Koller B, Clarke J, Delius H
EMBL, Postfach 102209, 69-Heidelberg, FRG.
EMBO J. 1985 Oct;4(10):2445-50. doi: 10.1002/j.1460-2075.1985.tb03954.x.
Partially spliced precursor mRNAs (pre-mRNAs) in the steady-state population of RNA from chloroplasts of Euglena gracilis were found by electron microscopy. The structure and the frequency of the pre-mRNAs of the psbA gene (the gene for the 32-kd protein of photosystem II), which is split by four introns in Euglena chloroplasts was analysed by electron microscopy. A chloroplast DNA (cpDNA) fragment containing the psbA gene from Euglena, was cloned into a pEMBL vector. The single-stranded recombinant phage DNA of the coding strand was prepared and hybridized with cpRNA. The majority of hybrids were formed with mature mRNA, but approximately 8% of the hybrids were formed with pre-mRNAs. The pre-mRNAs were either unspliced or incompletely spliced. A detailed analysis of the structure and the frequency of the pre-mRNAs of the psbA gene showed that the four introns are neither spliced out in a strictly random way, nor in a 5'-3' or 3'-5' direction. Introns 2 and 3 are preferentially spliced out first, intron 1 intermediately and intron 4 is generally spliced out last. However, this sequence is not a strict rule. We conclude that the introns can be spliced independently, each one at a different rate. The coding strand from a fragment of the psbA gene was separated and annealed with low mol. wt. cpRNA, which was isolated from an agarose gel. Small circular hybrids were found at the positions of the four introns, demonstrating for the first time covalently closed circular excised intron RNAs (iRNAs) in chloroplasts.
通过电子显微镜发现，纤细裸藻叶绿体RNA稳态群体中存在部分剪接的前体mRNA（pre-mRNA）。利用电子显微镜分析了psbA基因（光系统II 32-kD蛋白的基因）的pre-mRNA的结构和频率，该基因在纤细裸藻叶绿体中被四个内含子分割。将含有纤细裸藻psbA基因的叶绿体DNA（cpDNA）片段克隆到pEMBL载体中。制备编码链的单链重组噬菌体DNA，并与cpRNA杂交。大多数杂交体是与成熟mRNA形成的，但约8%的杂交体是与pre-mRNA形成的。这些pre-mRNA要么未剪接，要么剪接不完全。对psbA基因pre-mRNA的结构和频率进行的详细分析表明，四个内含子既不是以严格随机的方式剪接出去的，也不是按5'-3'或3'-5'方向剪接的。内含子2和3优先首先被剪接出去，内含子1其次，内含子4通常最后被剪接出去。然而，这个顺序并不是严格的规则。我们得出结论，内含子可以独立剪接，每个内含子的剪接速率不同。从psbA基因片段中分离出编码链，并与从琼脂糖凝胶中分离出的低分子量cpRNA退火。在四个内含子的位置发现了小的环状杂交体，首次证明了叶绿体中存在共价闭合的环状切除内含子RNA（iRNA）。
