The rearrangement of nitrogen-fixation (nif) genes during heterocyst differentiation in the cyanobacterium Anabaena offers a unique example of an environmentally induced cellular differentiation involving programmed genome rearrangement. When filaments of Anabaena are grown in the absence of a combined nitrogen source, 10% of the vegetative cells differentiate into heterocysts, the cell responsible for nitrogen fixation. This differentiation involves many morphological and biochemical changes including the expression of the nif genes. It has recently been shown that two different genome rearrangements occur near the nif genes of Anabaena during heterocyst differentiation: the excision of an 11 kb circular molecule (nifD excison) from the chromosome, and a second rearrangement with one DNA breakpoint adjacent to the nifS gene, but of unknown dimensions and topology. The long-term objectives of this project are to understand the regulation of the DNA rearrangement, the rearrangement mechanism, and the effects the rearrangement has on gene expression and on the development and function of heterocysts.
The specific aims of the proposed research are as follows: 1. A detailed characterization of the dimensions, topology, and mechanism of the nifS rearrangement by chromosome walking and mapping experiments designed to physically link the DNA breakpoints. 2. The analysis of the developmental expression of the genes affected by the nifS rearrangement with an emphasis on these genes adjacent to the rearrangement breakpoints. These experiments will use RNA isolated from various times during development for Northern and S1 nuclease protection analysis. 3. The identification and characterization of the gene(s) required for the nifS rearrangement, specifically the site-specific recombinase. This will be accomplished using the same strategy which allowed the identification of the nifD excisase gene. 4. The analysis of the developmental regulation of the """"""""excisase"""""""" gene involved in the nifD rearrangement and the corresponding gene for the nifS rearrangement. 5. Evaluation of new techniques that are capable of the identification of other rearrangements in the heterocyst genome.
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