Worm Breeder's Gazette 9(3): 25

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.

Progress Towards Isolation of the sqt-1 Gene

J. Kramer, J. Baxter, C. Basch and B. Edgar

We have attempted to 'Tc1 tag' the sqt-1 gene using a 
complementation screen with the recessive left roller allele sc13. 
Heterozygous unc-4(e120) 3) males were mated to 
TR679 hermaphrodites and the Fl progeny were screened for left rollers 
(putatively e120 sc13 / sqt-1 Tc1). Five left roller animals were 
detected among 16,000 Fl progeny. So far, four of these animals have 
been examined. All four of these animals segregate only Rol-Unc and 
Rol progeny. We had expected to see 1/4 wild-type progeny, since the 
null phenotype for sqt-1 is believed to be wild type. Instead, the 
putative sqt-1 Tc1 alleles all behave as recessive left roller 
mutations. Other labs have reported that Tc1 insertion mutations do 
not necessarily produce null phenotypes.
Two independent lines (mutant pairs) were established from each of 
the four original mutants. Each line was backcrossed to N2 five times, 
and the flanking markers unc-4(e104) and lin-29(n836) were crossed in 
and out. The backcrossed lines were examined for the presence of new 
Tc1 elements. Each of the lines contained only 0-3 new Tc1's. For 
three of the mutant pairs there was no new Tc1 element present in both 
members of the pair. Therefore, the left roller phenotype in these 
animals is not caused by a Tc1 insertion. It is possible that these 
mutations are the result of a Tc1 insertion and imprecise excision, or 
they may be due to other mutational events. In one of the mutant pairs,
BE143-BE144, there is a new Tc1 element present in both members of 
the pair. This Tc1 element is on a 3kb EcoRI fragment, and has been 
isolated from a size-selected sub-library of BE143 DNA cloned in 
lambda gt10. The insert from the phage clone has been subcloned into 
pUC18 and named pJK104. DNA flanking the Tc1 insert in pJK104 has been 
probed against genomic DNA's from N2, EM1002, and BE145 (a strain 
congenic for the sqt-1 region from TR679). It is clear that the Tc1 
element in pJK104 is not present in any of these strains, and 
therefore it has transposed in the BE143-BE144 strains.
pJK104 was used to isolate four overlapping cosmid clones, and Alan 
Coulson has kindly provided three more overlapping cosmids. The 
cosmids cover approximately 80kb. Since we have not isolated 
revertants of either BE143 or BE144, we do not know that this Tc1 
element is actually inserted into sqt-1. We would not be greatly 
surprised if sqt-1 were a member of the collagen multigene family. 
However, probing of the cosmid clones has shown that there are no 
collagen genes detectable within the 80kb covered by the cosmids. 
Either sqt-1 is not contained in these clones or it is not a collagen. 
We are also attempting to localize these clones relative to the 
deficiencies in the region of sqt-1. So far, various mnDf DNA's have 
been probed with pJK104 and one of the cosmid clones, with confusing 
results. Both the plasmid and cosmid clone repeatably show reduced 
hybridization intensity when probed to mnDf77 and mnDf87 DNA's, but 
show normal intensity with mnDf83 and mnDf89 DNA's. This is 
inconsistent with the fact that the latter two deficiencies extend 
beyond the former two deficiencies in both directions, according to 
the genetic mapping data. Internal controls have been used to equalize 
for the total amounts of DNA in different lanes. We are trying to 
clear up this discrepancy. Also, DNA's from strains carrying various 
sqt-1 alleles are being analyzed to determine if any alterations in 
restriction pattern are detectable by probing with the cosmid clones.
We have recently begun another screen for Tc1 transposition into sat-
l using Mike Finney's HH*6 mutator strain. Left roller-mutator animals 
are being constructed and will be screened for non-roller progeny.