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完整雀麦花叶病毒基因组的cDNA克隆及体外转录

cDNA cloning and in vitro transcription of the complete brome mosaic virus genome.

作者信息

Ahlquist P, Janda M

出版信息

Mol Cell Biol. 1984 Dec;4(12):2876-82. doi: 10.1128/mcb.4.12.2876-2882.1984.

DOI:10.1128/mcb.4.12.2876-2882.1984
PMID:6549346
原文链接:https://pmc.ncbi.nlm.nih.gov/articles/PMC369300/
Abstract

Complete cDNA copies of each of the brome mosaic virus genomic RNAs (3.2, 2.8, and 2.1 kilobases in length) were cloned in a novel transcription vector, pPM1, designed to provide exact control of the transcription initiation site. After cleavage at a unique EcoRI site immediately downstream of the inserted cDNA, these clones can be transcribed in vitro by Escherichia coli RNA polymerase to yield complete copies of the brome mosaic virus RNAs. Dideoxy sequencing of 5' transcript cDNA runoff products and direct sequencing of 32P-3'-end-labeled transcripts show that such transcripts initiate at the same 5' position as natural viral RNA and terminate within the EcoRI runoff site after copying the entire viral RNA sequence. When synthesized in the presence of m7GpppG, the transcripts bear the natural capped 5' terminus of brome mosaic virus RNAs. Such transcripts direct the in vitro translation of proteins which coelectrophorese with the translation products of natural brome mosaic virus RNAs. pPM1 should facilitate in vitro production of other viral and nonviral RNAs.

摘要

将雀麦花叶病毒基因组RNA（长度分别为3.2、2.8和2.1千碱基）的完整cDNA拷贝克隆到一种新型转录载体pPM1中，该载体旨在精确控制转录起始位点。在插入的cDNA下游紧邻的唯一EcoRI位点处切割后，这些克隆可通过大肠杆菌RNA聚合酶进行体外转录，以产生雀麦花叶病毒RNA的完整拷贝。对5'转录本cDNA末端终止产物进行双脱氧测序以及对32P-3'-末端标记的转录本进行直接测序表明，此类转录本与天然病毒RNA在相同的5'位置起始，并在复制整个病毒RNA序列后在EcoRI末端终止位点内终止。当在m7GpppG存在的情况下合成时，转录本带有雀麦花叶病毒RNA天然的帽化5'末端。此类转录本指导蛋白质的体外翻译，这些蛋白质与天然雀麦花叶病毒RNA的翻译产物共电泳。pPM1应有助于其他病毒和非病毒RNA的体外生产。

https://cdn.ncbi.nlm.nih.gov/pmc/blobs/a1c4/369300/afac032b1682/molcellb00154-0324-b.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/a1c4/369300/9e4531a09a53/molcellb00154-0322-a.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/a1c4/369300/3daee3d69ddf/molcellb00154-0323-a.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/a1c4/369300/ed753cafbaa2/molcellb00154-0324-a.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/a1c4/369300/afac032b1682/molcellb00154-0324-b.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/a1c4/369300/9e4531a09a53/molcellb00154-0322-a.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/a1c4/369300/3daee3d69ddf/molcellb00154-0323-a.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/a1c4/369300/ed753cafbaa2/molcellb00154-0324-a.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/a1c4/369300/afac032b1682/molcellb00154-0324-b.jpg

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