Worm Breeder's Gazette 9(2): 21

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 fem-3

T. Rosenquist and J. Kimble

Figure 1

We have cloned the fem-3 gene by Tc1 tagging.  The fem-3 product is 
required for spermatogenesis in both sexes and for negative regulation 
of tra-1 to permit male somatic development (Hodgkin, submitted).  
Eight spontaneous fem-3 alleles have been independently isolated in 
either of two strains active for Tc1 transposition (TR403 and TR679, 
obtained from P. Anderson) by intragenic reversion of the semidominant 
fem-3 allele q20 (Kimble, et. al., 1986, Symp. Soc. Dev. Biol., 44 (in 
press)).  Three TR403-derived alleles were backcrossed to N2 six times 
and then markers (unc-24 and dpy-20) flanking each side of fem-3 were 
recombined onto the backcrossed chromosome.  Southern analysis of 
Tc1 sequences revealed a novel 3.1 Kb  Eco R1 fragment in DNA 
extracted from alleles q102 and q104.  This fragment was cloned from 
q102.  The sequences flanking the novel Tc1 in q102 were used to 
select phage from an N2 genomic library (supplied by C. Cummins and P. 
Anderson).  Subclones of wild-type DNA corresponding to the Tc1 
insertion of 1.6 Kb was found in five of these alleles.  All of the 
insertions fall in a 1.5 Kb region that includes the insertion in q102 
(see map below).  Of the remaining two spontaneous fem-3 alleles, one 
is a complex DNA rearrangement affecting this same region and the 
other shows no detectable restriction map change.
Northern analysis of poly A+ RNA from adult hermaphrodites reveals a 
single 1.7 Kb transcript that hybridized to the DNA to which the 
insertion mutations have been mapped (see map below).  We therefore 
believe that this transcript must be fem-3.  Other transcripts which 
may represent genes adjacent to fem-3 have also been detected.  We are 
currently analyzing the pattern of transcription in this region 
through development and in males.
[See Figure 1]
The arrows above the line indicate points of insertions associated 
with fem-3 alleles.  Arrows below the line indicate transcripts seen 
in adult hermaphrodites detected with strand specific probes.  The 
arrows are placed under the HindIII restriction fragment that 
hybridizes to the transcript and do not indicate absolute position or 
any splicing pattern.

Figure 1