Worm Breeder's Gazette 13(3): 64 (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.

Molecular characterization of sma-2

Cathy Savage, Scott Townsend, Alyce L. Finelli,, Richard W. Padgett.

Waksman Institute and Department of Molecular Biology and Biochemistry, Rutgers University, Piscataway, NJ 08855.

We are interested in cell signalling by TGF-ß-like ligands in C. elegans. daf-4 has been shown to be a ser/thr kinase receptor that binds human BMP-2 ,a TGF-ß-like ligand (Estevez et al Nature 365: 644). In addition to its dauer constitutive phenotype, daf-4 mutants show several other phenotypes, suggesting that this receptor has multiple roles in nematode development. The genes sma-2 , sma-3 ,and sma-4 share two phenotypes in common with daf-4 :small body size (Sma) and specific male tail ray and spicule defects (Mab) (S. Baird, personal communication). The striking similarity in mutant phenotypes implicates sma-2 , sma-3 ,and sma-4 in this TGF-ß-like signal transduction pathway. To characterize these genes molecularly, we have begun by focusing on the molecular cloning of sma-2 .

sma-2 maps to the portion of chromosome m that has been completely sequenced (Wilson et al. Nature 368: 32). We have examined the predicted open reading frames (ORFs) in the interval containing sma-2 .Since no homologies to known components of TGF-ß signalling pathways were found in this interval, sma-2 is likely to encode a novel protein involved in TGF-,ß signal transduction. Using the genetic map position of sma-2 ,we targeted several predicted ORFs that may be encoded by the sma-2 gene. We used PCR to clone fragments containing these ORFs from sma-2 mutants and DNA sequence analysis to identify any alterations in the mutants. In this way, we have identified a point mutation in sma-2 ( e502 ),suggesting that the predicted ORF may be the product of the sma-2 gene. We are currently sequencing other sma-2 alleles to confirm this result. Further confirmation will come from transformation rescue with genomic DNA containing sma-2 .

We used a probe from the predicted ORF to screen Andy Fire's mixed stage and embryonic and Stuart Kim's cDNA libraries. Each of these libraries gave positive clones. The longest cDNA of 1.6kb was sequenced. This cDNA is mostly co-linear with the ORF ZK370 .2,but diverges significantly 3' to this ORF. The predicted protein product is about 400 amino acids long, and shares no significant homology to any protein in the database. There are also no hydrophobic stretches, suggestion that the protein may be cytoplasmic or nuclear.

Interestingly, the genome sequencing consortium has identified another predicted ORF, in the cosmid R13F6 ,that is homologous to ZK370 .2[WBG 13(#2)], indicating that sma-2 may be a member of a gene family. Using the putative sma-2 cDNA sequence, we examined the genomic sequence of R13F6 .In this way, we have identified protein coding sequence in R13F6 that is related (47% identical) through the full extent of the sma-2 cDNA. In addition, the amino acid alteration in sma-2 ( e502 )maps to a glycine residue that is also present in the R13F6 sequence, suggesting that this residue may be functionally important. Finally, R13F6 maps very near the location of sma-3 .An intriguing hypothesis is that sma-2 and sma-3 encode related signalling molecules required for TGF-ß-like signal transduction. We are currently injecting R13F6 and nearby cosmids to test for rescue of sma-3 .