Worm Breeder's Gazette 11(3): 44
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.
Neuronal degeneration in C. elegans can be caused by mutations in two genes, mec-4 and deg-1. Dominant alleles e1611, u214, and u231 of the mec-4 gene induce the degeneration of the touch receptor neurons. The dominant allele u38 of the deg-1 gene causes degeneration of a different group of neurons (including the PVC interneurons). Although different cells are affected by these mutations, the deaths induced appear physically similar--characterized by a vacuolar appearance of the dying cells before their disappearance. In addition, since mutations in the mec-6 gene suppress both mec-4 and deg-1 degenerations, it appears that the mec-4 and deg-1 gene products act similarly to induce degeneration in different cells. We are interested in deciphering the normal function of these gene products and in determining the nature of the changes that can culminate in abnormal cell death. We report here that mec-4 and deg-1 are members of a gene family and encode highly homologous proteins. The mec-4 gene was mapped to cosmid T20B9 by standard transposon tagging techniques. A 4 kb EcoRI restriction fragment within this cosmid was shown to be the site of three transposon insertions that are absent in revertants. In addition, a gamma ray-induced allele and an EMS-induced allele show polymorphisms in this fragment. 5x10+E6 plaques from Chris Martin's cDNA library (2-3 kb size fraction) were screened and a single 1.6 kb cDNA that included the sequences covered by the polymorphisms was isolated. We have sequenced this cDNA and the corresponding genomic DNA. The cDNA contains an open reading frame that encodes a protein of 494 amino acids. It contains a poly A tail, so we are confident that the 3' end of the gene is included in the cDNA clone. The 494 amino acids are encoded in 12 exons in the genomic DNA. We suspect that the mec-4 gene product may be larger than 494 amino acids since there are substantial open reading frames in the genomic sequence 5' to the position where the cDNA begins. We are currently attempting to identify the real 5' end of the gene through RACE amplification experiments. The deduced amino acid sequence of the mec-4 protein can be roughly divided into three domains. The first 430 amino acids include many cys and pro residues. In addition, this 'domain' has five sites for possible N-linked glycosylation. The next 30 amino acids are largely hydrophobic and are likely to constitute a trans-membrane domain. The carboxy terminal domain contains many charged residues. Although the deduced mec-4 protein does not have extensive homology with any entries in the current GenBank database, the mec-4 protein exhibits striking homology to the deg-1 protein sequence. These proteins are 50% identical over most of their lengths. We have seen evidence of other cross-homologous C. elegans genes on Southern blots. About eight cross-homologous bands are seen using a mec-4 probe. Similar cross-homology has been reported for the deg-1 gene (Wolinsky and Chalfie, WBG 10(2), Chalfie and Wolinsky, Nature, in press). Thus, it appears that the C. elegans genes that can mutate to cause degeneration are members of a gene family. We have constructed libraries of genomic DNA from the three dominant mec-4 mutants and have isolated clones of the e1611, u214, and u231 alleles. We are currently sequencing these alleles to determine the amino acid changes that are associated with degeneration. In addition, we have isolated other cross-homologous genes from C. elegans to continue our study of this gene family.