Identifying New Open Reading Frames Involved in Redox Regulation and Photosystem II Assembly in Thylakoid Membranes

Arizona State University, Scottsdale AZ

Investigators

Abstract

With the availability of ever increasing amounts of genomic sequence information, the old adage "the more we know, the more we know that we don't know" rings true. Of the thousands of genes in prokaryotes, close to half code for proteins of as yet unknown function. Whereas some of these may code for structural proteins, many unidentified open reading frames are expected to encode important regulatory proteins involved with assembly or stabilization of functional protein complexes. Surprisingly, over the past years it has become clear that many of these putative proteins of unknown function do not have easily recognizable counterparts even in relatively closely related organisms. Yet the functional identification of proteins involved in regulation or stabilization of physiological processes is of major importance in understanding the molecular physiology and metabolism of an organism as a function of its genomic information. Fairly comprehensive cosmid interruption libraries that each contain 30-45 kb fragments of genomic DNA from the yanobacterium Synechocystis sp. PCC 6803 into which transposons have been integrated at random sites will be used to identify new genes involved in photosynthesis. Such transposon interruption libraries are available for about 90 different cosmids, together covering more than 80% of the Synechocystis sp. PCC 6803 genome. Synechocystis strains lacking photosystem I, the major sink for electrons in the plastoquinone pool in the thylakoid membrane, will be transformed with these interruption libraries and transformants will be screened for high-light tolerance. Photosystem I-less strains are light-sensitive because over-reduction of the plastoquinone pool in the thylakoid membrane, which occurs at high light intensity, apparently is lethal. A second approach will use pseudorevertants (second-site mutants restoring a particular phenotype that can be selected for positively). The two approaches (random inactivation of genes in a relatively small region of the genome resulting in positively selectable phenotypes, and pseudorevertant mapping using genomic restriction maps) together provide an excellent way to link specific open reading frames to specific functions. As this approach uses the appearance of specific phenotypes as the first selection criterion and as identification of the affected gene is a simple second step, this project provides a powerful means to identify unknown open reading frames affecting electron flow around the plastoquinone pool in thylakoid membranes. Because a clear phenotype exists, work on mutants that do not segregate wild-type and mutant genome copies or that do not show a phenotype (often a source of frustration in targeted reverse-genetics approaches) is essentially eliminated. This project will contribute significantly to the identification of new open reading frames whose products are involved in processes such as redox regulation of the plastoquinone pool or assembly and stability of photosystem II. It is anticipated that the function of a number of unidentified genes relating to photosynthesis will be found that have not been identified by other means.