Class I and IVa β-tubulin isotypes expressed in adult mouse brain are glutamylated

We report the determination of the complete sequences for two chicken beta tubulin genes, beta 3 and beta 5. Taken with the previously published efforts, we have determined the primary structures of five of the seven beta tubulin genes in this vertebrate species. A comparison of these sequences unambiguously reveals that amino acid sequence variations among different beta tubulin gene products are distinctly clustered within an otherwise highly conserved framework of the beta tubulin molecule. To determine the extent to which this pattern of structural heterogeneity is conserved among vertebrates, we have isolated novel beta tubulin sequences from human and mouse cDNA libraries and compared these and all other known vertebrate beta tubulin sequences with the family of chicken polypeptide sequences. What emerges from such comparison is the recognition of distinct, evolutionarily conserved isotypes of beta tubulin that are distinguished primarily by their characteristic carboxyl-terminal variable region sequence and, to a lesser extent, by sequence in an amino-terminal variable domain as well. These correlations represent a convincing demonstration that multiple beta tubulin genes in vertebrates encode a family of closely related but structurally distinct beta tubulin isotypes and further serve to define the sequences of four classes of polypeptide isotypes that constitute that family.

The effect of the antimitotic drug taxol on the association of MAPs (microtubule-associated proteins) with microtubules was investigated. Extensive microtubule assembly occurred in the presence of Taxol at 37 degrees C. at 0 degrees C, and at 37 degrees C in the presence of 0.35 M NaCl, overcoming the inhibition of assembly normally observed under the latter two conditions. At 37 degrees C and at 0 degrees C, complete assembly of both tubulin and the MAPs was observed in the presence of Taxol. However, at elevated ionic strength, only tubulin assembled, forming microtubules devoid of MAPs. The MAPs could also be released from the surface of preformed microtubules by exposure to elevated ionic strength. These properties provided the basis for a rapid new procedure for isolating microtubules and MAPs of high purity from small amounts of biological material. The MAPs could be recovered by exposure of the microtubules to elevated ionic strength and subjected to further analysis. Microtubules and MAPs were prepared from bovine cerebral cortex (gray matter) and from HeLa cells. MAP 1, MAP2, and the tau MAPs, as well as species of Mr = 28,000 and 30,000 (LMW, or low molecular weight, MAPs) and a species of Mr = 70,000 were isolated from gray matter. Species identified as the 210,000 and 125,000 mol wt HeLa MAPs were isolated from HeLa cells. Microtubules were also prepared for the first time from white matter. All of the MAPs identified in gray matter preparations were identified in white matter, but the amounts of individual MAP species differed. The most striking difference in the two preparations was a fivefold lower level of MAP 2 relative to tubulin in white matter than in gray. The high molecular weigh MAP, MAP1, was present in equal ratio to tubulin in white and gray matter. These results indicate that MAP 1 and MAP2, as well as other MAP species, may have a different cellular or subcellular distribution.