Worm Breeder's Gazette 11(2): 67
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.
Our genetic analysis of let-23 suggests: (1) let-23 activity is necessary both for induction of 1 and 2 vulval precursor cells and for some negative feedback on the inductive process; and (2) let-23 is a complex locus acting in the development of several tissues (see CSH C. elegans 1989 Meeting Abstracts, p.108; Chamberlin and Sternberg, this issue). Complementary to this analysis, we set out to clone the locus to molecularly characterize how let-23 acts in the vulva and in multiple tissues. Our initial cloning efforts focused on finding molecular markers on either side of the gene. Joan Park previously had identified a Bergerac-specific Tc1 insertion, Tc(5A), which maps between rol-6 and unc-4 and is detected by probing with the cosmid T09F12. rol-6 maps an estimated 0.23 map units to the left of let-23; 0.5 map units to the right. We therefore constructed the strain rol-6(e187) 15)/vab-9(e1744) Tc(5A). By picking Rol non-Lethal recombinants (let-23(sy15) is a 100% penetrant lethal allele of let-23) and probing Southern blots with a subclone of T09F12, we mapped let-23 relative to rol-6 and Tc( 5A). Sixteen of twenty Rol non-Lethal recombinants carried Tc(5A). Given an approximate distance of 330kb between rol-6 and Tc(5A), this suggests let-23 lies 83 +/- 70 kb to the right of Tc(5A). As well as providing an estimate of distance, Tc(5A) defined a left boundary for let-23.Although there are no known DNA polymorphisms immediately to the right of let-23, there are several deficiency breakpoints that fall between let-23 and the next mapped marker, let-240. Since the right breakpoint of mnDf67 falls between these two markers and its left breakpoint falls between two markers, vab-9 and rol-6, whose positions on the cosmid contig have already been determined (J. Kramer WBG 10:1 and personal communication) we decided to clone the junction fragment of this deficiency and to use this fragment to 'jump' across mnDf67 establishing a DNA boundary to the right of let- 23. We probed genomic DNA from N2 and mnDf67/C1 with each of the six cosmids spanning the region between vab-9 and rol-6, and found that two cosmids, C25G9 and B0382, detect a 10kb BglII polymorphism unique to mnDf67 DNA. We isolated this 10kb band from a size-selected genomic lambda library and used it as a probe against DNA from 13 overlapping cosmids and 3 YACs extending approximately 400kb to the right of Tc(5A). One cosmid, C01G6, was detected by this probe. When C01G6 was used as a probe against mnDf67 DNA, polymorphisms were detected with several enzymes indicating that C01G6 contains the right breakpoint of mnDf67 and determines a right physical boundary for let- 23.The region from rol-6 to unc-4 is covered by one contig. However, there are two gaps covered by YACs but not covered by cosmids near let- 23: one just to the right of T09F12, another to the left of the Df67 breakpoint (see Figure). We estimated the extent of these gaps, 10kb and 65kb respectively, by sizing YACs that span them. The combined Tc( 5A), mnDf67, and YAC data positioned let-23 in a region of approximately 200kb that was centered around the cosmid T08E2. T08E2 is immediately flanked by several cosmids on each side and then by the two gaps. We subsequently probed Southern blots of the twenty let-23 alleles we have with the cosmids around T08E2. These blots have yet to yield definitive results due to the presence of several high-copy number repeats in the region. We next microinjected several of the cosmids to test for their ability to complement let-23 mutations. We have found that T08E2 rescues the lethal, vulvaless, and male tail phenotypes of let-23(sy97) . An adjacent cosmid to the left, W07A12, fails to rescue. Prior to our microinjection attempts, Koga and Ohshima (WBG, 11(1), p.37) had cloned by low-stringency hybridization with a v-ros probe two tyrosine kinase genes, kin-7 and kin-8, which map to the let-23 region defined above. In particular, kin-7 is contained in T08E2. We are presently narrowing down the region required for rescue to show whether in fact let-23 and kin-7 are the same gene. [See Figure 1]