Regulation of variable surface protein gene expression in paramecium

[506009] Forney本项目将研究草履虫可变表面蛋白表达的分子调控机制。四片草履虫（草履虫，stock 51）单株细胞系可以表达至少11种不同的表面蛋白，但在任何时候都只能在表面发现一种类型的蛋白。我们将进行一系列实验来验证表面蛋白基因编码区参与这种互斥表达调控的假设。51A和51B基因的部分将使用重组DNA技术结合形成嵌合体，然后将其转化为A和B基因缺失的突变体。正常情况下，A和B蛋白在细胞表面表现出互斥，因此嵌合蛋白将被检测，以确定它们是否可以与正常的A表面蛋白、B蛋白、两者或两者都共表达。我们的初步研究表明，51A和51B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。一项重要的初步实验表明，将B基因的-1649 ~ +885替换到A基因中，可使嵌合基因与野生型B基因共表达。这个显著的结果是第一个证据，部分编码区在控制互斥是重要的。其他实验将确定共表达所需的区域并研究其作用模式。通过使用钙调蛋白基因启动子同时转录A和B基因，将检验控制互斥的转录后机制的证据。序列特异性抗体将针对A和B蛋白产生，并用作分析表面蛋白结构和表达的工具。本项目将研究控制草履虫可变表面蛋白表达的分子机制。单株四片草履虫株系51可以表达至少11种不同的表面蛋白，但在任何时候表面上只能发现一种类型的蛋白。利用重组DNA技术，适当基因的嵌合体将形成并插入到生物体中，以便识别相关序列。初步证据表明，部分编码区在控制互斥中起重要作用。其他实验将确定共表达所需的区域并研究其作用模式。从这项工作中得到的信息可能会对理解高等生物中的这种遗传现象产生影响。＊＊＊

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. ***