Cloning and sequencing of cDNA encoding 4-aminobenzoate hydroxylase from an edible mushroom

食用菌4-氨基苯甲酸羟化酶cDNA的克隆和测序

基本信息

批准号：
06660114
负责人：
TSUJI Hideaki
金额：
$ 1.34万
依托单位：
The University of Tokushima
依托单位国家：
日本
项目类别：
Grant-in-Aid for General Scientific Research (C)
财政年份：
1994
资助国家：
日本
起止时间：
1994 至 1995
项目状态：
已结题

项目摘要

4-Aminobenzoate hydroxylase (EC 1.14.13.27) is a FAD-dependent monooxygenase which was found in an edible mushroom, Agaricus bisporus. We prepared three monoclonal antibodies recognizing the FAD-binding domain of the enzyme and showed that the antibodies can locate its FAD-binding domain. In order to elucidate the full primary structure of the enzyme, we tried to isolate a cDNA clone encoding the enzyme. The 613-bp DNA fragment encoding a part of the the enzyme was synthesized from poly(A)^+RNA by polymerase chain reaction and used as the probe in the following experiments. The size of the mRNA for the enzyme was shown to be approximately 1700 base by Northern blotting. DscDNA was prepared from poly(A)^+RNA,inserted into lambdagt10 arms, and then in vitro packaged. The cDNA library included 1*10^6 clones. Finally, four positive clones were isolated from the library by plaque hybridization. Of the clones, the clone with the longest insert cDNA was selected, the insert was subcloned into the Bam HI site of pUC19 plasmid and sequenced. The cDNA was shown to consist of 5'-untranslated region (14 bp), open reading frame (1380 bp), 3'-untranslated region (103 bp), and a poly A tail (20 bp) in a total nucleotide length of 1517 bp. The open reading frame coded for 460 amino acid protein with a molecular mass of 50974, and the deduced amino acid sequence included all of the amino acid sequences determined of 13 peptides which were isolated from the proteolytic products of the enzyme by high performance liquid chromatography. These findings demonstrate that the cDNA encodes the full length of the enzyme. At present, the development of an overexpression system of the cDNA is actively in progress.

4-氨基苯甲酸羟化酶 (EC 1.14.13.27) 是一种 FAD 依赖性单加氧酶，存在于食用蘑菇双孢蘑菇中。我们制备了三种识别该酶的 FAD 结合域的单克隆抗体，并表明这些抗体可以定位其 FAD 结合域。为了阐明该酶的完整一级结构，我们尝试分离编码该酶的 cDNA 克隆。通过聚合酶链式反应从poly(A)^+RNA合成编码部分酶的613-bp DNA片段，并用作以下实验中的探针。 Northern blotting显示该酶的mRNA大小约为1700个碱基。由poly(A)^+RNA制备DscDNA，插入lambdagt10臂，然后体外包装。 cDNA文库包含1*10^6个克隆。最后，通过噬菌斑杂交从文库中分离出4个阳性克隆。在克隆中，选择具有最长插入片段cDNA的克隆，将插入片段亚克隆到pUC19质粒的Bam HI位点并测序。该 cDNA 由 5'-非翻译区（14 bp）、开放阅读框（1380 bp）、3'-非翻译区（103 bp）和聚 A 尾（20 bp）组成，总核苷酸长度为1517bp。开放阅读框编码460个氨基酸，分子量为50974的蛋白质，推导的氨基酸序列包含了高效液相色谱法从该酶的蛋白水解产物中分离出的13条肽段测定的全部氨基酸序列。这些发现表明 cDNA 编码该酶的全长。目前，该cDNA过表达系统的开发正在积极进行中。