Worm Breeder's Gazette 11(2): 39
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 lin-29 gene of C. elegans activates a temporal developmental switch in the hypodermal cell lineage at the L4 molt. Seam cells of lin-29 mutant animals fail to exit the cell cycle and fail to undergo a switch from the synthesis of larval to adult cuticle. The lin-29 locus is tightly linked but to the right of a Tc1 insertion site (maP1) on chromosome II. Cosmid, lambda and YAC clones from this region were hybridized to Southern blots of lin-29 mutant DNAs. One YAC clone, Y11E9 (60 kb), identified two distinct allele-specific polymorphisms that affect a 7.3 kb EcoRI fragment. In lin-29(n836) an approximately 500 bp deletion removes an EcoRI site, fusing the 7.3 kb fragment to an adjoining 2.4 kb fragment generating a new 9.2 kb fragment (see figure). In lin-29(n1440) there appears to be an insertion into the 7.3 kb fragment, about 6.5 kb from the deletion site in n836, that creates a novel 8.3 kb fragment while the 2.4 kb fragment remains unaltered. To demonstrate that lin-29 sequences lie within the region delimited by these RFLPs, we constructed hermaphrodites of genotype unc-4(e120) 440)/sqt-1 36) and scored their progeny for wildtype recombinants. Four such recombinants were isolated from ~40,000 progeny. Homozygous lines derived from these recombinants were analyzed by Southern blotting, and in each case the polymorphic bands were replaced by the wildtype 2.4 and 7.3 kb EcoRI fragments. Thus, lin-29 sequences must, at least in part, reside within this region. Radiolabeled Y11E9 DNA was then used to screen cDNA libraries. 34 positives were isolated after screening ~100,000 plaques from J. Ahringer's 48 hour library. These positive clones were later rescreened with the polymorphic 9.2 kb band cloned from genomic n836 DNA. 11 of the clones were positive with this probe, were all ~1 kb in size and all cross-hybridized with each other. These cDNAs hybridize to the 2.4 kb EcoRI fragment in N2 DNA (see figure). Northern analysis using a representative cDNA as probe detected two transcripts, 1.8 and 2.5 kb in length. Although genetic analysis predicts lin-29 to be required only at the L4 molt, these transcripts are detected in RNA isolated from stages L1 through L4, suggesting that transcriptional control may not be solely responsible for regulation of lin-29. These transcripts were not detected in RNA isolated from adults or eggs. We are beginning analysis of these transcripts in the heterochronic genes lin-4, lin- 28, and lin-29. One of the isolated cDNAs has been sequenced and it predicts a protein containing three zinc fingers of the TFIIIA and Kruppel class (see figure), suggesting it may have a DNA binding function. As the cDNA sequenced was not full length, more such fingers may reside farther upstream. The fingerless portion of the protein revealed no significant homologies in searches of protein data bases. We are attempting to isolate full-length cDNAs corresponding to both of these transcripts. In addition, we have recently isolated genomic lambda clones that collectively span this region for use in transformation-rescue experiments. [See Figure 1]