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Ribozymes - Ribozymes are RNA molecules that act as enzymes and exhibit intramolecular catalysis (e.g., self-cleavage or self-splicing).

Ribozymes are of two types that can cleave RNA in a sequence specific manner.

In the first type a self splicing reaction occurs to remove an intron from the nuclear preribosomal RNA.

These ribozymes have a four base recognition sequence and are useful for specific cleavages of RNA in vitro.

The second type of ribozymes depends on the self cleavage reactions that occur during the replication of certain viroids and satellite RNAs.

Ribozymes can be targeted to different regions of a virus genome.

Ribozymes carry the antisense RNA concepts a step further by adding a catalytic domain, which cleaves and inactivates the target mRNA. Ribozymes are based on the discovery that some RNAs (certain transfer RNAs, preribosomal RNAs, satellite RNAs and viroid RNAs) are autocatalytic or can function like enzymes.

It has been demonstrated for the satellite RNA of tobacco ringspot virus, that the catalytic domain responsible for cleavage comprises 24 nucleotides and forms a hairpin loop like structure.

For this catalytic domain the cleavage site on the target RNA comprises only the three nucleotides GUC.

A ribozyme has been designed which consists of the 24-nucleotide catalytic domain flanked on both sides by antisense RNA selected to .align the catalytic unit with a GUC cleavage site on the target RNA.

In vitro cleavage of target mRNA by this ribozyme, directed by antisense RNA against three different GUC sites, has demonstrated its catalytic activity and site specificity.

Ribozymes should make antisense technology even more powerful and more broadly applicable as a means of down regulating and perhaps completely eliminating the functional expression of target genes.

Antisense RNA was originally discovered as a naturally occurring mechanism for down regulation of gene expression in bacteria.

By base pairing with the complementary sense portion of the target mRNA, antisense RNA inhibits the normal flow of information from DNA, through mRNA, to protein.

There is evidence that the resultant double stranded RNA impedes mRNA transport from the nucleus, encourages degradation of mRNA, or inhibits ribosome binding and translation of the mRNA.

Regardless of the exact mechanism, the net result is a reduction in synthesis of the target protein.

An antisense gene designed to down regulate expression of a target rice gene could be made by splicing together the coding sequences chosen to assure transcription to RNA at high levels in those cells expressing the target gene.

This technique has been demonstrated in other plant systems to down regulate expression of key enzymes and to generate physiological consequences.

Introduction of an antisense gene to tomato caused a dramatic reduction in polygalacturonase, an enzyme important in fruit ripening.

The transgenic tomatoes ripened more slowly. While the efficiency of antisense technology seems to vary with target protein, it is broadly applicable and should have a significant impact on plant genetic engineering.
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