Abstract. We describe the structure of a novel and unusually heterologous 13-tubulin isotype (MI31) isolated from a mouse bone marrow cDNA library, and a second isotype (MI33) isolated from a mouse testis cDNA library. Comparison of MI31 and MI33 with the completed (MI34, MI35) or extended (MI~2) sequence of three previously described 13-tubulin isotypes shows that each includes a distinctive carboxy-terminal region, in addition to multiple amino acid substitutions throughout the polypeptide chain. In every case where a mammalian interspecies comparison can be made, both the carboxy-terminal and internal amino acid substitutions that distinguish one isotype from another are absolutely conserved. We conclude that these characteristic differences are important in determining functional distinctions between different kinds of microtubule.The amino acid homologies between MI32, MI33, M134, and M135 are in the range of 95-97%; however the homology between MIll and all the other isotypes is very much less (78%). The dramatic divergence in MI31 is due to multiple changes that occur throughout the polypeptide chain. The overall level of expression of MI31 is low, and is restricted to those tissues (bone marrow, spleen, developing liver and lung) that are active in hematopoiesis in the mouse. We predict that the MI31 isotype is functionally specialized for assembly into the mammalian marginal band. MICROTUBULES are involved in a remarkable variety of cellular processes, including mitosis, morphogenesis, and the motion of cilia and flagella. With the discovery that vertebrate tubulins are encoded by multiple genes, the question arose as to the contribution of different tubulin gene products to the diversity of microtubule function. One hypothesis is that the microtubules involved in each cellular function are composed of special ¢t-and [~-tubulins (7, 27). A modification of this view is that some, but not all, ~t-and 13-tubulin isotypes contribute to the functional diversity of microtubules either through their differential polymerization, or by virtue of unique interaction with distinct microtubule-associated proteins. At the other extreme, one could imagine that all ¢x-and 13-tubulins function identically, and that the various genes have evolved for the purpose of delivering the different amounts of ct-and [3-tubulin protein needed in different cells. Some support for this idea comes from genetic evidence in Drosophila (11,12) and Aspergillus (20) where it has been shown that a mutation in a single tubulin gene affects many different microtubule functions. In addition, the expression of a chicken/yeast chimeric tubulin in mammalian cells results in its incorporation into both cytoskeletal and spindle microtubules without disruption of their function (3).With such questions in mind, we have been investigating the mammalian tubulin repertoire by exhaustive screening of cDNA libraries representing several different tissues. We recently reported the structure and patterns of expression of five ct-tubulin (17, 31) and three ...

Communicated by Ewald R. Weibel, Universität Bern, Bern, Switzerland, August 14, 1996 (received for review January 25, 1996