Worm Breeder's Gazette 10(1): 32

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.

Molecular Cloning of daf-1 IV

L.L. Georgi, P.S. Albert and D.L. Riddle

Mutations in daf-1 convey a dauer-constitutive phenotype with a 
maternal effect.  We found two apparent Tc1 insertion alleles of daf-1 
using the mut-4; background derived in the 
Waterston laboratory.
One spontaneous revertant was also selected for each of the two 
alleles and the sites of reversion were shown to be closely linked to 
daf-1 by appropriate genetic crosses.  Also, the revertants did not 
exhibit a dauer-defective phenotype, indicating that they did not 
harbor new dauer-defective mutations epistatic to daf-1.  To 
facilitate detection of novel, daf-1-associated Tc1-containing 
restriction fragments, strains with lower Tc1 copy number were derived 
by crossing to Bristol backgrounds.
Genomic DNA from lower copy number strains was probed with Tc1, 
revealing a 2.5 Kb  BamHI fragment present in both mutants and absent 
in the one revertant examined.  This fragment was recovered from an 
agarose gel and cloned in pUC18.  Subcloned flanking sequences were 
used to probe genomic Southern blots of the non-outcrossed mutant, 
revertant and parental mutator strains.  The flanking sequences 
hybridized to a 2.5 Kb  BamHI fragment in DNA from both mutants, 
corresponding to the fragment that was cloned and to a 0.9 Kb  BamHI 
fragment in the parental mutator strain and in the examined revertant (
corresponding to absence of Tc1).  The hybridizations also showed that 
the flanking DNA is single-copy.
DNA sequencing has revealed an open reading frame on both sides of 
the Tc1 insertion and a putative intron of ~50 bp.  An initial search 
of the sequence database, however, has not revealed any interesting 
similarities with known genes.  Analysis of the complete wild-type 
gene may produce further information.  Maybe it will be something new 
and different.