9305923 Cattolico Two enzymes, Rubisco (ribulose 1,5 bisphosphate carboxylase) and PRK (phosphoribulose kinase) are exclusively limited to Calvin cycle function. The structure of PRK differs among organisms. For example, Purple and Cyanobacteria have a PRK holoenzyme containing 6-8 subunits of 240,000 mwt. Chlorophytes display a dimeric, 90,000 mwt kinase. These organisms also significantly differ in the mechanisms by which PRK is regulated. To date, no published paper exists concerning the biochemistry or genetics of PRK in a marine plant. The laboratory had no success, after extensive study, using either chlorophytic or purple bacterial PRK hybridization probes in localizing PRK in a chromophytic alga. These data suggests that additional evolutionary signature(s) might exist for PRK. The primary goal of this project is to identify and characterize the PRK gene in a marine chromophytic plant. To this end investigators will isolate and purify PRK to homogeneity using the unicellular alga Heterosigma carterae as a model system. Microsequencing of the purified kinase should allow construction of appropriate oligonucleotide probes which can then be used to locate the organellar coding site of PRK. This site, once identified, will allow the gene to be cloned and sequenced. Putative domains of PRK gene function will be assessed, and phylogenies will be constructed in which the 9 known bacterial and chlorophytic plant sequences will be compared. The long term goals of this research will address questions concerning the effect of light on PRK protein (using antibodies) and gene (hybridization) regulation in this golden-brown alga. %%% The process by which all plants and some bacteria convert CO2 (a"greenhouse" gas) into more complex organic molecules is known as autotrophy. The Calvin cycle ranks as the most universal mechanism by which autotrophs fix CO2. Although 1/2 of the total carbon fixed on earth is processed by marine autotrophs that live in the l ighted strata of the ocean, information on Calvin cycle function in eukaryotes has been derived almost exclusively from terrestrial plants and green algae (Chlorophyta). The research presented in this proposal on the analysis of the Calvin cycle enzyme, phosphoribulose kinase represents a continued aggressive attempt to address biological questions of survival by ocean dwelling organisms at the molecular level. The lack of centers emphasizing molecular biotechnology transfer and student education in the ocean sciences is well recognized. ***
