Generation of an improved luciferase reporter gene plasmid that employs a novel mechanism for high-copy replication

We have engineered the reporter gene plasmid pXPG by incorporating a novel high-copy origin of replication and a modified luciferase gene into a pXP1-derived vector that efficiently blocks read-through transcription in eukaryotic cells. pXPG contains the Luc+ luciferase gene derived from pGL3 that lacks a peroxisomal targeting sequence, thereby allowing accumulation of luciferase protein in the cytoplasm rather than subcellular organelles of transfected eukaryotic cells. pXPG has distinct advantages over pGL3, because it contains SV40 polyadenylation signals that appear to be more efficient at blocking read-through transcription than the synthetic polyadenylation signal present in pGL3. pXPG contains a novel mutation near the origin of replication that increases plasmid copy number in Escherichia coli. This mutation alters the - 10 sequence in the RNA II promoter of the ColE1 origin of replication from TAATCT to TAATAT. As this sequence is a closer match to the consensus -10 element, we suggest that the mutation increases copy number by increasing the rate of transcription of the RNA II replication primer. This novel mechanism for increasing copy number may have more widespread applications than the commonly used pUC high-copy origin of replication mutation. Unlike pUC, which reverts to low copy number at 30 degrees C, the pXPG mutation supports a higher copy number at both 37 and 30 degrees C. (C) 2000 Academic Press.