Di Nocera P P, Dawid I B
Proc Natl Acad Sci U S A. 1983 Dec;80(23):7095-8. doi: 10.1073/pnas.80.23.7095.
Recombinant plasmids in which the sequence encoding the bacterial chloramphenicol acetyltransferase (CAT; acetyl-CoA:chloramphenicol 3-O-acetyltransferase, EC 2.3.1.28) has been placed under the control of Drosophila heat shock protein 70 (hsp 70) or copia promoters have been introduced into cultured cells of two Drosophila species (Schneider II line of Drosophila melanogaster and D. immigrans) as calcium-phosphate complexes. Within 1-2 days after transfection functional CAT enzyme was detected in cells exposed to either CAT recombinant. The expression of the bacterial information depends on the activity of the Drosophila promoters because plasmids in which the Drosophila DNA fragments were fused to the CAT coding sequence in inverted orientation did not support the synthesis of CAT enzyme activity. Low levels of CAT activity and of hybrid mRNA were detected in cells transformed with hsp-cat recombinants when the cells were maintained at room temperature, and both mRNA levels and CAT activity increased substantially after a brief exposure to 37 degrees C. hsp-cat mRNA has the same 5' terminus as authentic Drosophila hsp 70 messenger. These experiments document a practical system for the introduction and expression of isolated genes in cultured cells of Drosophila.
已将编码细菌氯霉素乙酰转移酶(CAT;乙酰辅酶A:氯霉素3 - O - 乙酰转移酶,EC 2.3.1.28)的序列置于果蝇热休克蛋白70(hsp 70)或copia启动子控制之下的重组质粒,作为磷酸钙复合物导入了两种果蝇(黑腹果蝇的Schneider II系和移民果蝇)的培养细胞中。转染后1 - 2天内,在接触任一CAT重组体的细胞中检测到了有功能的CAT酶。细菌信息的表达取决于果蝇启动子的活性,因为果蝇DNA片段以反向与CAT编码序列融合的质粒不能支持CAT酶活性的合成。当细胞在室温下培养时,用hsp - cat重组体转化的细胞中检测到低水平的CAT活性和杂交mRNA,短暂暴露于37℃后,mRNA水平和CAT活性均大幅增加。hsp - cat mRNA与真正的果蝇hsp 70信使RNA具有相同的5'末端。这些实验证明了一个在果蝇培养细胞中导入和表达分离基因的实用系统。