Worm Breeder's Gazette 8(3): 72
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.
Four different myosin heavy chain (MHC) isoforms are present in C. elegans muscle: myosins A and B in the body wall muscle cells myosins C and D in the pharyngeal muscle. The complete nucleotide sequence of the MHC B (unc-54) gene has been determined. Three additional MHC genes (myo-1,myo-2,myo-3) which hybridize to unc-54 nucleotide probes have been isolated and partially sequenced (1). Extensive sequence homologies indicate that these genes are members of a closely related gene family and would be expected to account for the known myosin heavy chain isoforms. We have used an immunological approach to identify the MHC isoforms encoded by these genes. The strategy was to express fragments of each gene in E. coli as fusion peptides for reaction with isoform-specific monoclonal antibodies. This approach was facilitated by two features of the nematode MHC genes. In all four genes, introns are limited in number and are clustered in the 5' region encoding the myosin head and toward the 3' end of the rod region. This results in a long stretch of uninterrupted sequence (3-4 kb) encoding most of the rod in each gene. In addition, as a result of the highly asymmetric codon usage in these and other nematode genes (2), most of the HindIII sites within these regions are in the same reading frame rAAG CTT = lys leu]. This coincidence simplified expression since all of the fragments could be ligated into a single vector of defined reading frame. HindIII fragments from each MHC gene were cut out of lambda 1059 and ligated into the expression vector pUR 288. This vector includes a restriction enzyme polylinker at the 3' end of the lacZ; the phasing of the HindIII site matches that of the MHC genes (3). Ampicillin resistant colonies were screened by a colony immunoassay using either rabbit polyclonal or mouse monoclonal antibodies and a peroxidase- conjugated secondary antibody. Fragments spanning most of the rod for each gene were identified from HindIII digests of DNA minipreps from positive clones. Each of the fragments encode high molecular weight hybrid proteins with -galactosidase (>140,000) which are readily resolved from bacterial proteins by SDS-PAGE. Immunoreactive fusion peptides were obtained for HindIII fragments varying in length from 51 bp to 2156 bp. The reactivity of the fusion products with individual monoclonal antibodies was tested by western blotting. The reactivity of MHC B specific monoclonal antibodies confirmed the identification of the unc- 54 gene. Antibody 28.2 recognizes a determinant expressed in the fusion peptide encoded by the 357 bp HindIII fragment from the S-2 region of the unc-54 gene. The location of this epitope is consistent with reactivity of 28.2 with the Unc-54 chymotryptic S-2 fragment (4). Two other MHC B antibodies, 5-2 and 5-3, react with different determinants in the 1054 bp sequence from the unc-54 gene. Antibody 5- 6, which is specific to the S-2 chymotryptic fragment of myosin A (4), reacts with a fusion peptide encoded by a 460 bp fragment from the hinge region of the myo-3 gene but not with fusion proteins encoded by homologous sequences from the other three genes. Similarly, the myosin-C specific antibody, 9.2.1, reacts preferentially with the 2156 bp fusion protein from myo-2. We have one antibody specific to MHC D, 5-17, but it did not react with any of the fusion peptides. Other antibodies specific to MHC A (5-14) and MHC B (5-8,5-22) also did not react with fusion peptides encoded by myo-3 or unc-54. The determinants recognized by these antibodies are likely to be outside the regions represented by the fusion peptides. Antibodies reactive with MHC A or MHC D (the A and D isoforms comigrate on Laemmli gels) however, recognize determinants in the 1577bp HindIII fragment from myo-1. These data suggest the following correlation between the MHC genes and the isoforms they encode: [See Figure 1]