Krippl B, Ferguson B, Rosenberg M, Westphal H
Proc Natl Acad Sci U S A. 1984 Nov;81(22):6988-92. doi: 10.1073/pnas.81.22.6988.
We have purified the human type C adenovirus E1A 13S mRNA gene product, expressed in Escherichia coli, and demonstrate that the protein exhibits genuine viral functions upon microinjection into mammalian cells. We show that the E1A protein activates expression of the adenovirus E2A gene and induces expression from the major late transcription unit of the adenovirus E1A deletion mutant, H5dl312. We use this functional assay to examine the stability of E1A protein microinjected into cells and find that E1A exhibits full function for at least 18 hours after its injection. In addition, the purified E1A protein was used to generate a high-titer monospecific rabbit antiserum. This antiserum was used to detect and localize E1A proteins within adenovirus-infected cells as well as within microinjected cells. The E1A protein is found to rapidly and quantitatively localize to the cell nucleus following microinjection into the cell cytoplasm. Thus, nuclear localization is an intrinsic property of the E1A polypeptide. The ability of the E1A protein to localize to the cell nucleus and to induce expression from the H5dl312 major late transcription unit is shown to be highly heat stable.
我们已经纯化了在大肠杆菌中表达的人C型腺病毒E1A 13S mRNA基因产物，并证明该蛋白在显微注射到哺乳动物细胞后具有真正的病毒功能。我们发现E1A蛋白可激活腺病毒E2A基因的表达，并诱导腺病毒E1A缺失突变体H5dl312的主要晚期转录单位的表达。我们使用这种功能测定法来检测显微注射到细胞中的E1A蛋白的稳定性，发现E1A在注射后至少18小时内都具有完整功能。此外，纯化的E1A蛋白被用于制备高滴度的单特异性兔抗血清。该抗血清用于检测腺病毒感染细胞以及显微注射细胞内的E1A蛋白并对其进行定位。发现将E1A蛋白显微注射到细胞质中后，它会迅速且定量地定位于细胞核。因此，核定位是E1A多肽的固有特性。E1A蛋白定位于细胞核并诱导H5dl312主要晚期转录单位表达的能力被证明具有高度的热稳定性。
