INTRON REQUIREMENT FOR EXPRESSION OF THE HUMAN PURINE NUCLEOSIDE PHOSPHORYLASE GENE
INTRON REQUIREMENT FOR EXPRESSION OF THE HUMAN PURINE NUCLEOSIDE PHOSPHORYLASE GENE
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DOI：
10.1093/nar/20.12.3191
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发表时间：
1992-06-25
影响因子：
14.9
通讯作者：
MCIVOR, RS
中科院分区：
文献类型：
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作者：
JONSSON, JJ;FORESMAN, MD;MCIVOR, RS
简化嘌呤核苷磷酸化酶（PNP）基因的工程，以确定内含子对人类PNP基因表达的影响。含有第一个内含子（完整或从2.9kb缩短至855 bp）、前两个内含子或所有五个PNP内含子的PNP小基因在瞬时转染鼠NIH 3 T3细胞后导致大量人PNP同工酶表达。在用含有最后三个内含子的PNP小基因转染后观察到低水平的人PNP活性。一个无内含子的PNP小基因构建体含有PNP cDNA融合基因组侧翼序列导致检测不到的人PNP活性。含有内含子的PNP小基因的异质、稳定的NIH 3 T3转染子（通过Southern分析验证）表达高水平的PNP活性，并含有通过北方分析可视化的适当加工的1.7 kb信息。无内含子PNP小基因（每个单倍体基因组40-45个拷贝）的稳定转染子不含可检测的人PNP同工酶或mRNA。在嵌合PNP启动子-细菌氯霉素乙酰转移酶（CAT）基因的上游或下游方向插入855 bp缩短的内含子1序列，与亲本PNP-CAT构建体相比，CAT表达增加了数倍。我们的结论是，人类PNP基因的表达在mRNA和蛋白质水平上是依赖于内含子序列的存在和PNP的表达水平直接随内含子的数目而变化。内含子1的不成比例的最大影响可以解释为在缩短的855 bp内含子1序列中保留的增强子样元件的存在。
Abbreviated purine nucleoside phosphorylase (PNP) genes were engineered to determine the effect of introns on human PNP gene expression. PNP minigenes containing the first intron (complete or shortened from 2.9 kb down to 855 bp), the first two introns or all five PNP introns resulted in substantial human PNP isozyme expression after transient transfection of murine NIH 3T3 cells. Low level human PNP activity was observed after transfection with a PNP minigene containing the last three introns. An intronless PNP minigene construct containing the PNP cDNA fused to genomic flanking sequences resulted in undetectable human PNP activity. Heterogeneous, stable NIH 3T3 transfectants of intron-containing PNP minigenes (verified by Southern analysis), expressed high levels of PNP activity and contained appropriately processed 1.7 kb message visualized by northern analysis. Stable transfectants of the intronless PNP minigene (40-45 copies per haploid genome) contained no detectable human PNP isozyme or mRNA. Insertion of the 855 bp shortened intron 1 sequence in either orientation upstream or downstream of a chimeric PNP promoter-bacterial chloramphenicol acetyltransferase (CAT) gene resulted in a several-fold increase in CAT expression in comparison with the parental PNP-CAT construct. We conclude that human PNP gene expression at the mRNA and protein level is dependent on the presence of intronic sequences and that the level of PNP expression varies directly with the number of introns included. The disproportionately greatest effect of intron 1 can be explained by the presence of an enhancer-like element retained in the shortened 855 bp intron 1 sequence.