Worm Breeder's Gazette 9(1): 22

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.

Further studies on unc-54

J. Kiff, D. Moerman and B. Waterston

Figure 1

We have expanded our collection of missense alleles of unc-54 that 
suppress unc-22 induced twitching to a total of 17 unc-22 suppressors. 
These mutations can be split into two classes, an s74-like class with 
t4 members (see Cell 29, 773-781) and a new class with 3 members, 
st130, st132 and st148.  This new class of mutations also act as 
dominant suppressors of unc-22(s12) and unc-22(st137::tc1), and a worm 
heterozygous for any of these mutations moves like N2 and has normal 
muscle structure.  However, an animal homozygous for st130, st132 or 
st148 is slow, does not lay eggs, and the muscle cells exhibit 
significant A-band disorganization.  It is this last trait that 
distinguishes this new class of mutation from mutations of the s74-
like class which affect movement but have little, or no affect on A-
band organization.
We have carried out an extensive series of reversion experiments 
using s74, s95, st130 and st132 to see if there are either inter- or 
intragenic sites that interact with these mutations.  The results 
summarized below.
[See Figure 1]
Only st150, an s95 revertant, proved to be an extragenic suppressor 
and it is probably a sup-3 allele.  The other revertants are the 
result of intragenic events.  The frequency at which intragenic 
revertants of st132 occur as compared to s95 (a member of the s74-like 
class) further suggests that st132 defines a new class of mutation in 
unc-54.  The st130 revertant and 2 of the st132 revertants are partial 
revertants while the other st132 revertants are full revertants to 
wild type.  This latter set has been divided into 2 groups - of 13 
tested mutations, 9 are dosage dependent, ie st132/st132, st132 
m/st132 +, and st132 m/st132 m have 3 discernable movement phenotypes, 
and 5 are dosage independent, ie, restoration of wild type movement is 
fully dominant.  The dosage response and the high reversion frequency 
of st132 indicates that compensatory second-site mutations in unc-54 
can occur.
The s95 intragenic mutations are full revertants and 5 of these were 
analyzed at the DNA level.  The s95 mutation is at position 2340 in 
the nucleotide sequence and changes CCA (gly) to ACA (arg) (J.M.B.  
183, 543-551).  Fortuitously s95 disrupts a HpaII site (CCCC to CCAC). 
Of the 5 intragenic revertants examined by Southern analysis, only 
one restores the HpaII site.  After cloning and sequencing the other 4 
revertants we were surprised to find that all 4 have the same 
nucleotide change at position 2342 thus altering ACA (arg) to ACT (ser)
.  Our inability to find compensatory mutations in another part of unc-
54 suggests that s95 may disrupt interactions with a molecule other 
than myosin.  The proximity of s95 to the putative ATP binding site (
EMB0 1, 945-951; P.N.A.S.  80, 4253-4257) makes ATP a likely candidate.

We have sequenced 3 other mutations located in the S1 region of the 
myosin heavy chain.  The null allele e1420 has a change at nucleotide 
3960 involving CAA (gln) to UAA (ochre).  Two other mutations from the 
s74-like class, s77 and st135, have been located.  The S1 region of 
st135 has 2 base changes, one at nucleotide 3855 changing CAC (his) to 
TAC (tyr), and a second at position 3640 changing GCC (ala) to ACC (
thr).  Since the (his) to (tyr) substitution is the least conservative 
change we suspect this is the important alteration.  The change in s77 
is at position 4272 and alters GGA (gly) to AGA (arg) which is 
particularly intriguing because the amino acid replaced is only 3 
residues from thiol-1.  The active thiol region has long been 
suspected of affecting ATP binding by the myosin heavy chain.
For those up on their unc-54 fine structure map you will note that 
the sequence data for e1420 and s77 shows a different order than was 
determined by genetic mapping.  In our genetic study the left/right 
position of these two alleles was determined on the basis of a single 
event, and that 'recombinant' lacked any flanking markers.  Presumably 
then the genetic data is in error.

Figure 1