G PROTEINS AND SIGNAL TRANSDUCTION IN NEUROSPORA CRASSA

异三聚体鸟嘌呤核苷酸结合蛋白(G蛋白)， 由α、β和伽马亚基组成，参与了 真核细胞对光、生长因子、激素和 神经递质。哺乳动物的阿尔法蛋白分为四类 根据氨基酸序列同源性，Gi、Gs、Gq和G12类。 结构性激活的Gi-α家族成员被牵连到 哺乳动物的细胞增殖和有丝分裂，可能通过 调节cAMP水平和/或离子通道活性。还有就是 越来越多的证据表明Gi-α蛋白可能控制蓝光反应 对细胞分化、向光性和生物钟很重要 在植物和脊椎动物中。我们已经分离到一个Gi-α家族基因(gna- 1)从多细胞真菌粗枝脉孢菌中分离得到。按顺序排列的 编码的GNA-1蛋白与GNA-1其他成员55%的同源性 Gi家族，并具有肉豆蔻酰化和百日咳毒素 修饰位点是这一组的序列特征。这个 GNA-1基因包含3个内含子，其中2个位于 美国大兵一家。此外，脉孢子虫的一种膜蛋白 体外用百日咳毒素标记GNA-1的大小。这些 结果表明，粗糙脉孢菌Gna-1是第一种微生物 阿尔法亚单位属于任何哺乳动物类别。类似于 在高等生物中，[cAMP]和[Ca~(2+)]都与 脉孢菌的细胞增殖和形态发生，而蓝光 诸如形态发生和重新设置生物钟之类的反应可能 涉及到[阵营]的变化。因此，我们假设GNA-1将 调节cAMP和/或钙离子水平，从而调节细胞增殖， 粗糙脉孢菌的形态发生和生物钟。具体的 目的是：1)在脉孢子虫中筛选与GNA-1相关的其他基因 Crassa，以确定gna-1是否属于基因家族。 2)过表达野生型gna-1，并创建空和 脉孢子菌GNA-1基因的结构性激活突变 活着。我们还将测试克隆的GNA-1基因的能力 GNA-1附近的补体形态突变，以确定是否 它们是等位基因。过度表达、无效和“致癌”突变体 将评估菌株的形态变化和cAMP的变化 和/或钙离子水平。3)高效表达和纯化Gna-1蛋白 生化分析(目标4)和抗体生成。抗血清 将用于GNA-1的定量和免疫定位 蛋白质和免疫抑制实验(目标4)。要设置 GNA-1与下游调控的重组分析 腺酰环化酶、cAMP磷酸二酯酶或钙离子等效应物 频道。GNA-1抗体将用于免疫抑制这些活性， 人类GI家族蛋白将被测试为GNA-1的替代品 化验结果。神经孢子虫GI同源物的功能研究 GNA-1将阐明人类GI家族α蛋白在 发育、有丝分裂和生物钟。

Heterotrimeric guanine nucleotide-binding proteins (G proteins), consisting of alpha, beta and gamma subunits, are involved in the response of eukaryotic cells to light, growth factors, hormones and neurotransmitters. Mammalian alpha proteins are grouped into four classes, Gi, Gs, Gq and G12, based on amino acid sequence identity. Constitutively-activated Gi-alpha family members have been implicated in cell proliferation and mitogenesis in mammals, possibly through regulation of cAMP levels and/or ion channel activity. There is also mounting evidence that Gi-alpha proteins may control blue light responses important for cell differentiation, phototropism, and the circadian clock in plants and vertebrates. We have isolated a Gi-alpha family gene (gna- 1) from the multicellular fungus Neurospora crassa. The sequence of the encoded Gna-1 protein is 55% identical to that of other members of the Gi family, and possesses the myristoylation and pertussis toxin modification sites which are the sequence hallmarks of this group. the gna-1 gene contains 3 introns, 2 of which are in positions conserved in the Gi family. In addition, a Neurospora membrane protein of the approximate size of Gna-1 is labeled by pertussis toxin in vitro. These results indicate that Neurospora crassa Gna-1 is the first microbial alpha subunit to belong to any mammalian class. Analogous to the situation in higher organisms, both [cAMP] and [Ca2+] are implicated in cell proliferation and morphogenesis in Neurospora, while blue light responses, such as morphogenesis and resetting the circadian clock, may involve changes in [cAMP]. Therefore, we hypothesize that Gna-1 will regulate cAMP and/or Ca2+ levels, and thus cell proliferation, morphogenesis and the circadian clock in Neurospora crassa. The Specific Aims are: 1) To screen for other genes related to gna-1 in Neurospora crassa, in order to determine whether gna-1 is part of a gene family. 2) To overexpress wild type gna-1, and to create both null and constitutively-activating mutations in the gna-1 gene in Neurospora in vivo. We will also test the ability of the cloned gna-1 gene to complement morphological mutations which map near gna-1, to determine if they are allelic. The overexpressing, null and "oncogenic" mutant strains will be assessed for changed morphologies and alterations in cAMP and/or Ca2+ levels. 3) To overexpress and purify the Gna-1 protein for both biochemical analysis (Aim 4) and antibody generation. the antisera will be used for quantitation and immunolocalization of the Gna-1 protein, and for immunoinhibition experiments (Aim 4). To set up reconstitution assays coupling Gna-1 to regulation of downstream effectors such as adenylyl cyclase, cAMP phosphodiesterase or Ca2+ channels. Gna-1 antibody will be used to immunoinhibit these activities, and human Gi family proteins will be tested as substitutes for Gna-1 in the assays. Investigation of the function of the Neurospora Gi homologue Gna-1 will elucidate the roles of human Gi-family alpha proteins in development, mitogenesis and the circadian clock.