Jankowski J M, Dixon G H
Biosci Rep. 1985 Feb;5(2):113-20. doi: 10.1007/BF01117057.
A series of plasmids containing new fusion genes in which the trout protamine gene is placed under the control of the complete herpes virus (HSV-1) tk promoter Pvu II-Bgl II fragment (pM8), or a shortened thymidine kinase (tk) promoter in which the region between the TATA box and the cap site is altered by using the Pvu II-Mlu I fragment (pM7), have been constructed. An additional recombinant plasmid was constructed in which the Bgl II-Ava II fragment of the protamine gene containing the entire protamine promoter but missing the protamine coding region was cloned into pBR322 between the Xho II 1666 and Hind III sites (pP5). For in vitro transcription, a HeLa cell lysate system was prepared and the RNA transcription products, after glyoxalation, were electrophoretically analyzed on 5% polyacrylamide gels. In constructing pM8 the DNA sequence between the tk promoter and the cap site was present while in pM7 it was deleted. Similar multiple transcripts were seen in both cases, indicating that the region between the promoter and the cap site has no effect upon transcription in vitro. The multiple transcripts appear to be due to the presence of a cryptic promoter in the complementary strand of the protamine gene. The activity of this cryptic promoter has been confirmed by comparison of the transcription of plasmid pP5, in which the protamine mRNA coding region has been deleted, with a previously described plasmid, pJBRP (Jankowski JM and Dixon GH (1984) Can. J. Biochem. Cell. Biol. 62, 291-300), containing the intact protamine gene.
构建了一系列含有新融合基因的质粒，其中鲑鱼鱼精蛋白基因置于完整的疱疹病毒（HSV-1）胸苷激酶（tk）启动子的Pvu II - Bgl II片段（pM8）控制之下，或者置于一个缩短的胸苷激酶（tk）启动子控制之下，该缩短的启动子中TATA框和帽位点之间的区域通过使用Pvu II - Mlu I片段进行了改变（pM7）。还构建了一个额外的重组质粒，其中将包含整个鱼精蛋白启动子但缺失鱼精蛋白编码区的鱼精蛋白基因的Bgl II - Ava II片段克隆到pBR322的Xho II 1666和Hind III位点之间（pP5）。对于体外转录，制备了HeLa细胞裂解物系统，并且将RNA转录产物进行乙二醛化后，在5%聚丙烯酰胺凝胶上进行电泳分析。在构建pM8时，tk启动子和帽位点之间的DNA序列存在，而在pM7中该序列被删除。在两种情况下都观察到了类似的多个转录本，这表明启动子和帽位点之间的区域对体外转录没有影响。这些多个转录本似乎是由于鱼精蛋白基因互补链中存在一个隐蔽启动子所致。通过比较缺失鱼精蛋白mRNA编码区的质粒pP5与先前描述的含有完整鱼精蛋白基因的质粒pJBRP（Jankowski JM和Dixon GH（1984年）《加拿大生物化学与细胞生物学杂志》62卷，291 - 300页）的转录情况，证实了这个隐蔽启动子的活性。
