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通过转染将内源性脑内A粒子基因的长末端重复序列导入真核细胞时,它可作为启动子发挥作用。

The long terminal repeat of an endogenous intracisternal A-particle gene functions as a promoter when introduced into eucaryotic cells by transfection.

作者信息

Lueders K K, Fewell J W, Kuff E L, Koch T

出版信息

Mol Cell Biol. 1984 Oct;4(10):2128-35. doi: 10.1128/mcb.4.10.2128-2135.1984.

Abstract

We describe experiments designed to determine whether an endogenous intracisternal A-particle (IAP) gene randomly selected from a mouse embryo library has the potential to be transcriptionally active. Assays for IAP gene transcription were done with permanently transformed rat cells and transiently transfected monkey and mouse cells. The rat cells, which had integrated IAP gene copies, contained IAP RNA. A start site within the IAP 5' long terminal repeat (LTR) was localized by S1 mapping. The promoter activity of the IAP LTR was also measured in cells 48 h after the introduction of recombinant plasmids in which bacterial chloramphenicol acetyl transferase (CAT) encoding sequences were under the control of the LTR. The IAP LTR promoted CAT activity in mouse and monkey cells. In mouse L-cells, the levels of CAT activity were 10 to 25% of those promoted by an analogous recombinant containing the Moloney murine sarcoma virus LTR as the promoter. In contrast to the Moloney murine sarcoma virus LTR, the IAP LTR was five- to eightfold more active in monkey cells than in mouse cells. The 5' and 3' LTRs were equally active, and promoter activity was dependent on having the orientation of the LTRs with respect to the CAT gene the same as their orientation with respect to the IAP gene. A 5'-flanking sequence containing a member of the highly repetitive R-sequence family increased CAT activity in COS cells 11-fold when present along with the LTR. Our results indicate that the LTR of an endogenous mouse IAP gene can function as an efficient promoter in heterologous as well as homologous cells.

摘要

我们描述了旨在确定从小鼠胚胎文库中随机选择的内源性脑内A颗粒(IAP)基因是否具有转录活性的实验。使用永久转化的大鼠细胞以及瞬时转染的猴和小鼠细胞进行IAP基因转录分析。整合了IAP基因拷贝的大鼠细胞含有IAP RNA。通过S1作图确定了IAP 5'长末端重复序列(LTR)内的一个起始位点。在引入重组质粒48小时后的细胞中,还测定了IAP LTR的启动子活性,其中细菌氯霉素乙酰转移酶(CAT)编码序列受LTR控制。IAP LTR在小鼠和猴细胞中促进了CAT活性。在小鼠L细胞中,CAT活性水平是含有莫洛尼鼠肉瘤病毒LTR作为启动子的类似重组体所促进活性水平的10%至25%。与莫洛尼鼠肉瘤病毒LTR相反,IAP LTR在猴细胞中的活性比在小鼠细胞中高五至八倍。5'和3' LTR具有同等活性,并且启动子活性取决于LTR相对于CAT基因的方向与它们相对于IAP基因的方向相同。当与LTR一起存在时,包含高度重复R序列家族成员的5'侧翼序列使COS细胞中的CAT活性增加了11倍。我们的结果表明,内源性小鼠IAP基因的LTR在异源细胞和同源细胞中均可作为高效启动子发挥作用。

https://cdn.ncbi.nlm.nih.gov/pmc/blobs/ec97/369031/2cea66dec14f/molcellb00152-0198-a.jpg

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