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Biomedical subjects

J W Littlefield

Publications and source records attributed to J W Littlefield.

At least 55 records · Page 3Linked to original sources
PubMed1976

Altered regulation of the rate of synthesis of dihydrofolate reductase in methotrexate-resistant hamster cells.

Dihydrofolate reductase was isolated from hamster cells sensitive to methotrexate and from a methotrexate-resistant subline which has an elevated enzyme level. The enzymes were compared by peptide map analysis, and no differences in primary structure could be detected. The rates of enzyme degradation and synthesis were determined in both cell lines by a novel approach based on the enzyme specific radioactivity (called radioaffinity labeling). Degradation of reductase was minimal in both cell lines, whereas the rates of synthesis were directly proportional to the steady state concentrations of enzyme. Thus the resistant cells synthesized reductase at a rate which was 140 times faster than that in sensitive cells. Therefore the high concentration of dihydrofolate reductase in the methotrexate-resistant cells is probably the result of an alteration of a cellular component which control the synthesis of the enzyme.

Animals↗
PubMed1976

Elevated dihydrofolate reductase messenger RNA levels in methotrexate-resistant BHK cells.

Polysomal RNA from cultured sublines of baby hamster kidney (BHK) cells directed protein synthesis in an in vitro system derived from wheat germ extract. One product of the in vitro synthesis was dihydrofolate reductase (DHFR), as confirmed by methotrexate-substituted Sepharose affinity chromatography followed by SDS-polyacrylamide slab gel electrophoresis and autoradiography of the proteins labeled with 35S-methionine. The DHFR synthesized in vitro comigrates in the gel with authentic BHK DHFR, indicating that the molecular weights and structures of the in vivo and in vitro enzymes are probably the same. Polysomal RNA obtained from the methotrexate-resistant BHK subline (A5), which possesses some 140 times higher DHFR levels than the methotrexate-sensitive parents subline (B1), directed the synthesis of approximately 70 times more DHFR per unit of total in vitro synthesized protein than did B1 polysomal RNA. Assuming then that the rates of translation of A5 and B1 DHFR mRNAs in the wheat germ cell-free system are the same, our results show that a major part of the high DHFR levels observed in A5 cells is due to the presence of elevated quantities of DHFR mRNA.

Cell Line↗
PubMed1974

Mutagenic effect of BUdR in diploid human fibroblasts.

It has only recently been possible to demonstrate the expected mutagenic effect of 5-bromodeoxyuridine (BUdR) in heteroploid hamster cells in culture. We have now extended this observation to diploid human fibroblasts utilizing techniques adapted from the work of Albertini and DeMars on X-ray mutagenesis at the hypoxanthine-guanine phosphoribosyltransferase (HGPRT) locus in these cells. In four separate experiments, fibroblasts from a female donor were exposed to 500 micrograms/ml ethylmethane sulfonate (EMS) or 3 micrograms/ml BUdR yielding survivals of 9% and 5%, respectively. After a 6-day expression period, survivors were plated in selection medium containing 0.3 micrograms/ml 8-azaguanine (8-AG). After 3-5 weeks, azaguanine-resistant colonies were isolated for characterization or stained for counting. The average spontaneous mutation rate/cell/generation was 0.6.10(-6). The average induced mutation rates for EMS and BUdR were 7.8.10(-6) and 6.3.10(-6)/cell/generation, respectively. Similar results were obtained in two experiments with an additional fibroblast line. Mutant colonies isolated following BUdR treatment demonstrated from 1.4 to 61.5% of the HGPRT activity of the parental line and showed at least 8% Barr bodies, excluding the possibility of contamination by Lesch-Nyhan cells. This demonstration of a BUdR effect comparable to that of an alkylating agent or X-irradiation opens the study of mutation due to base-analog substitution in diploid human cells.

Azaguanine↗
PubMed1973

Lambda-chain production in human lymphoblast-mouse fibroblast hybrids.

Mutant human lymphoblast cells deficient in hypoxanthine phosphoribosyltransferase (EC 2.4.2.8) activity were hybridized with thymidine kinase (EC 2.7.1.21)-deficient mouse fibroblasts. Hybrid cells were readily selected, as both parental lines were nonreverting and eliminated by hypoxanthine-amethopterinthymidine medium. Human lambda (lambda) chain was the only immunoglobulin chain produced by the lymphoblast parent, as determined by immunofluorescent techniques. Two independent hybrid clones chosen for detailed study synthesized human lambda chain, and continued to do so after prolonged culture. As in both parental lines, no human immunoglobulin heavy chains, complements C3 or C4, or alpha(1)-antitrypsin, or mouse immunoglobulin chains or complement C5 were detectable in the hybrids. Selection against thymidine kinase-containing hybrid cells with 5-bromodeoxyuridine did not eliminate positive lambda-chain reactivity, suggesting that the kinase and lambda-chain loci are not linked. The continued production of an immunoglobulin chain by human lymphoblast-mouse fibroblast hybrids contrasts with the extinction of other differentiated functions in several hybrid systems, and indicates that gene localization and linkage analysis for human immunoglobulin chains should be feasible with this system.

Animals↗
PubMed1973

Regulation of 5-methyltetrahydrofolate: homocysteine methyltransferase activity by methionine, vitamin B12, and folate in cultured baby hamster kidney cells.

Rapid growth of BHK cells in methioninedeficient medium required supplementation with homocysteine, B(12), and over 40-fold greater levels of folic acid than growth in methionine-supplemented medium. The activity of the B(12)-dependent 5-methyltetrahydrofolate: homocysteine methyltransferase was studied in extracts of BHK cells grown in media containing various concentrations of the compounds of the enzyme reaction. The methyltransferase activity increased over 4-fold when B(12)-deficient deficient medium was supplemented with optimal levels of B(12); this increase was not prevented by puromycin. Addition of homocysteine to growth medium containing methionine, B(12), and folic acid was without effect. However, methyltransferase activity increased 2.5- to 4.0-fold further beyond the highest levels obtained in the presence of methionine, B(12), and folic acid when homocysteine was substituted for methionine in the growth medium. This increase was blocked by puromycin and was not due to removal of feedback inhibition of activity by the product methionine. These results suggest that methyltransferase activity may be regulated in part by derepression of the enzyme's synthesis on substitution of the substrate homocysteine for the product methionine.

Animals↗
PubMed1972

Chemical mutagenesis at the phosphoribosyltransferase locus in cultured human lymphoblasts.

The presence of selectable genetic markers in long-term human lymphoblast cultures would facilitate cell hybridization experiments on the biosynthesis of immunoglobulins, as well as other studies. This work reports the induction with ethylmethane sulfonate of 6-thioguanine - resistant, phosphoribosyltransferase - deficient mutants in a lymphoblast line from a patient with infectious mononucleosis. These cells were unusually sensitive, with a D(0) value of 28 mug of ethylmethane sulfonate per ml; the sensitivity curve followed a biphasic pattern suggesting the presence of 3% resistant cells. Ethylmethane sulfonate increased the frequency of mutants resistant to 6-thioguanine over 100-fold, to about 2 x 10(-4); nitrosoguanidine was less effective. Almost all the mutants contained considerably less than 1% of the hypoxanthine-guanine phosphoribosyltransferase (EC 2.4.2.8) activity of wild-type cells. The mutation did not appear to result from loss of an X chromosome.

Alkanes↗