Worm Breeder's Gazette 11(5): 42


pal-1, a Homeobox Gene that Regulates a Homeobox Gene

David Waring and Cynthia Kenyon

pal-1 is a gene required for the formation of rays by the V6 cell in 
males.  As we have described previously, pal-1 effectively prevents 
the T cell from inducing V6 to generate alae and, thereby, allows V6 
to generate rays.  In the absence of the T cell (following laser 
ablation), pal-1 is not required for V6 ray formation.
Previously we presented evidence that pal-1 positively activates mab-
5.  Supporting this model we have found that pal-1 is suppressed by 
the mab-5 gain of function allele e1751 (formerly lin-21).  In unc-79(
e1061) 91) 51); 
90) males, only 5/100 sides had fewer than the wild-
type number of rays in the fan; and in those cases, it appeared that 
V6 generated a few rays but not the wild-type number (V6 makes no rays 
at all on 85% of the sides in pal-1(e2091); 90)).  
The e1751 lesion is a duplication of a region of DNA containing mab-5.  
As Salser and Kenyon describe in an abstract in this gazette, it seems 
likely that the e1751 phenotype is the result of inappropriate 
transcription of mab-5.  The fact that pal1+ activity is not required 
in the presence of the e1751 mutation suggests that pal-1 turns on mab-
5 at the level of transcription.  Whether or not it does so directly 
is unknown.
At last report we had narrowed the search for pal-1 to a 14 kb clone 
that carried the homeobox ceh-3.  Now we have a 5.5 kb, ceh-3 
containing clone, which rescues the pal-1 phenotype completely.  We 
have found 2 independent cDNA clones which are each 1.3 kb (from Chris 
Martin's miracle library).  One is just a few bases longer than the 
other.  The 5.5 kb clone that rescues pal-1 contains about 1 kb 
upstream of the 5' end of the cDNAs and about 400 basepairs downstream 
of the 3' end of the cDNAs.  For this reason we believe that ceh-3 is 
pal-1 (although we have not yet ruled out the alternative that there 
is a second gene on this clone).
These cDNAs encode a ~250aa protein that contains the ceh-3 
homeodomain.  As Burglin and Ruvkin showed, this homeodomain is 
homologous to the caudal gene of Drosophila.  Both cDNAs have part of 
what appears to be a trans-spliced leader sequence (the longer has 10 
bases homologous to the splice-leader), so we believe these cDNAs are 
virtually full length.  We have found no other conserved domains of 
any known significance, but we have found one curious homology to the 
mab-5 gene.  In the mab-5 cloning paper, Costa et al.  pointed out a 
serine rich region followed by a poly-alanine stretch.  Interestingly, 
pal-1 has a very similar region, which includes an identical stretch 
of 9 amino acids SAAAAAAAN (the San Box).  Such poly-amino acid 
stretches are common in homeodomain proteins, but their function is 
unknown.  While this homology may be insignificant, the fact that both 
pal-1 and mab-5 act in V6 to promote ray formation makes this homology 
mildly but not profoundly tantalizing.
Finally, to determine if pal-1 acts directly on mab-5 we are looking 
for pal-1 binding sites in the vicinity of mab-5.  (In principle, if 
pal-1 binds we have made a pal-1/glutatione-S-transferase fusion 
protein (glu-pal) and expressed it in E.  coli.  Our results are very 
preliminary; but we have been able to purify this fusion protein, and 
we have seen sequence specific binding (as judged by band shifts) to 
an oligo-nucleotide probe known to be bound in vitro by engrailed and 
several other homeodomain proteins (E.  Martin-Blanco and T Kornberg, 
personal communication).  We hope to be able to use the glu-pal fusion 
to identify authentic pal-1 binding sites near mab-5.