Worm Breeder's Gazette 6(1): 30

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.

Another Internal Deletion Mutant of unc-54

D.G. Moerman, D. Weidman, R.H. Waterston

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for the unc-22 region (Moerman, Rogalski and Baillie, unpublished 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.