Anabaena in a filamentous cyanobacterium that responds to nitrogen deprivation by elaborating along the filament semi-regularly spaced cells (heterocysts) specialized for N2-fixation. The pattern of heterocysts is initiated amd maintained by an inhibitory field emanating from developing and mature heterocysts. Dr. Wolk has isolated mutants that are defective early in development and have found cosmids that complement certain of these mutants. To study spatial aspects of gene expression in the developing filament, he has developed a vector that facilitates transcriptional fusions to luciferase (lux) and their maintenance in Anabaena. He proposes to: 1. Further develop and characterize the use of lux to monitor gene expression from individual cells. 2. Identify a gene that is expressed early in development, specifically in cells fated to become heterocysts. Candidate promoters will be fused to lux and light emission along the filament monitored through the course of development. 3. Subject Anabaena carrying the successful promoter/lux fusions to transposon mutagenesis and antisense RNA phenotypic mutagenesis to search for genes that regulate the expression of the promoter. Such genes will be identified by altered light emission. Dr. Wolk will focus on mutants that respond to N- stepdown by inducing light emission in all cells rather than only in developing heterocysts. 4. Clone genes that affect the expression of the promoter and, by fusing them to lux, localize their expression in time and space. Higher eukaryotes develop from single cells to collections of cells of varied types and functions, but inhomogeneous differentiation in prokaryotes is rare. Dr. Wolk proposes to exploit the differentiation of the filamentous cyanobacterium, Anabaena, to study these processes in a simple prokaryote where powerful genetic techniques can be utilized.
