Worm Breeder's Gazette 11(2): 37
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.
As part of our ongoing effort to identify genes for known muscle proteins, we have used an approach that starts with the purified protein and works backwards to the gene. To test this approach and gain experience with it we selected tropomyosin (Tm) a relatively abundant, easily purified, actin associated protein. A candidate Tm clone had previously been identified by Hiro Kagawa, but it was not known whether this gene produced the major muscle form or another form of Tm. Tropomyosin from total worm homogenate was brought to near 100% purity by salt extractions, ethanol and low pH precipitations, and FPLC column chromatography. The N-terminal was blocked so this material was treated with CNBr, and the fragments fractionated on a Laemmli acrylamide gel. The peptides were then transferred to an Immobilon-P membrane and Coomassie stained. Cleavage was partial with about 10 bands present ranging in molecular weight from 39kd to 15kd. 5 major bands were excised and given to the Washington U. Protein Chemistry Lab. to sequence. These yielded protein sequence data on two separate regions of Tm. Both regions had high homology to rabbit alpha skeletal muscle Tm and are presented below: [See Figure 1] Two degenerate oligonucleotides were made to the arrowed regions in the upper fragment. These oligos were used to amplify the predicted 80 bp PCR band from a cDNA library and genomic DNA. This PCR fragment was gel purified and then cloned into M13 and sequenced. This sequence yielded the expected protein sequence and is presented below: [See Figure 1] The 80 bp PCR fragment was hexamer labelled and used to probe the YAC library. The positive clones Y38D6 and Y68B7 map to the left of unc-54. This position and amino acid sequence agreed with the results independently obtained by Hiro Kagawa (this issue and personal communication) and indicates that Hiro has cloned and sequenced the major muscle tropomyosin gene. Deficiencies that map to the left of unc-54 were obtained from the CGC stock center and these were used as a substrate for PCR amplification. The arrested homozygous deficiency animals were digested with proteinase K and amplified using the oligos to Tm. Our results indicate eDf10, elete Tm. Tentative data indicates that eDf15 does not delete Tm while eDf16 does. The genes lev-11 and let-205 are known to be in this region. This project has shown the power of micro-sequencing of immobilized proteins, PCR amplification, the C. elegans YAC library, and the C. elegans genetic map with its deficiencies. These tools can lead the C. elegans researcher from their favorite protein to localize its gene.