Worm Breeder's Gazette 14(5): 48 (February 1, 1997)
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.
Laboratory of Molecular Biology NIDDK/NIH Building 5, Room B1-04 5 Center Dr MSC 0510 Bethesda, MD 20892-0510
The MyoD family of transcription factors are involved in the specification and development of vertebrate skeletal muscle. These factors contain a conserved basic helix-loop-helix (bHLH) domain that directs DNA binding and protein dimerization. In vertebrates, the MyoD family members heterodimerize with an ubiquitously expressed E protein to activate muscle specific expression. A single homolog of MyoD (CeMyoD) and E proteins (CeE/DA) has been found in C. elegans. Although CeMyoD is expressed in the nuclei of all body wall muscle cells and their precursor blastomeres, CeE/DA is not expressed in these cells. This result raises the question of whether there are other factors that heterodimerize with CeMyoD to direct proper body wall muscle development. To address this question, we have utilized the yeast two-hybrid system to identify gene products that interact with CeMyoD. We fused the bHLH domain of CeMyoD (aa 150-203) to the binding domain of Gal4 to screen a cDNA library containing fusions to the activation domain of Gal4 (the library was kindly provided by Bob Barstead). We isolated 3 potential interactors after screening 1,000,000 clones from the cDNA library. We have sequenced the inserts from these interacting clones. The following is a summary of each of the interacting clones: 1) pal-1. One of the clones identified in the screen is the homeodomain protein pal-1. It is involved in the development of the somatic daughters of the P lineage; the C+D lineages give rise to body wall muscle. Loss of pal-1 leads to a loss of posterior body wall muscle. The link between pal-1 and CeMyoD is not clear since the expression of pal-1 protein disappears prior to the onset of stable CeMyoD expression in the embryo. Additionally, pal-1 expression appears to be restricted from body wall muscle post-embryonically. (Thanks to Craig Hunter and Lois Edgar for sharing information on pal-1). 2) putative GTP binding protein. The second clone is similar to a putative GTP binding protein identified in C. elegans (CGP-1) and in Ascaris (AGP-1). This clone lies on Linkage Group V in cosmid TO4H1 and matches two clones from the Kohara EST library (yk20h4, yk20g2). We have used the cosmid sequence to design primers to PCR amplify a 2.5 kB portion of the genomic sequence upstream of the coding region of the gene to construct GFP and LacZ reporter constructs. The reporters are expressed in body wall muscle, pharyngeal muscle, intestinal muscle, and anal spincter muscle in adults and larvae. The earliest expression with these constructs is seen at the three-fold stage in the pharyngeal and anal spincter muscles. 3) cyclin D homolog. The sequence from the third clone has matches to three ESTs (yk118d3, CEESF44, CEESH01), but no matches to sequences in the genomic database. The predicted protein sequence is similar to Cyclin D. Cyclin D is a G1-cyclin and is involved in decision to differentiate or to re-enter the cell cycle. It is thought that Cyclin D acts as a growth factor sensor to tell cells to exit G1 and differentiate. In myogenesis, there is evidence that the myoD family members cooperate with G1 cyclins (Rb and Cyclin D) to activate skeletal muscle specific genes and to commit cells to muscle cell development. We have generated his-tagged fusion constructs to purify these proteins for use in generating antibodies, in biochemical studies to look at the interactions of these proteins with CeMyoD, and in band shift assays. We are characterizing the expression of these clones by in situ hybridization and their possible function by antisense RNA injection.