Worm Breeder's Gazette 10(2): 86
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.
This project started with a somewhat ill-fated attempt to identify the glp-1 gene within the 400kb Iin-12 to ep-7 contig. The glp-1 gene had been mapped between lin-12 and ep-7 and we hoped to test a large number of cosmids within the contig to see which one(s) rescued glp-1 mutations. To assay for rescue we were originally using a ts allele ( e2142) of glp-1 which lays dead (or doomed) embryos at the non- permissive temperature (25 C). The e2142 animals to be injected were born and raised at the permissive temperature of 15 C and then shifted up to 25 C as larvae. They were injected as late L4's or adults in the distal tip region of the gonad at 25 C and then maintained at that temperature. Since the embryonic lethality in glp-1 is a maternal effect, expression of glp-1 in the maternal gonad should yield rescued progeny. The choice of the distal tip region for injection was a result of studies with the sup-7 tRNA gene, which gives strikingly efficient maternal rescue of tra-3 amber alleles when injected into that region. To make a long story short none of the cosmids tried gave significant rescue of e2142 in this assay. It subsequently became clear from the data of J. Austin, J. Kimble, J. Yochem, and I. Greenwald that the glp-1 gene was likely to be partially or completely contained in the cosmid ZK506 and so we concentrated our subsequent efforts on the activity of that cosmid ( this cosmid lies to the right of lin-12, and does not overlap the lin- 12 gene). A more conservative approach toward assaying for expression was tried. For this, another allele of glp-1 was used (e2141). This ts allele exhibits both the sterile phenotype and maternal effect lethal phenotypes associated with glp-1. Like e2142, the e2141 stock will not grow at 25 C but unlike e2142 (for which many of the embryos fail to hatch even at 15 C) e2141 embryos almost all hatch at 15 C. Thus it was feasible to inject into e2141 animals raised at 15 C, and allow all the resulting F1 progeny to hatch at 15 C; expression of glp- 1 in the germ lines of the F1 progeny was then assayed by shifting to 25 C as larvae and scoring for fertility. This procedure resulted in recovery of four transformed lines (PD101- PD104) from twenty injected adults. No convincing examples of transient expression in the F1 germline without recovery of a transformed line were observed. Thus the transformation signal with this gene is comparable to heritable transformation signals with other genes, but significantly below levels of transient expression of early zygotic genes such as unc-54 or mec-3 (WBG10#1 pp28&60). The lack of a real burst of 'transient' rescuants in the F1 germline may turn out to hold true for all germline genes, and could result from the greater number of divisions for germline versus soma, which will eventually dilute injected DNA before it reaches the adult germline. Although they are all viable at 25 C, the transformed lines are all somewhat odd. For PD101 and PD102 the rescued (fertile) animals tend to be scrawny and unc, and some of the PD102 animals are Muv's. No homozygote line has been derived for either of these. For PD103 ( which is homozygote viable), the fertile adults look quite normal but most of the eggs fail to hatch at 25 C. These injections were performed as co-injections of ZK506 with a plasmid (pShZ1) containing a fusion construct between a Drosophila heat shock promoter and E. Coli -galactosidase. This was done in hopes of following the transforming DNA in individual animals. Animals from the transformed lines PD101 and PD102 have been stained for -gal activity after heat shock. Staining is in a pattern characteristic of the plasmid pShZ1, and presence of stain in the animals correlates to rescue of the glp-1 phenotype. DNA from strains PD102-104 have also been analyzed on southern blots (by S. Harrison) and shown to contain 2-12 copies of the injected cosmid. John Yochem sent a lambda phage with most or all of a lin-12 homolog gene mapping in the glp-1 region. This was injected into e2141 in order to select rescued lines as described above. The phage gives a very low transformation frequency (one transformed line in ~50 injected animals). It is not clear whether the low frequency reflects something missing in this lambda clone or just reflects a difference in transformation frequency between linear lambda and circular cosmid DNAs. The transformed line (PD110) expresses a co-injected HSP- gal fusion (confirming that transforming DNA is present). The PD110 line grows well at 25 C as a heterozygote and exhibits a very slow and scrawny homozygous phenotype. For the sake of future cloning of other maternal effect genes, we have gone back and repeated the distal tip injection experiments with ZK506. In the e2141 strain, a small number of rescued progeny were observed from this experiment (~.25 per injected tip); no convincing rescue was obtained after injection of e2142. These numbers (unlike those originally obtained for maternal rescue by sup-7) are insufficient for a really useful assay (since the rescued animals obtained from this sort of procedure would not be transformed.)