Worm Breeder's Gazette 11(3): 34

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 the daf-4 Gene

Miguel Estevez, Patrice Albert and D.L. Riddle

As is well known,C.  elegans must choose between two alternative L3 
larval fates at the L2 to L3 molt.  If conditions are favorable (ie.  
food and space are abundant) the animal molts into an L3 committed to 
becoming an adult within a day.  However, unfavorable environmental 
conditions induce the developmentally arrested dauer larva .  Dauers 
can survive for more than a month as they search for greener pastures 
in which to resume development toward adulthood.  Mutations defining 
the signal transduction pathway leading to dauer, or non-dauer 
development have been identified, and fall into two categories.  
Constitutive mutants form dauers at a restrictive temperature 
regardless of the presence of food.  Dauer-defectives, on the other 
hand, will not form dauers even when starved.  These isolated mutants 
and their interactions have been extensively characterized (Nature vol.
290, p.  688).  At this time, analysis of dauer induction at the 
molecular level is proceeding at breakneck speed.
daf-4, a late-acting dauer-constitutive gene in the genetic pathway, 
confers several curious phenotypes.  Like other dauer-constitutive 
mutants, daf-4 forms 100% dauers at 25 C.  In addition, daf-4 exhibits 
a small phenotype, and has been reported by Lewis Jacobson's lab to be 
defective in intestinal endocytosis.  It is also interesting to note 
that daf-12, a dauer-defective with a long phenotype, is epistatic to 
daf-4's constitutive phenotype, but not its small phenotype.  This 
might imply a role for the daf-4 gene product in pathways other than 
dauer induction.
Although genetic studies have provided insight into daf-4's relative 
place in dauer induction, molecular analyses of the gene will be 
required in order to further elucidate its function.  Toward this end, 
we have isolated two Tc1 induced daf-4 mutant alleles, and a revertant 
of each.  Genomic Southerns of DNA from the two mutants and their 
revertants were probed with Tc1 in the hope of finding a Tc1 band in 
the mutant alleles that was absent in the revertants.  An XbaIS) 
digest revealed an extra Tc1-containing band approximately 2.4 kb in 
size, and an EcoRIB) digest revealed an extra band approximately 1.9 
kb in size.  Both fragments were cloned.  Using sequences flanking Tc1 
from either fragment to probe Southern blots of mutant and revertant 
DNA's revealed a 1.6 kb upshift in the detected mutant bands relative 
to the revertant bands (this is consistent with the insertion of one 
Tc1).  The flanking sequences from both fragments detect the same 
bands on these Southern blots.  Sequencing of the XB fragments is now 
underway in order to identify the sites of Tc1 insertion in the two 
mutant strains.  Open reading frames have been found, but initial 
database searches have not revealed obvious homologies to known 
proteins.  Genomic and cDNA library screens have yielded 4 genomic 
clones, and 5 cDNA clones.  Sequencing of these clones will begin 
shortly.