Ribozyme that cleaves at 3' end or.....?

Brett Lindenbach brett at BORCIM.WUSTL.EDU
Fri Feb 26 17:17:01 EST 1999


>I want to express in yeast from a nuclear plasmid the following:
>
>sequence that self-cleaves at 3' end----RNA sequence of
>interest---sequence that self-cleaves at 5' end.
>
>In other words I am trying to make an RNA with defined 5' and 3' ends
>(it is a viral RNA so the ends are important for replication
>competence). The 3' end I can handle with the HDV antigenomic ribozyme,
>but what can be used to give the "perfect" 5' end?  [BTW I have tried
>the T7  promoter and T7 RNA polymerease expressed from a nuclear plasmid
>and that has not worked properly.]
>
>TIA for your help.
>
>Peter Russell
>Reed College
>yeast at reed.edu
>


Well, it sounds like you are going to be using it for replication.
So, try an inverted ribozyme at the 5'end, which will cleave the
viral minus strand upon synthesis (assuming the 5'NCR is not a
determinant for minus strand synthesis).  This strategy was used
by Andy Ball, PNAS V91, p12443 for launching FHV. Good luck.  BTW,
which virus are you trying?

Brett Lindenbach
brett at borcim.wustl.edu
Dept of Molecular Microbiology
Washington University School of Medicine
(314)362-2767





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