Intron requirement for expression of the human purine nucleoside phosphorylase gene

Abstract

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.

Tandem repeat variation in human and great ape populations and its impact on gene expression divergence

“…In addition, repeats in first introns were associated with greater expression divergence than repeats in other introns. This observation is consistent with previous work showing that most intronic regulatory regions occur in the first intron (Rohrer and Conley 1998), that the first intron has the highest divergence between human and chimpanzees (Gazave et al 2007), and that the first intron influences gene expression more than other introns (Jonsson et al 1992;Charron et al 2007).…”

Aberrantly splicedHTT,a new player in Huntington’s disease pathogenesis

“…In many species intron 1 length seems to be positively regulated with the expression level of the respective gene. 21,[24][25][26][27][28][29] Furthermore, neuronal genes and genes that are involved in development seem to have a higher content of non-coding DNA, which might assist in the tight regulation of their expression. 17 In plants, the propensity of these elements to influence gene expression was termed intron-mediated enhancement.…”

Analysis of the stimulatory effect of splicing on mRNA production and utilization in mammalian cells

“…Previous efforts to examine the effect of mRNA splicing on the level of gene expression have demonstrated that a number of intron containing genes, including the genes encoding ␤-globin, thymidylate synthase, or purine nucleoside phosphorylase, are very poorly expressed when transcribed as cDNAs but effectively expressed if one or more introns are present in cis (Buchman and Berg 1988;Deng et al 1989;Ryu and Mertz 1989;Jonsson et al 1992). On the other hand, cDNAs derived from some other intron containing genes have been reported to express at high levels in cultured cells even in the absence of any introns (Pasleau et al 1987;Brinster et al 1988;Malim et al 1988).…”