Crouse G F, Stivaletta L A, Smith M L
Department of Biology, Emory University, Atlanta, GA 30322.
Nucleic Acids Res. 1988 Jul 25;16(14B):7025-42. doi: 10.1093/nar/16.14.7025.
We have constructed a plasmid encoding a mouse dihydrofolate reductase (dhfr) minigene which produces dhfr transcripts with all of the 5' and 3' ends observed from the chromosomal mouse dhfr gene. The minigene contains 5' flanking regions, all dhfr coding sequences, one intervening sequence, 11.5 kb of 3' flanking regions beyond the termination codon, an E. coli plasmid origin of replication and antibiotic resistance, and an SV40 minimal origin of replication; the total size is 17.2 kb. When transfected into cells constitutively producing a temperature sensitive SV40 T antigen, the plasmid minigene replicates at the permissive temperature, but fails to replicate at the nonpermissive temperature. Therefore, transcription can be observed in the presence or absence of minigene replication. In addition, a stable divergently transcribed RNA is produced from the dhfr minigene promoter region, with the same 5' ends that are seen in the chromosomal divergently transcribed gene. We show that deletion of the sole remaining intron of the dhfr minigene significantly lowers the amount of dhfr transcript produced but does not affect the amount of divergent transcript. The promoter region for these transcripts contains four 48 bp repeats; reducing the number of these repeats lowers the amount of both dhfr and divergent transcripts produced from the minigene.
我们构建了一个编码小鼠二氢叶酸还原酶（dhfr）小基因的质粒，该小基因产生的dhfr转录本具有从染色体小鼠dhfr基因观察到的所有5'和3'末端。该小基因包含5'侧翼区域、所有dhfr编码序列、一个内含子序列、终止密码子下游11.5 kb的3'侧翼区域、一个大肠杆菌质粒复制起点和抗生素抗性，以及一个SV40最小复制起点；总大小为17.2 kb。当转染到组成型产生温度敏感型SV40 T抗原的细胞中时，该质粒小基因在允许温度下复制，但在非允许温度下不能复制。因此，无论小基因是否复制都能观察到转录。此外，从dhfr小基因启动子区域产生了一种稳定的反向转录RNA，其5'末端与染色体反向转录基因中的相同。我们表明，删除dhfr小基因唯一剩余的内含子会显著降低产生的dhfr转录本数量，但不影响反向转录本的数量。这些转录本的启动子区域包含四个48 bp的重复序列；减少这些重复序列的数量会降低从小基因产生的dhfr和反向转录本的数量。
