COMPARISON OF IN-VIVO ACTIVITIES OF 5'-CONNECTED AND 3'-CONNECTED CIS-ACTING RIBOZYMES - SELECTION OF INTRACELLULARLY ACTIVE RIBOZYMES USING THE GENE FOR DIHYDROFOLATE-REDUCTASE (DHFR) AS A SELECTIVE MARKER INESCHERICHIA-COLI

If ribozymes are to be exploited in vivo, it is necessary to select ri bozymes that are functional in the intracellular environment, Ribozyme s selected in the intracellular environment should retain their functi on in vivo as well as in vitro, We have devised a novel system for sel ection of active ribozymes from pools of active and inactive ribozymes using the gene for dihydrofolate reductase (DHFR) as a selective mark er, In our first attempt, a sequence encoding either an active or an i nactive ribozyme was connected upstream of the gene for DHFR. Each pla smid was designed such that, when the ribozyme was active, the ribozym e would cleave the target site and, as a result, the rate of productio n of DHFR would be high enough to endow resistance to trimethoprim (TM P). However, a critical defect may be associated with introduction of a ribozyme upstream of the DHFR gene because, during actual screening for active ribozymes on the 5' side from a pool of random sequences, t here is the danger of selecting sequences that are not related to the activity of ribozymes. Indeed, some upstream linker sequences affected the level of expression of the DHFR protein and, as a result, the res istance of Escherichia coil to TMP. Therefore, we newly constructed a 3'-connected ribozyme system, and activities in vivo of 5'-connected a nd 3'-connected ribozymes were compared. We found that the cleavage ef ficiencies in vivo were nearly identical for the two types of ribozyme , 24% for the 5'-side ribozyme and 23% for the 3'-side ribozyme, indic ating that polysomes did not seem to inhibit the action of the 3'-conn ected ribozyme, In both cases, when cells were transformed with a 1 : 1 mixture of active and inactive ribozyme-coding plasmids, it was main ly the cells that harbored the active ribozyme that survived in the pr esence of TMP.