Worm Breeder's Gazette 9(3): 22

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 tra-2

P. Okkema and J. Kimble

We have cloned the tra-2 gene by Tc1 tagging. The tra-2 gene product 
is thought to regulate the fem genes (or their products) to allow 
female development in both soma and germ line of XX animals (Hodgkin 
et al.,1985,CSHSQB,50,585-593). To isolate Tc1 insertions into tra-2, 
we selected for intragenic revertants of the tra-2(gf) allele q122. We 
backcrossed q122 (a gain-of-function allele of tra-2 that was induced 
by EMS on a Bristol chromosome) and a closely linked marker, e120, 
into two strains in which Tc1 actively transposes (TR403 or EM1002). 
Because XX animals heterozygous for q122 develop as females and XO 
animals develop as males, it is possible to construct male/female 
mating strains. Large populations of stable male/female strains (q122 
e120/ mnCl in TR403 or EM1002) were grown on Petri plates to permit 
mating. Five spontaneous recessive tra-2 alleles located in cis to 
q122 were isolated by shifting these animals to liquid culture to 
prevent mating and to select for self-fertility. Two of these alleles, 
q149 and q150, were backcrossed several times against N2. In addition, 
recombinants between each of these tra-2 alleles and linked markers on 
both sides of tra-2 were isolated to eliminate Tc1s which are not 
closely linked to tra-2. A radioactively labelled Tc1 probe was used 
to hybridize N2 DNA, q122 e120/mnCl (in Bristol) DNA, and DNAs 
isolated from a strain carrying the recombined q149 or q150 chromosome 
in Southern blots. A single novel 7.0kb BglII fragment was detected in 
q150 DNA. The fragment carrying this novel Tc1 was cloned, and a 
unique sequence flanking the q150 Tc1 insertion was subcloned to probe 
DNAs from other spontaneous tra-2 alleles. Hybridization with the 
unique probe showed that q149 contains a short deletion and that sc146,
a spontaneous tra-2 allele isolated in TR679 and kindly sent to us by 
Corrine Basch and Bob Edgar, contains a rearrangement. Interestingly, 
this TR679 derived spontaneous allele is not a Tc1 insertion. The same 
probe was then used to isolate 30kb of DNA in the region of tra-2 from 
a C. library provided by Claudia Cummins and 
Phil Anderson. We are currently mapping the tra-2 gene more precisely 
within this 30kb region and attempting to identify the tra-2