An Expression Vector for Multiple Ribozymes

Ribozymes are recognized as useful tools for the manipulation of genes because of their high specificity and the fact that they act without influencing the expression of genes that are unrelated targets (1 ,2 ). To date, many successful experiments with intracellular ribozymes have been reported. However, there are only few detailed descriptions of how to construct a ribozyme expression system. Each ribozyme is a kind of enzyme, and its tertiary structure is important for its enzymatic activity. For the expression of ribozymes in vivo, most researchers use expression systems of the type developed for the synthesis of proteins. For example, promoters resembling those recognized by RNA polymerase II (pol II systems) are used as part of the transcription system. Recently, it has been shown that such expression systems are not always suitable for ribozymes (3 ). The pol II type of promoter has a requirement for extra sequences at the 5′ and 3′ ends of the sequences to be transcribed, which are essential for correct and effective transcription. However, it is possible that such extra sequences might change the tertiary structure of ribozymes via, for example, inappropriate base-pairs with nucleotides in the ribozyme sequence. As a result, the ribozyme would be converted from its active conformation to an inactive conformation. Moreover, since the transcribed RNA can function as a messenger RNA, ribosomes bind to it, and such binding might inhibit the association of the ribozyme with its target RNA in the cytoplasm.