Analysis of the Calvin Cycle enzyme Phosphoribulose kinase in a marine autotroph

两种酶，Rubisco（核酮糖1,5二磷酸羧化酶）和PRK（磷酸核酮糖激酶）只局限于卡尔文循环功能。PRK的结构因生物而异。例如，紫色和蓝藻细菌有一个PRK全酶，含有6-8个亚基，240,000 mwt。绿藻显示一个二聚体，90,000 mwt激酶。这些生物在调控PRK的机制上也存在显著差异。迄今为止，还没有发表过关于海洋植物中PRK的生物化学或遗传学的论文。经过广泛的研究，实验室没有成功地使用绿藻或紫色细菌PRK杂交探针定位PRK在一种色藻中。这些数据表明，PRK可能存在其他的进化特征。本项目的主要目标是鉴定和表征海洋色植物中的PRK基因。为此，研究人员将分离和纯化PRK到均匀使用单细胞藻类异质藻作为模型系统。纯化激酶的微测序应该允许构建合适的寡核苷酸探针，然后可以用来定位PRK的细胞器编码位点。一旦确定了这个位点，就可以对该基因进行克隆和测序。将评估PRK基因功能的假设结构域，并构建系统发育系统，其中9个已知的细菌和叶绿素植物序列将进行比较。本研究的长期目标将解决有关光对金褐藻PRK蛋白（使用抗体）和基因（杂交）调控的影响的问题。所有植物和一些细菌将二氧化碳（一种“温室”气体）转化为更复杂的有机分子的过程被称为自养。卡尔文循环是自养生物固定二氧化碳的最普遍的机制。虽然地球上固定的总碳的1/2是由生活在海洋浅层中的海洋自养生物处理的，但真核生物的卡尔文循环功能的信息几乎完全来自陆生植物和绿藻（绿藻）。本提案中提出的关于卡尔文循环酶，磷酸核酮糖激酶分析的研究代表了在分子水平上解决海洋生物生存的生物学问题的持续积极尝试。缺乏强调分子生物技术转移和海洋科学学生教育的中心是公认的。＊＊＊

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. ***