Viable alleles of
unc-96 show normal movement but have a disorganized body wall muscle structure. By polarized light microscopy, there are highly birefringent "needles" near the ends of muscle cells, without any definite A or I bands. By EM, the most severe allele,
su151 has extra collections of thin filaments or intermediate filaments (IF), and masses of thick and thin filaments in abnormal locations. We have identified 2 new alleles. The first,
r291 , was isolated as a suppressor of
unc-105 and the second,
sf18 , by a non-complementation screen. To address whether the disorganization of
unc-96 is due to deterioration of the lattice as a result of contraction, we made a double mutant of
unc-96 with a mutation in the myosin heavy chain that decreases myosin activity (
unc-54(
s95)) but does not appreciably affect muscle structure.
unc-54(
s95);
unc-96(
su151) shows an increase in organization. This protection is also seen in
unc-54(
s95) / +;
unc-96(
su151) . We are in the process of examining muscle structure of
unc-96 mutants at early stages of development.
unc-98 , another muscle affecting gene, shows an identical polarized light phenotype to
unc-96 . We have constructed an
unc-96 unc-98 double and it shows enhanced muscle structure disorganization. After more finely mapping
unc-96 , we inspected the genomic sequence for IF protein candidates using ACeDB. One candidate, encoded on cosmids M6 and T19D7, is homologous to IF Protein A, a muscle specific IF isoform purified from Ascaris . We were able to achieve rescue using these cosmids or a 12 kb genomic fragment containing the gene. Surprisingly, we have been unable to detect the mutation sites in this gene by sequencing all of the coding sequence, most of the introns, and 1.7 kb of upstream sequence from
su151 and
r291 . A second IF protein is encoded by the cosmid F25E2 that lies only several cosmids to the right from M6. We were unable to rescue
unc-96 with this gene, and in addition, sequencing of
su151 failed to reveal a mutation site. A non-complementation screen is currently underway to better position the gene on the physical map using rearrangements. The ultrastructural phenotype and rescue with an IF protein gene suggests that
unc-96 truly encodes an IF protein or a protein that interacts with IFs.