RUI: Regulation of the Pyrimidine Pathway in Plants

嘧啶核苷酸是DNA和RNA生物合成的前体，是许多代谢过程中活化中间体的成分，这些代谢过程对植物和所有其他生物体的生长发育至关重要。从头合成嘧啶的基本生化步骤在原核生物和真核生物中都是保守的，但嘧啶途径的调控是通过几种不同的策略完成的，包括酶和代谢物的亚细胞区隔、基因复制和基因融合，以及不同的代谢控制机制。在植物中，对嘧啶生物合成的调控机制知之甚少，但大多数证据表明，调控的重要位点是该途径的第一步和/或第二步，涉及酶氨甲酰磷酸合成酶（CPSase）和天冬氨酸氨基甲酰基转酶（ATCase）。本研究将重点阐明植物中ATCase和CPSase活性的各种分子调控机制。作为实现这一目标的第一步，已经克隆了豌豆中编码三种不同atcase的cdna （pyrB1， pyrB2和pyrB3）。pyrB1和pyrB2基因已被证明在豌豆组织中有差异表达。这些基因编码ATCase蛋白，其初级序列相差25%，很可能具有非常不同的动力学特性。并可能不同程度地参与嘧啶途径活性的调节。目前正在研究这种可能性，使用多种方法，包括：1)在ATCase缺陷大肠杆菌菌株中表达的pyrB1和pyrB2产物的动力学表征，2)使用核酸和抗体探针研究编码ATCase的基因在不同植物组织中的表达以及对各种可能影响嘧啶生物合成的处理的响应。近年来，研究人员首次在拟南芥中克隆到编码CPSase的植物基因。编码该植物CPSase小亚基蛋白和大亚基蛋白（分别为carA和carB基因）的cdna的可用性将允许表征该基因及其在植物组织中的表达。在合适的大肠杆菌CPSase突变体中表达这些CPSase蛋白将有助于植物酶的生化和动力学表征。拟南芥CPSase蛋白在细菌中的过表达也有助于其纯化，用于生化研究和CPSase抗血清的生产。这些抗血清和编码植物CPSase蛋白的cdna将成为研究CPSase和嘧啶途径活性调控的宝贵工具。研究人员现在已经确定，与其他真核生物相比，植物中嘧啶途径的基本遗传组织更接近于细菌。尽管如此，植物仍然是唯一的主要生物群体，其中调节从头合成嘧啶的基本控制机制尚未阐明。ATCase和CPSase活性的亚细胞区隔、ATCase和CPSase本身的动力学和调控特性、代谢相互作用以及嘧啶和精氨酸途径活性的调控等许多问题将在本研究中得到解决。诸如此类的研究有助于更广泛地了解植物的初级和次级代谢，并为许多实际目的提供操纵植物生物化学的策略，包括开发除草剂或其他修饰植物生长或发育的化合物，以及生产植物衍生药物和其他有用的代谢物。除了这些研究带来的任何纯粹的科学进步外，几名本科生将参与这项研究。这些学生将在现代化的研究实验室中获得宝贵的训练，这将有助于他们为学士学位后的科学事业做好准备。

SLOCUM Pyrimidine nucleotides are precursors of DNA and RNA biosynthesis and are constituents of activated intermediates in many metabolic processes which are fundamental to growth and development in plants and all other living organisms. The basic biochemical steps in de novo pyrimidine synthesis have been conserved in prokaryotes and eukaryotes but regulation of the pyrimidine pathway is accomplished by several different strategies involving subcellular compartmentation of enzymes and metabolites, gene duplication and gene fusion, and different metabolic control mechanisms. In plants, little is known about the mechanisms which regulate pyrimidine biosynthesis, but most evidence suggests that the important sites of regulation are the first and/or second steps in the pathway, involving the enzymes carbamoylphosphate synthetase (CPSase) and aspartate transcarbamoylase (ATCase). This research will focus on the elucidation of various molecular mechanisms regulating the activity of ATCase and CPSase in plants. As a first step toward this goal, cDNAs encoding three different ATCases in pea (pyrB1, pyrB2 and pyrB3) have been cloned. The pyrB1 and pyrB2 genes have been shown to be differentially expressed in pea tissues. These genes encode ATCase proteins whose primary sequences differ by 25% and it is likely that they have very different kinetic properties. and may participate to different degrees in the regulation of pyrimidine pathway activities. This possibility is being investigated, using a variety of approaches which includes the following: 1) kinetic characterization of the pyrB1 and pyrB2 products expressed in an ATCase-deficient strain of E. coli, 2) use of nucleic acid and antibody probes to investigate expression of the genes encoding ATCase in different plant tissues and in response to various treatments that would be expected to impact pyrimidine biosynthesis. Recently, the first plant genes encoding CPSase in Arabidopsis were cloned by the investigators. Availabili ty of cDNAs encoding the small and large subunit proteins of this plant CPSase (carA and carB genes, respectively) will permit the characterization of this gene and its expression in plant tissues. Expression of these CPSase proteins in appropriate E. coli CPSase mutants will facilitate biochemical and kinetic characterizations of the plant enzyme. Overexpression of the Arabidopsis CPSase proteins in bacteria will also facilitate their purification for biochemical studies and the production of CPSase antisera. These antisera, and the cDNAs encoding the plant CPSase proteins, will be invaluable tools in investigations of the regulation of CPSase and pyrimidine pathway activities. The investigators have now established that the basic genetic organization of the pyrimidine pathway in plants more closely resembles that of bacteria than other eukaryotes. Nonetheless, plants remain the only major group of organisms in which the fundamental control mechanisms regulating de novo pyrimidine synthesis have not been elucidated. Issues such as subcellular compartmentation of ATCase and CPSase activities, the kinetic and regulatory properties of the ATCases and CPSases themselves, metabolic interactions and regulation of pyrimidine and arginine pathway activities and many other questions will be addressed in this study. Studies such as these contribute to a broader understanding of plant primary and secondary metabolism and suggest strategies for the manipulation of plant biochemistry for many practical purposes, including the development of herbicides or other compounds that modify plant growth or development, and production of plant-derived pharmaceuticals and other useful metabolites. Apart from any purely scientific advances resulting from these studies, several undergraduate students will participate in the research. These students will gain valuable training in a modern research laboratory which will help to prepare them for post-baccalaureate careers in science.