Worm Breeder's Gazette 7(2): 17
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.
Two sets of experiments have recently established the nature of sup- 5 and sup-7 mediated suppression. In the allele first approach Micheline Harris and Hazel Sive showed that unc-54(e1300) produces a COOH truncated myosin about 7,000 daltons smaller than the full length polypeptide. Suzanne Bolten cloned the DNA from this mutant using an unc-54 probe from Jon Karn to screen an e1300 library in. Jon's lambda1059 vector. We sent the clone to Jon who sequenced the 3' segment, and found a C+T mutation at position 8013 which changed a glutamine codon to an amber terminator. EMS at least in one instance has produced a GC to AT transition! In the suppressor first approach, we made crude tRNA preps from N2, sup-5 and sup-7 strains and sent them to Norma Wills in Ray Gesteland's lab in Salt Lake. Norma used the tRNAs in an in vitro translation system, which employs wheat germ extracts programmed with various messengers to test suppression of all 3 terminators. After initial negative results, we prepared nematode S-100 and Norma included this in her assays. It worked! Convincing suppression of a Brome Mosaic Virus coat protein UAG terminator with sup-7 tRNA was seen, and separation of the tRNAs by RPC-5 and Sepharose 4B chromatography produced a fraction greatly enriched for suppressor activity. The S-100 can come from either wild type or suppressor strains and works only with tRNA from either sup-5 or sup-7. In fact wild type yeast S-100 will substitute for C. elegans S-100. No suppression of other terminators was seen. The amino acid inserted by the suppressor is not yet known, nor do we know for certain that the suppression comes from a change in a tRNA anticodon . Currently we are trying to clone the suppressor genes using complementation of yeast amber mutations with a C. elegans library in an E. coli yeast shuttle vector, and ,screening lambda libraries with a partially purified tRNA preparation with suppressor activity. Nothing definitive yet but some encouraging results.