Worm Breeder's Gazette 10(1): 19

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sqt-1 is A Collagen Gene is sqt-1

J. Kramer, J. Johnson, B. Edgar and S. Roberts

Figure 1

We have isolated the sqt-1 gene by a combination of Tc1 tagging and 
chromosome walking/deficiency mapping.  In the last WBG we reported on 
five strains derived from TR679 that carry new recessive left roller 
alleles of sqt-1.  Four of the strains have been carefully analyzed 
for useful new Tc1's, Tc2's, and Tc3's.  Only one strain, BE143 sqt-1(
sc143), had a new element that could not be shown to be irrelevant (
Tc1-BE143).  This Tc1 element was shown to be the result of a recent 
transposition, and was not separated from sqt-1 after recombination 
with the flanking markers unc-4 and lin-29.  However, it seemed 
unlikely that it was actually inserted into sqt-1 because: it has the 
same phenotype as the other four TR679 derived strains, recessive left-
roller, which is not that expected for a sqt-1 null; none of the other 
three characterized strains have any detectable genomic alterations in 
the region where Tc1-BE143 is inserted; no transcripts are detectable 
using DNA flanking Tc1-BE143 as a probe.
The sqt-1(sc143) left roller allele was put back into a mutator 
background (Mike Finney's HH*6 strain) to look for non-roller 
revertants.  mut-2; sc143 animals were placed on race plates - 
bacterial lawn on one side, worms on the other.  Non-roller animals 
generally cross the plate faster than rollers, so screening the lawn 
enriches for non-rollers.  Four independent non-roller animals were 
recovered, backcrossed to remove mut-2, and genetically tested.  Two 
of the resultant strains still carry sc143, but also carry an unlinked 
recessive suppressor.  One strain carries an unlinked, dominant left 
roller allele (? sqt-3 ?), that supresses sc143.  Finally, one strain (
CH1) acts like a sqt-1 null strain, it fails to complement sqt-1 
recessive alleles, and gives a wild-type phenotype when heterozygous 
with other putative sqt-1 null alleles.  So far, only CH1 has been 
further characterized (see below ).
A cosmid contig was established from Tc1-BE143 (Thanks, Alan and 
John) and whole cosmid DNA's have been used as probes to genomic DNA's 
from several of the mnDf strains in the region.  Judging from relative 
hybridization intensities, Tc1-BE143 lies under Df's 77 and 87, but is 
outside of Df's 75, 76 and 86.  This indicates that it lies near, but 
not in the sqt-1 gene.  (Note: this approach has worked well with 
strains carrying small to moderate size deficiencies, but has been 
much harder to interpret when strains with large deficiencies (e.g., 
mnDf's 83 and 89) are used.  This problem is probably due to growth 
disadvantage of the heterozygotes carrying large deficiencies.) Both 
ends of Df86, the left ends of Df's 75, 76, and the right end of Df77 
have been identified in the contig as restriction fragments of altered 
size (see map).  sqt-1 must lie within the overlap region of these 
four deficiencies, since it is not complemented by any of them.  
mnDf86 is a deletion of approximately 85 kb, and the overlap region of 
the four deficiencies is approximately 40 kb.
Cosmid DNA's were probed under low stringency conditions with col-2 
and a single collagen gene was found in the contig, this collagen is 
contained on a 3.6kb EcoRI fragment, in cosmid C25B12, and is situated 
within the region of deficiency overlap.  The CH1 strain has a Tc1 
element inserted into this same 3.6kb EcoRI fragment (Tc1-CH1).  We 
are currently determining whether the Tc1 insertion is in the collagen 
gene, but are willing to predict that the collagen gene is sqt-1.  
None of the other TR679 or HH*6 derived sqt-1 alleles have any 
detectable alterations in this region of the genome.  So, only one out 
of six sqt-1 alleles generated in mutator strains is due to the 
presence of a transposable element.  Since sqt-1 alleles are generated 
in these strains at high frequencies (10+E-4), we are interested in 
knowing what the mutator generated alleles are the result of.
We have performed preliminary Northern blot analysis using the 3.6kb 
EcoRI fragment as a probe under very high stringency wash conditions (
0.03 M Na+, 20% formamide, 65 C).  We detect a transcript of approx.  
1.2kb in dauer, L4, and adult molt RNA's, no transcript Is detected in 
late embryo RNA.  These results are consistent with the temporal 
expression of the sqt-1 phenotype, but we cannot rule out possible 
cross-hybridization.  We are in the process of sequencing the wild-
type and mutant alleles of sqt-1 and will be able to generate gene-
specific probes from the sequence.  Also, we are making RNA's from CH1 
to determine if the 1.2kb transcript is present in this strain.
The evidence so far indicates that sqt-1 is simply another member of 
the collagen gene family, though obviously a rather crucial one, at 
least when it's there.
The map below indicates the position of vim-1, which is the putative 
vitelline membrane protein gene isolated by Paolo Bazzicalupo.  It 
seems possible that vim-1 could actually be ooc-3, since ooc-3 lies 
quite close to the left of sqt-1 and has a phenotype that could result 
from an abnormal vitelline membrane.  It appears, however, that vim-1 
lies under mnDf77, which is supposedly not the case for ooc-3.  This 
question merits further investigation.
[See Figure 1]

Figure 1