Ksenzenko V N, Kamynina T P, Pustoshilova N M, Kryukov V M, Bayev A A
Mol Gen Genet. 1982;185(3):520-2. doi: 10.1007/BF00334154.
Bacteriophage T5 was subjected to combined hydrolysis with the restriction endonucleases PstI and HindIII and the resulting fragments were inserted into the plasmid pBR322. Selection of transformants for Aps-Tcr-phenotype made it possible to screen the hybrid plasmids that contained promoter sequences in the cloned fragments. Two PstI/HindIII fragments, 720 bp (51% of the T5 DNA length) and 1,200 bp (70%) were cloned in this study. Tcr levels for these plasmids were as high as 18 micrograms/ml and 75 micrograms/ml, respectively. The presence of Escherichia coli RNA polymerase binding sites on both fragments was shown using the nitrocellulose filter assay. These binding sites are situated between 35 bp and 95 bp from the HindIII cleavage site on the 1,200 bp fragment; and within 420 bp from the HindIII site on the 720 bp fragment.
将噬菌体T5用限制性内切核酸酶PstI和HindIII进行联合水解，然后将所得片段插入质粒pBR322中。选择具有Aps-Tcr表型的转化体使得筛选在克隆片段中含有启动子序列的杂交质粒成为可能。本研究克隆了两个PstI/HindIII片段，分别为720 bp（占T5 DNA长度的51%）和1200 bp（占70%）。这些质粒的Tcr水平分别高达18微克/毫升和75微克/毫升。使用硝酸纤维素滤膜分析表明两个片段上均存在大肠杆菌RNA聚合酶结合位点。这些结合位点位于1200 bp片段上距HindIII切割位点35 bp至95 bp之间；以及720 bp片段上距HindIII位点420 bp范围内。
