Worm Breeder's Gazette 11(5): 65
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.
We have used the polymerase chain reaction (PCR) to isolate Tc1
transposon insertions into mlc-2, one of two regulatory myosin light
chain genes. Our goals were (i) to develop a general method for
identifying mutations in any sequenced gene and (ii) to establish the
phenotype of mlc-2 mutants.
Using one oligonucleotide primer from the target gene and another
from within Tc1, only molecules that contain both primer sites are
substrates for amplification. Such molecules are generated when Tc1
inserts within or nearby the target gene. Reconstruction experiments
using mixtures of wild-type and unc-54::Tc1 genomic DNA indicated that
insertion-containing molecules can be detected following Southern
blots when the mutant genomic DNA is present as a 10+E-5 or greater
fraction of total DNA.
To isolate mlc-2::Tc1 mutants, we took advantage of the increased
levels of Tc1 transposition in strain MT3126. This strain (
constructed by Mike Finney) contains the mutator activity of mut-2(
r459) crossed into a predominantly Bristol genetic background. We
established multiple independent populations of MT3126 and screened
each of them for the presence of mlc-2::Tc1 insertions. When
potential insertion-containing populations were identified, sibling
populations were subdivided in a manner that increased the frequency
of the mutant allele in the population. Following growth, the
subdivided populations were then retested for the mlc-2::Tc1 insertion.
After several rounds of subdivision and testing ('sib-selection'),
we identified single animals that contain Tc1 insertions within or
nearby mlc-2.We isolated three independent mutants using this
procedure. To determine the precise sites of Tc1 insertion, we
sequenced the DNA across the insertional junctions in each mutant
strain. Two insertions, mlc-2(r926::Tc1) and mlc-2(r927::Tc1), are
located at an identical position (but in opposite orientations) within
the third exon of mlc-2. The third insertion, rP2, is located
approximately 180 bp downstream of the predicted mlc-2 polyadenylation
site.
All three insertions are phenotypically wild-type as homozygotes.
Motility, hermaphrodite egg-laying, and male mating are normal. By
polarized light microscopy, the body-wall, pharyngeal, and sex muscles
are indistinguishable from wild-type. The homozygotes are not
temperature sensitive, and they have normal rates of development. mlc-
2 is one of two regulatory myosin light chain genes, designated mlc-1
and mlc-2. The protein sequences of mlc-1 and mlc-2 differ by a
single amino acid. mlc-1 null mutations are also phenotypically wild-
type (see Cummins et al., 1989 CSH Abstracts). We conclude that mlc-1
and mlc-2 likely encode redundant in vivo functions. This appears to
be the case despite the striking differences between these two genes
outside of their protein encoding regions. (For example, mlc-2 is
trans-spliced but mlc-1 is not.) We are currently attempting to derive
a mlc-1 utant.