Microtubules (MTs) are important cellular organelles that are structurally and functionally diverse. Moreover, different MTs are differentially sensitive to different depolymerizing agents and bind different MT associated proteins. It has been hypothesized that MT diversity may be caused by differences in the tubulin composition of MTs. Tubulin, the major structural protein of MTs, is composed of two subunits, alpha and beta tubulin. There are multiple genes for each of these subunits in all of the organisms that have been examined. Not only are there genetic isotypes of alpha and beta tubulin, but there are also posttranscriptionally modified tubulin isotypes, some of which have been shown to be localized to specific MT populations. In this application we propose to analyze the relationship between tubulin genetic isotypes and MT structure and function in the filamentous fungus Aspergillus nidulans. Three of the four Aspergillus tubulin genes, two beta tubulins and one alpha tubulin have been cloned and sequenced. The beta tubulins have been assigned to their genetic loci by DNA mediated integrative transformation, and the function of one of these genes has been examined by DNA mediated """"""""gene disruption."""""""" We propose to clone the remaining alpha tubulin gene, to use DNA mediated integrative transformation to assign the two as yet unassigned alpha tubulin sequences to their genetic loci and to use """"""""gene disruption"""""""" and in vitro mutagenesis to examine the functions of all of these genes. In addition we propose to use isotype specific antitubulin antibodies to localize specific tubulin to specific MTs by fluorescence immunocytochemistry and to construct chaemeric tubulin genes to determine whether tubulin genetic isotypes are interchangeable.
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