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首页> 外文期刊>Journal of Virology >Genome organization of RNA tumor viruses. I. In vitro synthesis of full-genome-length single-stranded and double-stranded viral DNA transcripts.
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Genome organization of RNA tumor viruses. I. In vitro synthesis of full-genome-length single-stranded and double-stranded viral DNA transcripts.

机译：基因组组织RNA肿瘤病毒。 I.体外合成全基因组长度的单链和双链病毒DNA转录物。

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Genome-length complementary DNA (cDNA) transcripts were synthesized in vitro by using purified virions of avian myeloblastosis virus. Moloney murine leukemia virus, and clone 124 mouse sarcoma virus. The size of the genomelenth cDNA transcripts was measured on either alkaline sucrose gradients or alkaline agarose gels. The longest cDNA transcripts synthesized by using avian myeloblastosis virus, Moloney murine leukemia virus, and clone 124 mouse sarcoma virus were 7, 9 and 6 kilobases (kb), respectively. The in vitro system used was capable of synthesizing double-stranded DNA, but the plus strands (same polarity as the viral RNA) were only 0.5 to 1.5 kb long. Lone Moloney murine leukemia virus cDNA transcripts were used as templates to synthesize the second plus strand. Essentially two strategies were employed as follows. (i) The 3' ends of the cDNA transcripts were extended by addition of 50 to 100 dAMP residues by terminal deoxynucleotidyl transferase. The (dA)n-tailed cDNA transcripts were used as templates along with an oligomer of dT as primer and Escherichia coli DNA polymerase to synthesize the plus strands. (ii) DNase-digested calf thymus DNA was used to prime the synthesis of plus strands on long cDNA with E. coli DNA polymerase I. In both cases, the synthesis of the plus strands was monitored by increased resistance of the cDNA templates to single-strand-specific S1 nuclease. The double-stranded DNA was fractionated on neutral sucrose gradients. Analysis of the double-stranded DNA synthesized by using oligo(dT) primer showed the plus strands to be about 5 to 6 kb long, whereas the plus strands synthesized by using DNase-digested calf thymus DNA primers were only 0.3 to 0.5 kb long. Double-stranded DNA synthesized by either method has an average size of 6 x 10(6) daltons. Double-stranded DNA was also synthesized by using cDNA transcripts as templates without the addition of any primers. In this case, the plus strands were covalently linked to the template strand and were not representative of the whole parent strand.
机译：通过使用禽血吸虫病病毒的纯化的病毒血管来合成基因组长度互补DNA（cDNA）转录物。莫尼鼠白血病病毒，克隆124只小鼠肉瘤病毒。在碱性蔗糖梯度或碱性琼脂糖凝胶上测量基因组列的CDNA转录物的大小。通过使用禽髓素病毒，Moloney鼠白血病病毒和克隆124小鼠肉瘤病毒合成的最长cDNA转录物分别为7,9和6千碱基（Kb）。所用的体外系统能够合成双链DNA，但是加号（与病毒RNA相同）仅为0.5至1.5kb。孤独的摩洛妮鼠白血病病毒cDNA转录物用作模板以合成第二加股。基本上雇用了两种策略如下。 （i）通过末端脱氧核苷酸转移酶加入50至100个潮湿残基，延长cDNA转录物的3'末端。 （DA）n尾CDNA转录物用作模板以及DT的低聚物作为引物和大肠杆菌DNA聚合酶，以合成加股。 （ii）DNA酶消化的小牛胸腺DNA用于在具有大肠杆菌DNA聚合酶I的长cDNA上填充加股的合成。在两种情况下，通过增加cDNA模板对单一的CDNA模板的抗性来监测加号的合成-Strand特异性S1核酸酶。双链DNA在中性蔗糖梯度上分离。通过使用寡核苷酸（DT）引物合成的双链DNA的分析显示加号为约5至6kb，而通过使用DNase-Difested CALF胸腺DNA引物合成的加股仅为0.3至0.5kb。通过任一方法合成的双链DNA的平均尺寸为6×10（6）道尔顿。通过使用CDNA转录物作为模板，还通过添加任何引物来合成双链DNA。在这种情况下，与模板链共价连接加链，并不代表全母链。

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    《Journal of Virology》 |1978年第3期|共15页
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    I M Verma;

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  • 收录信息 美国《科学引文索引》(SCI);
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