Worm Breeder's Gazette 9(3): 42

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The Isolation of unc-45 Lethal Mutations

L. Venolia and R.H. Waterston

Of genes affecting thick filaments in the body wall muscles of C. 
unc-45 III is one of the most difficult in which 
to isolate mutations. Onry 5 alleles have been isolated in general 
screens, compared to hundreds of alleles of unc-54, the major myosin (
myoB) gene. The 5 unc-45 alleles are all temperature sensitive (ts), 
recessive, and cause a reduction in thick filament number and 
organization at 25 C. It has been shown that unc-54(0);unc-45(ts) 
mutants are no worse off than unc-54 null mutants, suggest that unc-45 
acts at least in part through unc -
A precomplementation screen was performed to look for non-ts alleles 
of unc-45. aphrodites were mutagenized with 
EMS and mated with--unc-45(e286) males. Slow cross progeny were picked 
at 20 C and examined for Dpy and Slow progeny. Screening 18,000 cross 
progeny yielded 4 unc-45 isolates, 3 of which are homozygous lethal 
and one of which is ts. This EMS induced mutation frequency of 1/4,500 
genes is comparable to the general EMS induced forward mutation 
frequency of 1/2,000 genes (Brenner, 1974), and suggests that the 
lethal alleles may be loss of function alleles. The unc45(st601) 
allele has been the most completely characterized: it is fully 
recessive to wild-type and ts alleles, but is not itself ts. 
Recombination in the st601 to dpy-1 interval yielded a map distance of 
10, compared to the published e286-dpy-1 distance of 6.6 +/-.9 (More 
extensive data puts the e286-dpy-1 value at 7.4mu).
The finding of lethal alleles of unc-45 suggests that unc-45 
interacts with more than the unc-54 gene product, myoB. Mutants 
totally-lacking myoB are viable, having a few thick filaments composed 
of myoA. Thus, the failure by unc-45 to organize myoB into thick 
filaments should not, by itself, be fatal. All 3 lethal alleles (st601,
st603,st604) have been examined for their terminal phenotype. The 
embryo achieves 2-fold length with a slight overlap of head and tail. 
When the early 2-fold stage is reached, the embryos are capable of 
small movements, and the pharynx develops, but the embryos have almost 
never been observed to pump and they mostly fail to hatch. This 
phenotype is similar to the recently reported myo3(st378) hypomorphic 
phenotype, except that myo-3(st378) animals do pump and hatch (
waterston WBG 9:2, see Fire and Waterston this issue). myo-3 encodes 
the minor body wall muscle myosin, myo A. The similarity in phenotype 
of the myo-3(st378) homozygotes and the unc45(st601) homozygotes 
suggests that unc-45 is essential for myoA action, in addition to its 
interaction with myoB. The failure of the unc-45 lethal mutants to 
pump, might also suggest a role for unc-45 in pharyngeal muscle thick 
filament organization.