Worm Breeder's Gazette 11(2): 18
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.
We have constructed three translational fusions between unc-6 and the E. coli lacZ gene. The unc-6 gene encodes laminin B2 (Ishii et al WBG 10#3 p.39). The coding sequence begins with an apparent signal sequence which presumably functions in eventual secretion of the protein. In bacteria, it has been shown that secreted -gal is nonfunctional and can be deleterious to the cell. Thus we designed the fusion constructs to keep the -galactosidase region of the fusion proteins from being translocated through the membrane during synthesis. The first two constructs (pPD32.105 and pPD33.47) are fusions made so that only the first 10 amino acids of rmc-6 are present on the fusion protein. This 10 aa region should not function as a signal sequence. The constructs contain approximately 4kb of unc-6 5' flanking sequence, and carry a 3' region from the unc-54 gene. When pPD32.105 and pPD33.47 are injected into oocytes and F1 animals stained as adults we see a small set of cells stained which appear to be hypodermal. Generally there are 14 cells in the area of the retrovesicular ganglion and one cell in the preanal region. Occasional cells in the head are also stained, as well as cells in the ventral cord and cells in the vulva (four symmetrically located cells). Plasmid pPD32.105 contains a nuclear localization signal from SV40, resulting in nuclear staining, while pPD33.47 lacks the nuclear localization signal and results in more diffuse and often patchy staining of the cells. We have made three transformed lines by co-injecting pPD32.105 with the unc-22 antisense plasmid pPD10.46 and selecting for twitchers. In each case we see distinct staining of body wall muscle nuclei, which we attribute to the ability of the body wall muscle enhancer present in pPD10.46 to activate the unc-6 promoter in cis in the mixed tandem arrays. Expression of muscle constructs is not detected prior to the comma stage in embryogenesis, and thus we can look for unc-6 specific staining patterns in early embryos from the co-transformed lines. In all the lines we see staining early embryos: the two cell gut primordium stains around the 28 cell stage, while at approximately 240 cells, two clusters of 7-8 cells on opposite surfaces of the embryo stain. We believe that the latter are hypodermal precursors. Gut primordium staining is absent at the 240 cell stage, indicating instability of this particular fusion protein. We hoped that a construct including more sequences from within the gene might exhibit a more complete expression pattern. Thus, our third construct (pPD35.09) uses a fusion point in the fourth exon of unc-6, just past the domainVI/domainV junction in the laminin protein. To keep -gal on the cytoplasmic face of the membrane, we have used a new vector which includes a synthetic transmembrane domain as a 'stop transfer' sequence just before the -gal coding region. After injection of pPD35.09 into wild type animals, F1 animals showed a pattern of hypodermal staining much more extensive than that seen with the initial constructs. Most of the hypodermal cells in the head, the tail and in the ventral cord are stained, with additional staining in vulval and uterine cells (vulval staining is apparently limited to 'primary' cells derived from P6.p) and marginal cells in the pharynx. Striking is the absence of staining around the dorsal nuclei in hyp7. The intracellular localization of staining from pPD35.09 is consistent with the predicted transmembrane structure of the fusion protein: prominent staining is seen around the periphery of the nucleus ( presumably the rough endoplasmic reticulum) with some staining in clumps within cells (perhaps golgi) and along the outline of the cells (plasma membrane). Although unc-6 expression might be expected in most tissues making basement membrane, mosaic studies have implicated hyp7 dorsal nuclei as the focus for the unc-6 uncoordinated phenotype (Stern & Hedgecock, unpublished). Some possible reasons for lack of observed staining in adult dorsal hyp7 are: 1. pPD35.09 may lack a regulatory element specific for dorsal expression. 2. The fusion mRNA or protein could be preferentially lost, poorly translated or transported ventralward in hyp7. 3. Normal unc-6 expression in adults (after basement membrane is already constructed) may be at a very low level in dorsal hyp7. The transmembrane containing vector pPD34.110 may be useful for fusion studies with other genes encoding secreted or transmembrane proteins. The vector is diagrammed below. [See Figure 1]