Investigations have revealed that the regulation pf cytoskeletal protein genes respond to changes in cell morphology acccompanying growth and development. In particular, the production of tubulin messenger RNA responds to the level of unpolymerized tubulin protein in the cell. In most higher eucaryotic cells tubulin genes exist as complex multigene families. The Principal Investigator has started to characterize this multigene family in rat by cloning and sequencing different recombinant DNAs complementary to messenger RNAs isolated from different tissues. This has led to the identification of a neural specific Beta-tubulin mRNA. Further studies have revealed that tubulin mRNA production is regulated during brain development. This is manifested by a dramatic decrease in the neural specific messenger RNA accompanying the major morphological changes (i.e. neurite extention and synapse formation). Coincident with this decrease is the induction of synthesis of a larger Beta-tubulin messenger RNA, which is most predominant in the cerebellum. These studies imply that there is differential expression of the Beta-tubulin mutigene family during brain development. The proposes studies are designed to further characterize all the messenger RNAs expressed by the multigene family, in terms of their sequence complexity and tissue specificity. M13 subclones coeresponding to each of these messenger RNAs will be used to analyse Beta-tubulin gene expression both in vivo and iv vitro. The later studies will be performed in neuroblastoma cells in culture. Correlations will be made between the specific changes in Beta-tubulin gene expression accompanying changes in cell configuration, in particular microtubule organization, which occur during neurite outgrowth. When the specific pattern of expression are defined, a detailed analysis of the possible regulatory mechanisms will be undertaken. This will involve analysing in vivo isolated nuclear RNA and in vitro generated primary transcripts by S1-nuclease mapping techniques.
