Mutations in
unc-89 result in animals which move nearly as fast as wild type, but are thinner and more transparent. Muscle structure is abnormal with a thinner myofilament lattice, thick filaments not organized into A-bands, and no M-lines. The M-line is a structure in the center of A-bands that is believed to place thick filaments into proper register. Our goal is to identify suppressors of
unc-89 that might encode UNC-89-interacting proteins or components of the M-line. Because the phenotype of
unc-89 mutants is somewhat subtle, it would be difficult to distinguish an
unc-89 mutant from an animal with an improved phenotype. We have constructed
unc-89;
unc-22 double mutants to facilitate a suppressor screen. Both twitchin, encoded by
unc-22, and UNC-89, are unusually large ~750 kDa members of the intracellular branch of the Ig superfamily. By immunofluorescence, the two proteins localize to reciprocal areas of the A-band. UNC-89 colocalizes to the center, with mhc A, the minor myosin heavy chain of body wall muscle, while twitchin localizes to the remaining, major portion of the A-band. Double mutants of these two genes were made using several allelic combinations. All of the
unc-89 alleles used have a wild type-sized UNC-89 of reasonably high abundance (presumed missense mutations).The phenotype of the double in all cases is much more severe than either mutant alone. In some allelic combinations, movement is restricted to "head" motion as an adult ("paralyzed"), brood size is extremely reduced, and polarized light reveals greatly reduced birefringence. This paralyzed phenotype is characteristic of
unc-22(
s16);
unc-89(
e1460), as well as
unc-22(
s16);
unc-89(
su227) and
unc-22(
s16);
unc-89(
su240). Three additional alleles of
unc-89 were used to construct doubles. The
st86 double shows only 80% penetrance of the paralyzed phenotype. Neither
st79 or
ad539 are paralyzed, but both show muscle structure disorganization which is worse than either single mutant alone. A mutagenesis of
unc-22(
s16);
unc-89(
su227) yielded 2 animals with improved movement and a persistence of twitching, one of which is extragenic. Mutagenesis of
unc-22(
s16);
unc-89(
e1460) yielded 8 suppressors. Seven isolates are extragenic suppressors, two ofwhich act in a recessive manner. The eighth suppressor is intragenic, or closely linked to of the mutations. The mutation of one suppressed line, GB4, is located on X . A second, independently isolated suppressor, GB10, fails to complement GB4, and thus, these two mutations are likely to be alleles of the same gene. The suppressor from GB4, called sup
(sf1), suppresses
unc-89(
e1460), but not
unc-22(
s16); complete suppression occurs with either one or two copies of sup
(sf1). By polarized light,
unc-89(
e1460);sup
(sf1) have normal appearing muscle structure. By EM,
unc-89(
e1460);sup
(sf1) still have a thinner than wild type lattice, but the M-lines have been restored. By itself, sup
(sf1) has no obvious phenotype by the dissecting or polarized light microscope. sup
(sf1)X was mapped by STS mapping, and later placed within an ~3 map unit interval between
unc-1 and
dpy-3. We are currently using transgenic rescue to clone sup
(sf1).