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 eukary otic cells. pXPG contains the Luc+ luciferase gene derived from pGL3 that l acks a peroxisomal targeting sequence, thereby allowing accumulation of luc iferase protein in the cytoplasm rather than subcellular organelles of tran sfected eukaryotic cells. pXPG has distinct advantages over pGL3, because i t contains SV40 polyadenylation signals that appear to be more efficient at blocking read-through transcription than the synthetic polyadenylation sig nal present in pGL3. pXPG contains a novel mutation near the origin of repl ication that increases plasmid copy number in Escherichia coli. This mutati on alters the - 10 sequence in the RNA II promoter of the ColE1 origin of r eplication 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. T his novel mechanism for increasing copy number may have more widespread app lications than the commonly used pUC high-copy origin of replication mutati on. 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) 200 0 Academic Press.