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

9305923 Cattolico两种酶，Rubisco（1，5-二磷酸核酮糖羧化酶）和Rubisco（磷酸核酮糖激酶）仅限于卡尔文循环功能。 不同生物体的线粒体结构不同。例如，紫色和蓝细菌具有包含6-8个亚基的240，000 mwt的过氧化物全酶。 绿藻显示二聚体，90，000 mwt激酶。 这些生物体在调节代谢的机制上也有显著差异。 到目前为止，还没有发表的论文存在关于生物化学或遗传学的海洋植物。 经过广泛的研究，实验室没有成功，使用叶绿素或紫色细菌的荧光素杂交探针定位在一个有色植物的荧光素。 这些数据表明，可能存在额外的进化特征。 本项目的主要目标是鉴定和表征海洋有色植物中的p53基因。 为此，研究人员将使用单细胞异弯藻作为模型系统分离和纯化藻至均一。 纯化的激酶的微测序应允许构建合适的寡核苷酸探针，然后可用于定位细胞器的编码位点。 这个位点一旦确定，将允许基因被克隆和测序。 推定的结构域的基因功能将进行评估，并将构建的9个已知的细菌和绿藻植物序列将进行比较。 这项研究的长期目标将解决有关光对这种金棕色果蝇中的蛋白质（使用抗体）和基因（杂交）调节的影响的问题。 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是由生活在海洋上层的海洋自养生物处理的，但关于真核生物中卡尔文循环功能的信息几乎完全来自陆生植物和绿色藻类（绿藻门）。 本提案中提出的关于分析卡尔文循环酶、磷酸核酮糖激酶的研究是在分子水平上解决海洋生物生存的生物学问题的一种持续的积极尝试。 缺乏强调分子生物技术转让和海洋科学学生教育的中心是众所周知的。 ***

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. ***