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.

Studies on HSP16 Expression in Transgenic C. elegans Using lacZ Fusions

Eve Stringham, Don Jones and Peter Candido

Figure 1

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]

Figure 1