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L H Thompson

Publications and source records attributed to L H Thompson.

At least 145 records · Page 8Linked to original sources
PubMed1982

Role of DNA repair in mutagenesis of Chinese hamster ovary cells by 7-bromomethylbenz[a]anthracene.

The role of DNA repair in mutagenesis was studied in normal, repair-proficient Chinese hamster ovary cells and in two mutant strains that are deficient in excision repair. By using the mutagen 7-bromomethylbenz[a]anthracene (7-BrMeBA) and the technique of alkaline elution of DNA, the mutants were found to be defective at or before the incision step of excision repair. Dose--responses were determined for cell killing, mutation induction at three loci, and sister chromatid exchanges over a survival range of 1.0--0.1 after 7-BrMeBA treatment. The mutants were 5-fold more sensitive to killing than were the normal cells, but the degree of hypersensitivity to mutation induction varied depending on the mutant strain, the genetic marker, and the dose of mutagen. In each instance, the dose--response curve for mutations was essentially linear in the repair-deficient cells. In the normal cells, however, the curves for induced resistance to thioguanine and azaadenine were complex and were curvilinear with increasing slope at low doses. This behavior may be attributable to saturation of the excision repair system. No difference was seen in the efficiency of inducing ouabain-resistant mutations in the repair-deficient cells compared to the normal cells, indicating a qualitatively different behavior of this marker. These results are consistent with excision repair of 7-BrMeBA damage being error-free in Chinese hamster ovary cells. Sister chromatid exchange, another manifestation of DNA damage, also was induced with greater efficiency in the repair-deficient cells.

Animals↗
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↗
PubMed1982

Sister chromatid exchange and gene mutation.

The parallel induction of sister chromatid exchange (SCE) and single-gene mutation was quantified in Chinese hamster ovary cells following exposure to nine different physical or chemical agents representing a wide variety of inducing lesions. For each agent, the frequency of induced SCE was linearly related to the induction of mutation, but the efficiency of SCE formation relative to mutation induction differed for each. Studies in repair deficient cells suggested that unrepaired lesions enhanced the induction of both SCE and mutation but that the degree of enhancement was not necessarily the same for both endpoints.

Animals↗
PubMed1982

Defective removal of DNA cross-links in a repair-deficient mutant of Chinese hamster cells.

To further understand the relationships between DNA damage, DNA repair, and cellular end points such as survival and mutation, the repair capacity of a DNA repair-deficient mutant (strain UV-20) of Chinese hamster ovary cells was characterized in response to DNA cross-linking agents. This mutant, previously shown to be hypersensitive to killing by both ultraviolet light and the cross-linking agent mitomycin C, was also found to be extremely sensitive to cis-diamminedichloroplatinum, another DNA cross-linking agent. The efficiency of DNA cross-link removal after treatment with mitomycin C or cis-diamminedichloroplatinum was measured using the technique of alkaline elution and compared in wild-type Chinese hamster ovary cells and strain UV-20. Wild-type cells removed 80 or 95% of the cross-links within 24 hr after treatment with cis-diamminedichloroplatinum or mitomycin C, respectively. In contrast, UV-20 cells, which were equally as susceptible to cross-link damage as were wild-type cells, removed only a small proportion of the cross-links made by either agent. These results emphasize the importance of DNA repair processes in modulating the cytotoxic effects of chemicals that produce DNA cross-link damage and suggest that cross-link repair in Chinese hamster ovary cells is controlled by a pathway that also repairs damage from ultraviolet radiation.

Animals↗
PubMed1981

Genetic diversity of UV-sensitive DNA repair mutants of Chinese hamster ovary cells.

Mutant lines of Chinese hamster ovary cells that show hypersensitivity to killing and mutagenesis by UV light were analyzed by genetic complementation analysis to determine whether defects in different gene loci might underlie a common cellular phenotype,. To facilitate rapid screening of mutant clones, a procedure was devised that allowed presumptive complementation to be assessed on the basis of the frequency of UV-resistant cells after fusion by polyethylene glycol. Four classes were identified among 44 clones tested. By using drug-resistance markers for selection of hybrid cells in crosses between UV mutant and wild type, a mutant from each of the four classes was shown to behave as phenotypically recessive. Hybrids were also isolated from crosses between each of the pair combinations of the four mutants. All such hybrids were relatively resistant to UV killing, providing confirmation of the complementation classes. When mutants representing the four UV-complementation classes were tested with the polyaromatic hydrocarbon 7-bromomethylbenz(a)anthracene, complementation was again seen for all pair combinations. These results suggest that each class of mutants represents a biochemical defect that plays a common role in the repair of both UV-induced and chemically induced lesions in the DNA.

Animals↗
PubMed1981

Estrus and fertility in lactating sows and piglet performance as influenced by limited nursing.

Seven trials were conducted with 126 sows and their litters to determine the influence of limited nursing (LN), starting on day 21 of lactation, on estrous activity and conception rate during lactation and on piglet performance. One-half of the sows in each of the first three trials were allowed to nurse their piglets four times daily for 30-min periods for 12 days. In the last four trials, half of the sows nursed their piglets four times daily for the first 3 days and then three times daily for 4 days (7-day limited nursing). The remaining sows in each trial were allowed to nurse their piglets in a normal manner. All piglets were weaned at the end of the LN period. Piglets were weighed at the start of the study, at weaning and at 14 days postweaning. Creep intake was measured during the LN period. In the first three trials, eight of the 26 LN sows were in estrus before weaning, whereas no control (C) sows were in estrus during lactation (P greater than .01). The average interval from weaning to breeding was 1.7 and 6.6 days for LN and C sows, respectively (P greater than .01). In the last four trials, five of 37 LN sows were in estrus before weaning, whereas no C sows were in estrus during lactation (P greater than .07). The average interval from weaning to estrus was 3.3 and 4.3 days for LN and C sows, respectively, (P greater than .05). Conception rate did not differ between trials or between treatment groups. Weight gain during the treatment period was lower and creep feed intake greater (both P greater than .01) for pigs reared by LN sows than for piglets reared by C sows. Weight gained after weaning was greater (P greater than .05) for piglets reared by LN sows than for those reared by C sows in the first three trials, but was similar for the two groups in the following four trials. Piglet death loss was similar for the two groups throughout the experiment.

Animal Population Groups↗
PubMed1980

A screening method for isolating DNA repair-deficient mutants of CHO cells.

A simple procedure for isolating mutagen-sensitive clones of CHO cells was developed and applied in mutant hunts in which colonies were screened for hypersensitivity to killing by ultraviolet radiation (UV, ethyl methanesulfonate (EMS), or mitomycin C (MMC). Each of two UV-sensitive clones studied in detail had a D37 dose of 1.0 J/m2 compared to 7.0 J/m2 for the wild-type cells, and each was shown to have no detectable repair replication following exposure to UV doses of up to 26 J/m2. Although these mutants resemble xeroderma pigmentosum human mutants with respect to their repair defect and cross-sensitivity to the carcinogen 4-nitroquinoline-1-oxide, one of two clones (UV-20) is characterized by extreme hypersensitivity to MMC (80-fold as compared to the wild type). Clones having hypersensitivity to alkylating agents, but not UV, were obtained using MMC and EMS. In the latter case the two clones had significantly increased sensitivity to the killing action of 60Co gamma-rays.

Animals↗
PubMed1980

Validation of conditions for efficient detection of HPRT and APRT mutations in suspension-cultured Chinese hamster ovary cells.

Conditions for reliable and efficient assay of mutations affecting the activity of HPRT (hypoxanthine phosphoribosyltransferase EC 2.4.2.8) and APRT (adenine phosphoribosyltransferase EC 2.4.2.7) have been determined for a strain of CHO (Chinese hamster ovary) cells that has been adapted for rapid growth both in suspension culture and in monolayer. To facilitate measurement of mutation at the aprt locus, clones were derived that are presumptively heterozygous at that locus. At a limiting concentration of 8 microgram/ml of azaadenine, 14/16 of the resistant clones picked and tested had approximately 1/2 of the APRT activity of the wild-type cells. One such clone, strain AA8, was chosen for further studies and found to be readily mutable to resistance to 80 microgram/ml azaadenine. Most of the highly resistant colonies isolated (21/24) had very low in vitro APRT activity. The optimal conditions for detection of TGr and AAr mutations were determined for two critical parameters, expression time and cell density. Cultures treated with mutagen either in monolayer or in suspension were allowed to express mutations in suspension. The expression of mutations induced by UV light, EMS, and ICR-191 was complete by 3 days for AAr and by 4-5 days for TGr. The time required to reach a maximal frequency of mutants was essentially independent of the type of mutagen and the level of survival after treatment. Induced mutation frequencies for both loci were notably stable during the time intervals examined. With respect to cell-density conditions, both markers were detected at frequencies that were independent of the cell inocula over the range of 1 x 10(5) to 1 x 10(6) cells per 100-mm petri dish (i.e. 1.6 x 10(3) to 1.6 x 10(4) cells/cm2) containing 20 ml of medium. These results were obtained with both mutagenized populations and with reconstructed mixtures obtained by adding drug-resistant cells to varying numbers of wild-type cells. The rapid expression of mutations for both markers, particularly AAr, combined with the advantage that large inocula can be plated for selection of mutants, make this CHO strain an attractive system for the simultaneous measurement of mutations at the autosomal aprt and X-linked hprt loci.

Adenine↗
PubMed1980

Failure of the phorbol ester 12-O-tetradecanoylphorbol-13-acetate to enhance sister chromatid exchange, mitotic segregation, or expression of mutations in Chinese hamster cells.

The potent tumor promoter 12-O-tetradecanoylphorbol-13-acetate (TPA) was tested for its ability (a) to induce sister chromatid exchange, (b) to increase the rate of transition at the adenine phosphoribosyltransferase (apt) locus from the presumptive heterozygous state ((+/- to the homozygous state (-/ - or -), and (c) to enhance the frequency of mutations expressed after ultraviolet radiation mutagenesis. We have found no significant effect of TPA in any of these experiments. Sister chromatid exchange frequencies in both V79 and Chinese hamster ovary cells remained unchanged by TPA treatment under various conditions, a result inconsistent with the hypothesis that an important effect of TPA might be to increase the rate of chromosomal mitotic recombination (and hence segregation of recessive mutations) in a manner akin to increased chromatid recombination. We have also been unable to obtain evidence for mitotic recombination affecting the aprt locus in Chinese hamster ovary cells for which the rate of change to a high level of resistance to azaadenine was measured. The rate of 8.6 X 10(-7) mutation (and/or segregations) per cell generation assessed by fluctuation analysis was not increased by the continuous presence of TPA, 4 microgram/ml, in the medium. In the third set of experiments, mutant frequencies in Chinese hamster ovary cells after ultraviolet mutagenesis were measured for the markers ouabain resistance, thioguanine resistance, and azaadenine resistance, under conditions with and without pretreatment with TPA before mutant selection. No convincing enhancement in mutation expression was observed. In summary, these results argue that promotion by TPA does not proceed by a mechanism involving genetic recombination or the altered expression of newly mutated alleles.

Animals↗
PubMed1979

Isolation and characterization of revertants of the mammalian temperature sensitive leucyl-tRNA synthetase mutant tsHl.

Nine spontaneous and seven ethyl methanesulfonate induced revertants of the Chinese hamster ovary cell line mutant (tsHl), which possesses a temperature sensitive leucyl-tRNA synthetase, were isolated and characterized with respect to growth rate, leucyl-tRNA synthetase activity and thermolability, intracellular leucine pool size, and rRNA content. Although most revertants had increased leucyl-tRNA synthetase activity, and of those tested, all but one had increased thermostability, each appears to be unique. One revertant may be an intergenic suppressor since it appears to contain an elevated level of tsHl-like synthetase. There was no evidence for any of the revertants having increased rRNA and tRNA contents, however, many showed leucine pools two to three times larger than wild type cells. Since similar increases have been observed in tsHl cells they are believed to result from regulation of leucine pool size by the leucyl-tRNA synthetase and are of a magnitude sufficient to affect significantly the growth of revertants at 38.5 degrees C.

Amino Acyl-tRNA Synthetases↗
PubMed1979

Relationship between histidyl-tRNA level and protein synthesis rate in wild-type and mutant Chinese hamster ovary cells.

A preliminary investigation was carried out to determine how conditional lethal mutants affected in particular aminoacyl-tRNA synthetases may be used to study the role of tRNA charging levels in protein synthesis. The relationship between rate of protein synthesis and level of histidyl-tRNA in wild-type cultured Chinese hamster ovary cells was determined using the analogue histidinol to inhibit histidyl-tRNA synthetase activity. This response was compared with that obtained using a mutant strain with a defective histidyl-tRNA synthetase that phenotypically shows decreased rates of protein synthesis at reduced concentrations of histidine in the growth medium. The approach used was based on measuring the histidyl-tRNA levels in live cells. The percentage charging was estimated by comparing [14C]histidine incorporated into alkali-labile material in paired samples, one of which was treated with cycloheximide, five minutes before terminating during the incubation, to produce maximal aminoacylation. Wild-type cells under histidinol inhibition exhibited a sensitive, sigmoidal relationship between the level of histidyl-tRNA and the rate of protein synthesis. A decrease in the relative percentage of acylated tRNA (His) from 46% to 35% elicited a large reduction in the rate of protein synthesis from 90% to 30% relative to untreated cells. An unpredicted result was that the relationship between protein synthesis and histidyl-tRNA in the mutant was essentially linear. High acylation values for tRNA (His) were associated with rates of protein synthesis that were not nearly as high as in wild-type cells. These findings suggest that the charging charging levels of tRNA (His) isoacceptors could play a regulatory role in determining the rate of protein synthesis under conditions of histidine starvation in normal cells. The mutant appears to be a potentially useful system for studying the pivotal role of tRNA charging in protein synthesis, assuming that the altered response in the mutant is caused by its altered synthetase.

Animals↗
PubMed1979

DNA crosslinking, sister-chromatid exchange and specific-locus mutations.

Chinese hamster ovary cells were treated with the DNA-crosslinking chemicals, mitomycin C (MMC) and porfiromycin (POR), and their monofunctional derivative decarbamoyl mitomycin C (DCMMC). After exposure, the cells were studied for the induction of sister-chromatid exchanges (SCEs) and mutations at the hypoxanthine phosphoribosyltransferase and adenine phosphoribosyltransferase loci. The frequency of SCEs varied significantly in successive sampling intervals, requiring the weighting of each interval by the percentage of second-division mitosis in that interval to obtain the mean SCE frequency for each dose. All 3 compounds were potent inducers of SCEs but weakly mutagenic. All 3 chemicals by concentration were approximately equally effective in inducing SCEs or mutations. When the induced SCEs and mutations were compared at equal levels of survival, DCMMC was slightly more effective than MMC or POR in inducing SCEs and somewhat less mutagenic. These results indicate that the DNA interstrand crosslink is not the major lesion responsible for the induction of SCE or mutation by these compounds.

Adenine Phosphoribosyltransferase↗
PubMed1978

Evidence for structural gene alterations affecting aminoacyl-tRNA synthetases in CHO cell mutants and revertants.

Aminoacyl-tRNA synthetase (aaRS) activities in extracts of mutant strains of the Chinese hamster ovary line (CHO) were examined for alterations in thermal stability. Mutants having low activity for MetRS, AsnRS, or GlnRS contained aaRSs that were inactivated much more rapidly upon heating than those from wild-type cells. Revertant lines, isolated from cultures of these mutants (Asn-5, Met-2, and Gln-2) after treatment with nitrosoguanidine or ethyl methanesulfonate, had thermolabilities intermediate between mutant and wild-type, and consistently had higher activities than the mutants. With a modified in vivo aminoacylation procedure, two previously exceptional mutants. Arg-1 and His-1, showed pronounced reductions in the amount of arginyl-tRNA or histidyl-tRNA, respectively, under restrictive conditions, compared to wild type. Revertants of Arg-1 (like the mutant itself) had no measurable ArgRS in vitro activity (less than 0.4% of wild type) although in vivo aminoacylation in the one revertant tested was partially restored. These data provide evidence that the forward mutations have occurred in the structural genes of the aaRSs and that most of the reversions are probably the result of second-site point mutations in the aaRS genes.

Amino Acyl-tRNA Synthetases↗
PubMed1978

Six complementation classes of conditionally lethal protein synthesis mutants of CHO cells selected by 3H-amino acid.

Using a tritiated amino acid suicide procedure designed specifically to select conditional protein synthesis mutants, we have isolated and characterized a large number of such mutants of Chinese hamster ovary cells. All of the mutants are genetically stable and behave as recessives in somatic cell hybrids. Most of the new mutants are phenotypically dependent on the concentration of a specific amino acid as well as on temperature. In addition to identifying many additional leucyl- and asparagyl-tRNA synthetase mutants, complementation analysis has distinguished four new genetic classes representing methionine-, glutamine-, histidine-, and arginine-dependent mutants. Biochemical characterization of representative mutants from each of these six classes has identified the primary lesions as being defective aminoacyl-tRNA synthetases. Our selection results further demonstrate the high specificity of the 3H-amino acid procedure for isolating protein synthesis mutants. Reconstruction experiments performed with two representative mutants indicated a selection efficiency of approximately 10% under standard conditions.

Amino Acids↗