Duarte M, Mota N, Pinto L, Videira A
Instituto de Ciências Biomédicas de Abel Salazar, Lg. Prof. Abel Salazar 2, Universidade do Porto, Portugal.
Mol Gen Genet. 1998 Feb;257(3):368-75. doi: 10.1007/s004380050659.
We have isolated and characterised the nuclear gene that codes for the 30.4-kDa subunit of the peripheral arm of complex I from Neurospora crassa. The single-copy gene was localised on chromosome VI of the fungal genome by restriction fragment length polymorphism mapping. An extra copy of the gene was introduced into a strain of N. crassa by transformation. This strain was crossed with another strain in order to inactivate, by repeat-induced point mutations, both copies of the duplication carried by the parental transformant. Ascospore progeny from the cross were analysed and a mutant strain lacking the 30.4-kDa protein, nuo30.4, was isolated and further characterised. The mutant appears to assemble the membrane arm of complex I, while formation of the peripheral arm is prevented. Nevertheless, the mutant grows reasonably well--indicating that this well conserved protein is not essential for vegetative growth--and is able to mate with other strains both as male or female. Strains with multiple mutations are readily obtained from heterozygous crosses between different complex I mutants of N. crassa. On the other hand, homozygous crosses between several mutants, including nuo30.4, fail to produce ascospores. These results suggest that complex I plays an essential role during the sexual phase of the life cycle of the fungus.
我们已经分离并鉴定了编码粗糙脉孢菌复合体I外周臂30.4 kDa亚基的核基因。通过限制性片段长度多态性图谱分析，该单拷贝基因定位于真菌基因组的第六条染色体上。通过转化将该基因的一个额外拷贝导入粗糙脉孢菌的一个菌株中。将该菌株与另一个菌株杂交，以便通过重复诱导点突变使亲本转化体携带的重复序列的两个拷贝失活。分析杂交产生的子囊孢子后代，分离出一个缺乏30.4 kDa蛋白的突变菌株nuo30.4，并对其进行了进一步鉴定。该突变体似乎能够组装复合体I的膜臂，而外周臂的形成则受到阻碍。然而，该突变体生长得相当好——这表明这种高度保守的蛋白对于营养生长并非必需——并且能够作为雄性或雌性与其他菌株交配。通过粗糙脉孢菌不同复合体I突变体之间的杂合杂交很容易获得具有多个突变的菌株。另一方面，包括nuo30.4在内的几个突变体之间的纯合杂交未能产生子囊孢子。这些结果表明，复合体I在真菌生命周期的有性阶段起着至关重要的作用。
