Abstract
The protein ES20, derived from earthworm shock secretion, is a vomeronasally mediated chemoattractant for garter snakes (Jiang, X. C., Inouchi, J., Wang, D., and Halpern, M. (1990) J. Biol. Chem. 265, 8736-8744). Based on its 15-residue N-terminal amino acid sequence, degenerative oligodeoxynucleotide probes were synthesized and used to screen a cDNA library that was constructed in sense orientation using a Uni-ZAPTM XR vector and XL1-Blue MRF' host. A gene was cloned from a polymerase chain reaction as well as from the cDNA library. A combination of the forward degenerative primer and T7 primer was used to obtain gene-specific DNA fragments, from which probes were synthesized and successfully used in screening the cDNA library. The ES20 gene is about 700 base pairs long and encodes 208 amino residues. The ES20 gene was excised from a recombinant plasmid pSK-ES20, ligated to pQE30 expression vector, and transformed into Escherichia coli strain JM109. The selected recombinant plasmids were transformed into expression host cell, E. coli M15[pREP4]. Three transformants were selected, induced with isopropyl-1-thio-beta-D-galactopyranoside for fusion gene expression and an expressed 20-kDa fusion protein purified under denaturing conditions. This protein was refolded and gave a positive reaction against ES20-specific polyclonal antibodies. The fusion protein that had not been denatured remained as an aggregate and was an active chemoattractant for garter snakes.
Highlights
We have been studying chemosignal transduction in the vomeronasal system using garter snakes (Thamnophis sp.) as model subjects
When the vomeronasal organ is irrigated with an aqueous solution of ES20, it induces an increase in the firing rate of mitral cells in the accessory olfactory bulb, the postsynaptic target of receptor cells of the vomeronasal epithelium [1, 12, 13]
We report the cloning of the gene encoding ES20 and its deduced amino acid sequence
Summary
We have been studying chemosignal transduction in the vomeronasal system using garter snakes (Thamnophis sp.) as model subjects. As is the case for most terrestrial vertebrates, garter snakes possess dual olfactory systems, a main olfactory apparatus and a vomeronasal system. Garter snakes detect prey, such as earthworms (Lumbricus terrestris), with their vomeronasal systems (4 – 6). Several chemoattractive proteins to garter snakes have been isolated from earthworms [1, 10, 11]. When ES20 binds to its G protein-coupled receptors on the vomeronasal receptor cell membranes, the intracellular level of inositol 1,4,5-trisphosphate is increased while that of cAMP is decreased [14, 15]. Since a partial amino acid sequence of ES20 protein was previously reported [1], degenerative oligonucleotide primers were synthesized and utilized to clone the gene that encodes this chemoattractive protein. The fusion ES20 protein was recognized by polyclonal antibodies specific for the native ES20 protein and was active in a bioassay for chemoattractivity to garter snakes
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