title: Synthesis of a lacI gene analogue with reduced CpG content.
abstract: A lacI gene analogue with reduced CpG content has been synthesized. Codon usage in the lacI gene was manipulated to remove most CpG sites (82/95; 86%) while maintaining wild-type amino acid sequence. The double-stranded gene sequence was synthesized using standard beta-cyanoethyl phosphoramidite chemistry and subsequently cloned into pBR322. Bacterial promoter sequences with different levels of activity were attached upstream of the modified coding region to study its expression in E. coli. Production of lacI protein was confirmed in a lacI- E. coli strain by Western blot analysis and by measuring repression of the lacZ gene with the chromogenic lacZ indicator, 5-bromo-4-chloro-3-indolyl-beta-D-galactopyranoside (X-gal). The modified lacI gene construct can be used as a genetic target in cultured mammalian cells or in transgenic animals to avoid high levels of background mutation associated with methylated CpG sequences. The construction scheme described here provides a general approach to remove CpG sequences from gene constructs when methylation is undesirable.
authors.affiliation: Merck Research Laboratories, WP45-301, West Point, PA 19486, USA.
authors.forename: T R
authors.initials: TR
authors.lastname: Skopek
authors.forename: D R
authors.initials: DR
authors.lastname: Marino
authors.forename: K L
authors.initials: KL
authors.lastname: Kort
authors.forename: J
authors.initials: J
authors.lastname: Miller
authors.forename: T
authors.initials: T
authors.lastname: Pippert
mesh_terms: D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000096:biosynthesis / Q000235:genetics*; D001482:Base Composition; D001483:Base Sequence; D003001:Cloning, Molecular; D003062:Codon; D004269:DNA, Bacterial; D015226:Dinucleoside Phosphates; D004926:Escherichia coli / Q000235:genetics* / Q000378:metabolism; D029968:Escherichia coli Proteins; D015870:Gene Expression; D005798:Genes, Bacterial; D005813:Genes, Synthetic; D056968:Lac Repressors; D008969:Molecular Sequence Data; D010957:Plasmids; D012097:Repressor Proteins / Q000096:biosynthesis / Q000235:genetics*; D012689:Sequence Homology, Nucleic Acid
chemical_list: D001426:Bacterial Proteins; D003062:Codon; D004269:DNA, Bacterial; D015226:Dinucleoside Phosphates; D029968:Escherichia coli Proteins; D056968:Lac Repressors; D012097:Repressor Proteins; C539064:LacI protein, E coli; C015772:CpG dinucleotide