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首页> 外文期刊>Molecular and Cellular Biology >Construction of a modular dihydrofolate reductase cDNA gene: analysis of signals utilized for efficient expression.
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Construction of a modular dihydrofolate reductase cDNA gene: analysis of signals utilized for efficient expression.

机译：模块化二氢叶酸还原酶cDNA基因的构建：用于有效表达的信号分析。

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Dihydrofolate reductase (DHFR) modular genes have been constructed with segments containing the adenovirus major late promoter, a 3' splice site from a variable region immunoglobulin gene, a DHFR cDNA, and portions of the simian virus 40 (SV40) genome. DNA-mediated transfer of these genes transformed Chinese hamster ovary DHFR- cells to the DHFR+ phenotype. Transformants contained one to several copies of the transfected DNA integrated into the host genome. Clones subjected to growth in increasing concentrations of methotrexate eventually gave rise to lines containing several hundred copies of the transforming DNA. Analysis of the DHFR mRNA produced in amplified lines indicated the following. (i) All clones utilize the adenovirus major late promoter for transcription initiation. (ii) A hybrid intron formed by the 5' splice site of the adenovirus major late leader and a 3' splice site from a variable-region immunoglobulin gene is properly excised. (iii) The mRNA is not efficiently polyadenylated at sequences in the 3' end of the DHFR cDNA but rather uses polyadenylation signals downstream from the DHFR cDNA. Three independent clones produce a DHFR mRNA containing SV40 or pBR322 and SV40 sequences, and the RNA is polyadenylated at the SV40 late polyadenylation site. Another clone has recombined into cellular DNA and apparently uses a cellular sequence for polyadenylation. Introduction of a segment containing the SV40 early polyadenylation signal into the 3' end of the DHFR cDNA gene generated a recombinant capable of transforming cells to the DHFR+ phenotype with at least a 10-fold increase in efficiency, demonstrating the necessity for an efficient polyadenylation signal. Attachment of a DNA segment containing the transcription enhancer (72-base pair repeat) of SV40 further increased the biological activity of the modular DHFR gene 50- to 100-fold.
机译：已经用含有腺病毒主要晚期启动子，可变区免疫球蛋白基因的3'剪接位点，DHFR cDNA和部分猿猴病毒40（SV40）基因组的片段构建了二氢叶酸还原酶（DHFR）模块化基因。这些基因的DNA介导转移将中国仓鼠卵巢DHFR-细胞转化为DHFR +表型。转化子包含整合到宿主基因组中的一到几个拷贝的转染DNA。在甲氨蝶呤浓度增加的条件下生长的克隆最终产生了包含数百个转化DNA拷贝的品系。对扩增的品系中产生的DHFR mRNA的分析表明如下。 （i）所有克隆均利用腺病毒主要晚期启动子进行转录起始。 （ii）适当切除由腺病毒主要晚期前导序列的5'剪接位点和来自可变区免疫球蛋白基因的3'剪接位点形成的杂合内含子。 （iii）mRNA在DHFR cDNA 3'末端的序列上没有被有效的聚腺苷酸化，而是使用了DHFR cDNA下游的聚腺苷酸化信号。三个独立的克隆产生了包含SV40或pBR322和SV40序列的DHFR mRNA，并且RNA在SV40晚期聚腺苷酸化位点被聚腺苷酸化。另一个克隆已重组为细胞DNA，显然使用细胞序列进行聚腺苷酸化。将包含SV40早期聚腺苷酸化信号的区段引入DHFR cDNA基因的3'端，产生了一种重组体，能够将细胞转化为DHFR +表型，效率至少提高了10倍，这表明有效的聚腺苷酸化信号必不可少。包含SV40转录增强子（72个碱基对的重复序列）的DNA片段的附着进一步提高了模块DHFR基因的生物活性50到100倍。

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