Casey J L, Di Jeso B, Rao K, Rouault T A, Klausner R D, Harford J B
Cell Biology and Metabolism Branch, National Institute of Child Health and Human Development, Bethesda, Maryland 20892.
Ann N Y Acad Sci. 1988;526:54-64. doi: 10.1111/j.1749-6632.1988.tb55492.x.
Genomic DNA fragments corresponding to the promoter region of the human transferrin receptor were linked to either the full-length receptor cDNA or to the bacterial enzyme chloramphenicol acetyltransferase. These constructs were transfected into mouse and human cells, respectively. Gene expression was monitored 40-48 hours after transfection. Bal31 exonuclease was employed to produce 5' to 3' deletions of the promoter region. Deletion of DNA between -86 and -70 upstream of the receptor's mRNA start site resulted in a greater than 80% reduction in apparent promoter activity. DNA sequencing of the 150 bp upstream of the start site revealed that the promoter region contained several sequence elements more than 90% homologous to the consensus sequence for binding of the transcription factor Sp1. In addition, an 11 bp sequence identical to a segment of the enhancers of polyoma virus and adenovirus was located between -80 and -70. Internal deletions confirmed that this enhancer homologue was critical for full promoter activity. A 66 bp fragment encompassing the -80/-70 element augmented gene expression when the fragment was placed in either orientation upstream of the remainder of the transferrin receptor promoter.
将与人类转铁蛋白受体启动子区域相对应的基因组DNA片段与全长受体cDNA或细菌酶氯霉素乙酰转移酶相连。这些构建体分别转染到小鼠和人类细胞中。转染后40 - 48小时监测基因表达。使用Bal31核酸外切酶产生启动子区域5'至3'端的缺失。受体mRNA起始位点上游-86至-70之间的DNA缺失导致明显的启动子活性降低超过80%。起始位点上游150 bp的DNA测序显示,启动子区域包含几个与转录因子Sp1结合的共有序列有90%以上同源性的序列元件。此外,在-80至-70之间发现了一段与多瘤病毒和腺病毒增强子片段相同的11 bp序列。内部缺失证实,这种增强子同源物对完整的启动子活性至关重要。当一个包含-80 / -70元件的66 bp片段以任何方向置于转铁蛋白受体启动子其余部分的上游时,均可增强基因表达。