured in the presence or absence of the cis-acting recombination hotspot and in the presence or absence of the second trans-acting DNA binding protein. Minisatellite DNA had hotspot activity in two cell lines, but lacked hotspot activity in two closely related cell lines expressing a site-specific helicase that bound to DNA adjacent to the hotspot. Suppression of hotspot function occurred for both replicating and non-replicating recombination substrates. These results indicate that hotspot activity in vivo requires site occupancy by minisatellite DNA binding proteins.

Twenty nine hybrids retaining fragments of human chromosome 2 were characterized by reverse-FISH and by a panel of 106 STSs. Most of the hybrids are radiation hybrids retaining fragments of chromosome 2 as the only human contribution. The hybrid panel dissected chromosome 2 in 69 distinct physical regions, allowing a fine mapping of the sequences. These hybrids are particularly useful as starting points for generation, via Alu-PCR, of specific partial chromosome paints (PCP). We also report the mapping by FISH of 60 YACs located on chromosome 2. These resources can be advantageously used in cytogenetic investigations, with particular reference to cancer cytogenetics, as illustrated with the renal carcinoma cell line KRC/Y.

The interaction of chemical mutagens with mammalian cells is much more complex than that of gamma-irradiation because of the different ways in which chemical agents react with cell and medium components. Nevertheless, the system previously described for analysis of mutagenesis by gamma-radiation appears applicable to chemical mutagenesis. The approach involves measurement of cell survival, use of caffeine to inhibit repair, analysis of mitotic index changes, and quantitation of microscopically visible structural changes in mitotic chromosomes. The behavior of a variety of chemical mutagens and nonmutagens in this system is described and compared with that of gamma-irradiation. The procedure is simple and the results reasonably quantitative though less so than those of gamma-irradiation. The procedure can be used for environmental monitoring, analysis of mutational events, and individual and epidemiological testing. Mutational events should be classified as primary or secondary depending on whether they represent initial genomic insult, or genomic changes resulting from primary mutation followed by structural changes due to metabolic actions. While caffeine has multiple effects on the mammalian genome, when used under the conditions specified here it appears to act principally as an inhibitor of mutation repair, and so affords a measure of the role of repair in the action of different mutagens on cells in the G2 phase of the life cycle.

A novel gene, PiUS, was recently cloned and shown to increase phosphate uptake when expressed in oocytes, indicating that it may be an important regulator of cellular phosphate homeostasis. The phosphate wasting disease autosomal dominant hypophosphatemic rickets (ADHR) was previously mapped to chromosome 12p13 by linkage analysis. PiUS' role as a modulator of phosphate transport, as well as its intestinal and renal expression made the gene an appropriate candidate for ADHR. The purpose of our study was to determine the chromosomal localization of the human PiUS gene through the use of somatic cell hybrids and radiation hybrid mapping. In the present work, PiUS was localized to human chromosome 3p21.3 and is therefore not the ADHR gene.

In mammalian cells, nonhomologous end-joining is the predominant mechanism to eliminate DNA double strand breaks. Such events are at the origin of deletion mutagenesis and chromosomal rearrangements. The hallmark of Fanconi anemia, an inherited cancer prone disorder, is increased chromosomal breakage associated to over-production of deletions. Knowing that double strand breaks are at the origin of deletion mutagenesis, the question arises whether their processing is affected in FA. We set up a "host cell end-joining assay" to analyze the fate of double strand breaks into extrachromosomal substrates transiently replicated in normal and FA-D lymphoblasts. Although no difference in plasmid survival was found, blunt-ended breaks were sealed with significantly lower fidelity in FA cells, resulting in a higher deletion frequency and a larger deletion size. The results suggest that FA-D and FA-B gene products are likely to play a role in end-joining fidelity of specific DNA double strand breaks.