Separation of mutant and wild-type ribosomes

[Michael van Waes]

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!!

				Michael vW.
Forwarded message:
>
>              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.
>Language:       eng.
>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.