Worm Breeder's Gazette 11(5): 48
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.
We have previously described the cloning of crf-2, a gene with homology to nuclear hormone receptors. In situ hybridization experiments and developmental Northern blot analysis had indicated that crf-2 RNA is present primarily, if not exclusively, during the first half of embryogenesis (WBG, Vol. 11, #3). Subsequent Northern blot analysis has shown that the crf-2 message is present in N2 adults and in fem-2(b245ts) adults raised at restrictive temperature, but not in glp-1(e2141ts) adults raised at restrictive temperature. Thus crf- 2 is expressed maternally in the germ line. Whether or not it is also transcribed zygotically remains to be determined. In order to further define the crf-2 expression pattern a 6 kb fragment from the 5' end of the crf-2 gene has been fused to the lacZ gene in a derivative of one of Andy Fire's transformation vectors (see Burglin et al., this issue). Worms were transformed with this fusion gene in combination with a dominant rol-6 allele as a marker. Immunostaining of transgenic animals reveals expression of the crf- 2/lacZ fusion protein in nuclei of early embryos. Staining is first observed in 16-20 cell embryos, in most but probably not all cells. The crf-2/lacZ protein disappears in mitotic cells, and reappears after mitosis in a progressively restricted pattern as embryogenesis advances: fusion protein expression first appears to become concentrated primarily in the dorsal region of the embryo, but is ultimately found in only two to four cells near or in the cluster of neuroblasts in the presumptive head region before disappearing for good just as morphogenesis is beginning. Attempts to identify the exact cells which do or do not produce the fusion protein are currently underway. While the fusion gene expression pattern is consistent with the Northern blot and in situ hybridization analyses, we are also attempting to isolate a crf-2 cDNA, and hope eventually to generate antibodies against the actual crf-2 protein in order to compare its expression pattern with that of the fusion gene. These expression studies suggest that crf-2 is involved in some aspect of embryogenesis which is at least partially, if not entirely, under maternal control. Thus we expect (hope) that a crf-2 mutation will have a maternal-effect embryonic lethal or perhaps a zygotic lethal phenotype. The physical map location of crf-2 places it between dpy-5 and dpy-14 on Chromosome I, a region which the Ann Rose lab has saturated for lethal mutations. In order to take advantage of the wealth of information which the Rose group has collected, we have mapped crf-2 relative to the duplications which they have used in mapping their collection of lethals. A Bristol-Bergerac polymorphism has been detected two cosmids to the left of the crf-2 cosmid; the restriction patterns of the polymorphism (mgP29) are consistent with it being a Tc1 insertion. The Rose lab generously provided strains bearing duplications (Bristol-derived) with breakpoints at various sites between dpy-5 and dpy-14 as well as a strain (KR408) in which the dpy-5-unc-29 interval is derived from Bergerac (see Starr et aL, 1989, Genome 32: 365-372). The duplications were moved into the KR408 background. Since KR408 has the Bergerac allele of mgP29 while the duplications, if they cover the site of the polymorphism, have the Bristol allele, we were then able to map mgP29 relative to the duplications by genomic Southern blot analysis. This placed mgP29 outside of hDp15, hin hDp12 and hDp16. Additional probings of the blots with cosmids between mgP29 and crf-2 detected no evidence of a duplication breakpoint between these two markers, so crf-2 should also lie between the hDp37 and hDp12 breakpoints. We now plan to examine the terminal phenotypes and attempt transformation rescue of lethal genes from this region in efforts to correlate crf-2 with a genetically defined locus. Our initial candidate genes will include 8-10 loci from the Rose lethal collection which have been mapped relative to at least some of the duplications used above, though crf-2 could have been missed in their screens if maternal expression is required or sufficient since their screening strategy was not designed to detect maternal-effect genes. We also plan to look at some emb and zyg genes from the Stock Center collection which are less precisely mapped, but some of which do have maternal effects.