Worm Breeder's Gazette 12(3): 68 (June 15, 1992)
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 C. elegans member of the steriod/thyroid hormone receptor gene superfamily (WBG 11, #3). crf-2 mRNA is present in the maternal germline and in the early embryo. A crf-2 /lacZfusion protein is expressed during the first half of embryogenesis (WBG 11, #5). To determine if maternal expression of the fusion gene is necessary for all or part of the observed expression pattern transgenic males carrying the fusion gene were mated to fer-1 ( hc1 )hermaphrodites which had been raised at the restrictive temperature. The pattern of fusion protein expression observed in the cross-progeny was indistinguishable from that seen in the self-progeny of hermaphrodites carrying the fusion gene, indicating that zygotic expression of the fusion gene is sufficient to generate the pattern. We do not know yet if the fusion gene, like the native gene, is transcribed in the maternal germline.
In order to understand the role which crf-2 plays in embryogenesis we are attempting to obtain a mutation in the gene. Since the early expression suggests that a crf-2 mutant might have a maternal-effect or zygotic, perhaps lethal, phenotype we began by searching for lethal complementation groups which could correspond to crf-2 .The physical map location of crf-2 places it on Chromosome I between dpy-5 and bli-4 ,a region which contains a number of lethal complementation groups identified in Ann Rose's lab. The genetic interval in which crf-2 should lie was further defined by mapping flanking Bristol/Bergerac Tc1 polymorphisms relative to duplication endpoints (WBG 11, #5), placing the region of interest between the right-hand endpoints of hDp13 and hDp12 .Twelve complementation groups in the Rose collection, including both embryonic lethal and larval lethal loci, have ben mapped to this interval. The Rose lab kindly provided mutant strains of the genotype dpy-5 ( e61 )let-x unc-13 ( e450 ); sDp2 These strains segregate live Unc NonDpy and dead Dpy Unc progeny. We have tested for rescue of the lethal Dpy Unc class by cosmid AD7 ,which appears to contain the entire crf-2 gene, for 11 of the 12 loci (we were unable to observe a lethal class for let-377 ( h110 )and thus did not test this strain). This was initially done by directly transforming mutant strains with the cosmid. Fertile Dpy Unc lines were obtained from let-388 ( h88 ), let-531 ( h752 ),and let-532 ( h715 ).However, we were unable to demonstrate the presence of cosmid sequences in these lines, so we suspect that they arose from truncation of sDp2 (perhaps induced by the transformation procedure) rather than from rescue by the cosmid. We then crossed males bearing an array containing AD7 plus a marker to mutant hermaphrodites. Fertile Dpy Unc lines were recovered from two of the mutants ( let-531 ( h752 )and let-532 ( h715 )),but these strains also yielded Dpy Unc lines when crossed with N2 males, the lethal phenotype apparently being synthetic (similar behavior of these strains has been observed by S. McKay in the Rose lab). So we have concluded that crf-2 does not correspond to any of these complementation groups. If a crf-2 mutant actually has a lethal phenotype, this could indicate that the maternal expression is sufficient for viability and crf-2 was therefore not isolated in the Rose lab screens since they were not designed to detect maternal-effect lethals. Alteratively, since many of the lethals in their collection have been placed on the genetic map based on complementation by duplications which could contain discontinuities (see Browning et al, WBG 12, #2) our mapping of flanking polymorphisms might not have led us to the correct genetic internal. For example, if a hole in the duplications used (which were all derived from sDp2 )spans the location of crf-2 but not those of the flanking polymorphisms, a crf-2 mutation may have been mapped into a different genetic internal than the one selected. We therefore may not have tested the correct mutants. In similar experiments we also did not obtain rescue of several emb and zyg mutations broadly mapped to that region of Chromosome 1, some of which do exhibit maternal effects.
Since crf-2 does not appear to correspond to any existing mutations we have begun a screen for lethal mutations balanced by an extrachromosomal array, mgEx9 ,which carries the crf-2 cosmid (AD7) and the rol-6 ( su600 )marker. Following EMS mutagenesis of the strain rol-6 ( n1270 e187 ); mgEx9 ,4644 F2 Rollers were cloned and their progeny were screened for the absence or a markedly reduced frequency of NonRoller animals. Several candidate lines were isolated, and these fall into two major classes. The only viable animals segregated by lines in the first class are Rollers. Two of these lines (from 1 P0 )appear to carry recessive roller alleles, four lines (4 PO's) carry dominant roller alleles or integrated arrays, and four lines (1 P0 )may contain a mutation which gives a synthetic roller phenotype with rol-6 ( n1270 e187 );these lines are not being pursued further. Two lines (1 P0 )are still being evaluated and could represent zygotic lethal mutations balanced by mgEx9 .The second class of candidate lines (5, from 3 PO's) segregate reduced numbers of NonRollers that arrest as larvae, are sterile, or have markedly reduced brood sizes. These lines may carry maternal-effect lethal mutations (eg. the line in which all NonRollers are sterile) or incompletely penetrant zygotic lethal mutations balanced by the array. The candidate mutations are being mapped to verify that their genetic position is consistent with that expected for mutations balanced by mgEx9 .To determine if any correspond to crf-2 ,mutations that map appropriately will be tested for complementation by a fragment of cosmid AD7 containing only the crf-2 gene. If none of these mutations prove to be in crf-2 we plan to probe the crf-2 phenotype more directly using an antisense construct before undertaking additional mutant screens.