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Isolation and Characterization of a cDNA Clone for the gamma Subunit of Bovine Retinal Transducin

Hurley, James B. and Fong, Henry K. W. and Teplow, David B. and Dreyer, William J. and Simon, Melvin I. (1984) Isolation and Characterization of a cDNA Clone for the gamma Subunit of Bovine Retinal Transducin. Proceedings of the National Academy of Sciences of the United States of America, 81 (22). pp. 6948-6952. ISSN 0027-8424. http://resolver.caltech.edu/CaltechAUTHORS:HURpnas84

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Abstract

We have isolated and characterized a cDNA clone that encodes the gamma subunit of transducin, the guanine nucleotide binding regulatory protein found in vertebrate photoreceptors. The gamma subunit was separated from the alpha and ß subunits of transducin and purified to homogeneity by reversed-phase high performance liquid chromatography. The sequence of the first 45 amino acids at the amino terminus of this polypeptide was then determined by automated Edman degradation. Oligodeoxynucleotide probes corresponding to two nonoverlapping regions of this sequence were synthesized and then used to screen a bovine retinal cDNA library. One probe, Tgamma1, was a mixture of 32 different heptadecamers complementary to all possible mRNA sequences that could encode a portion of the Tgamma sequence; the other probe, Tgamma2, was a mixture of 128 different heptadecamers. Thirteen clones that hybridized with Tgamma1 were selected. Only one of these had an insert that also hybridized with Tgamma2. The DNA sequence of this insert encodes a 73-amino acid polypeptide that corresponds to the transducin gamma subunit on the basis of amino-terminal sequence, amino acid composition, and carboxyl-terminal sequence. The molecular weight of the mature gamma subunit is 8400. It appears to be synthesized as a discrete polypeptide and not as a domain of a larger precursor polyprotein. The transducin gamma subunit is very hydrophilic and acidic; it has 19 acidic and 11 basic amino acids as well as three cysteine residues. Furthermore, significant homology was found in comparisons of the nucleic acid sequence corresponding to the carboxyl terminus of the gamma transducin transcript with the sequences corresponding to the carboxyl terminus of ras oncogene products, suggesting a possible ancestral relationship between these genes.


Item Type:Article
Additional Information:Copyright © 1984 by the National Academy of Sciences Communicated by Lubert Stryer, July 23, 1984 We thank Jeremy Nathans for supplying us with his bovine retinal cDNA library, Dr. Suzanna Horvath and Carol Graham for synthesizing the oligodeoxynucleotides, Chin Sook Kim for preparation of reagents for the protein sequenator, Vince Farnsworth for performing the amino acid analyses, and Dr. R. Bruce Wallace for advice. This work was supported by a grant from the National Science Foundation to M.I.S., a Helen Hay Whitney Fellowship to J.B.H., National Institutes of Health Block Grant GM06965 to W.J.D., and National Institutes of Health Training Grant GM07401 to D.B.T. The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.
Subject Keywords:photoreceptor biochemistry; ras; signal transduction; molecular evolution
Record Number:CaltechAUTHORS:HURpnas84
Persistent URL:http://resolver.caltech.edu/CaltechAUTHORS:HURpnas84
Alternative URL:http://www.pnas.org/cgi/content/abstract/81/22/6948
Usage Policy:No commercial reproduction, distribution, display or performance rights in this work are provided.
ID Code:6085
Collection:CaltechAUTHORS
Deposited By: Archive Administrator
Deposited On:17 Nov 2006
Last Modified:14 Nov 2014 19:19

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