Induction and repair of gene conversion in UV-sensitive mutants of yeast

Summary Photoreactivation effect on UV-induced allelic recombination has been examined using various combinations of leu 1 alleles in UV-sensitive and wild type diploid yeast, Saccharomyces cerevisiae. The frequencies of UV-induced heteroallelic reversion in UV-sensitive strains, presumably lacking dark-repair, are strikingly enhanced compared to those in wild type at the same doses under dark condition. However, these enhanced frequencies of reversion are diminished by photoreactivation almost to the level of those in wild type. The induced frequencies of homoallelic reversion (mutation) of relevant alleles are apparently lower than those of heteroallelic reversion. Phenotypic analysis for linked gene leu 1 on UV-induced heteroallelic revertants has shown that most of the revertants are of the nonreciprocal type recombination (mitotic gene conversion). These results would indicate that most of the dark-repairable damage leading to mitotic gene conversion after UV-light is due to pyrimidine dimers.On leave of absence from Radiation Center of Osaka Prefecture, Shinke-cho Sakai, Osaka, Japan.     

Evidence for intrachromosomal gene conversion in cultured mouse cells

In this report we present an experimental scheme that facilitates the study of homologous recombination between closely linked genes in cultured mammalian cells. Two different Xho I linker insertion mutants of the herpes simplex virus type 1 thymidine kinase (HTK) gene were introduced into mouse LTK^? cells as direct repeats on a plasmid carrying a dominant selectable marker. Following stabilization of these sequences in the recipient cell, selection for TK⁺ was applied to detect recombinational events between different TK^? genes. TK⁺ segregants were observed at a frequency of 10^?4–10^?5 in lines harboring both mutant genes. Control lines carrying only one type of mutant HTK gene yielded TK⁺ cells at frequencies of 10^?7 or less. Physical analysis of the TK⁺ segregants has revealed the presence of an apparently normal HTK gene that is resistant to Xho I endonuclease digestion in each TK⁺ line examined. Analyses of the TK gene pairs before and after recombination suggest that at least 50% of the recombinants are the result of nonreciprocal exchanges of genetic information, or gene conversion events.     

Induction of glycophorin gene expression in cultured murine erythroleukemia cells

In order to identify the mRNA for mouse glycophorin, mRNA was isolated from immature erythroid cells obtained from the spleens of anemic mice, translated in vitro in mRNA-dependent rabbit reticulocyte lysate, and then immunoprecipitated with a specific antiserum. Glycophorin mRNA was shown to be present only in erythroid cells. In immunofluorescent and in vitro translation studies, it was shown that glycophorin mRNA is absent in uninduced murine erythroleukemia (MEL) cells, but is induced in dimethylsulfoxide-treated differentiating cells.     

Induction of adipose conversion by mouse serum in cultured cells and its characterization

Mouse serum (MS) effected a rapid accumulation of many lipid droplets by cultured cells in the growing or resting state. MS-induced adipose conversion in all of 12 randomly selected cell lines, including human, mink, rat, and mouse cells and almost all of the cells in a culture dish were converted. Under excessive amounts of MS, the cells became mature adipocytes, lost the ability to divide and soon died. However, proliferation of adipocytes induced by smaller quantities of MS was not different from that of control cells in calf serum (CS). When adipose conversion developed, oncorna virus producing cells ceased virus production and there was a clear connection between decrease of virus production and rate of adipose conversion. The adipose conversion of 3T3-FL cells grown for 7 days in the presence of bromodeoxyuridine (BUdR) was inhibited. Actinomycin D (actD) and cycloheximide also inhibited adipose conversion. It is suggested that the cells may have an inherent ability to differentiate into adipocytes.     

Fidelity of meiotic gene conversion in yeast

Summary Gene conversion was studied in a sample of 3869 unselected meiotic tetrads obtained from three diploids, respectively; heterozygous for a single ochre mutant, heteroallelic for a pair of ochre alleles, and heterozygous for an ochre specific suppressor. Although the genetic system were sufficiently sensitive to detect single base changes at the mutant codon level, none were found among 36 conversions (1+:3m) of the ochre mutants and 153 conversions (3S:1+) of the suppressor locus. These findings lead to the conclusion that the informational transfer in gene conversion occurred with complete fidelity. Gene conversion conserved and did not generate new genetic information. The error level of conversion was estimated as less than 10^-2/N.P.     

Mutagenesis in cultured mammalian cells. II. Induction of gene mutations in Chinese hamster cells

Mutations controlling the resistance to 6-mercaptopurine (6-M) and the ability to multiply in a medium with a low concentration of glucose (“glucose-independent” mutants) were induced in cultured Chinese hamster cells by N-nitrosomethylurea (NMU), 5-bromodeoxyuridine (BUdR), UV and X-rays. The chemical agents were found to be very active in induction of mutations to 6-M resistance (NMU and BUdR) and mutations of “glucose independence” (NMU). These agents increase the yield of mutations as compared to the spontaneous mutation rate by about two orders of magnitude. The induced rate of 6-M-resistant mutations by X-rays was 2.0 ? 10⁻⁷ per viable cell per roentgen. BUdR approximately equally increases the cell's sensitivity to both inactivating and mutagenic action of X-rays. The maximum induction of mutations to 6-M resistance by UV was observed at 100 erg/mm². This dose leads to 1 16-fold increase of the mutation frequency as compared to the spontaneous rate. Further increase of the UV dose up to 200 erg/mm² resulted in a lower yield of mutations per dose unit. The highest yield of mutations to 6-M resistance induced by NMU, BUdR and X-rays was observed if cells were plated in selective medium several generations after the mutagenic treatment. The maximum yield of mutations to 6-M resistance induced by UV and of glucose-independence induced by NMU was recorded if cells were transferred to selective media immediately after treatment. The kinetics of expression of mutations and the decline of their number observed after prolonged incubation of treated cells in nonselective conditions are discussed.     

Induction of gamma-glutamyltransferase in cultured brain cells

gamma-Glutamyltransferase is a membrane-bound enzyme widely distributed in animal tissues. This enzyme is involved in glutathione metabolism, but its exact biological function is still an open question. In rat brain cells in culture a depletion of L-cystine and L-glutamine or the addition of diethyl maleate to the culture medium leads to a decrease of intracellular glutathione concentration and to an increase of the specific activity of gamma-glutamyltransferase. The induction of the enzyme is inhibited by the addition of cycloheximide or actinomycin D to the culture medium.     

Unequal crossing-over and gene conversion at the amplified CUP1 locus of yeast

Summary Meiotic recombination was analyzed between two twelve-copy arrays of a gene amplification at theCUP1 locus ofSaccharomyces cerevisiae. Utilizing Southern analysis to identify spores with non-parental repeat arrays, we find that approximately 11% of a sample with 202 unselected tetrads possess at least one nonparental spore array. Both reciprocal and non-reciprocal changes are observed. The data suggest a model in which frequent mispairing among identical copies of the 2.0 kb repeat unit leads to the formation of unpaired loops containing integral numbers of repeat units. In this model, conversions involving the loops lead to non-reciprocal changes in arrays: about half are associated with reciprocal exchange, and net increases in repeat unit numbers occur about as frequently as net decreases. Thus, the known properties of gene conversion can account for all the segregations we observe.     

Induction by ionizing radiation of the gadd45 gene in cultured human cells: lack of mediation by protein kinase C.

The effect of ionizing radiation on the expression of two DNA-damage-inducible genes, designated gadd45 and gadd153, was examined in cultured human cells. These genes have previously been shown to be strongly and coordinately induced by UV radiation and alkylating agents in human and hamster cells. We found that the gadd45 but not the gadd153 gene is strongly induced by X rays in human cells. The level of gadd45 mRNA increased rapidly after X rays at doses as low as 2 Gy. After 20 Gy of X rays, gadd45 induction, as measured by increased amounts of mRNA, was similar to that produced by the most effective dose of the alkylating agent methyl methanesulfonate. No induction was seen after treatment of either human or hamster cells with 12-O-tetradecanoylphorbol-13-acetate, a known activator of protein kinase C (PKC). Therefore, gadd45 represents the only known mammalian X-ray-responsive gene whose induction is not mediated by PKC. However, induction was blocked by the protein kinase inhibitor H7, indicating that induction is mediated by some other kinase(s). Sequence analysis of human and hamster cDNA clones demonstrated that this gene has been highly conserved and encodes a novel 165-amino-acid polypeptide which is 96% identical in the two species. This gene was localized to the short arm of human chromosome 1 between p12 and p34. When induction in lymphoblast lines from four normal individuals was compared with that in lines from four patients with ataxia telangiectasia, induction by X rays of gadd45 mRNA was less in the cell lines from this cancer-prone radiosensitive disorder. Our results provide evidence for the existence of an X-ray stress response in human cells which is independent of PKC and which is abnormal in taxia telangiectasia.     

Induction of human high K(M) 5'-nucleotidase in cultured 293 cells

Human 293 cells were stably transfected with a plasmid introducing a receptor for the ecdysone analog muristerone. The cells were further stably transfected with muristerone-inducible expression vectors carrying either the cDNA for the human high K(M) 5'-nucleotidase or the coding sequence of the nucleotidase linked to the 5'-end of the sequence for the green fluorescent protein. Upon induction, both types of transfectants overproduced nucleotidase activity in a time- and dose-dependent manner. Western blots gave values close to the expected subunit molecular masses of 65 and 92 kDa, respectively, excluding processing of the induced proteins. Cells induced to overexpress the nucleotidase showed a decreased growth rate and contained smaller pools of each of the four common ribonucleoside triphosphates. They showed no increased resistance to the toxicity of 2-chlorodeoxyadenosine.     

Measuring the toxic effects of high gene dosage on yeast cells

 A novel method, which is rapid, reliable and quantitative, is presented for measuring the toxic effects on yeast cells of high dosage of any given gene. It is based on the possibility of monitoring the presence in cells of a plasmid carrying the ADE2 gene from Saccharomyces cerevisiae by direct observation of colonies, the construction of this particular plasmid being easily made by marked homologous recombination in yeast. Four yeast regulatory genes tested were found to result in various degrees of toxicity at high dosage. Possible implications of the measurement of gene toxicity for eukaryotic cell regulatory mechanisms and for the use of novel general approaches to gene selection, such as the gene-gene interference method, are discussed. Received: 18 March 1996 / Accepted: 23 August 1996     

Determination of radiation equivalence of ethyl methanesulphonate (EMS) for induction of gene conversion in diploid yeast.

A unit Rad-Equivalent Chemical (REC) has been suggested for purposes of quantitating the mutagenic hazards of chemicals. The usefulness of this approach is demonstrated by the establishment of a constant relationship between the forward mutation frequency and haploid genome size in various organisms for both radiation and chemical EMS. However, it is necessary to determine the radiation equivalence of chemicals in as many organisms and for as many end-points as possible. For end-points we are limited to forward mutations. Another relevant genetic end-point of interest in this regard is gene conversion which can also monitor any kind of DNA damage in a suitable diploid system. Hence, we have determined the REC value for EMS in diploid yeast with gene conversion as the end-point. This agrees well with the REC values estimated in a number of organisms with forward mutation as the end-point. This finding further underlines the generality of the REC concept.     

Induction of gene mutations and chromosomal aberrations by simian virus 40 in cultured mammalian cells

• Induction of gene mutations by SV40 was studied in aneuploid human and Chinese hamster cells. In Chinese hamster cells SV40-induced chromosome aberrations were also studied.

• SV40 penetrated into the cells of both lines and induced synthesis of the T antigen. The efficiency of infection in Chinese hamster cells was tested additionallby their ability to form colonies in medium lacking the serum growth factor. The maximal number of cells with growth factor independence was observed on the first day after infection. When hamster cells had been maintained in “factor-free medium” for the first two passages after infection a sub-line was isolated, which synthesized T antigen 60 days after exposure to SV40. This was considered to be an indirect proof of the integration of viral genome into host chromosome.

• A significant increase in the frequency of chromosomal aberrations was detected in SV40-infected Chinese hamster cells. It was observed on the first and second days after treatment. The most numerous were the chromosome and chromatid breaks, which were distributed randomly in 5 morphological groups according to the chromosome length.

• SV40-induced mutations of resistance to 8-AG and 6-MP in human and Chinese hamster cells respectively were detected, when cells were plated in selective medium one to five days after infection. Induction was detected in all the 4 experiments with human cells and in 9 out of 11 experiments with Chinese hamsters cells. Induction was highly significant according to the Wilcoxon test (P>0.99), when the results of all experiments carried out in human and Chinese hamster cells were summarized. Resistance was stable after prolonged cultivation of 13 isolated clones under non-selective conditions.

• It is suggested that viral genome integration, gene mutations and chromosomal aberrations may have common molecular mechanisms. The role of gene mutations in virus-induced carcinogenesis is discussed.

Induction of apoptosis in cultured colon cancer cells by transfection with human interferon beta gene

The growth of SW480 colon cancer cells following the transfection with the human interferon beta (hIFNbeta) gene entrapped in cationic multilamellar liposomes was effectively inhibited, but not that of the cells transfected with the gene from which the secretion signal sequence of hIFNbeta had been deleted. The amount of hIFNbeta secreted in the medium from SW480 cells transfected with hIFNbeta gradually increased and became maximum 3 days after the transfection, but no hIFNbeta was detected in the medium of the cells transfected with the secretion signal-deleted hIFNbeta. These findings indicate that the growth inhibition of SW480 cells after the transfection with hIFNbeta was caused by hIFNbeta secreted from the transfected cells. At that time, SW480 cells were induced to undergo apoptosis, which was identified by morphological aspects, viz., chromatin condensation, nuclear segmentation, and nucleosomal DNA fragmentation. The hIFNbeta-induced apoptosis was found to be linked to the activation of caspases 3 and 8 as evidenced by immunoblot, enzymological, and cell death inhibition analyses.