Worm Breeder's Gazette 10(3): 56
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.
unc-93 is a member of a group of interacting genes (unc-93,
sup-10, in
muscle structure and function. unc-93(e1500, n200) and sup-10(n983)
are rare, altered-function mutations that confer a distinctive
uncoordinated ('rubberband'), muscle-defective phenotype. The
rubberband phenotype suggests a defect in the regulation of muscle
contraction. Null alleles of unc-93, sup-10, and
sup-18 result in a wild-type phenotype alone and are recessive
suppressors of e1500 and n983. The wild-type null phenotype of four
of the five genes suggests they may be functionally redundant. sup-10
appears to encode a regulatory myosin light chain (see Cummins, Levin,
Albertson, Horvitz, & Anderson, WBG, 10(1): 38-39).
We cloned the unc-93 gene by transposon tagging. A 6.7 kb EcoRI
fragment containing a Tc1 insertion about 0.3 kb from one end was
cloned into a phage vector (see Figure). The cloned DNA, with the Tc1
removed, was used to probe Southern blots of total genomic DNA from 23
unc-93 mutants. Seven of thirteen mut-2-derived alleles showed
polymorphisms and six of the seven were insertions the size of Tc1.
Five of ten gamma ray-induced alleles showed polymorphisms; most of
these were complex rearrangements. [Additional polymorphisms could
have been missed because the probe probably does not include the
entire gene.] Three restriction fragments that display size
alterations in unc-93 mutants are shown in the Figure. The data
suggest that unc-93 sequences are located on both sides of the HindIII
site closest to the Tc1 insertion shown.
Probing with the 5.1kb EcoRI fragment containing unc-93 DNA showed
no cross-hybridizing bands on a genomic Southern at low stringency.
This result fails to support unc-97s membership in a homologous gene
family. In addition, the cloned unc-93 DNA does not hybridize to the
three known myosin light chain genes, mlc-1,
low stringency. [mlc-1 is sup-10;
bridizes with mlc-1;
sential myosin light chain gene and does not
cross-hybridize with mlc-1 or mlc-2.]Using the 5.1 kb EcoRI fragment
to probe a phage library of N2 genomic DNA, we isolated and
characterized two phage clones. These two clones overlap and each
contains about 13 kb of DNA. We sent the phages to A. Coulson and J.
Sulston at the MRC, and they identified an unc-93 contig of about 530
kb.
Using the 5.1 kb EcoRI fragment to probe a cDNA library (made by
Stuart Kim in our laboratory), we found five hybridizing cDNA clones
out of 165,000 screened. Four of the five clones contain a 250 bp
fragment and the fifth contains a 2650 bp fragment. The low frequency
of cDNA clones for unc-93 suggests that unc-93 may produce a low-
abundance RNA and perhaps a low-abundance protein. Preliminary
Northern analysis is consistent with this observation. [In contrast,
sup-10 encodes a high-abundance RNA.] The 250 bp clone hybridizes to
a 0.6 kb HindIII-HindIII genomic fragment (a fragment not found to
change in any unc-93 mutants). The 2650 bp clone hybridizes to the 5.
1 kb EcoRI fragment and to an adjacent 3.1 kb EcoRI fragment (to the
right in the Figure). Surprisingly, the two classes of cDNA clones do
not cross-hybridize at any appreciable level. The larger cDNA clone
is likely to represent an unc-93 transcript because it localizes to
the same region of the chromosome as unc-93 mutations. The status of
the smaller cDNA clone is unclear. Northern analysis should resolve
which cDNA clone(s) represent the unc-93 transcript(s). We will then
sequence the unc-93 gene and characterize it molecularly.
[See Figure
1]