A rarely commented on and not understood phenotype of
unc-54(
e675) mutants is that along with being paralyzed, these worms display a slight surface twitch. Another mutant allele of
unc-54 called
s291 exhibits this same phenotype. This mutant was isolated after a mutagenesis screen in which N2 hermaphrodites were mutagenized with 0. 07% formaldehyde and their F1 progeny were examined to see if any twitched in 1% nicotine. Unlike the majority of mutants from this type of screen, which are either alleles of unc-22IV or deficiencies for the
unc-22 region (Moerman, Rogalski and Baillie, unpublished results), mapping of
s291 demonstrates that it is an allele of
unc-54, a structural gene for a myosin heavy chain (mhc). Using the techniques developed by MacLeod et al. (1977) in their analysis of
e675 we have done a similar structural analysis of the
s291 mhc. One-dimensional sodium dodecyl sulfate (SDS)/polyacrylamide gels run on
s291 whole worms and
s291 purified myosin reveal a new mhc that is considerably shorter than N2 mhc's. Cyanylation fragments from the rod portion of this heavy chain molecule of
s291 are also shorter than N2 cyanylation fragments (on a 1-D SDS/polyacrylamide gel the
s291 alpha and beta fragments bracket paramyosin). To examine whether the shortened
s291 mhc is due to an internal deficiency we ran two-dimensional isoelectric focusing and SDS/polyacrylamide gels on complete cyanogen bromide (CnBr) digests of whole myosin from N2 and
s291 worms. The gel pattern is more complicated from whole myosin than the pattern observed by MacLeod et al. (1977) for CnBr digests of cyanylation fragments from rods, but the three carboxy-terminal fragments of interest (COOH-38K, 8K, 40K), and the 48K partial digestion product (8K + 40K) are easily discernable in gels from CnBr digested N2 whole myosin. The overall gel pattern of CnBr treated
s291 myosin is similar to the N2 pattern except that the 38K, 8K, 40K, and 48K digestion products are all missing. Instead, a new peptide of 41K (approx.) is present. This new peptide lies between the 38K and 40K peptides in the isoelectric focusing dimension. Our interpretation of these observations is that the
s291 deficiency is an internal deficiency that takes out the 8K peptide and part of the 38K and 40K peptides. The 41K peptide observed is the fusion product of the remainder of the 38K and 40K peptides. This gives an estimate of the size of the deficiency as 45K. Fine structure mapping studies show that
s291 maps to the left-hand side of the gene between
e1300 and
e1301. The heteroallele
s291/e675 appears to be lethal. The
s291 mhc is much less stable than the
e675 mhc. This is not surprising since it has a much larger deficiency, perhaps including as much as two thirds of the light meromyosin. Why worms homozygous for
e675 or
s291 twitch is not clear but we suspect that it is related to the fact that both mutants produce intact myosin heads that are not part of the myofilament lattice.