Worm Breeder's Gazette 13(3): 57 (June 1, 1994)

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.

Function of a Domain of the Myosin Heavy Chain Implicated in Familial Hypertrophic Cardiomyopathy

Craig A. Almeida, Kerry E. Swift, John J. Collins

Department of Biochemistry and Molecular, University of New Hampshire, Durham. NH 03820

We are investigating the function of a specific domain of the unc-54 gene product, the major myosin heavy chain (MHC) in C. elegans body-wall muscle. This domain is highly conserved in MHCs from diverse taxa, indicating it is important for MHC function. Supporting this, a missense mutation affecting this domain of the human B cardiac MHC results in the inherited heart disorder familial hypertrophic cardiomyopathy (FHC) (1,2). This mutation results in substitution of glutamine for an arginine residue that is invariant in all MHCs sequenced to date.

We have introduced the corresponding mutation into a cloned copy of the unc-54 gene [ unc-54 (R404Q)] in vitro and tested the effect in vivo. Extrachromosomal arrays containing unc-54 (R404Q)do not confer a dominant unc-54 mutant phenotype in wild-type animals (FHC exhibits autosomal dominant inheritance). unc-54 (R404Q)effects partial rescue when injected into unc-54 mutants. This is observed with both extrachromosomal arrays and arrays integrated via gamma-irradiation. These results leave open the question of the phenotype conferred by unc-54 (R404Q).To eliminate problems inherent to multicopy arrays, we sought to introduce the R404 Qmutation into the resident unc-54 gene by transposon-mediated "gap replacement" (3.4). unc-54 ( r360 )possesses a Tc1 insertion 438 bp downstream of the R404 site. However, unc-54 ( r360 )does not revert at a detectable frequency (<2.3X10 + E-6) in the genetic background in which it was isolated, suggesting the inserted element might not be excising. To activate excision of Tc1 , unc-54 ( r360 )was crossed into the mut-2 mutator background. The resulting strain, TW333 ,reverts from unc-54 ø to wild type at a frequency of 4X10 + E-4, indicating that Tc1 is excising from unc-54 in this strain (a prerequisite for gap replacement). A plasmid containing a portion of unc-54 (R404Q)was injected into TW333 and transformed lines were screened for motile, egg-laying revertants. We isolated five independent revertants: sequence analysis confirmed that one of these is homozygous for the unc-54 (R404Q)mutation (CGT->CAA). Southern blots demonstrate that the injected DNA is no longer present. The phenotype of this strain is essentially wild type, although its egg-laying ability may be somewhat impaired. We are now testing whether unc-54 (R404Q)suppresses twitching and/or hypercontraction of unc-22 and unc-105 mutants. Further studies are aimed at understanding the role of the R404 Qdomain in MHC function.

Literature Cited:

(1) Geisterfer-Lowrance. et al ., (1990) Cell 62 999-1006.

(2) Watkins, et al ., (1992) N. Eng. J. Med. 326: 1108- 1114.

(3) Gloor, et al ., (1991) Science 253: 1110-1117.

(4) Plasterk, R.H.A. and Groenan, J.T.M. (1992) EMBO J. 11: 287-290.