The purpose of this research project is to investigate the mechanisms that regulate expression of the individual genes of a multigene family. The experimental system includes the three psbA genes and two psbD genes of a cyanobacterium, Synechococcus sp. strain PCC 7942, also known as Anacystis nidulans R2. The overall goals are to determine the functional significance of expressing two forms of the psbA gene product, to examine changes in expression of the psbA and psbD genes in response to biologically significant environmental cues and inactivation of other members of the gene family, and to identify the mechanisms that regulate gene expression in this system. The products of psbA and psbD are the thylakoid proteins D1 and D2, respectively, which are thought to interact to form the reaction center of photosystem II. The three psbA genes encode two forms of the D1 protein whereas the two psbD genes encode an identical D2 protein. We have raised antisera that are specific for Form I or Form II of D1. We have shown that the psbA genes are differentially regulated and appear to respond to changes in incident light intensity. We also have shown that each of the psbD genes is functional and can individually support photoautotrophic growth. We will continue to examine the differential regulation of the psbA genes by using transcriptional and translational gene fusions as reporters of psb A expression. The gene fusions will be monitored in mutant genetic backgrounds that lack one or more members of the gene family. Transcript half-life measurements will indicate whether differential message stability affects expression of the gene family. New reporter genes will be tested to obtain one that allows visual screening of colonies on plates, making it possible to detect mutants affected in psbA regulation. The products of the psbA genes, Form I and Form II of D1, will be assayed using form-specific antibodies to probe membranes from wild-type and gene-inactivated mutant strains grown under different conditions. The psbA I will be altered by site-directed mutagenesis to target domains of the protein that are likely to be involved in protein maturation and stability. The regulation of the two psbD genes will be monitored using the methods that we have employed for the psbA gene family. Antibodies will be raised to the psbD product to allow assay of D2 levels in combination with different forms of D1 in the thylakoids of mutant strains. The start codon of the psbC gene, which overlaps the psbD1 open reading frame, will be identified experimentally using a reporter gene fused in-frame to putative amino terminal segments of psbC. Physiological properties of mutants expressing a single form of D1 and of other mutants lacking activity of specific genes from the psbA or psbD gene family will be examined. These experiments aim to determine whether loss of specific members of the gene families correlates with loss of adaptation to particular growth conditions.
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