We have tested the functional capacity of different beta tubulin isoforms in vivo by expressing beta 3-tubulin either in place of or in addition to beta 2-tubulin in the male germ line of Drosophila melanogaster. The testes-specific isoform, beta 2, is conserved relative to major metazoan beta tubulins, while the developmentally regulated isoform, beta 3, is considerably divergent in sequence. beta 3-tubulin is normally expressed in discrete subsets of cells at specific times during development, but is not expressed in the male germ line. beta 2-Tubulin is normally expressed only in the postmitotic germ cells of the testis, and is required for all microtubule-based functions in these cells. The normal functions of beta 2-tubulin include assembly of meiotic spindles, axonemes, and at least two classes of cytoplasmic microtubules, including those associated with the differentiating mitochondrial derivatives. A hybrid gene was constructed in which 5' sequences from the beta 2 gene were joined to protein coding and 3' sequences of the beta 3 gene. Drosophila transformed with the hybrid gene express beta 3-tubulin in the postmitotic male germ cells. When expressed in the absence of the normal testis isoform, beta 3-tubulin supports assembly of one class of functional cytoplasmic microtubules. In such males the microtubules associated with the membranes of the mitochondrial derivatives are assembled and normal mitochondrial derivative elongation occurs, but axoneme assembly and other microtubule-mediated processes, including meiosis and nuclear shaping, do not occur. These data show that beta 3 tubulin can support only a subset of the multiple functions normally performed by beta 2, and also suggest that the microtubules associated with the mitochondrial derivatives mediate their elongation. When beta 3 is coexpressed in the male germ line with beta 2, at any level, spindles and all classes of cytoplasmic microtubules are assembled and function normally. However, when beta 3-tubulin exceeds 20% of the total testis beta tubulin pool, it acts in a dominant way to disrupt normal axoneme assembly. In the axonemes assembled in such males, the doublet tubules acquire some of the morphological characteristics of the singlet microtubules of the central pair and accessory tubules. These data therefore unambiguously demonstrate that the Drosophila beta tubulin isoforms beta 2 and beta 3 are not equivalent in intrinsic functional capacity, and furthermore show that assembly of the doublet tubules of the axoneme imposes different constraints on beta tubulin function than does assembly of singlet microtubules.

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