A novel approach to design of cis-acting DNA structural element for regulation of gene expression in vivo

Abstract

Taking advantage of the degeneracy of the genetic code we have developed a novel approach to introduce, within a gene, DNA sequences capable of adopting unusual structures and to investigate the role of such sequences in regulation of gene expression in vivo. We used a computer program that generates alternative codon sequences for the same amino-acid sequence to convert a stretch of nucleotides into an inverted-repeat sequence with the potential to adopt cruciform structure. This approach was used to replace a 51-base-pair EcoRI-HindIII segment in the N-terminal region of the β-galactosidase gene in plasmid pUC19 with a 51-bp synthetic oligonucleotide sequence with the potential to adopt a cruciform structure with 18 bp in the stem region. In selecting the 51-bp sequence, care was taken to include those codons that are preferred in E. coli. E. coli DH5-α cells harbouring the plasmid containing the redesigned sequence showed drastic reduction in expression of the β-galactosidase gene compared to cells harbouring the plasmid with the native sequence. This approach demonstrates the possibility of introducing DNA secondary-structure elements to alter regulation of gene expression in vivo.