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M W Taylor

Publications and source records attributed to M W Taylor.

At least 127 records · Page 7Linked to original sources
PubMed1980

Inhibition of DNA chain elongation in a purine-auxotrophic mutant of Chinese hamster.

DNA synthesis has been examined in a purine-auxotrophic mutant cell line of Chinese hamster (V79 pur 1) under conditions of purine deprivation. At 6 h after the removal of purines from the growth medium, there is a decrease in semiconservative DNA replication. Alkaline velocity centrifugation of the DNA synthesized during a 1-min pulse under conditions of purine deprivation shows that approximately 50% of the newly replicated DNA is the size of Okazaki pieces. These are not incorporated into bulk DNA during a 1-h chase. If the purine supply is restored to the growth medium, these short DNA pieces are jointed to full-sized DNA within 1 h. DNA fiber autoradiolgraphy reveals a retardation in the rate of DNA replication fork movement but no apparent inhibition of initiation of synthesis on replication units within clusters actively engaged in replication. Our results indicate that purine deprivation specifically inhibits elongation of nascent dna chains.

Animals↗
PubMed1979

Characterization of hybrids between bovine (MDBK) and mouse (L-cell) cell lines.

Hypoxanthine-guanine phosphoribosyltransferase (HGPRT)-deficient mutants of a bovine kidney cell line (MDBK) were selected following mutagenesis with ethylmethane sulfonate or ICR-170G. MDBK mutants were hybridized to thymidine kinase-deficient L cells and selected in HAT medium. Parental and hybrid cells were characterized for isozyme patterns of lactic dehydrogenase malate dehydrogenase, glucose-6-phosphate dehydrogenase, and glutamate oxalate transaminase. Chromosomes of MDBK can be distinguished from mouse L cells by configuration and by fluorescent staining with Hoechst 33-258 stain. Hybrid cells contained both MDBK and L-cell chromosomes and had elevated DNA content. MDBK cells are normally restrictive for mengovirus replication. Both permissive and restrictive hybrids were found. Our data indicate that there was preferential loss of MDBK chromosomes in the hybrid cell lines.

Animals↗
PubMed1979

Lack of enhanced purine biosynthesis in HGPRT- and Lesch-Nyhan cells.

The rate of de novo purine biosynthesis was measured in a series of hypoxanthine guanine phosphoribosyl transferase deficient (HGPRT-) cells from a variety of sources, including human Lesch-Nyhan cells. Under optimum growth conditions, no enhanced purine biosynthesis was detected (in contrast to previous reports). An 'elevated' level of de novo purine biosynthesis could be detected in mutants following starvation for glutamine. However, this was the result of depression of purine biosynthesis in normal cells, with a resulting artifactual overproduction in mutants.

Animals↗
PubMed1978

Regulation of de novo purine biosynthesis in normal and 8-azaguanine-resistant Chinese hamster cells.

Azaguanine-resistant mutants of Chinese hamster ovary cells were isolated following mutagenesis with ICR-17OG. Of the eight mutant isolates examined, only one, Ag-5 had detectable hypoxanthine(guanine)phosphoribosyltransferase activity. Under normal conditions of growth, de novo purine biosynthesis in the mutants was not significantly different from wild type. However, when the cultures were starved for glutamine over a 2 h period before measuring 5'phosphoribosyl formylglycinamide (a relative measure of de novo purine biosynthesis), elevated levels of 5'-phosphoribosyl formylglycinamide accumulated in some of the mutants, and decreased levels in wild type and Ag-5. The level of purine biosynthesis could be related to the levels of glutamine in the pregrowth medium. The rate of purine biosynthesis correlated with 5-phosphoribosyl pyrophosphate levels, which were enhanced in the mutant (Ag-C) following the starvation period. No alterations were found in levels of 5-phosphoribosyl pyrophosphate synthetase or glutamine synthetase. The extent of feedback inhibition was normal in both mutant and wild type cells. These data suggest that the hypoxanthine (guanine) phosphoribosyltransferase locus is a regulatory gene.

Azaguanine↗
PubMed1978

Role of adenine phosphoribosyltransferase in adenine uptake in wild-type and APRT- mutants of CHO.

Adenine uptake in cultured Chinese hamster fibroblasts showed biphasic saturation kinetics. The transport system was highly specific for adenine and was competitively inhibited by adenosine. Utilizing mutant clones of Chinese hamster fibroblasts that have either reduced or negligible adenine phosphoribosyltransferase (APRT) activity, we found that (1) adenine was not accumulated against a concentration gradient in the absence of APRT activity and (2) after rapid initial uptake equal to that of the parent the rates of adenine accumulation found for the mutants correlated strongly with their residual APRT activities. Furthermore, using either artificially depressed phosphoribosylpyrophosphate pool size and APRT activities or the mutants with decreased APRT activity, we found that adenine transport was independent of phosphorylation by APRT. These studies suggest that adenine is transported as the free base by facilitated diffusion and is subsequently phosphorylated by APRT.

Adenine↗
PubMed1978

alpha-Amanithin-resistant mutants of Chinese hamster ovary (CHO) cells.

Spontaneous and EMS-induced alpha-amanitin-resistant CHO cells have been isolated and characterized. DNA-dependent RNA polymerase II in cell-free extracts from a mutant (ARM-1) was partially resistant to alpha-amanitin. Growing mutants for several generations in the presence or absence of alpha-amanitin did not change the pattern of inhibition. The mutants grew with a lag following transfer to medium with or without alpha-amanitin. The mutants have an altered RNA polymerase II, and possibly an altered cell membrane.

Amanitins↗
PubMed1978

Purification of adenine phosphoribosyltransferase by affinity chromatography.

The purine salvage pathway enzyme adenine phosphoribosyltransferase (AMP: pyrophosphate phosphoribosyltransferase EC 2.42.7) has been purified to greater than 85% homogeneity from crude rat liver 100,000 x g supernatant in one step by affinity chromatography. The enzyme binds to an AMP-agarose column and is eluted off the column by 1 mM 5-phosphoribosyl pyrophosphate with a 50 to 80% recovery. Enzyme kinetics indicate that the mechanism of the specific elution is due to competition of the product AMP and substrate 5-phosphoribosyl pyrophosphate for the same site on the enzyme.

Adenine Phosphoribosyltransferase↗
PubMed1977

Purineless death: ribosomal RNA turnover in a purine-starved ade- mutant of Chinese hamster cells.

When V79 pur 1, a purine-requiring auxotroph of a Chinese hamster cell line, is deprived of adenine, nucleic acid and protein synthesis decline rapidly. However, on continuous starvation RNA and DNA synthesis recommences to reach approximately 30% of the normal level between 12 to 24 h starvation. This is accompanied by a rise in the intracellular nucleotide pool. Utilizing mengovirus, which gives a productive infection in V79 pur 1 cells even under conditions of starvation, we can show that rRNA is preferentially degraded and provides the nucleotides for RNA synthesis. Thus "purineless" death in mammalian cells is accompanied by turnover of stable RNA.

Adenine↗
PubMed1977

Purine mutants of mammalian cell lines: III. Control of purine biosynthesis in adenine phosphoribosyl transferase mutants of CHO cells.

Spontaneous and mutagen-induced 2,6-diaminopurine-resistant mutants of Chinese hamster ovary (CHO-K1) cells were isolated. Such mutants fell into two classes: spontaneous and ethylmethane-sulfonate-induced mutants had approximately 5% wild-type adenine phosphoribosyl transferase (APRT) activity, whereas ICR-170G-induced mutants had barely detectable APRT activity. Since it has been reported that human hypoxanthine-guanine phosphoribosyl transferase (HGPRT) (Lesch-Nyhan syndrome) and APRT mutants over-produce purines, we examined the control and rate of purine biosynthesis in the Chinese hamster mutants. End product inhibition by adenine could not be demonstrated in such mutants, indicating that the active feedback inhibitor is a nucleotide rather than the free purine base, HGPRT activity was normal in all mutants examined except in one isolate. Purine biosynthesis as measured by the accumulation of the purine biosynthetic intermediate phosphoribosyl formylglycineamide was not elevated in the mutants as might have been predicted from work with Lesch-Nyhan cells. The data also suggest that our strain of CHO-K1 is physically or functionally haploid for the APRT locus.

Adenine Phosphoribosyltransferase↗
PubMed1976

Morphogenesis of picornaviruses: characterization and assembly of bovine enterovirus subviral particles.

Bovine enterovirus-I (BEV-I) infection results in the production of a low amount of infective virus. A large number of non-infectious virus particles can be detected in BEV-I lysates by haemagglutination. Attempts to isolate DI particles that might be responsible for this effect failed. However, infected cells were shown to contain large amounts of 80S particles as well as lesser amounts of 160S, 130S, 45S, 14S and 5S particles. The proportion of these subviral particles detectable by density gradient sedimentation depended on the ionic strength of the gradient buffer. At high ionic strength 130S particles were transformed into 160S particles, and 45S into 80S particles. The polypeptide composition of each virus particle was examined. Pulse-chase experiments confirmed that 80S particles were the predominant virus particles accumulating. No precursor-product relationship could be established for the 80S particle, although 5S and 14S particles were shown to be precursors of mature virus particles.

Animals↗
PubMed1975

Purine mutants of mammalian cell lines. II. Identification of a phosphoribosylpyrophosphate amidotransferase-deficient mutant of Chinese hamster lung cells.

A class of purine auxotrophs blocked early in the purine biosynthetic pathway was examined. The inability of these mutants to accumulate formylglycinamide ribotide (FGAR) in the presence of azaserine suggested that one or more of the first three enzymes of the pathway were either missing or defective. By direct enzyme assay, phosphoribosylpyrophosphate (PRPP) amidotransferase (E.C. 2.4.2.14) was found to be absent in extracts of mutant cells. Thus these cells were unable to convert PRPP to phosphoribosylamine (PRA). By reacting ribose 5-phosphate with ammonium ions, PRA was generated nonenzymatically in the incubation mixture, thus enabling us to test for the presence of the two enzymes required to convert PRA to FGAR. It was demonstrated that sufficient amounts of these enzymes, phosphoribosylglycineamide synthetase (E.C. 6.3.1.3) and phosphoribosylglycineamide formyltransferase (E.C. 2.1.2.2), were present in mutant extracts to allow synthesis of FGAR to occur once PRA was so provided.

Adenine↗