Analysis of signals controlling expression of the Chinese hamster ovary aprt gene.
Analysis of signals controlling expression of the Chinese hamster ovary aprt gene.
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中国仓鼠卵巢aprt基因表达控制信号分析。
DOI：
10.1128/mcb.8.6.2536-2544.1988
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发表时间：
1988
影响因子：
5.3
通讯作者：
Taylor,MW
中科院分区：
文献类型：
--
作者：
Park,JH;Taylor,MW
对中国仓鼠ovaryaprt基因5′端进行测序，并通过S1核酸酶保护和引物延伸试验确定转录起始位点。构建了覆盖相同区域的缺失突变体，并通过瞬时表达测定测量腺嘌呤磷酸核糖基转移酶（APRT）或氯霉素乙酰转移酶（CAT）活性。该基因使用一个单一的转录起始位点簇，缺乏共有序列，如TATA和CCAAT，这是真核启动子的一般组成部分。启动子序列的5′端缺失突变表明（i）APRT活性或转录延伸至位置-89没有降低（ii）额外的29个碱基对（bp）缺失使APRT活性和转录降低两倍;和（iii）转录起始位点之后的缺失（P5′ Δ +27）消除APRT活性和转录，表明直接位于主转录起始位点上游的60-bp片段参与基本转录，而29-bp片段参与基本转录。60 bp片段上游的60 bp片段刺激转录两倍。3′端缺失突变表明，在5′端前导序列和编码序列中缺失一个61 bp的片段，使anaprt-CAT基因转录本的翻译效率丧失。此外，在基因组3′端有两个多聚腺苷酸化信号，其中近端信号足以进行功能性多聚腺苷酸化。
The 5′ end of the Chinese hamster ovaryaprtgene was sequenced and transcription start sites were determined by both S1 nuclease protection and primer extension assays. Deletion mutants covering the same area were constructed, and adenine phosphoribosyltransferase (APRT) or chloramphenicol acetyltransferase (CAT) activity was measured by transient-expression assays. Theaprtgene uses a single cluster of transcription start sites and lacks consensus sequences such as TATA and CCAAT, which are general components of eucaryotic promoters. The 5′ deletion mutations of the promoter sequences demonstrated that (i) there is no decrease in either APRT activity or transcription extending to position -89 (relative to the main transcription start site); (ii) an additional 29-base-pair (bp) deletion decreases APRT activity and transcription twofold; and (iii) a deletion past the transcription start sites (P5′ Δ +27) abolishes both APRT activity and transcription, indicating that a 60-bp fragment immediately upstream of the main transcription start site is involved in basic transcription and a 29-bp fragment just upstream of the 60 bp-fragment stimulates transcription twofold. The 3′ deletion mutations showed that a deletion of a 61-bp fragment in the 5′ leader and coding sequence abolishes the efficient translation of anaprt-CAT gene transcript. In addition, there are two polyadenylation signals at the genomic 3′ end, with the proximal one being sufficient for functional polyadenylation.