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.

Mouse Monoclonals Meet Nematode Myosin Genes

D. Miller, F. Stockdale, J. Karn

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 
[See Figure 1]