Worm Breeder's Gazette 14(3): 20 (June 1, 1996)
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.
Wellcome Unit of Molecular Parasitology, Anderson College, University of Glasgow, Glasgow G11 6NU, U.K.
We are interested in understanding how the spatial and temporal patterns of the cuticle collagen genes are regulated. To this end we have been studying the regulation of the cuticle collagen gene dpy-7. dpy-7/lac-Z translational reporter gene fusions with as little as 310 bp of 5! flanking sequence produce exactly the same pattern of expression as larger constructs (up to 1265bp). However translational reporter gene fusions containing just 165 bp of 5! flank produce no b-galactosidase expression showing sequences between -310 and -165 are necessary for expression. b-galactosidase expression is first seen in hypodermal cells at the late comma stage and is seen in all postembryonic developmental stages including the adult. The precise pattern of expression has been resolved by an IFA double labelling experiment using anti-lac Z monoclonal antibodies to detect the nuclear localised b-galactosidase and monoclonal antibody MH27 to delineate hypodermal cell membranes. The dpy-7 reporter constructs are expressed in hyp-7 , P cells and most of the hypodermal cells of the head and tail (hyp-5, hyp-6 and hyp-10 stain particularly strongly). However there is no expression in the V (or other) seam cells. Throughout development there is an increasing number of hyp-7 nuclei staining, with expression presumably beginning some time after each nuclei joins the syncitium. Since dpy-7 is trans-spliced with SL1 we have mapped the transcription initiation site by using a 5! RACE strategy designed to be specific for pre-mRNA by using an oligo immediately 5! to the SL1 splice acceptor site in conjunction with the anchor primer in the final round of PCR. Transcription appears to initiate from numerous sites between -210 and -159 relative to ATG (-206 and -155 relative to SL1 splice acceptor). We have also cloned the C.briggsae dpy-7 homolog and can repair the C.elegans dpy-7 mutant phenotype by microinjection of the C.briggsae gene showing that the regulation (and also the function) of dpy-7 is conserved between the two species. Similarly C.elegans reporter gene constructs produce the same pattern of expression when transformed into C.briggsae and sequence between -310 and -165 is also necessary for expression in C.briggsae. Comparison of the 5! flanking sequences of dpy-7 between the two species shows a block of homology (H1) in which 66 out of 72 residues are conserved. This lies within the region necessary for reporter gene expression and immediately upstream of the transcription initiation sites ( - 202 to -273 C.elegans and -185 to -256 C.briggsae). This is the only significantly conserved region of sequence between the dpy-7 ATG and the next predicted upstream gene at -346 (by genefinder analysis). Using RT-PCR we cannot detect any dpy-7 transcripts which are SL2 trans-spliced which, together with our analysis of the dpy-7 functional promoter and transcription initiation sites, show that dpy-7 is not a downstream gene of a polycistronic transcription unit. Transcriptional lac/Z reporter gene fusions with as little as 145bp encompassing the H1 homology and the transcription initiation sites are sufficient to produce the same pattern of expression as the translational fusions although expression in the later stages (post L1) is poor. A number of constructs have been examined and the low level of later stage expression does not appear to be due to the loss of a particular region of sequence but seems to be a general feature of our transcriptional fusion constructs. The transcriptional fusions are all orientation dependant and so the element appears to function as a true tissue specific promoter element which drives expression in all hypodermal cells except the seam cells. A series of fine 5! deletions through the H1 region shows that a single 15bp deletion completely ablates the b-galactosidase expression pattern. This deletion disrupts an AGATAA motif (which is conserved in C.briggsae) suggesting that a member of the GATA transcription factor family may play a role in the regulation of dpy-7 expression.