Worm Breeder's Gazette 11(1): 40

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Sequence Analysis of unc-15 Mutants and its Intragenic Revertants

Keiko Gengyo and Hiroaki Kagawa

Figure 1

To understand the molecular mechanism of thick filament assembly, we 
are investigating the mutation site of EMS induced unc-15 mutants at 
the DNA level.  These mutant alleles are distinguished two categories. 
One is e1214, has no detectable paramyosin on SDS-PAGE gels and 
apparently null phenotype.  This mutant had amber nonsense mutation (Q 
to Amber) near the N-terminus.  The others are missense alleles which 
accumulate normal level's paramyosin, nevertheless result in the 
disruption of thick filament assembly.  The latter alleles are of 
variable phenotype, the most severely paralyzed allele, e73 caused the 
reversion of electric charge (E- to K+) by the point mutation.  
Further sequence analysis of two intragenic e73 revertants revealed 
that second mutations are exactly in the unc-15 gene, not in a closely 
linked gene, and also caused the reversion of electric charge (E- to 
K+).  These mutation might not disrupt the secondary structure of 
paramyosin, but only change the charge on the surface of alpha-helical 
coiled coil structure.  This might mean the assembly of paramyosin 
itself and/or paramyosin-myosin heavy chain result from charge 
interactions between the molecules.  We hope to calculate what kind of 
changes are induced by each of substituted amino acid with the 
computer program exploited by Dr.  McLachlan (McLachlan, A. D.  and 
Karn, J,1982, Nature 299, 226-231; McLachlan, A. D.  and Karn, J, 1983,
J.  Mol.  Biol.  164, 605-626; Kagawa, H., Gengyo, K., McLachlan, A. 
D., Brenner, S.  and Karn, J., 1989, J.  Mol.  Biol., 207, 311-333).  
Molecular organization of unc-15 alleles summarized.  More detailed 
epitope mapping of paramyosin is in progress.
[See Figure 1]

Figure 1