Masloff Sandra, Jacobsen Sabine, Pöggeler Stefanie, Kück Ulrich
Lehrstuhl für Allgemeine und Molekulare Botanik, Ruhr-Universität Bochum, D-44780 Bochum, Germany.
Fungal Genet Biol. 2002 Jul;36(2):107-16. doi: 10.1016/S1087-1845(02)00010-5.
The pro1 gene, controlling fruiting body development in the homothallic ascomycete Sordaria macrospora, encodes a C6 zinc finger protein with a typical DNA binding domain of GAL4-like C6 zinc finger proteins as well as a putative nuclear targeting signal. In the corresponding mutant pro1, the pro1 gene is deleted, and the transition of primordia into mature fruiting bodies is prevented. To further characterize the PRO1 polypeptide, the yeast system was used for identifying a transactivation domain in the N-terminal half of PRO1, which probably also functions in S. macrospora. The functional analysis was extended by using truncated versions of the pro1 gene in complementation transformations of a deltapro1 mutant. Interestingly, the 5' part of the pro1 gene encoding the DNA binding and transactivation domain as well as putative nuclear targeting signals was sufficient to restore fertility in the sterile pro1 mutant. In vitro mutagenesis verified that the DNA binding domain is essential for normal fruiting body development. This was concluded from transformation experiments with eight pro1 derivatives containing triplet substitutions in conserved codons of the DNA binding domain; some, but not all, failed in restoring the wild-type phenotype in mutant pro1. Using a PCR-based cloning strategy, pro1 homologs from the two related heterothallic species Neurospora crassa and Sordaria brevicollis were isolated, showing similarities in the predicted amino acid sequences of 91 and 90%, respectively. When a N. crassa pro1 cDNA clone was used in complementation transformations, we succeeded in restoring the wild-type phenotype to the S. macrospora pro1 mutant. These data suggest that pro1 homologs from heterothallic species can provide the pro1 function in homothallic ascomycetes. Based on the published sequence of the N. crassa genome, we identified hpro1A, another transcriptionally expressed gene, with a similarity of 40% to the pro1 genes, which is present as a single copy gene in N. crassa as well as in S. macrospora.
控制同宗配合子囊菌大孢粪壳菌子实体发育的pro1基因，编码一种C6锌指蛋白，该蛋白具有GAL4样C6锌指蛋白典型的DNA结合结构域以及一个假定的核定位信号。在相应的突变体pro1中，pro1基因被删除，原基向成熟子实体的转变被阻止。为了进一步表征PRO1多肽，利用酵母系统在PRO1的N端一半区域鉴定出一个反式激活结构域，该结构域可能在大孢粪壳菌中也发挥作用。通过在Δpro1突变体的互补转化中使用pro1基因的截短版本，扩展了功能分析。有趣的是，pro1基因编码DNA结合和反式激活结构域以及假定核定位信号的5'部分足以恢复不育pro1突变体的育性。体外诱变证实DNA结合结构域对于正常子实体发育至关重要。这是通过对八个在DNA结合结构域保守密码子中含有三联体替换的pro1衍生物进行转化实验得出的结论；其中一些（但不是全部）未能在突变体pro1中恢复野生型表型。采用基于PCR的克隆策略，从两个相关的异宗配合物种粗糙脉孢菌和短颈粪壳菌中分离出pro1同源物，其预测氨基酸序列的相似性分别为91%和90%。当将粗糙脉孢菌pro1 cDNA克隆用于互补转化时，我们成功地将野生型表型恢复到大孢粪壳菌pro1突变体中。这些数据表明，来自异宗配合物种的pro1同源物可以在同宗配合子囊菌中发挥pro1的功能。基于已发表的粗糙脉孢菌基因组序列，我们鉴定出hpro1A，另一个转录表达基因，与pro1基因的相似性为40%，它在粗糙脉孢菌和大孢粪壳菌中均作为单拷贝基因存在。
