Separation of mutant and wild-type ribosomes
Michael van Waes
bi__mvw at SELWAY.UMT.EDU
Tue Apr 19 16:56:20 EST 1994
Hi netters! There was a posting the other day asking about how to separate
wild type from mutant ribosomes. I coincidentally ran into this
paper, but I couldn't remember the original post's address. Thus, here it is
for everybody to enjoy and rejoice. Thank you!!
> Accession Number: 94089390.
>Author: Poot-R-A. Brink-M-F. Pleij-C-W. de-Boer-H-A. van-Duin-J.
>Author Affil.: Department of Biochemistry, Gorlaeus Laboratories, University
> of Leiden, The Netherlands.
>Title: Separation of mutant and wild-type ribosomes based on
> differences in their anti Shine-Dalgarno sequence.
>Source: Nucleic-Acids-Res. 1993 Nov 25. 21(23). P 5398-402.
>Pub. Type: journal-article.
>Abstract: We describe a system to isolate 30S ribosomal subunits which
> contain targeted mutations in their 16S rRNA. The mutations
> of interest should be present in so-called specialized 30S
> subunits which have an anti-Shine-Dalgarno sequence that is
> altered from 5' ACCUCC to 5' ACACAC. These plasmid-encoded
> specialized 30S subunits are separated from their
> chromosomally encoded wild-type counterparts by affinity
> chromatography that exploits the different Shine-Dalgarno
> complementarity. An oligonucleotide complementary to the 3'
> end of wild-type 16S rRNA and attached to a solid phase
> matrix retains the wild-type 30S subunits. The flow-through
> of the column contains close to 100% mutant 30S subunits.
> Toeprinting assays demonstrate that affinity column treatment
> does not cause significant loss of activity of the
> specialized particles in initiation complex formation,
> whereas elongation capacity as determined by poly(Phe)
> synthesis is only slightly decreased. The method described
> offers an advantage over total reconstitution from in vitro
> transcribed mutant 16S rRNA since our 30S subunits contain
> the naturally occurring base modifications in their 16S rRNA.
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