Worm Breeder's Gazette 11(2): 40
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.
Recently, several genes fitting the description of ''master switch' regulators of myogenesis have been identified. One of the best characterized genes of this set is MyoD. MyoD was cloned from a mouse fibroblastic cell line (10T1/2) that had previously been shown to form myoblasts when treated with the hypomethylating agent 5-azacytidine. Reintroduction and expression of the MyoD cDNA into 10T1/2 cells results in high frequency conversion of these cells to myoblasts that, in turn, are competent to form multinucleated myotubes when placed into differentiation media. Subsequent transfection experiments demonstrated that MyoD expression could convert a variety of other cell types into myoblast, including adipocytes, chondrocytes and melanocytes, albeit at frequencies lower than that seen in 10T1/2 cells. MyoD is a nuclear phosphoprotein with a region of amino acid sequence similarity to the oncogene myc and a growing list of developmental regulatory genes such as acheate-scute in Drosophila. This group of related genes has been termed the Helix-loop-helix (HLH) Family due to the putative secondary structure of the protein within the domain conserved among members. The HLH domain of MyoD, along with an adjacent amino-terminal basic (B) region, is necessary and sufficient for myogenic conversion in transfection assays. In addition, in vitro and in vivo studies suggest that MyoD acts by multimerization with either HLH proteins (through the HLH motif) and B- region mediated specific DNA binding. As the list of MyoD sequences from different organisms grew, it became apparent that the highly conserved B-HLH region was well suited for PCR amplification. Degenerate oligonucleotides were used to amplify genomic C. elegans DNA and amplified fragments of the appropriate size were cloned and sequenced. [See Figure 1] One of these clones had extensive sequence similarity to MyoD (see Figure 1) and was used as a probe to identify several genomic and a single cDNA clone in libraries constructed by Chris Link and Robert Barstead respectively. I have sequenced 4.5kb of genomic DNA and the entire 600bp CDNA. The results identify five exons and four introns comprising the carboxyl terminal two thirds of the gene; in the absence of a full length cDNA, the amino terminal end of the gene remains ambiguous. Although the B-HLH domain amino acid sequence of this C. elegans clone is very similar to the vertebrate MyoDs, the flanking regions show no apparent homology to vertebrate or Drosophila MyoDs. Preliminary analysis of expression of the nematode MyoD gene suggests it is a rare, 1200 base message present in embryonic and larval stages. The genomic phage harboring the putative C. elegans MyoD gene were sent to Alan Coulson and John Sulston. They fingerprinted the clones and matched them to a contig on the left arm of chrcmosome II. Correlating the genetic and physical map for the region suggests that MyoD is about a map unit (~650 kb) to the right of lin-31 placing it very close to unc-85. No previously mapped mutations in this relatively gene sparse interval have phenotypes indicative of a MyoD lesion, assuming that this gene serves a myogenic regulatory function in C. elegans.