Hoyle H D, Hutchens J A, Turner F R, Raff E C
Department of Biology, Indiana University, Bloomington 47405, USA.
Dev Genet. 1995;16(2):148-70. doi: 10.1002/dvg.1020160208.
In this study we examined two aspects of beta-tubulin function in Drosophila spermatogenesis: 1) beta-tubulin structural requirements for assembly of different categories of microtubules and 2) regulatory requirements for production of the correct tubulin protein level. In normal Drosophila spermatogenesis, the testis-specific beta 2-tubulin isoform supports multiple microtubule functions. Our previous work showed that another Drosophila isoform, beta 3, cannot support spermatogenesis, whereas a carboxyl-truncated form of beta 2, beta 2 delta C, can at least 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 whether beta 2 carboxyl sequences can rescue the functional failure of the beta 3 isoform in spermatogenesis, we constructed a gene encoding a chimeric protein, beta 3 beta 2C, in which beta 3 sequences in the carboxyl region are replaced with those of beta 2. Unlike either beta 3 or beta 2 delta C, beta 3 beta 2C can provide partial function for both assembly of axonemal microtubules and their organization into the supramolecular architecture of the axoneme. In particular, the beta 2 carboxyl sequences mediate morphogenesis of the axoneme doublet tubule complex, including accessory microtubule assembly and attachment of spokes and linkers. However, our data also reveal aspects of beta 2-specific function that require structural features other than the primary sequence of the isotype-defining variable regions, the C terminus and the internal variable region. Tests of fecundity in males that coexpress beta 2 and the chimeric beta 3 beta 2C protein showed that in Drosophila there are differential requirements for sperm motility in the male and in the female reproductive tract. Since some aspects of microtubule function in spermatogenesis are sensitive to the tubulin pool size, we examined the mechanisms for control of tubulin protein levels in the male germ cells. We found that both beta 2-tubulin mRNA accumulation and protein synthesis are dependent on gene dose, and that the level of expression is regulated by 3' noncoding sequences in the beta 2 gene. Our data show that the regulatory mechanisms that control tubulin pool levels in the Drosophila male germ line differ from those observed in cultured animal somatic cells. Finally, expression of transgenic constructs is consistent with early cessation of X chromosome expression in Drosophila spermatogenesis.
在本研究中，我们检测了果蝇精子发生过程中β-微管蛋白功能的两个方面：1）不同类别的微管组装对β-微管蛋白的结构要求，以及2）产生正确微管蛋白水平所需的调控要求。在正常的果蝇精子发生过程中，睾丸特异性β2-微管蛋白异构体支持多种微管功能。我们之前的研究表明，果蝇的另一种异构体β3不能支持精子发生，而β2的羧基截短形式β2δC至少在一定程度上可以提供β2的所有正常功能，但有一个功能除外：β2δC不能支持轴丝微管组织成轴丝的超分子结构。在此，为了测试β2羧基序列是否能挽救β3异构体在精子发生中的功能缺陷，我们构建了一个编码嵌合蛋白β3β2C的基因，其中羧基区域的β3序列被β2的序列所取代。与β3或β2δC不同，β3β2C既能为轴丝微管的组装及其组织成轴丝的超分子结构提供部分功能。特别是，β2羧基序列介导了轴丝双微管复合体的形态发生，包括辅助微管的组装以及辐条和连接体的附着。然而，我们的数据也揭示了β2特异性功能的一些方面，这些方面需要除了定义异构体的可变区域、C末端和内部可变区域的一级序列之外的结构特征。对共表达β2和嵌合β3β2C蛋白的雄性果蝇的繁殖力测试表明，在果蝇中，雄性和雌性生殖道对精子活力有不同的要求。由于精子发生过程中微管功能的某些方面对微管蛋白库大小敏感，我们研究了雄性生殖细胞中微管蛋白水平的控制机制。我们发现β2-微管蛋白mRNA的积累和蛋白质合成均依赖于基因剂量，并且表达水平受β2基因的3'非编码序列调控。我们的数据表明，果蝇雄性生殖系中控制微管蛋白库水平的调控机制与培养的动物体细胞中观察到的不同。最后，转基因构建体的表达与果蝇精子发生过程中X染色体表达的早期停止一致。
