Worm Breeder's Gazette 11(3): 13
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.
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.