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 transtection with a PNP mlnigene 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 lntroniess PNP mlnigene (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 chioramphenicol 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 wfth 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.
|Original language||English (US)|
|Number of pages||8|
|Journal||Nucleic acids research|
|State||Published - Jun 25 1992|
Bibliographical noteFunding Information:
We thank Steven R.Williams and Dr. David W.Martin, Jr. (Genentech, Inc.) for providing cloned genomic PNP DNA samples, Dr. Cary Mariash, Dept. of Medicine, Univ. of Minnesota for assisting with the videodensitometry measurements, and Dr. Brian Van Ness (Dept. of Biochemistry and Institute of Human Genetics, Univ. of Minnesota) for reviewing the manuscript. Timothy Kahl, Shawn Larsen, and Kirstin M.Thompson provided technical assistance. This work was supported by grant AI27416 from the National Institutes of Health and Basil O'Connor Starter Scholar Research Award No. 5-692 from the March of Dimes Birth Defects Foundation to RSM. JJJ received a NATO science fellowship and a stipend from the Gerald T. Evans fund, Dept. of Laboratory Medicine and Pathology, Univ. of Minnesota.