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

673
these mutants further supporting UNC-46’s role
in regulating transport of GABA into synaptic
vesicles. This technique can also be used to
quantitate neurotransmitter levels in whole
worm extracts. For example, unc-25 animals,
which are missing the enzyme glutamic acid
decarboxylase, have significantly decreased
GABA levels in their extracts compared to N2
animals. We currently are focusing our attention
on obtaining a purer vesicle preparation to
reduce the levels of GABA we see in the unc-47
mutants. Once established, this technique can be
used to characterize and identify genes involved
in the function and regulation of vesicular
transporters in C.elegans.
674. Analysis of Fluoxetine Resistant
Mutants of C. elegans
John M. Kemner, James H. Thomas
Department of Genetics Box 357360 University
of Washington, Seattle, WA 98195
Major depressive disorder is a significant public
health problem, affecting millions of individuals
worldwide. Only since the late 1950s have
chemotherapeutic agents been available for
treatment. One class of these drugs, serotonin
specific reuptake inhibitors (SSRIs), has been
particularly effective. Fluoxetine (Prozac) is a
representative SSRI that has been widely
prescribed. However, in spite of its rational
design, the clinically relevant mode of action
and basis of side effects of this drug remain
unclear.
We have documented a fluoxetine-induced
behavior in C. elegans, nose contraction, that
appears to be independent of 5-HT (nose
resistant to fluoxetine, nrf) (Choy & Thomas
1999). nrf genes fall broadly into two classes
based on the abundance of yolk in eggs. nrf-4,
nrf-5, nrf-6, and ndg-4 share a Peg (pale egg)
phenotype which the other nrf genes do not.
NRF-6 and NDG-4 are related multipass
membrane proteins; their expression in the
intestine is needed for fluoxetine-induced nose
contraction and is consistent with a model of
hydrophobic molecule transport. Additionally,
mRNA for a human NRF-6-related protein is
expressed in the prostate, a highly secretory
organ. This finding could not have been
predicted based on prior understanding of
SSRIs.
674
Additional information can be gained by
analysis of other classes of nrf mutants. None of
the nrf mutants isolated in our screen are fully
resistant to fluoxetine; higher concentrations of
drug and longer incubation times eventually
lead to nose contraction. Strong enhancement of
the Nrf phenotype is seen only in double
mutants between Peg and non-Peg genes, which
indicates the presence of two or more separately
functioning pathways of fluoxetine response in
C. elegans. Through mapping and
microinjection we have accomplished single
cosmid transgenic rescue of two non-Peg
mutants, nrf-2 and nrf-3. Also, three new
non-Peg nrf alleles including at least one new
gene, nrf-7, have been isolated. The molecular