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L A Chasin

Publications and source records attributed to L A Chasin.

At least 55 records · Page 3Linked to original sources
PubMed1983

Deletion of the diploid dihydrofolate reductase locus from cultured mammalian cells.

Gamma rays have been used to induce Chinese hamster ovary cell mutants in which the entire locus for dihydrofolate reductase (DHFR) has been eliminated. These mutants were isolated in two steps from a methotrexate-resistant clone (Flintoff, Davidson, and Siminovitch (1976). Somat. Cell Genet. 2, 245-262). The resistant cells contain amplified copies of a mutant dhfr gene that codes for a drug-resistant form of the enzyme. In the first step, methotrexate-sensitive mutants of the amplified line were selected. These mutants exhibit a reduced level of DHFR activity and contain a reduced number of dhfr genes. The remaining DHFR activity is methotrexate-sensitive. These mutants appear to be hemizygotes that have lost all copies of the amplified altered dhfr genes and retain one wild-type allele. In a second mutagenic step, mutants completely deficient in DHFR activity were isolated. Three of four of these mutants were the result of double deletions: they lack all traces of dhfr DNA sequences. The fourth mutant contains a deletion that extends through the 5' half of the dhfr gene. The hemizygotes for dhfr should be useful for the study of mutation at an autosomal mammalian locus without the complications of diploidy.

Alleles↗
PubMed1982

Multihormonal induction of alpha 2u-globulin in an established rat hepatoma cell line.

A subclone of the FU5-5 rat hepatoma cell line has been isolated which is inducible more than several hundred fold for the 20,000 dalton form of the major rat urinary protein alpha 2u-globulin. The basal relative synthetic rate (RSR) in growth medium containing 10% fetal calf serum was less than 2 X 10(-6) of total protein synthesis. Both dexamethasone and insulin were necessary for induction, and yielded a maximum induced RSR of 4-8 X 10(-3). Triiodothyronine (T3), dihydrotestosterone (DHT), rat growth hormone (GH), and estrogen, all of which have been shown to influence the induction of alpha 2u-globulin in the intact rat, were without effect on the cell line. A factor present in fetal calf serum was also necessary for maximum induction, since dexamethasone plus insulin in serum-free medium raised the RSR to only 3 X 10(-5); exogenous T3, GH, and DHT could not substitute for this serum factor. The kinetics of induction by dexamethasone were slow, with a lag of approximately 48 hr followed by a period of increasing RSR for 6-20 days. Removal of dexamethasone from induced cells led to an exponential decline in the RSR (t 1/2 15 hr). The concentrations of dexamethasone and insulin that could yield half maximum induction were 5 X 10(-8)M and 3 X 10(-11)M, respectively. Higher concentrations of insulin, although still in physiological range (10(-9)M), inhibited induction. At yet higher insulin levels, beyond the physiological range, alpha 2u-globulin synthesis returned to maximum values. The lack of DHT, T3, and GH requirement for alpha 2u-globulin induction in this cell line may mean that a regulatory aberrancy has occurred in this transformed cell line, or, alternatively, that these hormones act indirectly in the intact animal. This cell line should prove useful for the study of the molecular events associated with alpha 2u-globulin induction and for genetic approaches to the problem of multihormonal regulation of gene expression.

Alpha-Globulins↗
PubMed1981

Selective killing of methotrexate-resistant cells carrying amplified dihydrofolate reductase genes.

A method for the selective killing of methotrexate (MTX)-resistant cells has been developed. The selection is based on the incorporation of tritiated deoxyuridine into the DNA of MTX-resistant cells but not normal MTX-sensitive cells in the presence of the drug. A Chinese hamster ovary cell mutant that overproduces dihydrofolate reductase was used as an example of a MTX-resistant cell line. In this system, a 10,000-fold enrichment for wild-type MTX-sensitive cells could be achieved after 24 hr of exposure to the drug combination. This selection technique was applied to the isolation of MTX-sensitive segregants from hybrid cells formed between the MTX-resistant mutant and wild-type cells. The loss of MTX resistance and dihydrofolate reductase overproduction was always accompanied by the loss of a homogeneously staining region on chromosome 2 of the resistant parent that contains the amplified genes specifying this enzyme. While this region is always lost, other parts of chromosome 2 are almost always retained, suggesting that deletion rather than chromosome loss underlies marker segregation in this case. When the selection was applied to the resistant mutant itself, no MTX-sensitive revertants were obtained among 10(5) cells screened, attesting to the stability of gene amplification in this clone. It is suggested that this combination of drugs may be useful for the elimination of MTX-resistant tumor cells that develop after MTX chemotherapy.

Animals↗
PubMed1980

Isolation of Chinese hamster cell mutants deficient in dihydrofolate reductase activity.

Mutants of Chinese hamster ovary cells lacking dihydrofolate reductase (tetrahydrofolate dehydrogenase, 7,8-dihydrofolate:NADP+ oxidoreductase; EC 1.5.1.3) activity were isolated after mutagenesis and exposure to high-specific-activity [3H]deoxyuridine as a selective agent. Fully deficient mutants could not be isolated starting with wild-type cells, but could readily be selected from a putative heterozygote that contains half of the wild-type level of dihydrofolate reductase activity. The heterozygote itself was selected from wild-type cells by using [3H]deoxyuridine together with methotrexate to reduce intracellular dihydrofolate reductase activity. Fully deficient mutants require glycine, a purine, and thymidine for growth; this phenotype is recessive to wild type in cell hybrids. Revertants have been isolated, one of which produces a heat-labile dihydrofolate reductase activity. These mutants may be useful for metabolic studies relating to cancer chemotherapy and for fine-structure genetic mapping of mutations by using available molecular probes for this gene.

Animals↗
PubMed1979

Immortalization of normal liver functions in cell culture: rat hepatocyte-hepatoma cell hybrids expressing ornithine carbamoyltransferase activity.

Normal rat hepatocytes have been fused with highly differentiated rat hepatoma cells. Some of the hybrids express a physiologically significant level of activity of the urea cycle enzyme ornithine carbamoyltransferase (OCT), a liver-specific function not found in the hepatoma cells. These hybrids have 10% of the adult rat liver OCT specific activity, incorporate 3H-ornithine into protein arginine, and can be selectively grown in arginine-free medium supplemented with ornithine. Somatic cell hybridization of normal differentiated cells with highly differentiated neoplastic cells of the same tissue type may be useful as a general method for obtaining permanent cell lines with new tissue-specific phenotypes.

Animals↗
PubMed1979

Transformation of the gene for hypoxanthine phosphoribosyltransferase.

Purified DNA from wild-type Chinese ovary (CHO) cells has been used to transform three hypoxanthine phosphoribosyltransferase (HPRT) deficient murine cell mutants to the enzyme positive state. Transformants appeared at an overall frequency of 5 x 10(-8) colonies/treated cell and expressed CHO HPRT activity as determined by electrophoresis. One gene recipient, B21, was a newly isolated mutant of LMTK- deficient in both HPRT and thymidine kinase (TK) activities. Transformation of B21 to HPRT+ occurred at 1/5 the frequency of transformation to TK+; the latter was, in turn, an order of magnitude lower than that found in the parental LMTK- cells, 3 x 10(-6). Thus both clonal and marker-specific factors play a role in determining transformability. The specific activity of HPRT in transformant extracts ranged from 0.5 to 5 times the CHO level. The rate of loss of the transformant HPRT+ phenotype, as measured by fluctuation analysis, was 10(-4)/cell/generation. While this value indicates stability compared to many gene transferents, it is much greater than the spontaneous mutation rate at the indigenous locus. The ability to transfer the gene for HPRT into cultured mammalian cells may prove useful for mutational and genetic mapping studies in this well-studied system.

Animals↗
PubMed1978

Amplified dihydrofolate reductase genes are localized to a homogeneously staining region of a single chromosome in a methotrexate-resistant Chinese hamster ovary cell line.

Methotrexate-resistant Chinese hamster ovary cells selected for high resistance by progressive increments of methotrexate in the culture medium have levels of dihydrofolate reductase (tetrahydrofolate dehydrogenase, 7,8-dihydrofolate: NADP+ oxidoreductase, EC 1.5.1.3) 200 times that of sensitive cells and a corresponding increase in the number of copies of the dihydrofolate reductase gene. The resistant cells contain an expanded region on the second chromosome (homogeneously staining region) that is not present in sensitive cells. In situ hybridization of DNA complementary to dihydrofolate reductase mRNA shows that the dihydrofolate reductase genes are specifically localized to the homogeneously staining region of this chromosome in the resistant cells.

Cell Line↗
PubMed1978

Separation of linked markers in Chinese hamster cell hybrids: mitotic recombination is not involved.

A search for mitotic recombination was carried out using mutant subclones of cultured Chinese hamster ovary cells. Recombination events were sought between the linked loci specifying the enzymes hypoxanthine phosphoribosyl tranferase and glucose-6-phosphate dehydrogenase. It was shown by fluctuation analysis that markers at these two loci co-segregate from doubly heterozygous pseudotetraploid hybrid cells more than 90% of the time. The minority class of segregants, which had lost one marker without losing the other, were genetically analyzed to distinguish between the possibilities of mitotic recombination and deletion of chromosomal material. Nine clones in which a linkage disruption had occurred were studied, using further cell hybridization and segregation. In three cases, a recessive lethal loss of genetic information was indicated, suggesting the deletion mechanism. In six cases, it was demonstrated that no new linkage relationships had been established concomitant with linkage disruption. Thus, in all nine clones, the evidence indicated that mitotic recombination was not involved in the events that disrupted linkage between these two loci. If mitotic recombination takes place at all in this system, the rate must be less than about 10(-6) per cell per generation.

Animals↗
PubMed1977

Mutants of Chinese hamster ovary cells with altered glucose-6-phosphate dehydrogenase activity.

Two mutant clones of a Chinese hamster ovary cell line deficient in glucose-6-phosphate dehydrogenase (G6PD) activity have been characterized. In each case, there is evidence that a structural gene mutation has taken place. The first mutant produces 11% specific enzyme activity compared to wild-type parental cells, but this residual activity is much more heat sensitive than that of the wild type. The second mutant contains no residual activity, but a revertant was isolated that exhibits a partial restoration of G6PD activity with, again, an increased heat sensitivity. The selection of G6PD+ cells from G6PD- populations can be effected by exploiting the increased sensitivity of the latter to diamide, a compound that depletes the cell of reduced glutathione.

Clone Cells↗
PubMed1976

Mutant alleles for hypoxanthine phosphoriboxyltransferase: codominant expression, complementation, and segregation in hybrid Chinese hamster cells.

Chinese hamster ovary cell mutants resistant to the purine analogs 6-thioguanine or 8-azaguanine have been isolated following mutagenesis with ethyl methane sulfonate. The activities of hypoxanthine phosphoribosyltransferase (HPRT) in three such mutants have been found to exhibit an increased Km for the substrate 5-phosphoribosyl-1-pyrophosphate. The isoelectric point of the mutant enzyme activity has also changed in two mutants. Hybrid cells containing one mutant and one wild-type allele express both genes. Segregants that have lost only the wild-type allele can be selected on the basis of drug resistance. Two mutants exhibiting different alterations in HPRT activity can complement in a hybrid cell to yield a wild-type growth pattern and enzyme activity with intermediate electrophoretic and kinetic properties. The results suggest intracistronic complementation between structural gene mutants of HPRT.

Alleles↗
PubMed1975

Chromosome-wide event accompanies the expression of recessive mutations in tetraploid cells.

Mutants resistant to 6-thioguanine were induced in pseudotetraploid hybrid Chinese hamster cells homozygous wild-type at the locus for hypoxanthine phophoribosyl transferase but heterozygous for the linked marker glucose-6-phosphate dehydrogenase. About half of these mutants had concomitantly lost the wild-type allele for glucose-6-phosphate dehydrogenase, as expected if mutation plus chromosome segregation had occurred.

Alleles↗
PubMed1975

Isolation of mammalian cell mutants deficient in glucose-6-phosphate dehydrogenase activity: linkage to hypoxanthine phosphoribosyl transferase.

Mutants of Chinese hamster ovary cells deficient in glucose-6-phosphate dehydrogenase (D-glucose-6-phosphate: NADP 1-oxidoreducatse, EC 1.1.1.49) activity were isolated after mutagenesis with ethyl methane sulfonate. The mutants were induced at frequencies of about 10-4 and do not differ in growth properties from wild-type cells. They were isolated by means of a sib selection technique coupled with a histochemical stain of colonies for enzyme activity. The lack of enzyme activity is not due to a dissociable inhibitor, and is recessive in hybrid cells. Multiple mutants that lack hypoxanthine phosphoribosyltransferase activity (IMP:pyrophosphate phosphoribosyltransferase, EC 2.4.2.8) and adenine phosphoribosyltransferase activity (AMP:pyrophosphate phosphoribosyltransferase, EC 2.4.2.7) were isolated by further mutagenesis. By following segregation of wild-type phenotypes from heterozygous multiply marked hybrid cells, it was shown that the genes responsible for glucose-6-phosphate dehydrogenase activity and hypoxanthine phosphoribosyltransferase activity are linked in Chinese hamster cells, in agreement with the location of both on the X chromosome in humans. No linkage to adenosine phosphoribosyltransferase was found. The isolation of mutant cells carrying linked markers should prove useful for studying chromosomal events such as segregation, breakage, recombination, and X-chromosome reactivation.

Alleles↗
PubMed1974

Reversion of a Chinese hamster cell auxotrophic mutant.

A mutant cell strain derived from a Chinese hamster line by mutagenesis with ethylmethane sulfonate requires glycine for growth. In the wild type, glycine synthesis is catalyzed by serine hydroxymethyltransferase (EC 2.1.2.1). Cell fractionation by differential centrifugation and isopycnic sucrose gradient analysis reveals that the enzyme activity is found in both the mitochondrial and cytosol fractions. The specific activity in the mitochondrial fraction is about 20 times higher than in the cytosol, and is much more stable to thermal inactivation. The glycine-requiring mutant has lost all of the mitochondrial enzyme activity, while retaining the cytosol activity. The mutant is very stable but can be induced to revert by several chemical mutagens. One glycine-independent revertant induced by ethyl methane sulfonate was studied in detail. Serine hydroxymethyltransferase activity is again present in the mitochondrial fraction, at about [unk] of the wild-type level. However, the revertant mitochondrial enzyme exhibits an altered thermal sensitivity, with a half-life at 45 degrees of 55 min as compared to 180 min in the wild type. The half-life for the cytosol enzyme in all three strains is 7 min. Mixing experiments demonstrate that the heat lability of the revertant enzyme is not due to a dissociable factor in the extract. The data are consistent with the idea that the original mutation occurred in the structural gene for one isozyme of the enzyme and that the revertant has undergone a second mutation in this gene, partially restoring enzyme activity.

Acid Phosphatase↗
PubMed1970

Gene expression after transformation of Bacillus subtilis.

Sensitive assays of histidase activity were used to follow the production of this enzyme as directed by a gene newly introduced into cells of Bacillus subtilis by transformation. Histidase activity can be detected in histidase-negative recipient cells within 1 hr after the addition of deoxyribonucleic acid extracted from histidase-positive donors. Enzyme production continues for one to two additional hours and then ceases. Histidase production in the transformed cells is fully sensitive to catabolite repression. Catabolite repression is rapidly established after transformation of recipient cells that are resistant to this form of regulation.

Bacillus subtilis↗