Program of the 2001 International Worm Meeting - Sternberg Lab ...

671
671. EVIDENCE FOR A
CHA-1-SPECIFIC PROMOTER IN C.
ELEGANS
Dennis Frisby, Tony Crowell, John
McManus, Jim Rand
Program in Molecular and Cell Biology,
Oklahoma Medical Research Foundation.
Oklahoma City, OK. 73104
The cha-1 gene, encoding choline
acetyltransferase (ChAT), is required for
acetylcholine biosynthesis. Along with the
unc-17 gene, which encodes the vesicular
acetylcholine transporter (VAChT), cha-1 is
known to be expressed from a shared promoter
as a member of the cholinergic "operon". The
overall structure of this operon is conserved in
insects and mammals. We now have evidence
for a cha-1-specific promoter in C. elegans. A
putative, alternative 5’ exon was identified
between exons 2 and 3 of cha-1 by phylogenetic
scanning against C. remanei and C. briggsae. It
shares 69% identity and 92% similarity at the
amino acid sequence level with both species.
The structure of this putative exon (referred to
as 2.5) is reminiscent of a 5’ exon: it begins
with an ATG, it lacks an upstream splice
acceptor site, but it contains a consensus splice
donor site at its 3’ boundary. Furthermore,
comparison of the three species revealed two
conserved, apparently regulatory regions of 22
bp and 37 bp located approximately 300 bp
upstream of the apparent start codon. To test
whether exon 2.5 was expressed from a
cha-1-specific promoter, a 521 bp fragment from
the middle of exon 2 to the middle of exon 3
was cloned into the GFP expression vector,
pPD95.67, creating a translational fusion.
Neuronal expression of this construct was
observed in transgenic C. elegans, supporting
the existence of a cha-1-specific promoter.
Interestingly, expression appears to be targeted
to the secretory pathway, suggesting that exon
2.5 encodes an ER signal sequence.
672. A mod-5 SUPPRESSION
SCREEN FOR GENES INVOLVED
IN SEROTONERGIC
NEUROTRANSMISSION
Megan Higginbotham, Rajesh
Ranganathan, Bob Horvitz
HHMI, Dept. Biology, MIT, Cambridge, MA
02139, USA
672
Wild-type animals that have been food deprived
slow their locomotory rate in response to
bacteria (the enhanced slowing response).
Food-deprived mod-5(n3314) mutants
encountering bacteria slow even more than
food-deprived wild-type animals, exhibiting a
hyperenhanced slowing response. mod-5
mutants were originally identified as defective
in serotonin (5-HT) reuptake, and the mod-5
gene has recently been shown to encode a 5-HT
reuptake transporter. To identify additional
genes involved in serotonergic
neurotransmission, we designed a screen for
suppressors of the mod-5(n3314) mutation. The
screen took advantage of a second characteristic
of mod-5(n3314) mutants: hypersensitivity to
exogenous 5-HT. When placed in M9
containing 5-HT, mod-5(n3314) mutants stop
swimming sooner than wild-type animals.
Suppressors of mod-5(n3314) can be identified
as animals that continue to swim after
mod-5(n3314) mutants would have stopped.
We screened 18,350 haploid genomes and
obtained 61 independent mod-5 suppressors.
Eighteen of these isolates were found also to
suppress the hyperenhanced slowing response
exhibited by mod-5(n3314) mutants.
Interestingly, the strength of suppression of the
hyperenhanced slowing response did not strictly
correlate with the strength of suppression of
hypersensitivity to exogenous 5-HT. This
observation may indicate that the exogenous
5-HT and locomotion assays assess different
pathways that involve 5-HT neurotransmission.
To date, six suppressors have been mapped to
linkage groups: one to LG I, three to LG II, one
to LG V, and one to LG X. Further mapping
experiments are underway to determine the
identities of these genes.