ISOLATION AND CHARACTERIZATION OF THE NITRATE REDUCTASE STRUCTURAL GENE OF CHLAMYDOMONAS-REINHARDTII
ISOLATION AND CHARACTERIZATION OF THE NITRATE REDUCTASE STRUCTURAL GENE OF CHLAMYDOMONAS-REINHARDTII
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DOI：
10.1073/pnas.86.17.6449
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发表时间：
1989-09-01
影响因子：
11.1
通讯作者：
LEFEBVRE, PA
中科院分区：
文献类型：
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作者：
FERNANDEZ, E;SCHNELL, R;LEFEBVRE, PA
莱茵衣藻的硝酸还原酶结构基因已经通过使用来自莱茵衣藻的硝酸还原酶cDNA探针从基因组文库中分离出来。限制性片段长度多态性分析将衣藻克隆 (B6a) 定位到硝酸还原酶结构基因位点 nit-1。重叠插入片段覆盖了约 24 KB 的基因组区域，其中包含整个基因，跨度约为 5-8 KB。 B6a 克隆 DNA 片段的序列分析表明，在氨基酸水平上，与烟草和拟南芥硝酸还原酶和人 NADH 细胞色素 b5 还原酶的血红素和 FAD/NADH 结合结构域部分对应的区域具有高度的序列相似性。克隆基因作为硝酸还原酶的身份通过其在转化时补充 nit-1 突变的能力得到证实。硝酸盐还原酶基因在硝酸盐去抑制的细胞中产生 3.4 KB 的转录物；在存在铵的情况下生长的细胞中检测不到转录本。在含有假定调节基因 nit-2 突变的细胞中，发现 3.4 KB 转录物水平显着降低，表明野生型 nit-2 基因参与硝酸还原酶转录物水平的控制。
The nitrate reductase structural gene of Chlamydomonas reinhardtii has been isolated from a genomic library by using a nitrate reductase cDNA probe from barely. Restriction fragment length polymorphism analyses mapped the Chlamydomonas clone (B6a) to the nitrate reductase structural gene locus nit-1. Overlapping inserts cover a region of the genome of about 24 kilobases containing the entire gene, which spans approximately 5-8 kilobases. Sequence analysis of DNA fragments from the B6a clone demonstrated a high degree of sequence similarity at the amino acid level with regions corresponding to portions of the heme and FAD/NADH-binding domains of tobacco and Arabidopsis thaliana nitrate reductases and human NADH cytochrome b5 reductase. The identity of the cloned gene as nitrate reductase was confirmed by its ability to complement a nit-1 mutation upon transformation. The nitrate reductase gene produced a 3.4-kilobase transcript in cells derepressed with nitrate; the transcript was undetectable in cells grown in the presence of ammonium. In cells that contain a mutation in the putative regulatory gene nit-2, significantly lower levels of the 3.4-kilobase transcript were found, indicating that the wild-type nit-2 gene is involved in the control of nitrate reductase transcript levels.