Meneely PM et al. (1977) Worm Breeder's Gazette "further studies with the X-linked lethals."

Herman RK, Meneely PM

[Worm Breeder's Gazette, 1977]

Among 28,647 F1 progeny of X-irradiated N2 males, we have found 17 deficiencies of the unc-3 region, and have used these in mapping the lethals. Thirteen of these deficiencies fail to complement all mutants in the region. The map shown is based on the remaining four deficiencies and on three-point crosses involving unc-3, lethals; the positions found by recombination are consistent with the deficiency map. [See Figure 1] These fourteen essential genes are defined by twenty-three lethals; a fifteenth gene, let-5, is either to the left of unc-3 or to the right of let-2. Three of the lethals have been sent to us by other labs; one of these, e1471, provided by Andras Fodor, is the only allele of let-15. The other two alien lethals, e1470 and b246ts, provided by Andras and by Judy Kimble respectively, are alleles of let- 2. Counting these two, we have six alleles of let-2, all of which are temperature-sensitive. For five of the six, the restrictive temperature is 20 C; for the sixth, b246, it is 25 C. Complementation at 20 C is complex, with each mutant complementing at least one other; all fail to complement at 25 C. We have also looked at the epistatic interaction between the hermaphrodite specific lethal let-7 and the transformer mutants tra-1 and tra-2. Homozygotes for let-7 die at about L3, while hemizygous males live to adulthood. Since hemizygous hermaphrodites of the genotype unc-3 f1 also die as juveniles, the male survival does not appear to be due to having just one let-7 gene. We then checked if the survival was male-specific by using 2X transformer males; that is, we made tra/+; unc-3 and looked for Unc male self-progeny. They die at about the same stage as the homozygous hermaphrodites, suggesting that the interaction of let- 7 with the transformers is similar to that reported by Hodgkin and Brenner for the sex-influenced mutants dpy-21 and dpy-22. We conclude that the action of let-7 is on 2X individuals regardless of sexual phenotype, but that the critical region of the X is not the let-7 gene itself nor, in fact, any gene in the region deficient in mnDf1.

MacMorris MA et al. (1990) Worm Breeder's Gazette "Mosaic Expression of vit Transgene"

Broverman SA, Spieth J, Blumenthal TE, Greenspoon S, MacMorris MA

[Worm Breeder's Gazette, 1990]

A critical part of our analysis of the importance of the sequence elements VPE1 and VPE2 for vit promoter function has been the verification of correctly regulated expression. Stable transgenic lines containing a vit-2/6 fusion gene under the control of modified promoters have been tested for correct tissue, stage and sex specificity by in situ hybridization. In the course of the analysis of strains containing mutant promoters, we have made two surprising observations. (1) In strains in which most individuals have no detectable vit-2/6 RNA, there are occasional rare individuals ( approximately 5% in a strain with a point mutation in one of the two VPE2 elements) which have high levels of the RNA . (2) Individual worms have expression levels which vary dramatically within a single intestine. Since our initial discovery, we have looked at 13 strains with 11 different modified promoters affecting 6 different elements within the promoter. While 3 strains carrying the wild-type promoter region (247 bp) show expression in all intestinal cells, all strains carrying mutant promoters exhibit mosaicism to varying degrees. In each case the pattern is similar: the most anterior end of the intestine (int1 cells) has no or low hybridization signal, the middle region (approx int5 cells) shows the highest signal and more posterior intestinal cells show lowered levels. In strains that have mutant promoters with less drastically altered expression, the areas of positive signal extend farther anteriorly and posteriorly. In the least extreme cases (i.e. deletion of the most distal VPE1) the mosaicism is displayed only as a variable lack of signal in the first tier of intestinal cells. The signal changes continuously from regions of high to low intensity in most cases, with the exception of the int1 cells. The lack of expression in these cells is often delineated by an abrupt and distinct line of higher level expression beginning in the next set of cells. If we look at strains with an unmodified promoter at very early time points relative to the onset of vitellogenin synthesis (late L4 to early post-lethargus adults) we sometimes find a similar pattern, consistent with Pepper Schedin's observations on wild-type worms examined with a vit-5 specific probe. Our interpretation of the mosaic intestines is that the lowered affinity of binding for activator in these lines reveals an inherent spatial distribution of activator molecules within the worm intestine. The lowered level expression seen at the anterior and posterior ends of the intestine could be due to a single activator produced at the middle of the animal - creating a gradient in its distribution toward the head and tail, by gradients of two activators, both of which are required, one originating anteriorly and one posteriorly. At the earliest times in wild-type strains we see evidence of the activator distribution pattern, but normally all cells in the intestine quickly reach threshold levels of activator sufficient for wild-type promoter function. With debilitated promoters, higher thresholds are required and therefore mosaics can be found for an extended time. We interpret the occurrence of rare expressing animals within negative populations as evidence that promoter function depends on the binding of proteins to several elements to build a complex. Lowered levels of individual bound elements give a lowered chance of total complex function, yet once formed the complex gives 'normal' level expression.