Anabaena 7120 is a filamentous cyanobacterium that differentiates specialized N2-fixing cells called heterocysts along its filaments in response to limiting levels of fixed nitrogen in its environment. Heterocysts form in a simple pattern that is maintained during diazotrophic growth. The DNA in vegetative cells of Anabaena contains two elements that interrupt the coding regions of genes required for nitrogen fixation. During differentiation of heterocysts, these interrupting elements are excised by site-specific recombination between repeated sequences at the ends of the elements. The overall goal of this program is an understanding of the regulation of heterocyst differentiation, and the elucidation in molecular terms of the mechanism of these excision events in the context of heterocyst development. The availability of a system for the isolation of genes required for the development of functional heterocysts has made possible a more comprehensive study of the regulatory circuits that control differentiation and the developmental pattern in Anabaena. This work is relevant to both the programmed recombination events that occur in immuno-globulin-producing cells and to the recombinations between directly repeated sequences in humans that lead to deletion of essential genes. Elucidation of the mechanism of pattern formation and maintenance may also have relevance to the problem of spatial control of tissue differentiation in higher eukaryotes, especially as this relates to abnormalities in human fetal development. The projects to be pursued include the isolation of mutants that fail to differentiate heterocysts normally or that fail to rearrange their nif DNA; the classification and complementation of such mutants; analysis of the genes isolated by complementation by DNA sequencing, protein expression in E. coli, and cellular localization by immunocytochemistry; the isolation and characterization of new developmental mutants that arise in populations of cells engineered to overproduce heterocysts; the regulation of the gene encoding excisase studied by using gene fusions and the selection of non- expressing mutants; and the determination of the role in heterocyst development of two newly-isolated genes that affect pattern formation.
