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Genetic Test for Involvement of Intervening Sequences in Transport of Nuclear RNA

机译：介入RNA转运的介入序列的遗传测试。

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The construction of a recombinant virus in the late region of simian virus 40 is presented. The small intervening sequence of late 19S RNA (0.760 to 0.765 map unit) was cloned and inserted into the EcoRI site (1.0 map unit) in the late region of simian virus 40. This is a mutant virus that now has two intervening sequences, one at the normal position (0.760 map unit) and another out of the context of its flanking sequence and now at 1.0 map unit. The recombinant appears poisonous, as repeated attempts to plaque it as a virus with a standard helper virus were unsuccessful. The transcription of this recombinant was, therefore, studied after direct DNA transfection onto CV-1 cells. Nuclease S1 analysis of mutant RNA indicates that the major nuclear transcript was a spliced but nuclear 16S RNA species. Normally, 16S RNA is not found in the nucleus. This result was shown to be an artifact of the DNA transfection protocol. When the glycerol shock was done after infection with virus, a similar alteration in the makeup of nuclear RNA was seen. A transient stock of this double-intron mutant was finally obtained, using a nonrevertable helper virus. The transcriptional analysis of this mutant showed that unspliced 19S RNA was not transported and remained within the nucleus, whereas spliced 19S and 16S RNAs were transported. We conclude that the retention of nuclear transcripts within the nucleus is not simply due to the presence of intronic sequences, as spliced 19S and 16S RNAs which contain the second intron were efficiently transported.
机译：提出了在猿猴病毒40的晚期区域中重组病毒的构建。克隆了晚期19S RNA的小插入序列（0.760至0.765位图单元），并将其插入猿猴病毒40晚期区域的 Eco RI位点（1.0位图单元）。现在有两个插入序列，一个位于正常位置（0.760个图单元），另一个不在其侧翼序列的上下文中，而现在处于1.0个图单元。重组体似乎是有毒的，因为反复尝试用标准辅助病毒将其斑块化为病毒是不成功的。因此，在将DNA直接转染到CV-1细胞后，研究了该重组体的转录。突变RNA的核酸酶S1分析表明，主要的核转录本是一个剪接但有核的16S RNA物种。通常，在细胞核中未发现16S RNA。该结果显示为DNA转染方案的人工产物。当病毒感染后进行甘油休克时，可观察到核RNA组成的类似变化。最终，使用不可逆的辅助病毒最终获得了该双内含子突变体的瞬时储备。该突变体的转录分析表明，未剪接的19S RNA不被转运并保留在细胞核内，而剪接的19S和16S RNA被转运。我们得出结论，核转录本在细胞核内的保留不仅仅是由于内含子序列的存在，因为包含第二个内含子的19S和16S RNA剪接RNA被有效地转运了。

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