Worm Breeder's Gazette 10(2): 32

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Antimorphic Mutations in the Head Region of the unc-54 Myosin Heavy Chain Gene

Amy Bejsovec and Philip Anderson

Figure 1

We are investigating the molecular basis of dominant antimorphic 
mutations at the unc-54 locus.  These dominant-uncoordinated, 
recessive lethal mutations disrupt the assembly of thick filaments in 
the body wall muscle.  By genetic and ultrastructural criteria, the 
mutant gene product interacts with both wild-type myosin heavy chain B,
the unc-54 gene product, and myosin heavy chain A, the minor heavy 
chain class found in body wall muscle thick filaments.  The strong 
disruptive effect is mediated by very low levels of full length 
protein - depending on the allele the amount ranges from less than 2% 
to approximately 20% of wild-type levels.  Following EMS mutagenesis, 
these antimorphic missense alleles arise at a frequency equal to that 
of recessive null alleles at the unc-54 locus.
To determine what portions of the myosin heavy chain are affected by 
these mutations, we have employed a technique developed by Myers et al.
(Science 230: 1242, 1985) for detecting single base changes in 
genomic DNA.  Base changes have been localized for 10 independently 
isolated EMS-induced alleles; all 10 show changes in the region of the 
gene encoding the globular head.
[See Figure 1]
One allele, unc-54(r342), carries 2 separate point mutations - one 
in the head and one in the rod portion of the gene.  Both mutations 
have been sequenced and only the one located in the head creates an 
amino acid substitution.  We have used the polymerase chain reaction (
PCR) technique (Saiki et al., Science 239: 487, 1988) to amplify and 
then sequence without cloning the affected region from mutant genomic 
DNA.  Mutant sequences confirm the accuracy of mismatch positions 
estimated by the Myers technique.  The head mutations sequenced so far 
create non-conservative substitutions for amino acids that are highly 
conserved among many myosin heavy chain sequences (including the yeast 
heavy chain).  Thus we have mutationally identified parts of the 
globular head that are important in thick filament assembly.
[See Figure 1]

Figure 1