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.

Identification of an Apparent Analogue of the Myogenic Regulatory Gene, MyoD, in C. elegans

Michael Krause and Harold Weintraub

Figure 1

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.

Figure 1