Worm Breeder's Gazette 11(2): 53
These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author.
The gene pal-1 is required in males for the generation of rays by the V6 cells. As we have reported, pal-1 stimulates the production of rays by overcoming cell signals from the T cell that would otherwise inhibit ray production. (The Pal-1 V6 phenotype is suppressed by ablation of T.) The effect of pal-1 is to stimulate the function of the gene mab-5 and inhibit the function of lin-22 in V6 or its descendants (Cell, in press Jan. 12, 1990). We have rescued pal-1 animals by injection of a 14 kb fragment of DNA that appears to contain ceh-3 [one of the scores of homeoboxes found in C. elegans, ( Burglin et al. Nature 341:239)]. pal-1 maps very close to ced-4 on the left arm of linkage group III. Twelve recombinants between unc-79 and dyp-17 did not separate ced-4 and pal-1. unc-79 and dpy-17 are 0.25 map units apart. Thus with 95% confidence we know that ced-4 and pal-1 are less than or equal to 0.05 map units apart. This suggested that the genes might be within 50 kb of each other (using the conservative estimate of 1 Mb per map unit). ced-4 has been cloned and mapped to a contig by Junying Yuan. We therefore sent to England for a series of cosmids in the region (Thank you John and Alan) and began injecting to look for pal-1 rescue. We injected several of these cosmids (we used the gene unc-31 and the unc- 31-bearing cosmid C14G10 as a coinjection marker) and obtained heritable but unstable rescue of the Pal-1 phenotype with the cosmid W05E6. According to the cosmid map W05E6 completely spans the cosmid AC10 from which Burglin et al. cloned the homeobox ceh-3. It didn't take a Ph.D. to realize that this would be a good place to start looking for the pal-1 gene. Using the published ceh-3 protein sequence as a guide, we designed a degenerate oligonucleotide that should recognize the ceh-3 sequence, and used it to probe Southern blots of W05E6. We were thereby able to generate a restriction map of the cosmid and localize the hybridization to a 1.8 kb BamHI/SalI fragment. (Since we have not sequenced the hybridizing sequence we can not say unequivocally that this is ceh-3). We then subcloned a 14 kb (KpnI partial) fragment that contains the 1.8 kb Bam/Sal fragment (pWK14). We injected this 14 kb subclone into pal-1 animals and rescued the Pal-1 phenotype. As noted above pal-1 males are missing rays in their fans. Rescued animals had wildtype fans. In addition we saw one animal (out of 5) with an extra ray located in the body of the animal about halfway between the distal end of the gonad and the tail. Our numbers are still quite small so we cannot yet say how frequent the production of ectopic rays is. The generation of such ectopic rays might be due to negative control element(s) that are missing from the pWK14 plasmid or to overexpression of the gene caused by high copy number of the injected DNA, etc. In conclusion we have localized the pal-1 gene to a 14 kb fragment with the ability to rescue the Pal-1 phenotype. This fragment probably contains the homeobox ceh-3. Thus the question remains: Does pal-1 have a homeobox? We cannot be certain, but it looks like it might. If so this would suggest that the activity of pal-1 somehow overcomes the effect of cell signals by influencing transcription.