REGULATION OF BETA-TUBULIN FUNCTION AND EXPRESSION IN DROSOPHILA SPERMATOGENESIS

In this study we examined two aspects of beta-tubulin function in Dros ophila spermatogenesis: 1) beta-tubulin structural requirements for as sembly of different categories of microtubules and 2) regulatory requi rements for production of the correct tubulin protein level. In normal Drosophila spermatogenesis, the testis-specific beta 2-tubulin isofor m supports multiple microtubule functions. Our previous work showed th at another Drosophila isoform, beta 3, cannot support spermatogenesis, whereas a carboxyl-truncated form of beta 2, beta 2 Delta C, can at l east to some extent provide all of beta 2's normal functions, save one : beta 2 Delta C cannot support organization of axonemal microtubules into the supramolecular architecture of the axoneme. Here, to test whe ther beta 2 carboxyl sequences can rescue the functional failure of th e beta 3 isoform in spermatogenesis, we constructed a gene encoding a chimeric protein, beta 3 beta 2C, in which beta 3 sequences in the car boxyl region are replaced with those of beta 2. Unlike either beta 3 o r beta 2 Delta C, beta 3 beta 2C can provide partial function for both assembly of axonemal microtubules and their organization into the sup ramolecular architecture of the axoneme. In particular, the beta 2 car boxyl sequences mediate morphogenesis of the axoneme doublet tubule co mplex, including accessory microtubule assembly and attachment of spok es and linkers. However, our data also reveal aspects of beta 2-specif ic function that require structural features other than the primary se quence of the isotype-defining variable regions, the C terminus and th e internal variable region. Tests of fecundity in males that coexpress beta 2 and the chimeric beta 3 beta 2C protein showed that in Drosoph ila there are differential requirements for sperm motility in the male and in the female reproductive tract. Since some aspects of microtubu le function in spermatogenesis are sensitive to the tubulin pool size, we examined the mechanisms for control of tubulin protein levels in t he male germ cells. We found that both beta 2-tubulin mRNA accumulatio n and protein synthesis are dependent on gene dose, and that the level of expression is regulated by 3' noncoding sequences in the beta 2 ge ne. Our data show that the regulatory mechanisms that control tubulin pool levels in the Drosophila male germ line differ from those observe d in cultured animal somatic cells. Finally, expression of transgenic constructs is consistent with early cessation of X chromosome expressi on in Drosophila spermatogenesis. (C) 1995 Wiley-Liss, inc.