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首页> 外文期刊>Molecular and Cellular Biology >Transient expression of a mouse alpha-fetoprotein minigene: deletion analyses of promoter function.
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Transient expression of a mouse alpha-fetoprotein minigene: deletion analyses of promoter function.

机译：小鼠甲胎蛋白小基因的瞬时表达：启动子功能的缺失分析。

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The constitutive transcription of a mouse alpha-fetoprotein (AFP) minigene was examined during the transient expression of AFP-simian virus 40-pBR322 recombinant DNAs introduced into HeLa cells by Ca3(PO4)2 precipitation. We tested three constructs, each of which contains the AFP minigene and pBR322 DNAs inserted in the late region of simian virus 40 and found that the relative efficiency of AFP gene expression was dependent on the arrangement of the three DNA elements in the vector. The transcripts begin at the authentic AFP cap site and are properly spliced and polyadenylated. To define a sequence domain in the 5' flanking region of the AFP gene required for constitutive expression, sequential 5' deletion mutants of the AFP minigene were constructed and introduced into HeLa cells. All AFP deletion mutants which retained at least the TATA motif located 30 base pairs upstream from the cap site were capable of directing accurate and efficient AFP transcription. However, when the TATA sequence was deleted, no accurately initiated AFP transcripts were detected. These results are identical to those obtained from in vitro transcription of truncated AFP 5' deletion mutant templates assayed in HeLa cell extracts. The rate of AFP transcription in vivo was unaffected by deletion of DNA upstream of the AFP TATA box but was greatly affected by the distance between the simian virus 40 control region and the 5' end of the gene. The absence of any promoter activity upstream of the TATA box in this assay system is in contrast to what has been reported for several other eucaryotic structural genes in a variety of in vivo systems. A sequence comparison between the 5' flanking region of the AFP gene and these genes suggested that the AFP gene lacks those structural elements found to be important for constitutive transcription in vivo. Either the AFP gene lacks upstream promoter function in the 5' flanking DNA contained within the minigene, or the use of a viral vector in a heterologous system precludes its identification.
机译：在通过Ca3（PO4）2沉淀引入HeLa细胞的AFP-猿猴病毒40-pBR322重组DNA的瞬时表达过程中，检查了小鼠甲胎蛋白（AFP）小基因的组成性转录。我们测试了三个构建体，每个构建体均包含AFP小基因和插入猿猴病毒40晚期区域的pBR322 DNA，发现AFP基因表达的相对效率取决于载体中这三个DNA元件的排列。转录本从真实的AFP帽位开始，并正确剪接和聚腺苷酸化。为了在组成性表达所需的AFP基因的5'侧翼区域中定义序列结构域，构建了AFP小基因的顺序5'缺失突变体，并将其引入HeLa细胞。所有至少保留位于帽位点上游30个碱基对的TATA基序的AFP缺失突变体均能够指导准确而有效的AFP转录。但是，当删除TATA序列时，未检测到准确启动的AFP转录本。这些结果与从HeLa细胞提取物中测定的截短的AFP 5'缺失突变体模板的体外转录获得的结果相同。体内AFP转录的速率不受AFP TATA盒上游DNA缺失的影响，但受猿猴病毒40调控区与基因5'端之间距离的影响很大。在该测定系统中，TATA盒上游不存在任何启动子活性，这与多种体内系统中其他几种真核结构基因的报道相反。 AFP基因的5'侧翼区域与这些基因之间的序列比较表明，AFP基因缺少那些对体内组成型转录很重要的结构元件。 AFP基因在小基因中所含的5'侧翼DNA中缺少上游启动子功能，或者在异源系统中使用病毒载体无法对其进行鉴定。

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