Worm Breeder's Gazette 11(4): 65
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.
Genetic analysis suggests that let-23 is required in the vulva for both an inductive function necessary for vulval differentiation and an inhibitive function necessary for proper specification of vulval fates (WBG, 10(2), p.81). Mutations in let-23 result in the Vulvaless and Hyperinduced phenotypes in the vulva, larval lethality, abnormal male spicules, sterility, and transformation of P12.p to P11.p fate. Some of our let-23 alleles uncouple these phenotypes from one another, suggesting multifunctionality at the locus (CSH C. elegans 1989 Meeting Abstracts, p. 108). The let-23 gene maps to the region around the cosmid T08E2, and microinjection of that cosmid rescues let- 23 mutant phenotypes (WBG, 11(2), p. 67). Furthermore, T08E2 contains a tyrosine kinase gene, kin-7, cloned by low-stringency hybridization with a v-ros probe (WBG, 11(1), p. 37). We now show that let-23 and kin-7 are the same gene. We inject using the rol-6 dominant selection scheme developed by Craig Mello. We coinject DNA from the let-23 region (abbreviated LDNA) along with rol-6(dom) DNA (abbreviated ROL) into the strain let-23( mn23) unc-4(e120)/mnC1(dpy-10(e128) unc-52(e444)), where mn23 is a 100% penetrant lethal let-23 allele and mnC1 is a balancer for the region. We score rescue by two criteria which, in both positive and negative control experiments, always correlate with each other. First, if stable Roller lines, which should be mn23 e120/mnC1; array( LDNA+ROL), segregate Rol Uncs, (genotypically mn23 e120/mn23 e120; array), then this suggests that the LDNA rescues the let-23 lethal phenotype (in all but one case the vulvaless and sterile phenotypes of mn23 are also rescued). Second, rescue of the F1 progeny of the injected hermaphrodites is indicated by the presence of F1 Rollers which are Unc. These Rol Uncs are often sterile (indicating transient rescue), though sometimes they are fertile. We have shown that a few cosmids and plasmid constructs also rescue the Vulvaless allele sy97 and the lethal allele mn216.The 15 kb genomic plasmid pK7-13.8, which contains the kin-7 gene and roughly 3 kb on either side, rescues let- 23 (see Figure). This result suggests that let-23 is contained in pK7- 13.8. The plasmid YO#NG213, which deletes about 4 kb of 3' genomic sequence from pK7-13.8 (including approximately 800 bp from the C- terminus of cDNA sequence but not including the kinase domain), also rescues. This observation suggests that no sequences 3' of the kinase domain are needed for rescue. However, the constructs pIS6 and pK7- DTK both of which delete an additional 1 kb (in genomic and cDNA sequence) from the C-terminus of YO#NG213 and lack the kinase domain, fail to rescue. Thus, a deletion within kin-7 that lacks the kinase domain, but still maintains about half the N-terminal end of cDNA sequence, fails to rescue. This observation suggests that the kinase domain is required for rescue and that no sequences 5' of kin-7 on pK7- 13.8 are needed for rescue. Finally, the plasmid pK7-5.5 which contains the kinase domain and C-terminal sequences but lacks 10 kb of 5' pK7-13.8 sequences fails to rescue. Thus, the N-terminal half of kin-7 is needed for rescue. By these rescue results, therefore, the let-23 locus and the kin-7 tyrosine kinase gene are the same. We can furthermore show that the two key negative results (pK7-DTK and pK7-5.5) are due to deletions in let-23 and not due to faulty plasmids (e.g. gratuitous point mutations) since coinjection of pK7-5.5 with pK7-DTK results in rescue. The one stable line we have segregates Rol Uncs, although at a lower frequency than seen with above constructs, and these are mostly sterile. The coinjection also results in transient F1 Rol Uncs, although again at a lower frequency. The presence of repeat elements in the region have stymied our attempts to detect allele specific polymorphisms on Southern blots. Nonetheless, we have localized a point mutation in the null allele sy5 to the kinase domain using hydroxylamine modification of C mismatches ( the protocol kindly provided by Bob Barstead), further supporting our injection results. Analysis of let-23 sequence suggests that let-23 encodes a receptor tyrosine kinase related to the EGF Receptor family. We hypothesize based on genetic results and its inferred molecular structure that let- 23 might be the receptor in the vulva precursor cells for an intercellular signal required for vulval differentiation. [See Figure 1]