Vector expression of adenovirus type 5 E1a proteins: evidence for E1a autoregulation.
Vector expression of adenovirus type 5 E1a proteins: evidence for E1a autoregulation.
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5 型腺病毒 E1a 蛋白的载体表达：E1a 自动调节的证据。
DOI：
10.1128/mcb.5.10.2684-2696.1985
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发表时间：
1985
影响因子：
5.3
通讯作者：
Ziff,EB
中科院分区：
文献类型：
--
作者：
Smith,DH;Kegler,DM;Ziff,EB
我们瞬时表达腺病毒C型E1 a蛋白在野生型或突变体形式的质粒载体，具有不同的组合E1 a和猿猴病毒40增强子元件，并含有SV 40的DNA复制起点，可以在COS 7细胞中复制。我们测量了载体编码的E1 a mRNA的水平和蛋白产物的转换调节特性。三种载体在COS 7细胞中编码E1 a mRNA的水平相当：（i）编码wt 289-氨基酸E1 a蛋白的质粒（这补充了E1 a缺失突变体dl 312，用于在复制和非复制条件下早期区域E2 a的表达）;（ii）wt 243-氨基酸E1 a蛋白的载体（这弱地补充了ddl 312，并且仅在ddl 312的高多重性的条件下）;（iii）突变体pSVXL 105，其中289个氨基酸的E1 a蛋白的氨基酸残基38至44（包括两个高度保守的残基）被3个新的氨基酸取代（这也有效地补充了DD 1312）。第四种载体，突变体pSVXL 3，其接头取代移动了阅读框架，从289个氨基酸的蛋白质的氨基末端编码截短的70个氨基酸片段，不能互补1312。令人惊讶的是，pSVXL 3在COS 7细胞中过表达E1 a mRNA约30倍，与其他载体相比。pSVXL 3的过表达可通过与awtE 1a载体共转染来逆转。我们认为wtE 1a蛋白通过最近描述的289-和243-氨基酸E1 a蛋白产物的转录抑制功能来调节其自身mRNA的水平，而pSVXL 3不能负性自调节。
We transiently expressed adenovirus type C E1a proteins in wild-type or mutant form from plasmid vectors which have different combinations of E1a and simian virus 40 enhancer elements and which contain the DNA replication origin of SV40 and can replicate in COS 7 cells. We measured the levels of E1a mRNA encoded by the vectors and the transition regulation properties of the protein products. Three vectors encoded equivalent levels of E1a mRNA in COS 7 cells: (i) a plasmid encoding thewt289-amino acid E1a protein (this complemented the E1a deletion mutantdl312 for early region E2a expression under both replicative and nonreplicative conditions); (ii) a vector for thewt243-amino acid E1a protein (this complementeddl312 weakly and only under conditions of high multiplicities ofdl312); (iii) a mutant, pSVXL105, in which amino acid residues 38 through 44 of the 289-amino acid E1a protein (which includes two highly conserved residues) are replaced by 3 novel amino acids (this also complementeddl312 efficiently). A fourth vector, mutant pSVXL3 with which linker substitution shifts the reading frame to encode a truncated 70-amino acid fragment from the amino terminus of the 289-amino acid protein, was unable to complementdl312. Surprisingly, pSVXL3 overexpressed E1a mRNA approximately 30-fold in COS 7 cells in comparison with the other vectors. The pSVXL3 overexpression could be reversed by cotransfection with awtE1a vector. We suggest thatwtE1a proteins regulate the levels of their own mRNAs through the recently described transcription repression functions of the 289- and 243-amino acid E1a protein products and that pSVXL3 fails to autoregulate negatively.