The long range objective of this proposed research is an understanding of the regulation of the assembly of tubulin and to a lesser extent, actin. Four areas are to be considered; 1) The genetic complexity of actin and tubulin, 2) The role of microtubule associated proteins in tubulin polymerization, 3) The function of centrioles in nucleating aster formation and particularly determining whether there is a specific RNA associated with centrioles, and 4) The integration of microtubule assembly with other events in the cell cycle, utilizing Xenopus oocyte maturation as a model system. The genetic complexity of tubulin and actin will be studied using cDNA clones isolated from embryonic chicken brain mRNA to answer the following questions: how many genes are there for tubulin and actin, if multiple how are they arranged on chromosomes, are they selectively expressed? We will determine the amino acid sequence of alpha and beta tubulin from the nucleic acid sequence, study the regulation of tubulin synthesis, isolate genomic clones to study the organization of the tubulin genes, and initiate collaborative genetic studies in Drosophila and yeast. Post-translational modification of microtubule associated protein will be examined by comparing the structure of the proteins synthesized in vivo with those translated from mRNA in vitro. Several studies are also proposed on the physiological significance of the phosphorylation of these proteins and tubulin. Using new sources of centrioles, new methods for their purification and better methods of RNA identification, we will attempt to determine if there is a specific RNA associated with centrioles. The physiological significance of tyrosylation and phosphorylation of tubulin and associated proteins will be correlated with the stage of the cell cycle during Xenopus oocyte maturation. The turnover and compartmentalization of stable derivatives of tubulin will be assessed in vivo after microinjection into oocytes and eggs.
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