Cloning of flagellar genes in Chlamydomonas reinhardtii by DNA insertional mutagenesis.
Cloning of flagellar genes in Chlamydomonas reinhardtii by DNA insertional mutagenesis.
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作者：
L. Tam;P. Lefebvre
衣原体是研究许多细胞过程的流行遗传模型系统。在这份报告中，我们描述了一种新的方法来分离衣原体基因使用克隆的硝酸还原酶基因（NIT1）作为插入诱变剂。通过玻璃珠转化将含有NIT1基因的线性化质粒引入nit1突变体细胞中。在3000个Nit+转化体中，74个表现出广泛的表型的运动缺陷，这表明DNA转化是诱变细胞的有效方法。对于15个这样的运动突变体回交到nit-突变株，运动表型与Nit+表型共分离，表明这13个突变体的运动缺陷可能是由质粒的整合引起的。进一步的遗传分析表明，这些突变体中有三个含有先前鉴定的基因座的等位基因：mbo 2（仅向后移动），pf 13（瘫痪鞭毛）和vfl1（可变鞭毛数）。其他三个异常鞭毛数量突变体没有映射到任何先前描述的基因座突变产生类似的表型。分离mbo 2和vfl1突变体中整合质粒侧翼的基因组序列，并将其用作探针以获得野生型基因组克隆，其在转化成细胞后补充运动缺陷。我们的研究结果表明，这种新的方法克隆衣原体突变基因的潜力。
Chlamydomonas is a popular genetic model system for studying many cellular processes. In this report, we describe a new approach to isolate Chlamydomonas genes using the cloned nitrate reductase gene (NIT1) as an insertional mutagen. A linearized plasmid containing the NIT1 gene was introduced into nit1 mutant cells by glass-bead transformation. Of 3000 Nit+ transformants examined, 74 showed motility defects of a wide range of phenotypes, suggesting that DNA transformation is an effective method for mutagenizing cells. For 13 of 15 such motility mutants backcrossed to nit- mutant strains, the motility phenotype cosegregated with the Nit+ phenotype, indicating that the motility defects of these 13 mutants may be caused by integration of the plasmid. Further genetic analysis indicated that three of these mutants contained alleles of previously identified loci: mbo2 (move backward only), pf13 (paralyzed flagella) and vfl1 (variable flagellar number). Three other abnormal-flagellar-number mutants did not map to any previously described loci at which mutations produce similar phenotypes. Genomic sequences flanking the integrated plasmid in the mbo2 and vfl1 mutants were isolated and used as probes to obtain wild-type genomic clones, which complemented the motility defects upon transformation into cells. Our results demonstrate the potential of this new approach for cloning genes identified by mutation in Chlamydomonas.