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PMID: 6180433 Published · ppublish English Journal Article Research Support, U.S. Gov't, P.H.S.

Purification of low-abundance messenger RNAs from rat liver by polysome immunoadsorption.

Proceedings of the National Academy of Sciences of the United States of America ·Vol. 79 ·No. 13 ·1982-07-00 ·Pages 4015-9

Kraus JP, Rosenberg LE

Abstract

We have purified three low-abundance hepatic mRNAs to near homogeneity by polysome immunoadsorption. The mRNAs coding for the precursor of ornithine transcarbamoylase [carbamoylphosphate:L-ornithine carbamoyltransferase, EC 2.1.3.3], the precursor of the beta-subunit of propionyl-CoA carboxylase [propionyl-CoA:carbon dioxide ligase (ADP-forming), EC 6.4.1.3], and cystathionine beta-synthase [L-serine hydro-lyase (adding homocysteine), EC 4.2.1.22], representing approximately 0.20, 0.02, and 0.015% of total hepatic mRNA, respectively, were purified 450- to 6,300-fold. We used the following steps: interaction of rat liver polysomes with an IgG fraction of monospecific antisera raised against each polypeptide; immobilization of polysome-antibody complexes on a protein A-Sepharose column; removal of the bulk of polysomes by extensive washing; dissociation of ribosomal subunits and elution of specific mRNA with EDTA; and isolation of the eluted mRNA by chromatography on an oligo(dT)-cellulose column. It seems likely that this procedure will permit isolation of other low-abundance mRNAs and subsequent cloning of their respective cDNAs.

MeSH Terms
Animals Antigen-Antibody Complex Carboxy-Lyases/genetics Cystathionine beta-Synthase/genetics Enzyme Precursors/genetics Hydro-Lyases/genetics Immune Sera Liver/enzymology Male Methylmalonyl-CoA Decarboxylase Ornithine Carbamoyltransferase/genetics Poly A/genetics Polyribosomes/metabolism Protein Biosynthesis RNA/genetics RNA, Messenger/genetics,isolation & purification Rats
Chemicals
Antigen-Antibody Complex Enzyme Precursors Immune Sera RNA, Messenger Poly A RNA Ornithine Carbamoyltransferase Carboxy-Lyases Hydro-Lyases Cystathionine beta-Synthase Methylmalonyl-CoA Decarboxylase
Authors & Affiliations
2 authors, click to expand affiliations / ORCID
Kraus J P
Rosenberg L E
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30 references, click to expand
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Article Info
Journal
Proceedings of the National Academy of Sciences of the United States of America
Abbr.
Proc Natl Acad Sci U S A
ISSN
0027-8424
DOI
10.1073/pnas.79.13.4015
Published
1982-07-00
Pages
4015-9
Language
English
Region
United States
NLM ID
7505876
PMCID
PMC346567
Subset
IM
Grants
NIADDK NIH HHS · AM 09527 · United States
External Links
PubMed source DOI: 10.1073/pnas.79.13.4015 PMC Full Text View journal details
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