The
ama-1 gene, which is located 0.05 map units to the right of dpy- 13 on LG IV, encodes the amanitin-binding subunit of RNA polymerase II. The gene was originally defined by the dominant mutation,
m118, which conveys resistance to -amanitin (Sanford, Golomb, and Riddle, 1983, J. Biol. Chem. 258, 12804-12809). Twenty amanitin-sensitive mutants that carry recessive-lethal alleles of
ama-1 were isolated following EMS mutagenesis of resistant parents (Rogalski et al. WBG: Vol. 8, No. 3, and Vol. 9, No.l). We are working on a fine-structure analysis of
ama-1 and have mapped 14 of these lethal mutations in five-factor crosses with respect to
m118,
dpy-13, e of genotype f dpy- 13
(e184) 8 mX) +/unc-17
(e245) + + unc-S
(e53) are put into I ml liquid cultures with 3% OP50 and 20 g/ml -amanitin. Only worms in which recombination between the resistance mutation (
m118) and the lethal mutation (mX) has occurred will grow in the amanitin. If the lethal mutation is to the right of the resistance mutation, then the resistance-bearing chromosome is +
dpy-13(
e184) 8) ), and recombinants carrying this chromosome will be either Dpy or Unc-5. If the lethal mutation is to the left of
m118, then the recombinant chromosome is
unc-17(
e245) +
ama-1(
m118) +, and animals carrying this chromosome will be either Unc- 17 or semi-Dumpy. There is an average of 500,000 worms in each experiment. A different strategy is being employed to order lethal mutations with respect to each other. This requires the construction of heteroallelic lethal strains, so mDp1 red for viability (see Rogalski et al., this issue). For example, amanitin- resistant progeny are to be selected among progeny of mDp1 45) 45) 84) 8
m328) +/ +
dpy-13(
e184) 8
m329) unc-S
(e53) heterozygotes. The figure shows the
ama-1 fine-structure map. Allele names are given along with phenotypes exhibited at 20 C (
em1=embryonic lethal, ell=early larval lethal,
m11=mid-larval lethal, sa=sterile adult). The map is 0.018 map units long, and the
m118 mutation is near the
dpy-13 end. The lethals have not yet been ordered with respect to each other, so positions are based on recombination frequencies with
m118. We are unable to separate 4 of the adult sterile mutations from
m118. So far, all of the early larval lethal mutations map to the right of the
m118 mutation, and only the mid-larval and embryonic lethal mutations map to the left of
m118. This represents the first fine-structure map of an essential gene in C. ope that this will be useful to guide the DNA sequence analysis of selected mutant
ama-1 alleles. {figure 1}