Worm Breeder's Gazette 10(1): 82

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Another Look at 32P mutagenesis

R. Hoskins, M.M. Shen and A. Spence

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.