STRUCTURE AND EXPRESSION OF MAMMALIAN TUBULIN GENES

这一建议涉及哺乳动物的结构、进化和表达。 微管蛋白基因。这些基因编码α-和β-亚基 微管是一种在许多重要的生命活动中发挥重要作用的结构 细胞器--纤毛、鞭毛、有丝分裂纺锤体和细胞骨架-- 所有真核细胞。建议的方法将涉及1) 人β-微管蛋白基因的DNA序列分析 编码不同的β-微管蛋白亚型。2)a)多肽半抗原的合成 对应于小的羧基末端区域，该区域是 单个β-微管蛋白亚型，b)这些半抗原在半抗原/载体中的用途 用于制备β-微管蛋白同型特异性单抗的结合物 C)这些血清在免疫荧光实验中的应用 确定两个或两个以上的β-微管蛋白亚型是否可以在 单个微管。3)a)小鼠α-亚基编码基因克隆的分离 和构建的全长cDNA文库中的β-微管蛋白序列 胚胎和成体PolA+mRNA，b)基因特异性的产生 3‘-非翻译区亚克隆探针，c)这些探针的原位使用 利用分期动物冷冻切片进行杂交实验 个体微管蛋白的组织特异性发育调控研究 基因。4)利用Beta-微管蛋白基因/BPV穿梭载体 人β-微管蛋白基因序列的鉴定 决定细胞质中β-微管蛋白水平变化的关键 响应细胞内大小调节而发生的mRNAs 单体池。5)a)构建编码Tau和MAP的克隆探针 序列，b)使用这些探针i)确定数量和 编码这些蛋白质的序列的进化保守性，II)到 评估Tau和/或MAP mRNA水平对水平变化的反应 3)研究微管蛋白单体的发育规律。 蛋白质的原位杂交。

This proposal concerns the structure, evolution and expression of mammalian tubulin genes. These genes encode the Alpha- and Beta-subunits of microtubules, structures that perform an essential role in many vital organelles--cilia, flagella, the mitotic spindle and the cytoskeleton--of all eucaryotic cells. The proposed approach will involve 1) characterization by DNA sequence analysis of human Beta-tubulin genes that encode distinct Beta-tubulin isotypes. 2)a) Synthesis of peptide haptens corresponding to small carboxyterminal regions that are unique to individual Beta-tubulin isotypes, b) use of these haptens in hapten/carrier conjugates for the generation of Beta-tubulin isotype-specific monoclonal antisera, c) application of these sera in immunofluorescence experiments to determine whether two or more Beta-tubulin isotypes can coassemble in a single microtubule. 3)a) Isolation of cDNA clones encoding mouse Alpha- and Beta-tubulin sequences from cDNA libraries constructed from whole embryo and adult polyA+ mRNA, b) generation of gene-specific 3'-untranslated region subcloned probes, c) use of these probes in in situ hybridization experiments using cryostat sections of staged animals to study the tissue-specific developmental regulation of individual tubulin genes. 4) Use of Beta-tubulin gene/BPV shuttle vectors for the identification of sequences within human Beta-tubulin genes that are critical in determining changes in the level of cytoplasmic Beta-tubulin mRNAs that occur in response to modulation of the size of intracellular monomer pools. 5)a) Construction of cloned probes encoding Tau and MAP sequences, b) use of these probes i) to determine the number and evolutionary conservation of the sequences encoding these proteins, ii) to assess the response of Tau and/or MAP mRNA levels to changes in the level of tubulin monomer, iii) to study the developmental regulation of these proteins by in situ hybridization.