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