Multiple tubulin genes and mechanisms of posttranslational modification are thought to establish the properties and functions of microtubules in different cell types, but the details are still not known for a single cell or microtubule system. Avian erythrocytes contain a unique cytoskeletal structure, the marginal band, which is composed of one of these isotypes. The goal is to characterize tubulin expression and the structure of the erythrocyte tubulin gene in chicken erythrocytes and determine if a constitutive tubulin found in undifferentiated erythroblasts can substitute for the specific isotype during the formation of this unique cytoskeletal structure. Specific aims are to (1) characterize the pattern of expression of constitutive and specific tubulins during erythrocyte differentiation; (2) analyze the gene of the specific tubulin for enhancer elements that activate its expression; (3) analyze function by transfecting cells with tubulin genes and examining cytoskeleton formation by immuno-light and electron microscopy. NIH-3T3 cells will be transfected with constructs containing the specific CB6 tubulin gene, and conversely early erythroblaslts will be transfected with the constitutive CB3 tubulin gene to alter the ratio of endogenous tubulin isotypes to determine if a constitutive isotype can substitute in marginal band formation. A positive effect in band disruption will imply a unique function for the specific tubulin gene. This project will address the fundamental question of what, if any, is the functional distinction between highly-similar isotypes of cellular proteins, encoded for by separate, developmentally-regulated genes.
