TRANSCRIPTIONALLY REGULATED GENES IN A SYMBIOSIS

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The major goal of the proposed research is to provide a more complete understanding, at the molecular level, of interactions between the eukaryotic water fern Azolla and its endosymbiont, the filamentous cyanobacterium Anabaena. Molecular and ultrastructural techniques will be used to achieve this goal. Two genes that are transcriptionally regulated when Anabaena is in the symbiotic state will be characterized in greater detail in the proposed work. These are the g1nA and psbA genes, which encode glutamine synthetase and the 32-kD protein of photosystem II. We plan to determine the structure and analyze the expression of these genes in the endosymbiont. The g1nA gene will be identified and isolated from a genomic library of endosymbiont DNA. The 5'-flanking and amino-terminal regions of this gene will be identified and sequenced. The g1nA promoter(s) used by the endosymbiont for transcription will be identified. Glutamine synthetase will be localized within individual cells of cyanobacterial filaments with immunoelectron microscopy. With respect to the psbA genes, the number of genes comprising this multi-gene family will be determined. All members of this gene family will be isolated from a genomic library. Unique DNA fragments that can serve as gene-specific probes for the different members of the psbA gene family will be identified. Specific gene copies that are transcribed in the endosymbiont will be identified. Also, it will be determined whether different sized psbA transcripts found in endosymbiont RNA are the result of specific processing, transcription of different gene copies, or both. It will be determined whether psbA genes are transcribed in heterocysts. Also, the location of 32-kD proteins within cells of cyanobacterial filaments will be elucidated with immunoelectron microscopy. This work will provide answers to basic questions regarding the heterocyst differentiation process of cyanobacteria,k and indeed the mechanism of cellular differentiation in the broadest sense: where are GS and the 32-kD proteins located, and how are their genes regulated. The proposed work should add interesting insight as to the mechanisms of interaction between eukaryotic and prokaryotic genomes resulting in the control of gene expression. This may provide fundamental information on controls of gene expression that could also hold true for host-pathogen or other medically important associations.