Discrimination of a single base change in a ribozyme using the gene for dihydrofolate reductase as a selective marker in Escherichia coli.
For use of ribozymes in vivo, it is desirable to select functional ribozymes in the cellular environment (in the presence of inhibitory factors and limited concentrations of mandatory Mg2+ ions, etc.). As a first step toward this goal, we developed a new screening system for detection in vivo of an active ribozyme from pools of active and inactive ribozymes using the gene for dihydrofolate reductase (DHFR) as a selective marker. In our DHFR expression vector, the sequence encoding either the active or the inactive ribozyme was connected to the DHFR gene. The plasmid was designed such that, when the ribozyme was active, the rate of production of DHFR was high enough to endow resistance to trimethoprim (TMP). We demonstrated that the active ribozyme did indeed cleave the primary transcript in vivo, whereas the inactive ribozyme had no cleavage activity. Cells that harbored the active-ribozyme-coding plasmid grew faster in the presence of a fixed concentration of TMP than the corresponding cells that harbored the inactive-ribozyme-coding plasmid. Consequently, when cells were transformed by a mixture that consisted of active- and inactive-ribozyme-coding plasmids at a ratio of 1:1, (i) mainly those cells that harbored active ribozymes survived in the presence of TMP and (ii) both active- and inactive-ribozyme-harboring cells grew at an identical rate in the absence of TMP, a demonstration of a positive selection system in vivo. If the background "noise" can be removed completely in the future, the selection system might usefully complement existing selection systems in vitro.
Fujita
S
S
National Institute of Bioscience and Human Technology, Ministry of International Trade and Industry, Tsukuba Science City, Japan.
Koguma
T
T
Ohkawa
J
J
Mori
K
K
Kohda
T
T
Kise
H
H
Nishikawa
S
S
Iwakura
M
M
Taira
K
K
D001483:Base Sequence; D004926:Escherichia coli / Q000235:genetics; D005800:Genes, Fungal; D008969:Molecular Sequence Data; D016337:RNA, Catalytic / Q000737:chemistry*; D012333:RNA, Messenger / Q000378:metabolism; D012641:Selection, Genetic; D013329:Structure-Activity Relationship; D013762:Tetrahydrofolate Dehydrogenase / Q000235:genetics*; D014296:Trimethoprim Resistance
D016337:RNA, Catalytic; D012333:RNA, Messenger; D013762:Tetrahydrofolate Dehydrogenase