Search PubMed⌕ Search

Biomedical subjects

M W Taylor

Publications and source records attributed to M W Taylor.

At least 109 records · Page 6Linked to original sources
PubMed1984

Induction of gamma-interferon activity by elevated temperatures in human B-lymphoblastoid cell lines.

When the human B-lymphoblastoid cell lines GM3299 and GM4408 were heat shocked, elevated levels of antiviral activity were detected. This antiviral activity has been characterized as the result of de novo synthesis of interferon (IFN) gamma. Levels of antiviral activity as high as 20,000 units/ml per 10(6) cells were found after heat treatment. By using anti-IFN-alpha and anti-IFN-gamma sera, it was shown that the high antiviral activity in GM3299 after heat shock is due to a synergistic effect of constitutively produced IFN-alpha and induced IFN-gamma.

B-Lymphocytes↗
PubMed1983

High-frequency mutation at the adenine phosphoribosyltransferase locus in Chinese hamster ovary cells due to deletion of the gene.

Evidence for a two-step model to explain the high-frequency expression of the recessive phenotype at the autosomal adenine phosphoribosyltransferase (APRT; EC 2.4.2.7) locus in Chinese hamster ovary (CHO) cells was given by Simon et al. [Simon, A. E., Taylor, M. W., Bradley, W. E. C. & Thompson, L. (1982) Mol. Cell. Biol. 2, 1126-1133]. This model proposed a high-frequency event, leading to allelic inactivation or a loss of gene function, and a low-frequency event, causing a structural alteration of the APRT protein. Either event could occur first, resulting in two classes of heterozygotes. We have analyzed the low-frequency event that gave rise to the class 2 aprt heterozygote D416 and the high-frequency event that led to APRT- cells derived from D416. Genomic Southern blots of Msp I- or Hpa II-digested DNA from wild-type CHO, aprt heterozygote D416, and two APRT- cell lines derived from D416 indicate a loss of a specific Msp I/Hpa II recognition sequence at one aprt locus in the heterozygote that correlates with the production of the electrophoretically altered APRT protein found in D416. The APRT- mutants are homozygous for the loss of this Msp I/Hpa II site. By using an additional CHO gene as an internal control, it was determined that the APRT- mutants contain only a single copy of the altered aprt gene. Thus, the high-frequency event that produces APRT- mutants derived from D416 is not an inactivation event but rather a deletion of the wild-type aprt gene.

Adenine Phosphoribosyltransferase↗
PubMed1983

Mechanism of mutation at the aprt locus in Chinese hamster ovary cells: analysis of heterozygotes and hemizygotes.

A two-step model to explain the high frequency of mutation at the diploid adenine phosphoribosyltransferase (aprt) locus in CHO cells has been proposed previously (Simon et al., Mol. Cell. Biol. 2:1126-1133, 1982). This model indicates that two distinct classes of aprt heterozygotes can be isolated. Class 1 heterozygotes, the most abundant class, were defined as those which arose spontaneously and were capable of undergoing mutation to the APRT- phenotype only at a low frequency (putative point mutation). Class 2 heterozygotes arose from a mutation and gave rise at a high frequency to APRT- cells. This high-frequency event has been identified as a deletion of the wild-type allele (A. E. Simon and M. W. Taylor, Proc. Natl. Acad. Sci. U.S.A. 80:810-814, 1983). In this paper we report further analysis of class 1 heterozygotes with respect to genetic structure, gene products, and karyotype. Our study indicated that class 1 heterozygotes contain two different types of mutants. About half have only one copy of the aprt gene and an unaltered karyotype, indicating that a deletion (similar to the high-frequency second-step event observed for class 2 heterozygotes) rather than a loss of the chromosome was responsible for the generation of the aprt+/- genotype. The remainder of the previously designated class 1 heterozygotes still contained two copies of the aprt gene (within the limits of the quantitation technique used) and arose presumably by a point mutation. One of this group, D423, was characterized with respect to aprt gene products and found to produce an electrophoretic variant in addition to the wild-type protein. APRT- mutants derived from D423 retained the same number of aprt gene copies as D423 and still synthesized a protein that comigrated with wild type, unlike APRT- mutants derived from class 2 heterozygotes. D423 and the other heterozygotes with two aprt genes therefore did not fit into either class 1 or 2 and are now designated class 3. The model we present suggests that only one of the two aprt alleles present in wild-type cells can undergo the deletion.

2-Aminopurine↗
PubMed1983

A comparison of purine and pyrimidine pools in Bloom's syndrome and normal cells.

Nucleotide content of normal and Bloom's syndrome fibroblasts and lymphocytes were examined by reversed phase HPLC. The ATP/ADP ratio in primary cultures of normal human fibroblasts was at least three fold higher than in the primary cultures of Bloom's syndrome fibroblasts. After three months in culture the ratios of ATP/ADP of the Bloom's cells approach those of normal fibroblasts. Individual nucleotide measurements showed that initial differences did not reflect excess ADP, but rather very low levels of ATP in Bloom's syndrome fibroblasts. The amount of ATP increased gradually during culture. However, even after three months in culture, significant differences were noted in ATP levels between Bloom's syndrome fibroblasts and normal fibroblasts. Thus a defect in Bloom's syndrome is correlated with a defect in purine biosynthesis or ATP generation.

Adenine Nucleotides↗
PubMed1982

Rapid and reliable method for the analysis of nucleotide pools by reversed-phase high-performance liquid chromatography.

A rapid and reliable protocol for the simultaneous separation of ribo-, deoxyribo- and cyclic nucleotides has been developed using high-performance liquid chromatography on a C18 microBondapak column and isocratic elution with ammonium phosphate buffer 0.2 m, pH 5.1). Resolution of deoxyribonucleotides has been confirmed by performing resolution before and after periodate oxidation. The general order of elution is ribonucleotides, deoxyribonucleotides and cyclic nucleotides. While periodate oxidation improved the clarity of separation of deoxyribonucleotides by eliminating ribonucleotides, incorporation of methanol in the eluent shortened the retention time of cyclic nucleotides. The application of this method to a complex biological system is reported.

Chromatography, High Pressure Liquid↗
PubMed1982

Regulation of de novo purine biosynthesis in Chinese hamster cells.

Regulation of de novo purine biosynthesis was examined in two Chinese hamster cell lines, CHO and V79. De novo purine biosynthesis is inhibited at low concentrations of adenine. The mechanism of inhibition was studied using the RNA and protein synthesis inhibitors actinomycin D, cycloheximide, and azacytidine. Although all three inhibitors rapidly inhibited de novo purine biosynthesis in vivo, neither adenine nor the RNA and protein synthesis inhibitors could be found to have an effect in vitro on either phosphoribosylpyrophosphate (PRPP) synthetase or amido phosphoribosyltransferase, the first enzymes of the de novo pathway. However, in the presence of actinomycin D, cycloheximide, and azacytidine, there was a 50% or greater reduction in PRPP concentrations. This reduction in PRPP levels is correlated with a 2-fold increase in purine nucleotides in the acid-soluble pool. It is proposed that in the presence of the metabolic inhibitors there is an increase in nucleotide pools due to degradation of RNA, with a resulting feedback inhibition on de novo purine biosynthesis. In contrast to a previous report (Martin, D. W., Jr., and Owen, N. T. (1972) J. Biol. Chem. 247, 5477-5485), we could find no evidence for a repressor type mechanism in these cells.

Adenine↗
PubMed1982

Regulation of purE transcription in a purE::lac fusion strain of Escherichia coli.

A purE::lac fusion strain was isolated by using a special Mu phage developed by M. Casadaban. In the presence of adenine (100 micrograms/ml), beta-galactosidase synthesis was repressed by greater than 90%. beta-Galactosidase activity could be detected 6 to 8 min after the removal of adenine and increased linearly for at least 20 min. purR- mutants were isolated and synthesized 1.7- to 1.8-fold-higher levels of beta-galactosidase compared with purR+ cells. Azaserine derepressed purE transcription approximately 1.7-fold by lowering purine nucleotide pools. Glutamine and pyrimidine supplementation or starvation had no effect on purE transcription. A comparison of the rate of de novo purine biosynthesis and purE transcription indicated that the in vivo rate of de novo purine biosynthesis was more sensitive to the inhibitory effects of adenine than was transcription at the purE locus.

Adenine↗
PubMed1982

Mechanism of adenine toxicity in Escherichia coli.

The mechanism of adenine toxicity in an hpt gpt strain of Escherichia coli that is extremely sensitive to adenine inhibition was investigated. Adenine-resistant derivatives had secondary mutations in adeninephosphoribosyltransferase or the purR repressor. Growth studies with various purine salvage pathway mutants and the ability of guanosine to prevent adenine toxicity indicated that adenine exerts its toxic effects by depleting guanine nucleotide pools. In the presence of adenine, ATP pools increased twofold in wild-type cells and stabilized after 5 min. In contrast, ATP pools continued to rise in hpt gpt cells up to 25 min and increased sevenfold after adenine addition. hpt gpt cells were shown to have higher levels of adeninephosphoribosyltransferase than did the wild-type cells. In response to adenine addition, GTP pools dropped three- to fourfold in all strains tested. Although GTP levels returned to near normal values in wild-type cells after 35 min, no restoration of GTP pools was observed in the hpt gpt strain during this period. Measurements of guanine pools before and after the addition of adenine indicated that guaninephosphoribosyltransferase plays an important role in maintaining GTP pools by converting the free guanine to GMP during guanine nucleotide depletion.

Adenine↗
PubMed1982

Genetic instability at the adenine phosphoribosyltransferase locus in mouse L cells.

Resistance to adenine analogs such as 2,6-diaminopurine occurs at a rate of approximately 10(-3) per cell per generation in mouse L cells. This resistance is associated with a loss of detectable adenine phosphoribosyltransferase activity. Other genetic loci in L cells have the expected mutation frequency (approximately 10(-6)). Transformation of L cell mutants with Chinese hamster ovary cell DNA results in transformants with adenine phosphoribosyltransferase activity characteristic of Chinese hamster ovary cells. No activation of the mouse gene occurs on hybridization with human fibroblasts. That this high frequency event is the result of mutation rather than an epigenetic event is supported by antigenic and reversion studies of the 2,6-diaminopurine-resistant clones. These results are consistent with either a mutational hot-spot, a locus specific mutator gene, or a site of integration of an insertion sequence.

Adenine Phosphoribosyltransferase↗
PubMed1982

Model involving gene inactivation in the generation of autosomal recessive mutants in mammalian cells in culture.

We present evidence for a two-step model for expression of the recessive phenotype at the diploid adenine phosphoribosyl transferase (aprt) locus in Chinese hamster ovary cells. This model proposes a high-frequency event leading to allelic inactivation and a low-frequency event leading to a structural alteration of the APRT protein. Either event can occur first, resulting in two types of heterozygous cells. The proposed model is based on analysis of Chinese hamster ovary presumptive aprt heterozygotes and APRT- mutants, derived by two different laboratories. The major class of heterozygotes (class 1) had approximately 50% parental APRT activity, 50% immunologically precipitable APRT protein, and only wild-type enzyme as based on two-dimensional gel electrophoresis and thermal inactivation studies. We propose that one allele at the aprt locus has been inactivated in these heterozygotes. APRT- mutants derived from any single class 1 heterozygote arose at a low frequency and contained either no immunologically detectable APRT protein or an APRT enzyme which was, in most cases, demonstrably altered. The second class of heterozygotes, consisting of two independent isolates, gave rise to APRT- cells at a high frequency (10(-3) to 10(-5). These heterozygous cell lines had 50% of parental APRT activity and only wild-type spot, or wild-type and an electrophoretic variant spot, on two-dimensional gels. These aprt heterozygotes appear to have arisen by mutation at one allele. APRT- mutants derived from either heterozygote of this class had all lost the wild-type activity, consistent with the proposed model.

Adenine Phosphoribosyltransferase↗
PubMed1981

Selection for purine regulatory mutants in an E. coli hypoxanthine phosphoribosyl transferase-guanine phosphoribosyl transferase double mutant.

We have studied the relationship betwen purine salvage enzymes, 6-mercaptopurine resistance, and the purR phenotype in E. coli. Mutants resistant to 6-mercaptopurine were found to have defects in HPRT, the purR repressor, or in both. Analysis of these mutants led to the isolation of a hypoxanthine phosphoribosyl transferase-guanine phosphoribosyl transferase double mutant (hpt- gpt-) that is extremely sensitive to adenine. Two classes of adenine resistant mutants were isolated from this strain. The first class was deficient in APRT (apt-) while the second class represented purine regulatory mutants (purR-). There is thus selection for the purR phenotype in a hpt- gpt- background.

Adenine↗