Worm Breeder's Gazette 11(4): 30

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.

Cloning and Sequencing of a Putative unc-87 cDNA

Susan D. Goetinck and Robert H. Waterston

In Volume 11,#2 of the WBG we reported the identification of a 
cosmid, K01B11, that detects a polymorphism associated with the unc-87 
phenotype.  Here, we report more evidence consistent with the proposal 
that this cosmid contains at least part of the unc-87 gene.  We also 
describe the cloning and sequencing of a putative unc-87 cDNA, and 
make observations on the primary structure of the predicted amino acid 
st1005 is a spontaneous allele of unc-87 isolated in a BO mut-6 
background.(1)  Southern blots containing DNAs prepared from st1005 
and coisogenic revertants, were hybridized with K01B11.  The pattern 
observed was consistent with a 2.5 kb insertion into the wildtype gene 
causing the mutation.  The wildtype member of this polymorphism is a 5.
9 kb BglII fragment; K01B11 also contains a 5.9 kb BglII fragment.
The 5.9 kb BglII fragment was subcloned from the cosmid.  We used 
Northern analysis to determine if this genomic clone contained coding 
region, and whether any message detected was expressed in a pattern in 
st1005 consistent with what one might expect for the unc-87 message in 
a mutator-induced allele.  Total RNA was prepared from mixed-stage 
populations of st1005, a coisogenic revertant, and N2.  Thirty  g of 
each were analyzed on Northern blots.  Hybridization of the genomic 
clone detected a 1.4 kb transcript in N2 and the coisogenic revertant 
but nothing in the strain carrying st1005.  A control hybridization 
with an unrelated probe indicated that intact st1005 RNA was present 
on the blot.  These results suggest that the 1.4 kb transcript might 
be the unc-87 message.  Inability to detect a transcript also suggests 
that st1005 is a hypomorphic, and possibly a null allele of unc-87.We 
next set out to obtain a cDNA, knowing that we could test any positive 
clones for the expected hybridization pattern on Northern blots.  The 
genomic clone was used to screen Bob Barstead's cDNA library.  We 
identified a cDNA clone large enough to account for a full length 
message.  This clone was hybridized to a Northern blot, and the 
pattern observed was the same as that obtained with the genomic clone.
This cDNA clone has been sequenced.  A portion of the trans-spliced 
leader SL1(2) present at the 5' end indicates that the cDNA contains 
the entire coding region.  The sequence predicts a 358 amino acid 
protein.  A diagonal comparison of the predicted amino acid sequence 
with itself revealed seven regularly spaced motifs of approximately 41 
residues.  These motifs have identities ranging from 27 to 60 percent, 
with an average identity of 42 percent.  A consensus derived from 
these motifs was used to search the NBRF protein database.  Fifty 
percent identity exists between approximately the first 35 amino acids 
of the consensus and a similar length stretch of the protein SM22alpha,
a 22 kD peptide isolated from chicken gizzard smooth muscle.(3)  The 
function of the chicken protein is not known, but it appears to be 
smooth muscle specific.  The entire predicted amino acid sequence was 
used to search a translation of the DNA database, and no other 
significant similarity was detected.
DNA from other alleles of unc-87 will be examined by the PCR and/or 
chemical mismatch analysis.  Detection of DNA rearrangements or 
sequence changes in the region covered by the cDNA will provide 
further support that we have in fact identified the unc-87 gene, and 
not a linked gene affected by the st1005 rearrangement.  Future plans 
include production of fusion protein to generate antibodies, 
sequencing of the genomic clone, and transformation rescue.