Zhang D, Lefebvre P A
Department of Genetics and Cell Biology, University of Minnesota, St. Paul 55108, USA.
Genetics. 1997 May;146(1):121-33. doi: 10.1093/genetics/146.1.121.
In Chlamydomonas reinhardtii, the genes required for nitrate assimilation, including the gene encoding nitrate reductase (NIT1), are subject to repression by ammonia. To study the mechanism of ammonia repression, we employed two approaches to search for mutants with defective repression of NIT1 gene expression. (1) PF14, a gene required for flagellar function, was used as a reporter gene for expression from the NIT1 promoter. When introduced into a pf14 mutant host, the NIT1;PF14 chimeric construct produced a transformant (T10-10B) with a conditional swimming phenotype. Spontaneous mutants with defective ammonia repression of the NIT1 promoter were screened for by isolating cells that gained constitutive motility. (2) Insertional mutagenesis was performed, followed by screening for chlorate sensitivity in the presence of ammonia ion. One insertional mutant and six spontaneous mutants were allelic and defined a new gene, FAR1 (free from ammonia repression). FAR1 was mapped to Linkage Group I, 7.7 cM to the right of the centromere. The far1-1 mutant strain was used to clone DNA adjacent to the site of plasmid insertion, which was then used as a hybridization probe to clone the FAR1 gene from wild type.
在莱茵衣藻中，硝酸盐同化所需的基因，包括编码硝酸还原酶（NIT1）的基因，会受到氨的抑制。为了研究氨抑制的机制，我们采用了两种方法来寻找NIT1基因表达抑制缺陷的突变体。（1）PF14是鞭毛功能所需的基因，用作NIT1启动子表达的报告基因。当导入pf14突变体宿主时，NIT1;PF14嵌合构建体产生了具有条件游动表型的转化体（T10-10B）。通过分离获得组成型运动性的细胞，筛选出NIT1启动子氨抑制缺陷的自发突变体。（2）进行插入诱变，然后在铵离子存在下筛选氯酸盐敏感性。一个插入突变体和六个自发突变体是等位基因，并定义了一个新基因FAR1（不受氨抑制）。FAR1被定位到连锁群I，着丝粒右侧7.7 cM处。far1-1突变体菌株用于克隆与质粒插入位点相邻的DNA，然后用作杂交探针从野生型中克隆FAR1基因。
