Worm Breeder's Gazette 11(2): 74
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.
The sex determination gene her-1 encodes at least two XO-specific
RNAs, a moderately abundant 0.8 kb transcript, and a much rarer 1.2 kb
transcript (WBG 10 no.3, 11.88, CSH meeting abstracts, 1989). These
transcripts are aberrantly expressed in XX animals carrying a her-1(gf)
allele (n695 or y101), but are absent in XO animals homozygous for
the diepoxybutane-induced her-1 null allele (hv1y101). Our current
model of the exon/intron structure, based on Northern analysis as well
as sequence of several cDNAs and portions of the genomic DNA, is shown
below. We cloned three her-1 cDNAs by probing a lambda gt11 him-8
embryonic cDNA library (Schauer and Wood, WBG 10 no.3, 11.88) with
fragment 'C'. Exhaustive screening with a probe specific for the
large transcript ('A') was negative, and we expect the 1.2 kb mRNA's
low abundance may preclude its isolation from conventional libraries.
To enrich for cDNAs containing the upstream exon(s) we primed 1st-
strand cDNA synthesis using oligonucleotides complementary to a 5'-end
sequence in our longest phage cDNA and amplified the tailed products
in vitro by an 'anchored'-PCR(1, 2, 3) (this should also generate full
length 5' cDNAs from the smaller transcript). We also tried
substituting primers containing sequences from the SL1 and SL2 splice
leaders (generously provided by the Hirsh lab, Synergen Inc., Boulder,
CO) for the upstream anchor. Assaying the PCR products by Southern
analysis (probe 'A' vs. probe 'B') showed that both her-1 transcripts
are trans-spliced to SL1 (the SL2 primer cross-hybrids to a limited
extent in PCR-S. Bektesh and X.-Y. Huang, pers. comm.). This
conclusion was confirmed by sequencing anchor-generated clones for the
smaller transcript. Cloning of the larger-transcript sequence is in
progress.
The longest open reading frame (ORF) in the smaller transcript
begins at the site of SL1 addition (+23). Translation from an ATG
codon at +100 would yield a potential protein of Mr = 7.4 kDa (64 a.a.)
; another ATG at +75 is out of frame with the ORF and precedes a TGA
terminator. Searches of the current protein and DNA databases found
no sequences with significant homology. We conjecture that one or
more upstream exons are spliced into this same ORF to extend the amino-
terminal portion of the protein (the other two frames terminate
quickly after +23). To assay for expression of such proteins we are
raising rabbit antisera against E. coli-her-1 fusion proteins.
[See Figure 1]