Worm Breeder's Gazette 11(5): 21

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Molecular cloning of lin-3 and transgenic lin-3 Muvs

Russell Hill and Paul Sternberg

Figure 1

lin-3 is required for vulval induction; therefore lin-3 is either 
necessary for the generation of the inductive signal by the anchor 
cell or for the response to the inductive signal by the VPCs.  lin-3 
is interesting to us because genetic experiments indicate that lin-3 
acts early in the pathway of vulval induction and because lin-3 
appears to be essential for vulval induction.  Other lin-3 phenotypes 
include larval lethality, hermaphrodite sterility, and a Mab defect 
that includes crumpled spicules.
The physical location of the lin-3 locus was identified by 
transposon tagging as described previously (WBG 11.3).  We have 
defined a region sufficient for rescue of lin-3 by microinjecting 
genomic subclones in the vicinity of two lin-3 RFLPs, syP1 and syP2.  
lambda PS#2 rescues the lethal phenotype of the lin-3 lethal alleles 
n1059, s751, sy51, sy52, and sy53.  lambda PS1, lambda PS2, and pRH9 
can all rescue the vulvaless phenotype of lin-3.  Surprisingly these 
transgenes also cause a variable fraction of lin-3(+),  
and lin-3(null) animals to a have a multivulva (Muv) phenotype.  The 
multivulva phenotype could be caused by over-expression of wildtype 
lin-3 or some mutant effect of the transgenic lin-3 DNA.  pRH10 and 
pRH22 have no ability to rescue and do not cause a multivulva 
phenotype.  pRH19, which is a truncated derivative of pRH9, rescues 
less well than pRH9.  pRH19 was used to isolate a lin-3 cDNA because 
it does not overlap the poxy and abundant fol-1 (friend of lin-3) 
transcript (which was previously detected by reverse northern analysis 
and shown to reside in a region not necessary for rescue of lin-3).  
The lin-3 cDNA was isolated from a library made by Chris Martin.  This 
cDNA detects both lin-3 RFLPs on southern blots and is single copy in 
the genome.  The 3' end of this cDNA extends outside of pRH19 but is 
within pRH9.
We conclude that pRH9 contains the lin-3 coding region and that 
pRH19 is a crippled 3' deletion of the gene.  We are sequencing lin-3 
and examining the genetic properties of the transgenic lin-3 Muvs.[See 
Figure 1]

Figure 1