Effect of intron A from human cytomegalovirus (Towne) immediate-early gene on heterologous expression in mammalian cells

Chapman et al. (1991). Nucleic Acids Res. DOI: 10.1093/nar/19.14.3979 Citations: 294

Whether introns downstream of the hCMV promoter boosted expression was unclear. This paper compared secretion from CMV IE1 plasmids with and without intron A. Intron A raised HIV-1 gp120 secretion about 50-fold, and mutating its NF1 site reduced the gain.

Parts used

hCMV Intron A

Key findings

  • Chapman et al. cloned and sequenced a 2.4 kb fragment of the human cytomegalovirus Towne strain immediate-early gene containing intron A (823 bp), revealing a perfect consensus NF1 binding site at nucleotides 1488-1501 and an 18 bp regulatory element in exon A. The sequence analysis extended the known hCMV Towne IE1 gene by 533 bases and showed 97% homology with the AD169 strain in both coding and non-coding regions.

  • Expression vectors containing intron A demonstrated 2.3 to >300-fold increases in secreted glycoprotein levels compared to intron-deleted vectors in transiently transfected COS7 cells. Factor VIII heavy chain (gp92) expression increased from 43% to 100% of maximum, HIV-1 gpl20 expression rose from <0.3% to 100%, and full-length Factor VIII increased from 31% to 100% when intron A was present, demonstrating broad enhancement across diverse mammalian glycoproteins.

  • Site-directed mutagenesis of three base pairs in the intronic NF1 consensus sequence reduced Factor VIII gp92 expression to 69% and Factor VIII gp80 expression to 59% of wild-type intron A levels. Gel shift assays confirmed reduced NF1 protein binding to the mutant oligonucleotide compared to wild-type sequence, demonstrating that the NF1 binding site partially but not completely accounts for intron A-mediated expression enhancement in monkey kidney cells.

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Abstract

A 2.4 kb fragment of hCMV (Towne strain), containing the 5' end of the major immediate-early gene, has been cloned, sequenced, and used to construct a series of mammalian cell expression plasmids. The effects of regulatory regions present on this fragment were assessed using human glycoproteins as reporter molecules. We compared secreted levels of Factor VIII, t-PA, and HIV-1 envelope glycoproteins in cells transfected with plasmids in which intron A of the immediate-early gene was present or absent. Secretion of several glycoproteins was significantly higher when cells were transfected with intron A-containing plasmids. Mutation of three basepairs in the strong nuclear factor 1 (NF1) binding site in intron A led to reduced transient expression levels, but not to the level observed in the absence of intron A. Reduced expression from NF1 mutant plasmids was roughly correlated with reduced binding in vitro of NF1 proteins to a synthetic oligonucleotide containing the mutation. The evidence indicates that sequences in intron A positively regulate expression from the hCMV immediate-early enhancer/promoter in transformed monkey kidney cells.