Worm Breeder's Gazette 11(4): 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.

Genetic and Molecular Analysis of spe-4

Steve L'Hernault and Michele Arduengo

Mutation of the spe-4 gene disrupts C.  elegans sperm meiosis so 
cells that appear by Nomarski optics to be spermatocytes actually 
contain four haploid nuclei.  This phenotype is not changed when 
either of two EMS induced alleles of spe-4 are placed in trans to the 
noncomplementing chromosome I deletion sDf6, suggesting that this gene 
might have a sperm-limited function (S.  L'Hernault et al., 1988 
Genetics 120: 435).  We have undertaken a molecular analysis of the 
spe-4 gene in order to better understand its role in spermatogenesis.
Initially, our strategy was to map both breakpoints of sDf6, which 
we knew contained at least part of spe-4, and sDf5, which was outside 
spe-4 but included unc-13, a gene that is very close to spe-4.  We 
obtained a number of cosmids between unc-15 and lin-10 and probed 
Southern blots of genomic DNA's prepared from all three spe-4 alleles (
balanced to sDf5), veral different balancers) 
and wild type.  We have not found any novel restriction fragments 
associated with sDf5 or the right breakpoint of sDf6 but have 
confirmed the apparent location of the left sDf6 breakpoint as under 
C05B12 (an unc-15 cosmid), as first discovered by I.  Maruyama.  
However, the cosmid C44E1 identified an allele specific deletion of 
~200 b.p.  associated with the spontaneous spe-4 allele q347 (provided 
by Tim Schedl); this mutation arose on a Bristol chromosome I.  We 
have two-factor mapped spe-4(q347) relative to unc-13 to obtain 
recombinants for molecular characterization.  We analyzed 2,951 F2 
from spe-4(q347) 091)/++ F1 animals and only one 
nonSpe Unc recombinant was obtained.  This recombinant was picked and 
allowed to propagate, and it did not contain the ~200 b.p.  deletion 
in its DNA, further suggesting the association between this deletion 
and spe-4.The cosmid C44E1 has been extensively characterized by I.  
Maruyama because it contains the unc-13 gene; this gene encodes a 6 kb 
RNA.  We have probed Southern blots with oligo labeled restriction 
fragments from this cosmid and have localized the above-mentioned 200 
b.p.  deletion to a 2.1 kb HindIII fragment.  This fragment has been 
cloned and its insert only hybridizes to an abundant 1.3 kb male 
specific RNA on total RNA male vs female Northerns (male=RNA from Mog 
fem-3 gf; female= RNA from fem-1 lf).  We will shortly begin trangenic 
rescue experiments with C44E1 and various subclones in order to 
determine if spe-4 is on this cosmid.