Worm Breeder's Gazette 11(5): 50
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.
Four distinct hsp16 genes reside at two loci, hsp16A and hsp16B, in the C. elegans genome. The genes hsp16-1 and hsp16-48 reside at hsp16A (IV), while hsp16B contains hsp16-2 and hsp16-41. At each locus the gene pairs are transcribed in opposite directions. Initial studies of hsp16 expression were reported in WBG 11, #2 . When pPC16.48-1 animals (in which the hsp48 promoter directs transcription of lacZ) were heat shocked and stained for -gal activity, very intense and extensive expression was observed in nuclei of body muscle and hypodermis (constructs contain the SV40 nuclear localization signal). This included muscle and hypodermal cells of the vulva, spermatheca, uterus, and anus. In addition, 2-3 nuclei ( gland cells) in the pharynx stained frequently. Intestinal staining occasionally was quite prominent, particularly in larvae. When the hsp48-1 intergenic region was inverted such that the hsp-1 promoter directed transcription, expression in the muscle and hypodermis was secondary to, i.e. weaker and less consistent than, intestinal expression. Many more nuclei appeared to stain in the head, particularly within the pharynx itself. Also some nerve ganglia just anterior to the terminal bulb stained. An XbaI fragment which extends from the hsp16-48 heat shock elements (HSE's) to the hsp16-1 HSE's in pPC16.1-48 was flipped to see if the expression pattern could be converted to resemble more closely that of pPC16.48-1 worms. Our initial results suggest that to a degree this may have happened. Like pPC16.1-48 worms, intestinal expression was strong in the XbaI inverted construct. However, expression in general body musculature was much more extensive and there was limited expression in the pharynx, as in pPC16.48-1 worms. Translational fusions made by inserting lacZ in-frame into the second exon of hsp16-1(pPCZ1) produced the most general expression pattern. In addition to expression in the body musculature and hypodermis, muscle and epithelia in the pharynx itself also stained. Neural expression was occasionally fairly strong, extending down the ventral cord from ganglia in the head. Intense intestinal expression was usually observed in these animals and sometimes included nuclei of the pharyngeal intestinal valve. In males, nuclei of M-derived muscle in the tail and hypodermal nuclei of the rays were stained. Coelomocytes in the male as well as the hermaphrodite also stained. Expression in embryos for all of these constructs can be quite intense from gastrulation on. We have not observed expression in earlier stage embryos. A transcriptional fusion (pPC16.48-51) containing a fragment of the hsp16-48/1 intergenic region which eliminates the HSE's and TATA box of the hsp16-1 gene but maintains a stretch of alternating purines and pyrimidines present in the center of the intergenic region was injected and lines established. Expression in embryos was still very strong in these lines and could include almost all nuclei at gastrulation stage. In larvae and adults, somatic expression was much weaker and infrequent. While many worms showed some body muscle expression, only occasionally did intestinal nuclei stain. Collectively, these results suggest that the hsp16-48 promoter contains elements which drive expression in the general body musculature and hypodermis, while hsp16-1 seems to confer greater expression in the intestine, neurons and specialized muscle and hypodermis of the vulva and anus. We have also examined expression at the hsp16-41/2 locus. Two gene fusions provided by Dennis Dixon, pHS16.25 and pDX16.31, which are analogous constructs to pPC16.48-1 and pPCZ1 respectively, were injected and transgenic lines established. pHS16.25 worms stained very intensely in pharyngeal muscle and epithelia, including the pharyngeal intestinal valve, and in ganglia in the head. Neural expression could extend along the ventral cord, and ganglia in the tail also stained. Intestinal and hypodermal expression were prominent in these animals, while body muscle expression was weak and nearly always confined to a region around the pharynx. Heavy staining was observed around the vulva, in the pharynx and in neurons of pDX16. 31 worms. In addition, body muscle occasionally stained and weak expression in the intestine was usually observed. Dennis reported similar findings with these gene fusions in the East Coast Meeting abstracts last year. When we stained worms containing a transcriptional fusion (pPC16.41-51) which eliminates the TATA box and HSE's of hsp16-2, expression was observed in the intestine and more infrequently in neural nuclei and hypodermal nuclei of the vulva. Again, as in pPC16;48-51 lines. intense staining was still observed in numerous nuclei in gastrulating embryos. Thus, these transcriptional fusions appear to be lacking elements necessary for high level expression in the somatic tissues of later stages. In summary, these results suggest that hsp16-41 expression may be greatest in the intestine. LacZ constructs carrying the hsp16-2/41 promoter show intense expression in the pharynx, while those carrying the hsp16-1/48 promoter are expressed most intensely in body wall muscle. Thus, there appear to be not only intralocus differences in gene expression but also interlocus ones. [See Figure 1]