CLONING AND EXPRESSION OF THE RHODOBACTER-SPHAEROIDES REACTION CENTER-H GENE
CLONING AND EXPRESSION OF THE RHODOBACTER-SPHAEROIDES REACTION CENTER-H GENE
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DOI：
10.1128/jb.168.2.953-961.1986
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发表时间：
1986-11-01
影响因子：
3.2
通讯作者：
KAPLAN, S
中科院分区：
文献类型：
--
作者：
DONOHUE, TJ;HOGER, JH;KAPLAN, S
通过使用类似荚膜红细菌基因的限制性片段作为异源杂交探针，已经鉴定并克隆了反应中心H多肽的球形红细菌结构基因(puhA)。通过部分DNA序列分析和在球形红球菌偶联转录-翻译系统中合成免疫反应性Mr-28,000反应中心H多肽，证实了1.45-kilobase BamHI限制性片段上puhA的存在。 puhA 基因上游大约 450 个碱基对的 DNA 足以在体外表达该蛋白。使用内部 puhA 特异性探针进行 Northern RNA-DNA 印迹分析，鉴定出在已知影响反应中心复合物和光合膜的细胞内容物的生理条件下存在于不同细胞水平的至少两个明显单顺反子的转录物。使用特定上游限制性片段探针进行的 Northern 印迹分析表明，1,400 核苷酸的 puhA 特异性 mRNA 在 1,130 核苷酸转录物的上游具有 5'' 末端。在缺乏可检测的细菌叶绿素和光合膜的化学异养生长的细胞中，puhA 特异性 mRNA 和免疫反应中心 H 蛋白均可检测到。
The Rhodobacter sphaeroides structural gene (puhA) for the reaction center H polypeptide has been identified and cloned by using restriction fragments for the analogous Rhodobacter capsulatus gene as a heterologous hybridization probe. The presence of puhA on a 1.45-kilobase BamHI restriction fragment was confirmed by partial DNA sequence analysis and by the synthesis of an immunoreactive Mr-28,000 reaction center H polypeptide in an R. sphaeroides coupled transcription-translation system. Approximately 450 base pairs of DNA upstream of the puhA gene were sufficient for expression of this protein in vitro. Northern RNA-DNA blot analysis with an internal puhA-specific probe identified at least two, apparently monocistronic, transcripts present at different cellular levels under physiological conditions known to affect the cellular content of both reaction center complexes and photosynthetic membrane. Northern blot analysis with specific upstream restriction fragment probes revealed that the 1,400-nucleotide puhA-specific mRNA had a 5'' terminus upstream of the 1,130-nucleotide transcript. Both puhA-specific mRNA and immunoreactive reaction center H protein were detectable in chemoheterotrophically grown cells which lacked detectable bacteriochlorophyll and photosynthetic membrane.