Both G W, Bellamy A R, Street J E, Siegman L J
Nucleic Acids Res. 1982 Nov 25;10(22):7075-88. doi: 10.1093/nar/10.22.7075.
Using Simian-11 rotavirus RNA, a strategy has been developed for the production of full length cloned copies of the genes of double-stranded (dsRNA) viruses. Genomic RNA segments were polyadenylated and reverse transcribed to yield a mixture of full length cDNA copies of both possible polarities. The cDNAs were annealed, filled in to complete any partial copies, tailed and inserted into the PstI site of pBR322 using dG/dC tailing. Cloned rotavirus cDNA gene copies were assigned to genomic RNA segments by Northern hybridization. The complete sequence of gene 8 which codes for NCVP3, a non-structural protein of SA11 rotavirus, was determined from a cloned gene copy. It is 1059 bases in length and has an open reading frame which could code for a protein containing 317 amino acids. The apparent 5' and 3' terminal non coding regions are 46 and 59 bases in length, respectively. The sequence ATGTGACCOH at the 3' end of the plus strand is conserved in four of the eleven genes examined. The cloning procedures used should be generally applicable to viruses with segmented dsRNA genomes.
利用猿猴轮状病毒11型（Simian-11 rotavirus）RNA，已开发出一种策略用于生产双链（dsRNA）病毒基因的全长克隆拷贝。基因组RNA片段被聚腺苷酸化并进行逆转录，以产生两种可能极性的全长cDNA拷贝的混合物。将cDNA退火，补齐以完成任何部分拷贝，加尾并使用dG/dC加尾法插入pBR322的PstI位点。通过Northern杂交将克隆的轮状病毒cDNA基因拷贝分配到基因组RNA片段。从一个克隆的基因拷贝中确定了编码SA11轮状病毒非结构蛋白NCVP3的基因8的完整序列。它长度为1059个碱基，有一个开放阅读框，可编码一个含317个氨基酸的蛋白质。正义链3'端的明显5'和3'末端非编码区分别为46和59个碱基长。在所检测的11个基因中的4个基因中，正义链3'端的序列ATGTGACCOH是保守的。所使用的克隆程序通常应适用于具有分段dsRNA基因组的病毒。
