In addition to let-240 and let-31, several remaining genes, including mel-8, mel-9, mel-11, egl-28, and egl-37 , had been mapped to an overlapping genetic interval, but had not been precisely positioned relative to the chromosomal deficiencies used to locate kin-15 and kin-16 (Hodgkin et al., 1988). Complementation tests with relevant chromosomal deficiencies indicates that none of these genes map to the kin-15 / kin-16 interval (Morgan and Dumezweni, unpublished observations).
The only remaining candidate of the existing mutations is eat-3, which is required for normal pharyngeal pumping (Avery, 1993). Initial complementation tests with eat-3 and the kin-15/kin-16 transgenic plasmid were unsuccessful due to technical difficulties; further mapping studies are in progress. However, considering the pharyngeal function of eat-3 and the hyp7 expression of kin-15 and kin-16, it is unlikely these are corresponding genes.
Since none of the existing mutations appear to be allelic to kin-15 or kin-16, I chose to pursue a PCR-based screen for transposon insertion alleles (Rushforth et al., 1993), since approaches based on homologous recombination are currently impractical in C. elegans. Unlike traditional mutant screens, a physical screen makes no assumptions about the nature of the null phenotype, insuring that mutant alleles will be identified.
To expedite this reverse genetic approach, several labs have recently begun to establish frozen banks of Tc1 insertion alleles by propagating and freezing lines with an active Tc1 transposable element (see Zwaal et al., 1993). Ron Plasterk and Yuji Kohara's labs kindly agreed to screen their TC1 insertion banks using PCR primers specific for kin-15 and kin-16 and successfully isolated two Tc1 insertion alleles for each gene (Figure 1, Y. Andachi and Y. Kohara, pers. comm.). The kin-15 transposon insertions are within exons (ms9 and ms4), while the kin-16 transposon insertions are outside the coding regions, at the start of an intron (ms3) or in the 3' untranslated region (ms8).
As seen with other Tc1 insertions alleles, none of the kin-15::Tc1 and kin-16::Tc1 strains exhibit any apparent abnormalities. Rushforth et al. (1993) reported that the transposon sequences of several mlc-2::Tc1 alleles were largely removed during RNA splicing, even when inserted into exons. The resulting nearly wild-type mRNAs, while containing small insertions, may therefore still encode functional proteins. The kin-15::Tc1 and kin-16::Tc1 strains probably appear phenotypically normal because the transposon sequences are also removed during RNA splicing, although this has not been examined. Alternatively, kin-15 and/or kin-16 may not be required for nematode development, physiology or morphology or the two tandem, homologous genes may be functionally redundant. Fortunately, a relatively straightforward method has been developed for isolating null mutations once a Tc1 insertion allele is available.