Worm Breeder's Gazette 14(4): 65 (October 1, 1996)
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.
Depts. of Genetics and Anesthesiology, Case Western Reserve University and University Hospitals of Cleveland, Cleveland, Ohio 44106.
Mutations in the unc-1 gene supress the altered anesthetic
sensitivities of the unc-79 and unc-80 mutants. We have previously
reported (WBG 14; 1 p.56.) that the cosmid C04D1 rescues the unc-1(e580)
mutant. Since, the overlapping cosmid K03E6 has been sequenced , we
microinjected that and found that it rescues unc-1(e580) also. So, we
concentrated on the region common to the two cosmids. The smallest stably
rescuing fragment (5.6 Kb) contains two genes. The plus strand encodes a
protein with homology to Stomatin - a cation channel modulator found in
all tissues including neurons. The minus strand encodes a protein with
strong homology to Neurocalcin - a neuron specific calcium binding
protein. The two genes are
transcribed in opposite directions and Genefinder predicts that their ORFs
partially overlap.
When we probed genomic DNA from the different unc-1 alleles with
subsets of the 5.6 Kb rescuing fragment, the only probe which identified
differences in several unc-1 alleles is a 2.6 Kb genomic fragment
containing the neurocalcin gene along with the last one and a half exons
of the stomatin gene. However, another probe which has only the stomatin
exons common with this probe does not detect these differences in the
unc-1 alleles ; indicating that these changes probably lie outside the
stomatin coding region.
We have isolated cDNAs for both these genes from mixed stage C.
elegans cDNA libraries ( kindly provided by Peter Okkema and Bob Barstead
). The cDNAs are currently being sequenced. Preliminary data indicate that
the coding regions of the two genes do not actually overlap, as was
suggested by Genefinder. In addition, we have isolated a 2.3 Kb deletion
mutant fc51, which lacks the entire coding region of the neurocalcin
.This mutant contains the entire coding region ( as is indicated by the
cDNA ) of the stomatin gene. Sequence analysis shows that fc51 is a
discontinuous deletion . A stretch of 40bp internal to the deletion
breakpoints is present in this mutant. However, these 40 bp lie outside
the coding region of neurocalcin. fc51 behaves like the null class of
unc-1 alleles. Nothern blots with RNA from several unc-1 alleles when
probed with the neurocalcin cDNA show that the neurocalcin transcript is
missing in the fc51 mutant. We have also confirmed this result by RT-PCR.
However, to make matters more complicated (or more interesting ),
preliminary data indicate that the amount of the stomatin transcript is
also substantially reduced in the fc51 mutant. So perhaps, the
transcription of both the genes is co-ordinately regulated by an element
that lies in the region taken out by the fc51 deletion. For both the
genes, the size of the transcript is consistent with the size of the cDNA
isolated for that gene. Taken together, these observations make it seem
likely that the unc-1 is the neurocalcin gene. In humans, the main protein
that neurocalcin interacts with is S100B, which is found in elevated
levels in patients with Alzheimer's disease and Trisomy 21. In each
case, the patients have an increased sensitivity to volatile anesthetics.
Currently, our efforts are directed towards finding out which of
these two genes represents unc-1. So, we are microinjecting a fragment
containing only the neurocalcin gene into an unc-1(e580) mutant.
Ultimately, it is possible that only localizing some of the mutations in
unc-1 to either of these genes may be the only conclusive proof of which
gene is unc-1. The sequencing is also currently underway.