Worm Breeder's Gazette 15(3): 27 (June 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.

A Second Abd-B-like-homeobox on LG IIIR: implications for how nob-1 mutations disrupt posterior embryonic patterning

Kimberly Van Auken, Colleen Cary, Bill Wood

Dept. MCD Biology, University of Colorado, Boulder, CO 80309-0347

        nob-1 IIIR, identified in screens for genes that control
posterior embryonic patterning, is defined by three alleles: ct223 and
ct351 are severe loss-of-function mutations that result in the embryonic
lethal Nob (no back end) phenotype, and ct230 is a weak loss-of-function
mutation that results in viable animals with mildly deformed tails.  We
recently reported the cloning and characterization of two nob-1 cDNAs
(kindly provided by Yuji Kohara) [WBG 15(1):29, 1997; 15 (2):28, 1998]. 
The smaller of the two cDNAs, which contains a 1.35 Kb insert, can
encode a protein containing a homeodomain similar to those of the
posterior paralog group of Hox genes, including Abd-B  and the
vertebrate Hox 9-13 genes.  The second, nob-1B, a longer clone of 2.2
Kb, appears to be alternatively spliced: it contains an additional 25
amino acids from an extended second exon, and the third intron, between
the codons for amino acids 50 and 51 in the homeodomain, is not removed.
Therefore, this transcript should not encode a functional homeodomain.
        To understand the molecular basis for the observed phenotypes,
we are using PCR and newly generated genomic sequence (thanks to Alan
Coulson and the C. elegans G.S.C.) to amplify and sequence the nob-1
region for each mutant allele.  So far, we have determined that the
viable ct230 allele, is a 192 bp deletion that removes the last 3 bp of
exon 2 (or the last 78 base pairs of extended exon 2 in the larger cDNA)
and 189 bp of the second intron.  Barring a cryptic splicing event, this
mutation should result in truncated messages that terminate before the
        ct223 and ct351 also appear to be deletions, although their
precise endpoints are not yet known.  ct223 is a deletion from the
predicted translational start site to at least 1.5 Kb upstream, which
could eliminate essential transcriptional regulatory elements resulting
in lack of all nob-1 transcripts.  In contrast, the ct351 deletion
begins in the second intron and extends at least 4.0 Kb in the 3'
direction past the predicted end of nob-1.  Interestingly, in addition
to the nob-1 homeobox, ct351 also deletes a second homeobox that resides
approximately 2.5 Kb 3' of nob-1.  The latter sequence is predicted to
encode another homeodomain of the posterior paralog group with 88%
sequence similarity to Drosophila Abd-B.  This is in comparison to the
homeodomains of nob-1 and egl-5, which are approximately 72% similar to
Abd-B.  We do not yet know if the second homeobox is part of a new gene
(Genefinder fails to predict one), but preliminary RNA blot analysis
shows that this homeobox is transcribed.  We predict that ct351 should
result in loss not only of nob-1 transcripts, but also of transcripts
containing the second homeobox.  Since ct223 and ct351 result in similar
loss-of-function phenotypes, and ct223 appears to be a deletion at the
5' end of nob-1, we hypothesize that these two homeobox genes share some
5' regulatory sequences, if not 5' coding sequence, as well.  Thus, the
viable mutation may only affect one of the homeodomains, while the
lethal mutations affect both.
        Our current experiments are directed towards determining the
precise nature of the lesions in ct223 and ct351, and how each mutation
affects transcript production.  We also plan to isolate full-length
transcripts containing the second Abd-B-like homeodomain to determine
their structure, and to begin using RNAi to explore the roles of each
transcript in posterior embryonic patterning.  We are also searching for
more homeobox genes in this region, so far without success; however,
sequencing in this region is still unfinished.