Worm Breeder's Gazette 14(3): 47 (June 1, 1996)
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.
|1||Department of Molecular Biology and Biochemistry, Rutgers University, New Brunswick, NJ 08855.|
|2||Department of Biochemistry, New York University, Medical School, New York, NY 10016.|
Degenerins are proteins postulated to encode Na+ ion channel subunits that can mutate to induce necrotic-like cell death. Touch cell degenerins mec-4 and mec-10 are hypothesized to be subunits of a mechanically-gated ion channel expressed in the touch receptor neurons. Several !new! degenerin family members have been identified by the C. elegans sequencing project. We are interested in deciphering the functions of these degenerins. Cosmid R13A1 on chromosome IV harbors a predicted gene (R13A1_4) with a high degree of similarity to degenerins (62%). We analyzed the spatial and temporal pattern of R13A1_4 expression by generating stable transgenic strains harboring fusions of candidate 5! regulatory regions of R13A1_4 to lacZ and GFP. R13A1_4 is expressed in about 35 neurons that have cell bodies situated primarily in the ventral cord and nerve ring. Staining is most intense in L1 and L2 animals, although it is evident at late embryonic stages and persists into adulthood. The neuronal processes of some stained cells are clearly detectable, which has facilitated identification of some of the cells that express this degenerin family member. The PDA, PDB, DA, DB, DD, VD, VC, VB, HSN, ASH, BDU, SABD, PVM, PVC, AVB, AVA, AVD, AVE, AVJ, AVG, AIB, AIN and AIZ neurons express R13A1_4placZ and R13A1_4pGFP. Additional neurons in the nerve ring also strongly express this gene. The posterior touch receptor neurons (PLMs) clearly do not express these fusions. To get hints about the potential mutant phenotype of R13A1_4, we fused R13A1_4 regulatory regions to the toxic mec-4(d), so that the cells in which this new degenerin family member is expressed undergo degeneration. These ablation studies help both in identification of the cells in which a degenerin is expressed and in determination of possible mutant phenotypes (provided that the degenerin is needed for cell function). Animals harboring the R13A1-4 ablation fusion are extremely uncoordinated. The most severely affected animals are nearly paralyzed, cannot back up and coil when prodded with a pick. Transgenic animals also appear to be touch abnormal and somewhat hypercontracted at the posterior.. When we compared the genetic and physical maps we realized that R13A1 mapped in the vicinity of unc-8. unc-8 had been proposed to encode a member of the degenerin gene family (Shreffler et al., 1995) because dominant gain-of-function mutations cause swelling of ventral cord neurons, a phenotype suppressed by mutations in mec-6 (which also suppress swelling and death induced by toxic alleles of degenerin family members mec-4, mec-10 and deg-1). The phenotype of the R13A1_4pmec-4(d) ablation fusion line very closely resembles the phenotype of the semi-dominant unc-8(n491) allele. This ablation phenotype is fully suppressed by mec-6 mutations. Several lines of evidence support that R13A1_4 is unc-8. First, injection of cosmid R13A1 into the semi-dominant unc-8(e15) background strongly suppressed the Unc phenotype in transgenic animals (E. Wolinsky and W.S., unpublished results). Second, we PCR amplified fragments including R13A1_4 from both wild type and from semi-dominant unc-8(n491) and found that when the PCR products were injected into N2 animals, only transgenic lines containing amplified unc-8(n491) DNA were uncoordinated. Third, we identified Tc1 transposon insertions within R13A1_4 in transposon-tagged unc-8 strains (provided by E. Wolinsky and W.S.). Coupled with the expression pattern of R13A1_4, these data strongly argue that R13A1_4 is indeed unc-8. We are currently sequencing the mutant unc-8 alleles e15, e49 and n491 to identify the semi-dominant mutations. Since the motorneuron swelling phenotype of unc-8 mutant strains harboring these alleles is distinct from degeneration induced by mec-4(d), the mutations might map to sites other than those where dominant mutations are located in toxic mec-4(d)alleles. Identifying these mutations should provide insight into the function of degenerins.