Worm Breeder's Gazette 12(2): 48 (January 1, 1992)

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.

A New C. elegans Degenerin Homologue

Mingxia Huang, Marty Chalfie

Department of Biological Sciences, Columbia University, New York, NY 10027

Two genes, deg-1 and mec-4 ,can be mutated to induce neurodegeneration in C. elegans (Chalfie and Wolinsky, Nature 345: 410, 1990; Driscoll and Chalfie, Nature 349: 588, 1991). The proteins predicted from partial cDNAs from these genes, degenerins, are approximately 50% identical. Because additional cross-hybridizing sequences are found in C. elegans genomic DNA, we have been interested in determining whether additional degenerin genes exist.

We were able to amplify several segments from genomic DNA using PCR and degenerate oligos for two conserved regions in mec-4 and deg-1 (CGDPRF...ADFGGQ). Although a mec-4 probe hybridized with two of the fragments (one was the correct size for mec-4 ),a deg-1 probe did not. The lack of deg-1 hybridization may signify that one of the conserved regions used to generate the oligos might be interrupted by an intron in deg-1 (it is not in mec-4 ).The second band showing mec-4 hybridization was cloned and sequenced and had extensive homology to mec-4 and deg-1 .This clone was used to probe Chris Martin's 2-3 kb cDNA library and four cDNAs were found among 6 x10 +E6screened. These cDNAs all represented the same transcript. The largest cDNA (2.3 kb) defines an ORF of 730 amino acids with two in frame stop codons about 150 bp from the 3' end. We are not sure if this cDNA clone contains the complete message since the two most upstream methionines are only 40 bp from the 5' end of the clone and no upstream stop codons are seen in frame. We are calling this gene deg-2 .

deg-2 share extensive sequence identity with mec-4 and deg-1 (54% identity with mec-4 over 469 aa sequence and 47% identity with deg-1 over a 282 aa region - only partial cDNAs are available for these genes). Overall there is a better match (fewer gaps introduced and more extensive homology) between mec-4 and deg-2 than between either and deg-1 .Like mec-4 ,the deg-2 predicted protein contains a hydrophobic domain near but not at the amino terminus (this was predicted for the mec-4 protein from a comparison of genomic sequences of the C. elegans and C. briggsae genes by Monica Driscoll) and two Cys-rich domains and a second hydrophobic region; the second Cys-rich and hydrophobic regions are also predicted from the smaller deg-1 cDNA. The C-termini of all three proteins are not similar, but all are highly charged. Thus, the degenerins may be membrane proteins that span membranes twice. One putative glycosylation site is conserved in all three predicted sequences. All three sequences also contain the Ala near the beginning of the second hydrophobic domain that is mutated in mec-4 and deg-1 to cause neurodegeneration. We are currently obtaining a full length genomic clone so that we can mutate this Ala to determine whether this deg-2 mutation will also result in a dominant degeneration of neurons or other cells.

deg-2 was mapped to YACs Y51E1 , Y75D9 ,and Y58B1 by Alan Coulson; this positions the gene between xol-1 and kin-9 on the X chromosome, approximately 1 map unit to the right of deg-1 .Hybridization to the YAC grid revealed several weaker hybridizing spots (representing, minimally, DNA on chromosomes I, IV, and V). These YACs may identify additional homologues. Interestingly, the deg-2 probe did not hybridize in these experiments to YACs containing mec-4 or deg-1 .We are pursuing the analysis of these other putative degenerin genes and examining the pattern of hybridization of mec-4 and deg-1 probes to the YACs.