Hallmann A, Rappel A, Sumper M
Lehrstuhl Biochemie I, Universität Regensburg, D-93053 Regensburg, Germany.
Proc Natl Acad Sci U S A. 1997 Jul 8;94(14):7469-74. doi: 10.1073/pnas.94.14.7469.
With only two different cell types, the haploid green alga Volvox represents the simplest multicellular model system. To facilitate genetic investigations in this organism, the occurrence of homologous recombination events was investigated with the intent of developing methods for gene replacement and gene disruption. First, homologous recombination between two plasmids was demonstrated by using overlapping nonfunctional fragments of a recombinant arylsulfatase gene (tubulin promoter/arylsulfatase gene). After bombardment of Volvox reproductive cells with DNA-coated gold microprojectiles, transformants expressing arylsulfatase constitutively were recovered, indicating the presence of the machinery for homologous recombination in Volvox. Second, a well characterized loss-of-function mutation in the nuclear nitrate reductase gene (nitA) with a single G --> A nucleotide exchange in a 5'-splice site was chosen as a target for gene replacement. Gene replacement by homologous recombination was observed with a reasonably high frequency only if the replacement vector containing parts of the functional nitrate reductase gene contained only a few nucleotide exchanges. The ratio of homologous to random integration events ranged between 1:10 and 1:50, i.e., homologous recombination occurs frequently enough in Volvox to apply the powerful tool of gene disruption for functional studies of novel genes.
单倍体绿藻团藻仅由两种不同的细胞类型构成，是最简单的多细胞模型系统。为便于对该生物体进行遗传学研究，人们对同源重组事件的发生情况展开了调查，目的是开发基因替换和基因破坏的方法。首先，通过使用重组芳基硫酸酯酶基因（微管蛋白启动子/芳基硫酸酯酶基因）的重叠无功能片段，证明了两个质粒之间的同源重组。在用包被了DNA的金微弹轰击团藻生殖细胞后，回收了组成型表达芳基硫酸酯酶的转化体，这表明团藻中存在同源重组机制。其次，选择了核硝酸还原酶基因（nitA）中一个特征明确的功能缺失突变作为基因替换的靶点，该突变在5'剪接位点有一个单一的G→A核苷酸交换。只有当包含部分功能性硝酸还原酶基因的替换载体仅含有少数核苷酸交换时，才能以相当高的频率观察到同源重组介导的基因替换。同源整合事件与随机整合事件的比例在1:10至1:50之间，即同源重组在团藻中发生的频率足以应用强大的基因破坏工具来对新基因进行功能研究。
