Worm Breeder's Gazette 10(3): 55
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I have localized the fem-1 gene to a region not greater than 6 kb by means of transformation rescue. Polymorphisms present in two fem-1 alleles, e2044 and e2382, proved useful in directing my attention to the relevant region of the rescuing cosmid C17E1. The two alleles are deletions which extend in opposite directions from the fem-1 region, and which overlap by about 5kb as indicated in the figure. It seemed likely that this 5 kb region would contain at least part of fem-1 and indeed, a phage clone spanning the region (CB#1059) was able to rescue fem-1(null) mutations upon injection into oocytes. The figure shows the results of a series of injection experiments with plasmid subclones of the rescuing phage. The experiments were done as described in the last WBG (p.29) except that the injected females were fem-1(e1965). I scored XX progeny as rescued if they were self- fertile, and XO as rescued if they were somatically normal males, though some of them were partially or completely feminized in the germline. I suggest that the smallest clone that I have tested to date, CB#1099, contains all sequences essential for fem-1(+) function, on the grounds that I have obtained 1 self-fertile line homozygous for e1965 and carrying CB#1099 as an extrachromosomal array. I cannot exclude the possibility that sequences necessary for full expression of the gene reside outside of the fragment carried on this clone, especially since it did not seem to rescue as efficiently as larger clones. However, the numbers involved are small and differences between them might be meaningless, or might reflect nothing more than reduced mitotic stability of the smaller subclones. The apparent excess of rescued male progeny over hermaphrodites, which is fairly pronounced with the smaller subclones, disappears if I discount those male progeny which remain feminized in the germline ( that is, if I use germline phenotype as the criterion of rescue in both XX and XO animals). The more frequent occurrence of somatically- rescued males may be due to instability of the injected DNA, leading to dilution of its products to inadequate levels in the germline (cf. Fire and Priess WBG 10:2 p. 86). Other hypotheses are probably not worth worrying about unless this one can be excluded. A transcript of about 2.5 kb (2.7 in the last WBG), present in males and hermaphrodites is confined to the 6 kb region present in CB#1099 and I believe it to be the major fem-1 mRNA. I have isolated a 1kb putative fem-1 cDNA from a library constructed by D. Pilgrim. I am presently, and predictably, sequencing the genomic region which is sufficient to rescue fem-1 mutants. [See Figure 1]