Worm Breeder's Gazette 10(1): 82
These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author.
Mutagenesis of C. elegans following incorporation of [32P]- phosphate was first reported by Babu and Brenner (Mutation Res. 82: 269 ( 1981)). Among the mutants obtained were unc-36(e873), which later proved to harbor the translocation eT1, and unc-31(e928), which carries a 3 kb deletion at the unc-31 locus. We have produced a new set of [32P]-induced mutations in genes of interest to us, in the hope that DNA alterations detectable by Southern blotting might be efficiently produced. Our protocol was as follows: Plates of phosphate-free NGM agar (15 ml) where spread with 0.2 ml of 0.5 M phosphate buffer (pH 6.5) and 1 mCi carrier-free [32P]-phosphoric acid to give a specific activity of 10 Ci/mol phosphate. The plates were seeded with E. coli NA22 (K12) and kept at room temperature for two days. Synchronized L1 larvae were prepared by allowing eggs to hatch overnight in M9 buffer, transferred to the plates of radioactively- labelled bacteria, and incubated at 20 C until most animals were young adults. Mutagenized adults were then transferred to OP50-seeded NGM plates and incubated for screening of their progeny. In a complementation screen to produce unc-30 and unc-31 alleles on the balancer chromosome nT1, the strain nT1/sDf22 was mutagenized and F1 Unc progeny were cloned. One unc-30 mutant and two unc-31 mutants were found among 7500 wild-type F1 progeny. Southern blotting shows that both new unc-31 alleles carry DNA rearrangements at the locus, neither carries a simple deletion. No rearrangements have been detected in 11 EMS alleles of unc-31. Complementation screens for new unc-4 alleles have been carried out using either EMS or [32P] as the mutagen. As an initial step, an unc-4 allele was introduced on the balancer chromosome mnC1 by screening for F1 Uncs after EMS mutagenesis of the strain mnDf12/mnC1. The strain rol-6(e187) 9)/mnC1[unc-4(e2151)] was then constructed as the parent for the screens. In two EMS experiments, 64,500 wild-type F1 animals were screened, yielding five true-breeding unc-4 alleles, for an unusually low frequency of 8x10+E-5. However, 16 other F1 Uncs, displaying the Unc-4 phenotype, failed to breed true; we are unable to explain the high frequency of these apparent somatic mosaics. All five of the new EMS-induced alleles are homozygous viable, four having the phenotype of the strong canonical allele unc-4(e120), while the fifth allele is weaker. In a single experiment with [32P], 30,500 wild-type F1 animals were screened, yielding seven F1 Uncs. All seven bred true, but only three of these 4 were homozygous viable unc-4 alleles, all strong, resulting in a frequency of 1x10+E-4. One of these three has displayed recombination suppression with the linked Rol marker, and may be a translocation. The remaining four mutants are homozygous lethal. Two of these appear to be deficiencies, failing to complement the flanking markers mab-3 and egl-43; the third mutant complements both markers, and the fourth has not yet been tested. Mutations in fem-1, btained as feminizing suppressors in a mab-1; k) or mab-11; k) background after EMS mutagenesis (Hodgkin, WBG, this issue). Similar results were found using [32P] mutagenesis. Approximately 4000 mab-11( e2008); 09) animals were mutagenized and a screen of their F2 progeny, representing 40,000 F1 chromosomes, yielded one fem-1 allele, two fem-3 alleles, and two weak tra-1 alleles of the unusual class described by Hodgkin (this issue). A third tra-1 allele of the same class was recovered upon screening the F1 progeny of 1000 mutagenized animals of genotype mab-1(e1228); 09) ( 800O F1 chromosomes screened). Our results suggest that the frequencies of viable mutants induced by [32P] and EMS at each locus are similar, but that the types of mutational events obtained are different. All three alleles of unc-31 have readily detectable DNA rearrangements at that locus. We hope that [32P]-induced alleles may prove generally useful in locating genes on the physical map.