GENETIC AND BIOCHEMICAL ANALYSIS OF PHYCOBILISOMES

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The aim of this proposal is to understand the regulatory mechanisms involved in controlling the expression of genes encoding the components of the phycobilisome, the major light- harvesting complex in cyanobacteria and red algae. Ultimately this research will extend our understanding of how light is perceived and converted into biochemical signals which alter expression of specific genes. The technology developed and exploited during the course of this work may expand our knowledge of the ways in which cyanobacteria can be genetically manipulated and benefit the analyses of such processes as nitrogen fixation, photosynthesis, acclimation to stress and utilization and degradation of organic compounds. Levels of specific light harvesting, pigmented phycobiliproteins are exquisitely responsive to light quality in some cyanobacteria. This modulation, probably the consequence of changes in the activity of specific phycobiliprotein genes, results in more efficient absorption of prevalent wavelengths of light by the phycobilisome. With information now available on the organization, nucleotide sequences and transcriptional characteristics of phycobiliprotein and linker polypeptide (nonpigmented components of the phycobilisome) genes, we can develop both in vivo and in vitro systems for analyzing the function of the individual phycobilisome polypeptides, and the ways in which the genes for these polypeptides are regulated. Gene transfer techniques (conjugation, transformation) will be used to target lesions into specific phycobiliprotein or linker genes (or genes clustered with the phycobiliprotein and linker genes). Characterization of these mutants will help define the role of these genes in the biosynthesis of the phycobilisome. Gene transfer technology will also enable us to determine which sequence elements (cis acting factors), probably located 5' to the site of transcription initiation, are important to regulated phycobiliprotein gene expression. furthermore, by fusing these regulatory sequences to genes encoding proteins which are toxic to the cyanobacterium, we may be able to select regulatory mutants (organisms which cannot respond to changing light quality). To complement in vivo examination of gene regulation, we will develop an in vitro transcription system (that reflects in vivo regulation) and use it to define the individual components required for regulated expression of the biliprotein genes. These may include both unique species of RNA polymerase and trans acting factors which bind regulatory sequences. Combining in vitro and in vivo technology should enable us to define the molecular processes involved in phycobilisome biosynthesis.