Worm Breeder's Gazette 1(1): 18
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.
Our work has concentrated on the two complemetation groups - unc-15 and unc-54 - believed to contain the structural genes for paramyosin and a myosin heavy chain respectively. Because anomalous complementation has been observed between alleles of unc-15, e.g. E1216 complements E73 and E1402 but fails to complement E1214 and E843, more detailed mapping has been done with each isolate. Advantage was taken of the polarized light phenotype to allow these strains to be mapped against E61(dpy-5) and E51 or E1091(unc-13) in three factor crosses. E73 was investigated most extensively and mapped to the left of unc-13 and 0.1% or less from it. Results with all the other alleles including E1216 are consistent with this position. Feasibility of fine structure mapping within the two complementation groups has been demonstrated. For the unc-15 locus the double heterozygote e61 e73/ + e1214 was constructed and one such animal placed on each of 100 large petri plates. The plates were allowed to overgrow (3-4 generations), the worms washed off, sedimented and placed on the clear half of a plate with a lawn of E. coli on the other half. After 1-2 hours worms which had not reached the lawn were removed and the remaining worms screened for recombinants. Three of the plates yielded wild type animals, giving an approximate frequency of recombination of 2 x 10+E-6. Results were consistent with the mutation order of e61 e73 e1214. It is unlikely that these animals represented revertants. For the unc-54 locus the double heterozygote e1301/e675 was constructed and recombinants looked for in the same way as in the unc- 15 experiment. One difference is however that E1301 is a ts allele and to take advantage of this to generate a large number of heterozygotes in the first generation, the PO's were kept at 15 C for a week before the plates were shifted to 25 C. A recombinant frequency of about 2 x 10+E-5 was found. Reversion frequency for E675 and E1301 is less than 10+E-6. Ordering of the mutations was not possible as no closely linked marker was available but the ease with which recombinants were detected and the frequency with which they occurred is encouraging . Other work on the unc-54 locus has concentrated on defining the nature of the defects in the mutants in biochemical terms. Using cyanylation cleavage (Degani and Patchornik, 1974) of purified myosin we have identified two high molecular weight peptides as fragments of the unc-54 heavy chain. E675 contains a heavy chain about 10,000 daltons less than the wild type protein as well as heavy chains of the normal molecular weight (Epstein et al., 1974) in a ration of about 3- 4 to 1. Cyanylation of E675 myosin shows these partial cleavage products are both smaller by 10,000 daltons. By labelling the NH2- termini of these large fragments with 14CN and then treating the labelled fragments with CN-Br, we were able to show the amino-termini of the E675 peptides were identical to the corresponding N2 fragments. Analysis of all the peptides, labelled and unlabelled, reveals E675 to be lacking a small peptide, presumably from the COOH-terminal portion of the molecule. Myosin from other unc-54 alleles has also been subjected to cyanide cleavage. Many alleles, e.g. E190, show a virtual absence of both the unique fragments while several of the other peptides are proportionally greatly increased. These peptides are presumed to derive from other myosin heavy chains. In addition to such null-os, others,e.g. E1258, show a pattern intermediate between the N2 and the null-os with some of the two unique fragments present but with a variably increased proportion of the other peptides. The ts alleles E1157 and E1301 show a pattern nearly like the wild type. These alleles probably contain missense mutations. Together these results give strong support to the hypothesis that unc-54 contains a structural gene for a myosin heavy chain and that the heavy chain of E675 is a nonsense fragment. The residual myosin at the normal molecular weight in E675 appears to be the product of other structural genes. Notes on strains: E1402 is a ts allele of unc-15, while E1157 and E1301 are ts alleles of unc-54. E843 also appears to be an allele of unc-15 rather than unc-54 (Epstein et al., 1974). There is no evidence for a chromosomal rearrangement. E903, derived from a + e843/e61 + animal, however, is an allele of unc-54 and complements E843. The generation of E903 remains obscure. E677 fails to complement E723 and E890 and thus all three belong to unc-60 V. Also unc-82 IV now has two isolates, E1220 and E1323. These map about 3% from dpy-13 and between this and unc-31.