Worm Breeder's Gazette 10(3): 52

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.

Cloning of Putative Opsin Genes of C. elegans

John Boom, Kevin Lobo, Michael J. Smith and A.H. (Jay) Burr

Because the 520-600 nm wavelength range of photosensitivity of C.  
elegans (Burr 1985, Photochem.  Photobiol.  41, 577-582) is within the 
range expected for rhodopsin-like visual pigments, we investigated the 
possibility that the C.  elegans genome contains an opsin gene.  We 
used a 1.6 kb Drosophila ninaE opsin cDNA [cRh1], provided by Charles 
Zuker, to probe Southern blots of C.  elegans genomic DNA digested 
with EcoRI.  At a moderate stringency level (62 C; 1xSSPE) the probe 
identified 4 strongly and several weakly hybridizing bands ranging in 
size from 1.5 to 7.7 kb.  Some opsin genes cross-hybridize at moderate 
to high stringencies (e.g.  bovine rod-cell opsin cDNA [cRhB-rod] with 
vertebrate rod-cell opsin genes and cRh1 with the Drosophila Rh2 gene).
Many require low stringencies (e.g.  cRhB-rod with vertebrate cone-
cell opsin genes and cRhB-rod with Drosophila opsin genes), while 
others do not cross-hybridize (e.g.  cRh1 with the Drosophila UV-
sensitive opsin genes Rh3 and Rh4).  Though other signal proteins of 
the opsin family (adrenergic receptors and muscarinic acetylcholine 
receptors) have several structural and functional features in common 
with opsins, their genes do not cross-hybridize with opsin genes even 
within the same species (human) at low stringencies.  In the light of 
these observations, our results at moderate stringency suggest that C. 
elegans has opsin gene(s) with relatively close resemblance to the 
ninaE gene of Drosophila.  We have screened a C.  elegans genomic 
library in Lambda Charon 4 phage (constructed by T.  Snutch) with cRh1 
under conditions identical to those used for the genomic blots.  
Thirteen positive phage giving strong hybridization signals at 
moderate stringency were grouped into 5 classes (designated s401-s405) 
based on the restriction fragment patterns produced by combined 
digestion with EcoRI and HindIII.  Representative phage clones have 
been sent to Alan Coulson and John Sulston to be localized on the 
chromosomal contig map.  The 5 classes were further characterized 
using probes produced by clevage of cRh1 with PstI.  The 0.8 kb 
segment contains the major 3' portion of the coding region of the 
ninaE opsin (Zuker, et al.  1985, Cell 40, 851-858), including the 
sequences encoding the second extracellular loop and the 11-cis 
retinal binding site, both of which are highly conserved in opsins but 
not the other signal proteins.  This probe hybridizes strongly with 7 
different restriction fragments derived from s401-s405.  The 0.6 kb 
PstI segment of cRh1 contains 5' noncoding sequences and the 5' third 
of the coding region, including the highly conserved first cytoplasmic 
loop, the putative binding site for G-protein in all of the signal 
proteins of the opsin family.  It hybridizes weakly with the same 
fragments detected by the 0.8 kb probe and weakly with 2 unique 
fragments in s403.  Thus, restriction and hybridization data suggest 
that our cloned DNA fragments may represent as many as 5 different 
opsin genes of C.  elegans which may have greater sequence identity to 
the Rh1 (Drosophila ninaE) gene than that between Rh1 and some other 
opsin genes.