Worm Breeder's Gazette 15(1): 74 (October 1, 1997)
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|
A close relative? Following several failed attempts by ourselves and others to identify a C. briggsae homologue of the C. elegans her-1 gene by low-stringency hybridization, we set out to find one by synteny. A C. elegans probe derived from a putative protease inhibitor gene (ZK287.4) about 12 kb to the left of her-1 was cloned from P. Okkema's cDNA library by PCR and used to isolate a genomic clone from D. Baillie's C. briggsae genomic library (with about 80% identity at the nucleotide level). A briggsae genomic sequence from this clone was then used to probe the briggsae fosmid grids made by the Wash. U. Genome Sequencing Center, and this probe identified a set of fosmids that form a contig. Preliminary sequencing in St. Louis has so far identified homologues of three other predicted genes present on the C. elegans cosmid ZK287, indicating that we are in the right region. If a her-1 homologue is identified, we will compare its genomic sequence, expression, and function to the C. elegans gene, e.g. by probing Northern blots made with RNA from briggsae wild-type hermaphrodites and a male-incidence-high mutant, mih-3, (kindly supplied by D. Baillie and backcrossed twice), to determine whether small and large transcripts are found and whether they are sex-specific as in C. elegans, and by testing whether the briggsaegene, like C. elegans her-1, causes dominant masculinization in transgenic animals.
A distant relative? An EST sequence with significant similarity to her-1 was recently reported in a cDNA library made from adult males of the filarial parasitic nematode Brugia malayi by the Filarial Genome Project. The sequence of this cDNA was determined and sent to us by Ibrahim Kamal; we have confirmed it by PCR sequencing from a B. malayi cDNA library provided by S. Williams at Smith College. The B. malayi sequence predicts an amino acid sequence which is 42% similar and 35% identical to that of C. elegans HER-1, with similarity distributed over the entire protein after the first Cys residue. More significantly, 14/14 of the C. elegans Cys residues are conserved, as are 12 of the 13 residues that are altered by sequenced lf mutations in C. elegans her-1 (1). Nested PCR from the B. malayi cDNA library using primers to SL1 and the region corresponding to exon 4 of C. elegans her-1 gave one band of the size expected for the full-length cDNA, suggesting that the Brugia mRNA is trans-spliced and that there is no smaller trans-spliced transcript as there is in C. elegans. Primers from regions corresponding to exons 1 and 4 from the C. elegans gene were then used to amplify a sequence by PCR from a B. malayi genomic library obtained from Ulrike Wagner, Giessen, Germany. Preliminary sequencing of the product indicates that the Brugia gene has 3 introns. Intron 1 is at a different position than in C. elegans; introns 2 and 3 are at the same positions, but intron 2 is much smaller than in C. elegans [where it contains the P2 promoter for small transcript (2)]. The PCR product has also been used to isolate clones from the genomic library for further sequencing. In preliminary experiments based on earlier work with the C. elegans gene (2), a construct carrying the unc-54 promoter, the B. malayi coding sequence, and the C. elegans her-1 3'UTR has been injected into hermaphrodites of a him-8;her-1(0) mutant strain (PA43). Some masculinization was observed in the resulting transgenic lines, suggesting at least limited conservation of function in the B. malayi gene product. Confirmation of this result as well as further genomic sequencing are in progress.
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 and others mentioned above for materials and unpublished data.