The effects of differential polyadenylation on expression of the dihydrofolate reductase-encoding gene in Chinese hamster lung cells.
The effects of differential polyadenylation on expression of the dihydrofolate reductase-encoding gene in Chinese hamster lung cells.
复制标题
差异聚腺苷酸化对中国仓鼠肺细胞二氢叶酸还原酶编码基因表达的影响。
DOI：
10.1016/0378-1119(95)00381-f
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发表时间：
1995
期刊：
影响因子：
3.5
通讯作者：
Melera,PW
中科院分区：
文献类型：
--
作者：
Yang,H;Hussain,A;Melera,PW
三种不同大小的mRNA从中国仓鼠肺（CHL）细胞系DC-3F中存在的两个DHFR（编码二氢叶酸还原酶）等位基因中的每一个表达。由于DHFR基因中存在的多聚腺苷酸位点的差异利用和位于一个等位基因的第三聚腺苷酸信号内的遗传多态性，每个等位基因产生的转录物的相对缺失显著不同。我们试图确定多聚腺苷酸化的这种差异是否会影响从任一等位基因表达的DHFR mRNA的稳态水平，并且在更一般的意义上，询问含有多聚腺苷酸位点的基因中3′端RNA加工的差异是否会影响基因表达的最终水平。开发了一种基于SV 40启动子的瞬时表达系统，产生嵌合猫：：DHFR转录本，以再生与两个DHFR等位基因中的每一个相关的体内mRNA聚腺苷酸化模式。结果表明，从这些构建体中的每一个在体外表达的聚腺苷酸化RNA的总量是相同的，而不管它们之间发生的聚腺苷酸信号的差异利用。此外，从每个等位基因在体内表达的DHFR mRNA的个体周转率的测量，如通过脉冲追踪标记和放线菌素D抑制研究所确定的，显示转录物半衰期没有显着的等位基因特异性差异。最后，在亲本DC-3F细胞中测量DHFR poly（A）+mRNA的稳态水平表明，在正常生长期间，两个等位基因以相同的程度表达。因此，即使在体内DHFR转录物的3′端加工中发生显着的等位基因特异性差异，这种差异似乎不会影响DHFR基因表达的稳态水平。
Three differently sized mRNAs are expressed from each of two DHFR (encoding dihydrofolate reductase) alleles present in the Chinese hamster lung (CHL) cell line, DC-3F. The relative abundancy of the transcripts produced from each allele differs dramatically as a result of differential utilization of the multiple poly(A) sites present in the DHFR gene and a genetic polymorphism located within the third poly(A) signal of one allele. We sought to determine whether such differences in polyadenylation affect the steady-state levels of DHFR mRNAs expressed from either allele and, in a more general sense, to ask whether differences in 3′ end RNA processing in a gene containing multiple poly(A) sites affects the final level of gene expression. An SV40 promoter-based transient expression system producing chimeric cat::DHFR transcripts was developed to regenerate the in vivo mRNA polyadenylation patterns associated with each of the two DHFR alleles. The results demonstrate that the total amount of polyadenylated RNA expressed from each of these constructs in vitro is the same regardless of the differential utilization of the poly(A) signals that occurs between them. Moreover, measurement of the individual turnover rates of the DHFR mRNAs expressed in vivo from each allele, as determined by pulse-chase labeling and actinomycin D inhibition studies, revealed no significant allele-specific differences in transcript half-lives. Finally, measuring the steady-state levels of DHFR poly(A)+mRNA in parental DC-3F cells demonstrated that both alleles are expressed to the same extent during normal growth. Thus, even though dramatic allele-specific differences in 3′ end processing of DHFR transcripts occur in vivo, such differences do not appear to influence the steady-state levels of DHFR gene expression.