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The rad10, rad16, rad20, and swi9 mutants of the fission yeast Schizosaccharomyces pombe, isolated by their radiation sensitivity or abnormal mating-type switching, have been shown previously to be allelic. We have cloned DNA correcting the UV sensitivity or mating-type switching phenotype of these mutants and shown that the correcting DNA is encompassed in a single open reading frame. The gene, which we will refer to as rad16, is approximately 3 kb in length, contains seven introns, and encodes a protein of 892 amino acids. It is not essential for viability of S. pombe. The predicted protein is the homolog of the Saccharomyces cerevisiae RAD1 protein, which is involved in an early step in excision-repair of UV damage from DNA. The approximately 30% sequence identity between the predicted proteins from the two yeasts is distributed throughout the protein. Two-hybrid experiments indicate a strong protein-protein interaction between the products of the rad16 and swi10 genes of S. pombe, which mirrors that reported for RAD1 and RAD10 in S. cerevisiae. We have identified the mutations in the four alleles of rad16. They mapped to the N-terminal (rad10), central (rad20), and C-terminal (rad16 and swi9) regions. The rad10 and rad20 mutations are in the splice donor sequences of introns 2 and 4, respectively. The plasmid correcting the UV sensitivity of the rad20 mutation was missing the sequence corresponding to the 335 N-terminal amino acids of the predicted protein. Neither smaller nor larger truncations were, however, able to correct its UV sensitivity.

We have constructed a variety of pUC-based vectors designed for maintenance in Schizosaccharomyces pombe. These can be used for both gene bank construction and subcloning. Plasmids pUR18 and pUR19 are modifications of pUC vectors containing the Sc. pombe ars1 and ura4 sequences and retaining the lacZ XGal blue-white selection system for screening for DNA inserts. These vectors have been used to construct representative Sc. pombe and Saccharomyces cerevisiae genomic libraries. To assist in the creation of gene deletions, we have constructed another two plasmids. Combined with the technique of partially filling-in 5' overhangs created with restriction enzymes, these plasmids simplify the replacement of all or part of an open reading frame by a functional ura4 gene. Furthermore, such constructs can be excised with SfiI as a linear fragment for use in Sc. pombe transformations. When integrated into the Sc. pombe genome, the site of integration can be easily mapped by pulsed-field gel electrophoresis using the presence of a novel NotI site.

We have used the SV40-based shuttle vector pZ189 to determine ultraviolet mutation spectra in SV40-transformed cell lines from two patients with Cockayne's syndrome (CS) and ataxia telangiectasia (AT). The shuttle vector was UV-irradiated, transfected into the cells and recovered two days later, after many rounds of replication had occurred. Plasmid DNA was used to transform indicator bacteria in which plasmids containing a mutation in the supF gene resulted in white colonies. Mutant plasmids were analysed both by agarose gels and by DNA sequencing. In contrast to published spectra for xeroderma pigmentosum cells, the types of mutation induced by UV mutation in the CS and AT cell lines were similar to each other and to published spectra for normal cell lines. There were however, some differences in the sequence distribution of the mutations.

We have carried out molecular and biochemical analyses on several spontaneous and mutagen-induced ouabain-resistant mutants from the mouse lymphoma cell line L5178Y. Mutant cells were much more resistant than wild-type cells to the toxic effects of ouabain. The gross structures and copy numbers of two ouabain-resistant genes were unaltered in the mutants, as was the expression of the Na,K-ATPase gene. Uptake of 86Rb+ was more resistant to ouabain in the mutants than in wild-type cells. There was no evidence for an inducible uptake mechanism. The Na,K-ATPase activity in extracts of the mutants was, in all cases examined, more resistant than wild-type cells to the inhibitory action of 10(-3) M ouabain. At 10(-5) M ouabain, the Na,K-ATPase activity in three out of eleven mutants was actually more sensitive to ouabain than in wild-type cells. These data suggest that the ouabain-resistance of the mutants probably results from single base-changes in the alpha subunit of the Na,K-ATPase gene, rather than from amplification or overexpression of ouabain-resistance genes. The latter types of alterations have been described for ouabain-resistant lines derived in other laboratories.

We describe the use of a simplified bacterial fluctuation test to detect induced mutation, either incorporating liver microsomal or whole liver cell preparations. We have evaluated both types of test using three agents. The fluctuation assay seems somewhat slower, simpler and more sensitive than a conventional plating test with microsomes. A whole cell preparation appears marginally more effective than a microsomal fraction for metabolic activation of 9,10-dimethyl-1,2-benzanthracene, but rather less effective for benz(a)pyrene and 2-acetamidofluorene. Ultimately the usefulness of activation by whole cells may depend upon whether the method can give a correlation with carcinogenicity that is more quantitative than microsome methods and better reflects organ and species specificity.

Escherichia coli strain WP2 and its repair-deficient derivatives are suitable strains for mutagen screening. In these strains, agents which cause base substitution mutations can be shown to increase the frequency of Trp+ revertants. In addition, agents causing many types of DNA damage can be detected through increased killing of the repair deficient derivatives. Four ways of performing tests are described: (a) Spot tests in which a small amount of the agent under test is placed directly on a selective agar plate. Trp+ revertants are counted and increased sensitivity of repair-deficient strains determined from the size of the zone of inhibition of cell growth. (b) Treat and plate tests, where a strain is treated with the agent under test and subsequently plated to determine survival or frequency of Trp+ revertants. (c) A simplified fluctuation test which shows exceptional sensitivity in measuring mutation with low levels of mutagens. (d) Use of a liver microsomal fraction in conjunction with treat and plate tests to detect metabolically activated mutagens. The merits and defects of these systems are discussed. Common pitfalls in evaluating tests and procedures for avoiding them are described.

The lexA gene suppresses the spontaneous inviability of recA strains of Escherichia coli without affecting their repair deficiency. We have taken advantage of this to construct a uvrA recA lexA triple mutant CM871 that combines extreme repair deficiency with near wild-type growth. We have used this strain in conjunction with an isogenic strain WP67 uvrA polA and isogenic wild-type strain WP2 in a differential killing test. Dilute suspensions in buffer of the repair-deficient strains and repair-proficient control are incubated for 2 h at 37 degrees C with test compound in the presence or absence of a S9 metabolising system. Survival is determined by Miles Misra plating. For each strain three 10 microliter spots of each of three concentrations of test compound and of the untreated control are placed on a nutrient agar plate. Following overnight incubation survivors are counted. Results with reference compounds are given and the advantages and disadvantages of the test are discussed.

L5178Y wild-type and TK+/- (3.7.2c) cells were treated with sodium fluoride over a range of concentrations (10-500 micrograms ml-1) and treatment times (4, 16 and 48 h) covering less than 10-100% survival. The mutant frequency at five genetic loci (resistance to ouabain, 6-thioguanine, excess thymidine, methotrexate and 1-beta-D-arabinofuranosyl cytosine) was assayed in wild-type cells and trifluorothymidine in TK+/- cells. No significant induced mutation at any locus was observed after 4 h of treatment. Sixteen hours of treatment with high concentrations of sodium fluoride did not induce resistance to ouabain, but resulted in some significant induction of 6-thioguanine, 1-beta-D-arabinofuranosyl cytosine and methotrexate resistance, although the results were variable between experiments and no dose-response was observed. At the thymidine kinase locus, a dose-related increase in mutant frequency to excess thymidine and trifluorothymidine resistance was observed. The maximum induction was approximately eight times the control frequency after TK+/- cells were treated with the highly toxic concentration of 500 micrograms ml-1 of sodium fluoride for 16 h. These observations, and an analysis of the colony size of trifluorothymidine-resistant mutants in TK+/- cells, suggest that sodium fluoride is clastogenic to dividing cultured mammalian cells at high, toxic concentrations. Further work is desirable to investigate the mechanism by which chromosomes are damaged at high concentrations of fluoride, since without such a mechanistic understanding, extrapolation of our data to the human situation must be insecure. Nevertheless, the knowledge available at present gives no reason to expect any genotoxic effects in human tissues at levels of fluoride ions to which they are currently exposed in the general population.

As a mutagen screening procedure we have used a modification of the Luria and Delbrück fluctuation test in which the individual tubes are scored by eye for the presence or absence of a mutation. The test is simple and extremely sensitive, detecting concentrations of mutagens up to 100-fold lower than conventional tests. Measuring mutation to tryptophan independence in Escherichia coli strain WP2 we have found that methyl methanesulphonate (0.5 mug/ml), mitomycin C (0.0015 mug/ml), dichlorvos (5 mug/ml, and K2CrO4 (0.5 mug/ml) are all positively mutagenic in the test, whereas NiCl2 is negative. Chronic exposure to low levels of mutagens using this method appears to induce more mutations than might be predicted by extrapolation from short exposure experiments at higher doses. The procedure is applicable to any system which involves mutation to prototrophy from a non-leaky auxotrophic requirement and should prove valuable in detecting and investigating the effects of low doses and chronic exposures.

Mutation frequency decline (MFD) is an irreversible loss of newly-induced suppressor mutations occurring in excision-proficient Escherichia coli during a short period of incubation in minimal medium before plating on broth- or Casamino acids-enriched selective agar. It is known that MFD of UV-induced mutations may occur before DNA containing pre-mutagenic lesions is replicated, but we conclude that MFD can also occur after the damaged DNA has been replicated on the basis of the following evidence. (1) Mutation fixation in rich medium (i.e., loss of susceptibility to mutation frequency decline) with ethyl methanesulphonate mutagenesis begins immediately, whereas with UV it is delayed for 20--30 min. (2) The delay in mutation fixation after UV can be explained neither by inhibition of DNA replication nor by a delay in the appearance of error-prone repair activity in the irradiated population. (3) MFD at later times after UV irradiation is more rapid and is less strongly inhibited by caffeine than is MFD immediately after irradiation. (4) Excision is virtually complete 20 min after 3 J m-2 UV but at that time virtually all mutations are still susceptible to MFD. We have presented evidence elsewhere that in bacteria there is an alternative error-free excision-dependent type of post-replication repair of potentially mutagenic daughter strand gaps. We suggest that this process is inhibited at tRNA loci in the presence of nutrient broth or Casamino acids, possibly because of a broth-dependent change in the structure of the single-stranded region including the tRNA locus.

E. coli WP2 and its repair-deficient derivatives were treated with the pyrrolizidine alkaloids, heliotrine and monocrotaline in the presence of a liver microsomal fraction. The doubly repair-deficient strains WP100 uvrA recA and CM611 uvrA exrA showed considerable killing. The singly repair-deficient strains WP2 uvrA, CM561 exrA and CM571 recA showed slight killing. In strains WP2 and WP2 uvrA induced reversion to Trp+ was not detected with either monocrotaline or mitomycin C. These results are entirely consistent with liver activation converting pyrrolizidine alkaloids into bifunctional alkylating agents.