Worm Breeder's Gazette 15(2): 30 (February 1, 1998)
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||Dept. of MCD Biology, University of Colorado, Boulder, CO 80309-0347|
|2||Genome Sequencing Center, Washington U. Sch. of Med., St. Louis, MO 63108|
In previously described preliminary experiments, we had identified and begun sequence analysis of two overlapping C. briggsae (Cb) fosmids likely to include the her-1 region, based on hybridization to a Cb probe made from the homologue of a protease inhibitor gene near her-1 in C. elegans (Ce) [WBG 15(1): 74]. Recent sequencing has identified Cb homologues, in conserved order, of all the predicted genes on Ce cosmid ZK287, including her-1. Using primers from sequences in the regions corresponding to Ce exons 1 and 4, we amplified a partial Cb-her-1 cDNA by RT-PCR using RNA isolated from a Cb male-incidence-high mutant strain, mih-3 (kindly supplied by D. Baillie and backcrossed twice). The Cb HER-1 protein (174 amino acids) predicted from the cDNA sequence is only 57% identical and 74% similar to Ce HER-1 (175 amino acids). The identities are fairly evenly distributed throughout the molecule except in the putative signal sequences present in both proteins. All the 13 amino acid residues altered by sequenced Ce-her-1(lf) mutations (1) are conserved, as are all 14 Cys residues and the positions of the three introns. Cb introns 2 and 3 are unusually long (3.9kb and 1.9kb, respectively). Ce intron 2 is also very long (3.5kb) and contains a second promoter (P2), but intron 3 is much smaller (0.5kb). The Ce-her-1 gene produces two male-specific transcripts, the larger rarer one (1.2kb), which is necessary and sufficient for masculinizing function, driven from the P1 promoter upstream of exon 1 and the smaller more abundant one (0.8kb), which has no known function, driven from the P2 promoter in intron 2 (2). The smaller but not the larger is trans-spliced to SL1, and is also present at detectable levels in hermaphrodites. RNA blots probed with the partial Cb-her-1 cDNA show that Cb produces a transcript of about 1.2 kb that is present at a substantially higher level in RNA from a mixed population of mih-3 animals than in RNA from wild-type Cb hermaphrodites, although it is detectable in the latter. No smaller Cb transcript was detected in either population, and RT-PCR using SL1 and Cb exon 4 primers with Cb-mih-3 RNA template showed no indication of an SL1-trans-spliced product. We are currently comparing Cb and Ce upstream and intron sequences for candidate regulatory elements. 1. Perry, M.D., C. Trent, B. Robertson, C. Chamblin, and W. B. Wood (1994) Genetics 138:317-327. 2. Perry, M.D., W. Li, C. Trent, B. Robertson, A. Fire, J. Hageman, and W. B. Wood (1993) Genes & Devel. 7:216-228. Thanks to David Baillie for materials and unpublished data. MNRIITLLTIFGLIGWLEASFSNSQIKEAAQKCCTPNRYECCMDMIKFGTPIKCGYERDLK M R + + G G+ E + + IK+AA+KCCT NR ECC++++KFGTPI+CGY+RD K M-RYLPIFVFLGSFGYTETTLTKELIKDAAEKCCTRNRQECCIEIMKFGTPIRCGYDRDPK IPALVYNCMQQELFAQEPEKRMNLDDSVCCTVFGQDLNDPNRRCESICKTTMQSPSLDAAT +P VY C+Q LFA+EP+K++NLDDSVCC+VFG D ND RRCE+ CK M SPS+DAAT LPGYVYKCLQNVLFAKEPKKKINLDDSVCCSVFGNDQNDSGRRCENRCKNLMTSPSIDAAT KLQKIKDCTLSENVLYQCFTKCQMLRRQDIKIEVLHFNEYCNTTYLQKRPIH Cb her-1 +L IK C+L +NVLY+CF KC+ LR+ IKIEVL F EYCN T++QKR + RLDSIKSCSLLDNVLYKCFEKCRSLRKDGIKIEVLQFEEYCNATFIQKRTFRGV Ce her-1