Intron requirement for expression of the human purine nucleoside phosphorylase gene.
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.
Jonsson
J J
JJ
Institute of Human Genetics, University of Minnesota, Minneapolis 55455.
Foresman
M D
MD
Wilson
N
N
McIvor
R S
RS
D016475:3T3 Cells; D000818:Animals; D001483:Base Sequence; D015152:Blotting, Northern; D015139:Blotting, Southern; D015500:Chloramphenicol O-Acetyltransferase / Q000235:genetics / Q000378:metabolism; D003001:Cloning, Molecular; D004742:Enhancer Elements, Genetic / Q000235:genetics; D005786:Gene Expression Regulation / Q000235:genetics*; D006367:HeLa Cells; D006801:Humans; D007438:Introns / Q000235:genetics*; D051379:Mice; D008969:Molecular Sequence Data; D011683:Purine-Nucleoside Phosphorylase / Q000235:genetics* / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism
D011993:Recombinant Fusion Proteins; D015500:Chloramphenicol O-Acetyltransferase; D011683:Purine-Nucleoside Phosphorylase