CHARACTERIZATION AND EXPRESSION OF TUBULIN GENES

我们实验室的研究表明，微管蛋白基因家族在 单细胞真核生物莱茵衣藻由2β微管蛋白组成 编码相同蛋白产物的基因，以及2个α微管蛋白基因 编码的基因产物略有不同。为什么这种单倍体生物 维持每种微管蛋白类型的23个基因，以及它们所扮演的角色 在生命周期中的每一个基因，都是我们现在可以 地址。使用分子生物学和遗传学的三种方法将是 用于了解微管蛋白基因的功能。1)确定 基因表达的协调度，特定的DNA探针 将用于量化每个基因的转录本水平 在各种条件下微管蛋白组装和微管蛋白 基因表达是受调控的。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的表达； 鞭毛的截断，这引发了大量的 微管蛋白(和其他鞭毛基因)的表达；并诱导 鞭毛或细胞质微管解聚，减少 微管蛋白基因的表达。2)定位微管蛋白基因的区域 对修饰的表达、转化的调控很重要 微管蛋白-lacZ融合基因进入衣藻，将被用来检测它们的 在上述条件下的表达式。这些转变 实验将使检查可能的控制区成为可能 编码序列中的3‘和5’基因。特别感兴趣的是 一个可能的控制区将是第三个插入序列 β微管蛋白基因，两个基因之间的保守率为89%。3) 将利用对抗微管药物的抗药性来筛选突变体 微管蛋白基因。对突变体的检查将使我们能够确定 分析细胞过程中微管蛋白基因的功能 在个体突变体中被扰乱。变异型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。一个 一些微管蛋白结合药物，如秋水仙胺和长春花碱，已经 被广泛用作化疗药物。拟议的研究 应该为抗微管蛋白药物的作用提供重要的洞察力， 以及更普遍的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.