Molecular biology of G protein alpha subunits from bovine photoreceptors and the nematode Caenorhabditis elegans

Author: Lochrie, Michael Alan

Year: 1991

Degree: Dissertation (Ph.D.)

Advisor: Simon, Melvin I.

Committee Members: Simon, Melvin I.; Wold, Barbara J.; Lester, Henry A.; Tanouye, Mark; Kennedy, Mary B.; Sternberg, Paul W.

Option: Biology

DOI: 10.7907/zqph-5443

Abstract

This thesis examines the molecular biology of G protein alpha subunits from bovine photoreceptors and the nematode Caenorhabditis elegans.

Chapter one describes the nucleotide sequence of the bovine cone photoreceptor transducin alpha subunit (Tc[alpha]). Analysis of the sequence defined regions homologous to other GTP binding proteins which may be involved in guanine nucleotide binding, allowed the positions of amino acids which are ADP-ribosylated by pertussis toxin and cholera toxin to be determined, and led to the prediction that G proteins are posttranslationally modified with lipids, which serve to anchor G protein alpha subunits to membranes. Comparison of the Tc[alpha] amino acid sequence with other alpha subunit sequences as they became available provided the first indications that G proteins would be more numerous and diverse than previously thought. The diversity observed among G protein subunits and its structural and functional implications are reviewed in the introduction.

In Chapter 2 the characterization of G protein alpha subunits in the nematode C. elegans is described. Two genes were isolated and their DNA sequences were determined. The protein products of these genes appear to be unique to C. elegans. A cDNA encoding a homolog of Go[alpha] was also isolated and sequenced. Thus, C. elegans has identifiable homologs of mammalian G proteins as well as G proteins that may be unique to it. The chromosomal positions of the genes were determined. Each maps to a unique location near mutations that could be in G protein alpha subunits.

In Appendix 1 the characterization of photoreceptor specific gene expression in human retinoblastoma cultures is described. These cells express cone photoreceptor-specific genes, but not rod photoreceptor specific genes. Therefore they may provide a system for studying the DNA elements required for the expression of genes specifically in cone cells.

Appendix 2 surveys systems for the heterologous expression of transducin alpha subunits in E. coli, yeast, and insect cells. The E. coli expression system offers the most promise for obtaining adequate amounts of active, pure protein.

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