Worm Breeder's Gazette 14(5): 68 (February 1, 1997)

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.


Claire Bonnerot, Nathalie Pujol, Danielle Thierry-Mieg

CRBM, CNRS, BP 5051, 34090 Montpellier Cedex 1, France.

Everybody has played with unc-32(el89) worms, but the molecular
identification of this gene has proven elusive.  We still are not sure,
but have some preliminary evidence that it could correspond to one of
the three presently known vacuolar ATPase genes, C05D12.1 on II,
F35H10.4 on IV and ZK637.8 on III.  In 1988, John Sulston rescued the
el89 allele by transformation with cosmid ZK637.  Four more alleles,
among which two lethals, were isolated in our lab.  The locus became
complex (wbgll.5p93).  The first injections of subclones of ZK637
pointed to the glutathione reductase as the preferred candidate, but
some of the transformants turned out to be gene specific suppressors; in
addition, no mutations were found by sequencing the GR gene.

More subclones of ZK637 were recently injected at 10 ng/ul, together
with pRF4 (100 ng/ul) and/or pPD93-97 (GFP expressed under myo-3 in the
body muscles, at 20 ng/ul).  The results are presented in the table
below.  The data indicate that an 8.2kb fragment (E5-lin9) is able to
rescue at least one Unc and one Let allele.  We noticed however, that
most arrays rescuing the Uncs are unable to rescue the Lets when crossed
in.  There may be a certain dosage needed to rescue the lethals, so that
negative results in the table may become positive when more arrays are
tested (the present number tested is given in parenthesis).  Also, even
when the lethals are rescued, the adults are usually sterile.  The
minimal rescuing fragment can encode a vacuolar proton pump homologue.
In collaboration with Yuji Kohara, we showed that at least three
alternative transcripts of this gene are expressed in the worm: this
could account for the complexity of the locus.


Although the unc-32 locus may well turn out to control the pH of some
vesicles, two observations prevent us from being overconfident that we
have finally identified the gene: 
1- The construct E5-Xk, where the proton pump should be inactivated by a
frameshift in the third exon common to the two cDNAs sequenced, rescues
the el89 allele.
2- An identical mutation, G1339A, was found in two independent alleles,
el89 and fl20, but it lies in the middle of a large intron (1036 or 502
bp long, depending on the alternative splicing).