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.

Mutants with Altered Muscle Structure: The unc-15 and unc-54 Loci

R.H. Waterston, A.R. Macleod, R.M. Fishpool

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 
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.