We have isolated suppressors of the temperature sensitive allele,
unc-4 (
e2322ts ),by relying upon a chemotaxis attraction assay to select for animals with improved movement. Although
unc-4 (
e2322ts)animals cannot crawl backward at restrictive temperature (25 C), they can crawl forward almost as well as wild type and thus can traverse a bare NGM plate to reach food if given enough time [See Figures]. If forward movement is also blocked by including
unc-24 (
e138),which only slightly inhibits backing, then less than 2% of the
unc-4 unc-24 animals can get to food in an overnight test.
unc-4 -suppressedanimals ought to reach the bacteria, however, because virtually 100 % of
unc-24 mutants are successful Typically, about 104 F1 progeny of EMS-treated
unc-4 ;
unc-24 L4 'swere placed on the bare side of a 100 mm selection plate and left overnight at 25 C. Animals reaching the bacterial lawn were tapped on the head to check for backward movement. From 5 x 105 F1 's,we obtained four intragenic
e2322ts revertants and four strong, dominant, extragenic suppressors (frequency/haploid genome = 4 x 10-6). (Not surprisingly, we also obtained
unc-24 suppressors!) All four of the
e2322ts extragenic suppressors map to the
bli-4 -
unc-15 interval on I which suggests that they may be allelic. We have provisionally assigned these
unc-4 suppressors to a new locus, bkn- 1 (for "backing again .") None of the extragenic suppressors display a detectable phenotype in an
unc-4 (+)background and none suppress the null allele,
unc-4 (
wd1).
e2322ts contains a Leu to Phe substitution in helix-2 of the homeodomain (from sequencing cDNA derived by reverse transcription PCR). It is conceivable, therefore, that specific mutations in the promoter region of a presumptive
unc-4 target gene could compensate for such a minor defect in the
unc-4 homeodomain. This model would also explain the failure to suppress
unc-4 (
wd1) in which no detectable
unc-4 mRNA is produced.