CHARACTERIZATION AND EXPRESSION OF TUBULIN GENES

我们实验室的工作表明，微管蛋白基因家族在细胞中表达， 单细胞真核生物莱茵衣藻由2 β微管蛋白组成 编码相同蛋白质产物的基因和2个α微管蛋白基因 编码略有不同的基因产物。 为什么这个单倍体生物 每种微管蛋白都有23个基因， 在生命周期中由每一个基因所决定，是我们现在可以 地址. 将介绍三种利用分子生物学和遗传学的方法， 用来了解微管蛋白基因的功能。 1)确定 基因表达的协调程度，特异性DNA探针 将用于定量每个基因的转录水平 在多种条件下，微管蛋白蛋白组装和微管蛋白 基因表达受到调控。 这些条件包括通过 细胞周期，在特定阶段触发微管蛋白基因表达; 切断鞭毛，引发了大量的增加， 微管蛋白（和其他鞭毛基因）的表达;并诱导 鞭毛或细胞质微管的解聚， 微管蛋白基因的表达。 2)定位微管蛋白基因的区域 重要的是调节表达，转化修饰的 将微管蛋白-lacZ融合基因导入，衣原体将用于检查其 在上述条件下表达。 这些变换 实验将有可能检查可能的控制区域， 该基因位于编码序列的3'和5'端。 其中很有前景 一个可能的控制区将是第三插入序列， β微管蛋白基因，这是89%的保守性之间的2个基因。 第三章 抗微管药物的耐药性将用于选择突变体， 微管蛋白基因 对突变体的研究将使我们能够确定 每个微管蛋白基因的功能，通过分析细胞过程 在个体突变体中被破坏。 改变的和野生型基因的比较 应该有助于确定药物结合所需的微管蛋白区域。 一 一些微管蛋白结合药物，如秋水仙胺和长春碱， 发现广泛用作化疗剂。 拟议研究 应该提供抗微管蛋白药物作用的重要见解， 以及更普遍重要的基因调控问题， 在真核系统中表达。

Work from our laboratory has shown that the tubulin gene family in the unicellular eukaryote Chlamydomonas reinhardtii consists of 2 beta tubulin genes coding for identical protein products, and 2 alpha tubulin genes coding for slightly different gene products. Why this haploid organism maintains 23 genes for each of the tubulin types, and what roles are played by each of the genes during the life cycle, are questions we can now address. Three approaches using molecular biology and genetics will be used to understand the functions of the tubulin genes. 1) To determine the degree of coordination in the expression of the genes, specific DNA probes will be used to quantitate the level of transcripts from each of the genes under a variety of conditions in which tubulin protein assembly and tubulin gene expression is regulated. These conditions include passage through the cell cycle, which triggers tubulin gene expression at a specific stage; amputation of the flagella, which triggers a massive increase in the expression of the tubulin (and other flagellar genes); and induced depolymerization of flagellar or cytoplasmic microtubules, which decreases expression of the tubulin genes. 2) To locate regions of the tubulin genes important for regulation of expression, transformation of modified tubulin-lacZ fusion genes into, Chlamydomonas will be used to examine their expression under the conditions described above. These transformation experiments will make it possible to examine possible control regions of the genes both 3' and 5' to the coding sequence. Of particular interest as a possible control region will be the third intervening sequence of the beta tubulin genes, which is 89% conserved between the 2 genes. 3) Resistance to anti-microtubule drugs will be used to select mutants in the tubulin genes. Examination of mutants will allow us to determine the function of each of the tubulin genes by analyzing the cell process disrupted in individual mutants. Comparison of altered and wild type genes should help identify regions of the tubulins required for drug binding. A number of tubulin-binding drugs, such as colcemid and vinblastine, have found widespread use as chemotherapeutic agents. The proposed research should provide significant insight into the action of anti-tubulin drugs, as well as the more generally important question of the regulation of gene expression in a eukaryotic system.