Regulation of variable surface protein gene expression in paramecium

9506009 Forney本项目将研究控制草履虫可变表面蛋白表达的分子机制。 四脲草履虫（Paramecium tetraurelia）的单个细胞系，原种51可以表达至少11种不同的表面蛋白，但在任何一个时间在表面上仅发现一种类型。我们将进行一系列的实验来验证这一假设，即表面蛋白基因的编码区参与了这种相互排斥的表达的调节。51 A和51 B基因的部分将使用重组DNA技术连接以形成嵌合体，然后将嵌合体转化为具有A和B基因缺失的突变体。通常，A和B蛋白表现出与细胞表面的相互排斥，因此将测定嵌合蛋白以确定它们是否可以与正常A表面蛋白、B蛋白、两者或两者都不共表达。我们的初步研究表明，51 A和51 B基因在转录水平上受到调控，但A基因上游的序列不足以控制这种转录的调控。一个重要的初步实验已经表明，将B基因的-1649至+885替换到A基因中导致嵌合基因与野生型B基因的共表达。这个显著的结果是第一个证据，表明编码区的一部分在控制互斥中是重要的。额外的实验将定义共表达所需的区域并研究其作用模式。 控制互斥的转录后机制的证据将通过使用钙调蛋白基因启动子同时转录A和B基因来检查。将产生针对A和B蛋白的序列特异性抗体，并用作分析表面蛋白的结构和表达的工具。 %本计画将探讨草履虫表面可变蛋白表达的分子机制。 一株四脲草履虫原种51可以表达至少11种不同的表面蛋白，但在任何一个时间在表面上只发现一种类型。 使用重组DNA技术，将形成适当基因的嵌合体并插入生物体中，以识别相关序列。 初步证据表明，编码区的一部分是重要的控制互斥。 额外的实验将定义共表达所需的区域并研究其作用模式。 这项工作所产生的信息可能对理解高等生物中的这种遗传现象有影响。 ***

9506009 Forney This project will investigate the molecular mechanism that controls the expression of variable surface proteins in Paramecium. A single cell line of Paramecium tetraurelia, stock 51 can express at least 11 different surface proteins, yet only a single type is found on the surface at any one time. We will perform a series of experiments to test the hypothesis that the coding regions of surface protein genes are involved in the regulation of this mutually exclusive expression. Portions of the 51A and 51B genes will be joined using recombinant DNA techniques to form chimeras which will then be transformed into mutants that have deletions of the A and B genes. Normally, the A and B proteins exhibit mutual exclusion from the cell surface, therefore the chimeric proteins will be assayed to determine if they can be coexpressed with the normal A surface protein, the B protein, both or neither. Our preliminary studies have shown that the 51A and 51B genes are regulated at the level of transcription, yet the sequences upstream of the A gene are not sufficient to control the regulation of this transcription. An important preliminary experiment has shown that substitution of -1649 to +885 of the B gene into the A gene results in coexpression of the chimeric gene with the wild type B gene. This remarkable result is the first evidence that a portion of the coding region is important in controlling mutual exclusion. Additional experiments will define the region required for coexpression and investigate its mode of action. Evidence for post-transcriptional mechanisms that control mutual exclusion will be examined by simultaneously transcribing both the A and B genes using the calmodulin gene promoter. Sequence specific antibodies will be produced against the A and B proteins and used as tools to analyze both the structure and expression of surface proteins. %%% This project will investigate the molecular mechanism(s) that control the expression of variable surface proteins in Paramecium. A single line of Paramecium tetraurelia stock 51 can express at least 11 different surface proteins, yet only a single type is found on the surface at any one time. Using recombinant DNA techniques, chimeras of appropriate genes will be formed and inserted into the organism in order to identify relevant sequences. Preliminary evidence indicates that a portion of the coding region is important in controlling mutual exclusion. Additional experiments will define the region required for coexpression and investigate its mode of action. It is possible that information resulting from this work could have implications for understanding such genetic phenomenon in higher organisms. ***