Worm Breeder's Gazette 15(2): 22 (February 1, 1998)
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.
| 1 | Washington University School of Medicine, St. Louis, MO 63110 |
| 2 | Oklahoma Medical Research Foundation, Oklahoma City OK 73104 |
unc-10 mutants exhibit a variety of behavioral defects including
locomotory, pharyngeal pumping and defecation abnormalities.
Futhermore, these mutants are resistant to the
acetylcholinesterase inhibitor aldicarb. In concert, these
phenotypes suggested that unc-10 could encode a synaptic
component. Previous genetic mapping of unc-10 positioned it
left of center on chromosome X. However, the gene was only
roughly mapped relative to cloned genes. Our interest in a gene
encoding a rab3 GTP-binding protein effector which physically
mapped in the unc-6 unc-18 interval led us to test if the gene
might be mutated in unc-10 animals. pRIM4, a 17.5 kb genomic
subclone of C34H12 containing the C. elegans homolog of Rab3
interacting molecule (Rim) rescued the behavioral phenotypes of
both unc-10(e102) and unc-10(md330) . unc-10 + pRIM4 trangenic
animals also exhibited aldicarb sensitivity comparable to wild
type animals. Additionally, we examined ten unc-10 alleles
(e102, js244, md293, md330, md1117, md1972, md1995, md2122, ox64
and ox67) for polymorphic lesions using PCR. md330 and md1117
both delete large portions of the coding region of the Rim gene.
Specifically, md1117 consists of an 8.6 kb deletion that removes
the entire coding region of the gene (500 bp upstream of the ATG
to 100 bp downstream of the termination codon). Finally, we
refined the map position of unc-10, positioning it between unc-6
and dpy-7; a position that corresponds to the physical location
of the Rim gene. Specifically, from unc-10(e102)/unc-6(n102)
dpy-7(e88) 6 of 16 Dpy non Unc recombinants carried unc-10 and
6 of 10 Unc non Dpy recombinants carried unc-10. Thus, we
conclude that unc-10 encodes the C. elegans homolog of rab3
effector Rim. Rim was isolated by Thomas Sudhof's group in a
two-hybrid screen using an activated form of rab3 as bait (Wang
et al. 1997. Nature 388:593). Vertebrate Rim binds to
activated (GTP-bound forms of Rab3), but not to GDP-Rab3 or
other Rab proteins. Most interestingly, Rim is localized to the
presynaptic nerve terminal. Structurally, Rim is a very large
protein containing a zinc finger domain, a proline rich domain,
a PDZ domain, and two C2 domains. The highest conservation
between the C. elegans gene and vertebrate Rim occurs in the C2
domains. Similar C2 domains are also found in synaptotagmin
(snt-1), unc-13, and rabphilin-3A. Our great interest in the
gene stems for its potential use as a novel synaptic marker.
Since PDZ domains have been demonstrated to localize components
synaptically, we engineered GFP::Rim-PDZ domain constructs.
Unfortunately, these fusion proteins containing only the Rim-PDZ
domain fail to localize to synaptic regions. Production of
antisera and other GFP constructs are in progress.
Interestingly, the phenotype of unc-10 Rim mutants are much more
severe than those of rab-3 (Nonet et al. 1997. J. Neurosci
17:8061) or aex-3 mutants (a nucleotide exchange factor for
rab-3; Iwasaki et al. 1997. Neuron 18:613). This suggests that
some Rim functions are probably independent of rab-3 activity.
Through deletion of various domains of Rim, we intend to define
the functional regions of this protein. For example, is the
rab-3 binding domain (zinc finger) required for Rim function? We
thank Terese Rakow and Bill Schafer for sharing unc-10 mapping
data and Erik Jorgensen for unc-10 alleles.