Jankowski J M, Dixon G H
Can J Biochem Cell Biol. 1984 May;62(5):291-300. doi: 10.1139/o84-041.
An in vitro approach has been used to study trout protamine gene expression using various recombinant plasmids containing trout protamine genes as templates in the HeLa cell lysate transcription system. The specific RNA transcript which is protected against S1 nuclease digestion by hybridization to the protamine gene sequence is alpha-amanitin sensitive (1 micrograms/mL), showing that RNA polymerase II is involved. The sizes of transcripts from templates linearized with Bam HI, Rsa I, and Hpa II (all downstream from the putative TATA box) are consistent with those predicted from the known sequence of the protamine gene. Digestion at an Alu I site only 14 base pairs (bp) upstream from TATA box has no effect on the accuracy of transcription in vitro; however, cutting at an Ava II site 9 bp downstream from the TATA box (reading from the first T) abolishes transcription. Chimeric plasmids, in which a herpes simplex virus (HSV-1) thymidine kinase (tk) promoter is tandemly inserted upstream from the trout protamine DNA sequences or as a replacement of the natural protamine promoter, were constructed. Use of these plasmids allowed an examination in a single assay of eight different putative promoter sequences (TATAAAA, TATAAA, TACAAA, TATATA, TATTTAA, CATATTA, TATATTAT, and TATTTAT) that are localized in either the protamine or the tk genes. The canonical TATAAAA promoter (the natural protamine promoter) was the strongest one and, in its presence, none of the others were used significantly for transcription. However, when this promoter was removed the weaker promoters were able to promote transcription.
一种体外方法已被用于研究鲑鱼精蛋白基因的表达，该方法使用各种含有鲑鱼精蛋白基因的重组质粒作为模板，在HeLa细胞裂解物转录系统中进行。通过与精蛋白基因序列杂交而免受S1核酸酶消化的特定RNA转录本对α-鹅膏蕈碱敏感（1微克/毫升），表明RNA聚合酶II参与其中。用Bam HI、Rsa I和Hpa II（均在假定的TATA框下游）线性化的模板产生的转录本大小与根据精蛋白基因已知序列预测的大小一致。在距TATA框仅14个碱基对（bp）的Alu I位点进行切割对体外转录的准确性没有影响；然而，在TATA框下游9 bp（从第一个T开始读）的Ava II位点进行切割会消除转录。构建了嵌合质粒，其中单纯疱疹病毒（HSV-1）胸苷激酶（tk）启动子串联插入鲑鱼精蛋白DNA序列上游或替代天然精蛋白启动子。使用这些质粒可以在一次检测中检查位于精蛋白或tk基因中的八个不同的假定启动子序列（TATAAAA、TATAAA、TACAAA、TATATA、TATTTAA、CATATTA、TATATTAT和TATTTAT）。典型的TATAAAA启动子（天然精蛋白启动子）是最强的启动子，在其存在的情况下，其他启动子均未被显著用于转录。然而，当去除该启动子时，较弱的启动子能够促进转录。
