Somatic Cell Genetics最新文献

A temperature-sensitive (ts) mutant, tsBN2, which was derived from BHK21 and is defective in the regulatory mechanism for chromosome condensation, was transformed to the temperature-resistant (ts+) phenotype by means of DNA-mediated gene transfer with hamster and mouse DNA. Treatment of mouse DNA with the restriction enzymes EcoRI, HindIII, PstI and SalI, but not with XhoI, almost completely abolished the transforming activity. A fluctuation test, originally devised by Luria and Delbrück, was used to estimate the reversion and transformation frequencies of tsBN2 cultures.

Amphotericin B-resistant mutants isolated from Chinese hamster V79 cells (1) are defective in cholesterol synthesis and more sensitive to an oxygenated sterol analog, 7-ketocholesterol, than their parental cell line. We isolated 7-ketocholesterol-resistant mutants from an amphotericin B-resistant mutant, AMBR-1. The 7-ketocholesterol-resistant mutants had regained increased level of free cholesterol, and they showed somewhat similar dose-response curves to amphotericin B as that of V79. Sterol synthesis from acetate, but not from mevalonate, in 7-ketocholesterol-resistant clones was threefold higher than that of AMBR-1. 7-Ketocholesterol-resistant clone, unlike AMBR-1, could form colonies in the presence of lipoprotein-depleted serum. The results are discussed in terms of probable change in the sterol biosynthetic pathway by the different lesions.

We have used a genetic approach to study the differentiation of B lymphocytes. The cultured murine cell line 70Z/3 resembles pre-B cells in containing the heavy chain of the immunoglobulin IgM, mu, as an internal protein in the absence of light chain, L. However, overnight incubation with the B cell mitogen lipopolysaccharide (LPS) induces the cells to mature to a B lymphocyte-like state by the induction of L chain synthesis and the appearance of IgM on the cell surface. We have used immunoselection against surface-bound IgM to isolate LPS uninducible variants of 70Z/3. These fall into two complementation groups, LPS A and LPS B. LPS A variants predominated and were found at a frequency of 1/1200. These cells were completely unresponsive to LPS. LPS B was represented by a single variant in which a subset of cells was induced to display wild-type levels of membrane-bound IgM, and the proportion of induced cells increased with prolonged incubation with LPS. We detected no structural defects in either variant group, but LPS B may represent a defect in the decision to differentiate in response to LPS.

Mouse LA9 cell lines were selected for increased resistance to either HQNO or myxothiazol, inhibitors of electron transport which bind to the mitochondrial cytochrome b protein. Two phenotypically distinguishable HQNO-resistant mutants were recovered while the myxothiazol-resistant isolates had a common phenotype. All three mutant phenotypes were transmitted cytoplasmically in cybrid crosses. Biochemical studies further established that for all three mutant types, resistance at the cellular level was paralleled by an increase in inhibitor resistance of mitochondrial succinate-cytochrome c oxidoreductase, the respiratory complex containing cytochrome b. As with the previously described mitochondrial antimycin-resistant mutant, the initial biochemical and genetic studies indicated that these mutations occur within the mitochondrial cytochrome b gene. This conclusion was strongly supported by the results of mtDNA restriction fragment analyses in which it was found that one HQNO-resistant mutant had undergone a small insertion or duplication in the apocytochrome b gene. Finally, all four mitochondrial cytochrome b mutants have been analyzed in both cell plating studies and succinate-cytochrome c oxidoreductase assays to determine the pattern of cross-resistance to inhibitors of cytochrome b other than the one used for selection.

The chromosomal locations of the genes for the common alpha subunit of the glycoprotein hormones and the beta subunit of chorionic gonadotropin in humans and mice have been determined by restriction enzyme analysis of DNA isolated from somatic cell hybrids. The CG alpha gene (CGA), detected as a 15-kb BamHI fragment in human DNA by hybridization to CG alpha cDNA, segregated with the chromosome 6 enzyme markers ME1 (malic enzyme, soluble) and SOD2 (superoxide dismutase, mitchondrial) and an intact chromosome 6 in human-rodent hybrids. Cell hybrids containing portions of chromosome 6 allowed the localization of CGA to the q12 leads to q21 region. The greater than 30- and 6.5-kb BamHI CGB fragments hybridizing to human CG beta cDNA segregated concordantly with the human chromosome 19 marker enzymes PEPD (peptidase D) and GPI (glucose phosphate isomerase) and a normal chromosome 19 in karyotyped hybrids. A KpnI-HindIII digest of cell hybrid DNAs indicated that the multiple copies of the CG beta gene are all located on human chromosome 19. In the mouse, the alpha subunit gene, detected by a mouse thyrotropin (TSH) alpha subunit probe, and the CG beta-like sequences (CG beta-LH beta), detected by the human CG beta cDNA probe, are on chromosomes 4 and 7, respectively.

A mouse monoclonal antibody (2B2) recognizes an antigen which is present on most human peripheral blood leukocytes but is absent from most proliferating cells. The antibody precipitated two surface-labeled membrane glycopolypeptides with molecular weights of 86,000 and 145,000, and it was strongly mitogenic to normal human lymphocytes. Somatic cell hybrids have been used for assigning the genes coding for these membrane glycoproteins to human chromosome 21. The assignment was based on correlation of antigen expression on mouse-human T-lymphocyte hybrids with the presence of human chromosomes in the same hybrid clones.

Hamster-human hybrids which contained an inactive human X chromosome were treated by 5-azacytidine. Hypoxanthine guanine phosphoribosyltransferase derepressed hybrids were selected and derepression of three other loci, phosphoglycerate kinase, alpha-galactosidase, and glucose-6-phosphate dehydrogenase were studied. Among 32 hybrids selected for hypoxanthine guanine phosphoribosyltransferase, two were found to be reactivated at four X loci. The independence or nonindependence of the reactivation events will be discussed. No correlation was found between the time of replication and the expression or nonexpression of the X chromosome genes: X chromosomes reactivated at four loci remained late replicating; conversely early replication can exist without the expression of some X genes.

Mouse-Chinese hamster somatic cell hybrids containing various combinations of mouse chromosomes were analyzed for the presence of the mouse c-Ha-ras (1) sequences after restriction endonuclease digestion and hybridization with a 32P-labeled Ha-ras specific probe according to the procedure of Southern (2). The presence of the mouse c-Ha-ras containing fragment was correlated with the presence of mouse chromosome 7 in the hybrids.

Karyotypes of recessive mutants at the autosomal adenine phosphoribosyltransferase (Aprt) locus in a clone of the near-diploid mouse CAK cell line have been analyzed. The Aprt located on chromosome 8. One copy of chromosome 8 was morphologically abnormal in the parental clone (CAK-B3-Toyr13) from which Aprt- mutants were isolated. Among 22 mutants, there were ten in which one copy of chromosome 8 had been lost. Four of these were monosomic, and in the others duplication of the remaining homolog had occurred. These findings indicate that newly induced recessive mutations in cultured mammalian cells can be expressed as the result of loss of one chromosome carrying a wild-type allele with or without duplication of the homolog carrying the mutant allele. Loss and duplication would not be detected in cell lines lacking morphologically marked chromosomes.

Peptide expression influenced by human chromosome 21 was examined by comparing two-dimensional electrophoretograms of a human-hamster hybrid cell containing human chromosome 21 with its parent hamster cell and a revertant of the hybrid which had segregated the chromosome 21 genes for SOD-1, GARS, and a cytotoxic cell-surface antigen. Certain peptides were found in the hybrid but not in the hamster cell. Some, but not all, of these peptides segregated with markers for chromosome 21. Hamster peptides were also found which apparently were suppressed in the hybrid. Finally, one peptide was identified which was unique to the revertant cell. These findings may be of potential relevance to Down's syndrome.