Worm Breeder's Gazette 10(1): 18

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Sequencing of dpy-13 IV, a Collagen-like Gene

N. von Mende, D.Mck. Bird, P.S. Albert and D.L. Riddle

Figure 1

The dpy-13 gene is located 0.05 map units to the left of ama-1 IV, a 
gene that has been cloned and sequenced (see Bird and Riddle, this 
issue).  As we reported at the C.  elegans Meeting last May, we 
isolated three spontaneous dpy-13 mutants in Tc1 'high-hopper' strains 
(given to us by Moerman and Waterston) then used cosmid sequences (
provided by Sulston and Coulson) to identify a small region 10 kb from 
ama-1 that carried a 1.6 kb Tc1 insert in each of the three mutants, 
but lacked the insert in two revertants tested.  Hybridization of the 
dpy-13 clone, a 1.25 kb HindIII/EcoRI fragment, to the genomic DNA 
from the EMS-induced dpy-13 mutants, e184 and e458, revealed deletions 
of about 30 bp and 250 bp, respectively.  The left endpoint of a 
deficiency, mDf4, that included ama-1 was known from genetic analysis 
to be nearby, and this was located 8.2 kb to the left of dpy-13.On a 
northern blot with wild-type RNA, the dpy-13 clone detected a 
transcript at 1.1 kb.  An additional band of 1.25 kb was detected in 
RNA from one of the Tc1 insertion mutants.  The 1.25 kb species may 
represent missplicing resulting from the Tc1 insert.  The normal size 
1.1 kb transcript also found in the dpy-13::Tc1 could represent 
alternative splicing, or it might be the transcript of a related gene. 
Even at high stringency, the 1.25 kb probe hybridizes weakly to more 
than a dozen fragments in digests of genomic DNA.  This homology 
extends throughout the fragment.
The Tc1 insertion sites were found to be clustered near the HindIII 
site, and DNA sequencing revealed that this is the 5' end of the gene. 
Nearly all of the gene has now been sequenced.  Including three short 
(~50 bp) introns, the distance from the initiator methionine codon to 
the stop codon is 1.15 kb.  Transcription is from left to right on the 
genetic map.  The DNA sequence reveals an even closer relationship to 
collagen genes than we reported at the C.  elegans Meeting.  There is 
a general similarity in the deduced amino acid sequence (Figure 1), 
especially to col-2 (Kramer et al., 1982, Cell 30: 599-606).  Both 
genes encode five short helical regions with glycine as every third 
amino acid.  When hybridized to genomic DNA at moderate stringency, 
both dpy-13 and col-2 detect the same bands, which presumably 
represent a subset of collagen genes in C.  elegans.  The region 
around ama-1 appears to be rich in collagen genes; ama-1 is flanked by 
two such genes that have not been identified genetically.  The dpy-13 
gene, however, seems to have a unique function, since recessive 
mutations affecting body length are detectable in this gene.

Figure 1