Isolation of recombinants between T7 and T3 bacteriophages and their use in vitro transcriptional mapping
Isolation of recombinants between T7 and T3 bacteriophages and their use in vitro transcriptional mapping
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T7 和 T3 噬菌体之间重组体的分离及其在体外转录图谱中的应用
作者：
H. Beier;M. Golomb;M. Chamberlin
从在各种基因（基因1至基因19）中携带琥珀标记的T3和T7亲本间的杂交中分离出多种T3 X T7重组体。这些重组体的遗传组成通过参考所选择的标记并且还直接通过分析从T3 X T7重组体体内翻译的蛋白质来确定。虽然T3和T7蛋白密切相关，但大多数T3和T7蛋白在大小上略有不同，因此可以通过蛋白质分析确定基因的遗传起源。由T3和T7 RNA聚合酶从T3 X T7重组噬菌体DNA读取的主要转录物变化，这取决于存在T3或T7染色体的哪些区域。T7 RNA聚合酶不能以可观的速率利用T3聚合酶所采用的主要启动子位点，匡威亦然。因此，用T3或T7聚合酶获得的重组噬菌体DNA的转录模式允许确定基因组上不同启动子位点的同一性。T3 X T7重组DNA的转录分析与早期的观察一起已被用于绘制T3和T7的遗传图谱上的7种主要T3和T7 RNA物种中的5种的启动子位点。T7和T3 RNA种类IIIa、IV和V的启动子位点分别起源于基因7、9和10的起始处; T7和T3 RNA种类I和II的启动子位点分别位于基因11和基因13的左侧。没有发现T3 X T7重组体，其噬菌体RNA聚合酶的特异性与物种IIIa和I的相应启动子位点不相关（其转录覆盖60%的基因组）。这意味着由重组基因组指定的RNA聚合酶能够读取由野生型噬菌体基因组上的酶从主要启动子正常读取的序列中编码的所有信息。这表明噬菌体聚合酶对启动子位点选择的体外特异性也在体内保持。
A variety of T3 X T7 recombinants were isolated from crosses between T3 and T7 parental phages carrying amber markers in various genes (gene 1 to gene 19). The genetic constitution of these recombinants was determined by reference to the selected markers and also directly by analysis of the proteins translated from the T3 X T7 recombinants in vivo. Although T3 and TM phages are closely related, most T3 and T7 proteins differ slightly in size, and hence the genetic origin of a gene can be determined by protein analysis. The major transcripts read by T3 and T7 RNA polymerases from T3 X T7 recombinant phage DNAs vary, depending on which regions of the T3 or T7 chromosome are present. T7 RNA polymerase is unable to utilize major promoter sites employed by T3 polymerase at an appreciable rate, and the converse is also true. Hence the transcriptional pattern for a recombinant phage DNA obtained with the T3 or T7 polymerase allows a determination of the identity of the different promoter sites on the genome. The transcriptional analysis of T3 X T7 recombinant DNAs together with earlier observations has been used to map the promoter sites for five out of seven major T3 and T7 RNA species on the genetic maps of T3 and T7. The promoter sites for the T7 and T3 RNA species IIIa, IV, and V originate at the beginning of genes 7, 9, and 10, respectively; the promoter sites for the T7 and T3 RNA species I and II are located to the left of gene 11 and gene 13, respectively. No T3 X T7 recombinants were found for which the specificity of the phage RNA polymerase was not correlated with the corresponding promoter sites for species IIIa and I (the transcription of which covers 60% of the genome). That means that the RNA polymerase specified by the recombinant genome is able to read all of the information encoded in sequences read normally from major promoters by the enzyme on the wild-type phage genome. This suggests that the in vitro specificity for promoter site selection by the phage polymerases is also maintained in vivo.