Isolation of specific messenger RNA by adsorption of polysomes to matrix-bound antibody

Schütz, Günther; Kieval, Shalom; Groner, Bernd; Sippel, Albrecht E.; Kurtz, David T.; Feigelson, Philip

doi:10.1093/nar/4.1.71

Cited by 57 publications

(39 citation statements)

References 26 publications

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“…The contaminant sequences are overemphasized in this autoradiogram since the probe was labeled to such a high specific activity and since exposure of the main band is well beyond the linear range of the film. The trypsin inhibitor probe hybridized intensely to a 770-nucleotide region (lane 4 (10,27). While the principle is the same, the elution ofA20 above the EDTA background was easily detected from the GARSepharose columns and the problem of fines generates by alkalitreated cellulose was avoided.…”

Section: Resultsmentioning

confidence: 99%

“…More recently, an immunoadsorption procedure utilizing anti-antibody coupled to p-aminobenzyl-cellulose has been used to adsorb antibody-tagged specific polysomes. After batch-washing of the matrix, the bound polysomes were dissociated with buffer containing 20 mm EDTA (27). In adapting this procedure, I have found that Sepharose is an appropriate support for the anti-antibody (in this case goat anti-rabbit) and that application of polysomes, washings, and elution can be conveniently performed in a manner analogous to standard column techniques for antigen-antibody purification by affinity chromatography.…”

Section: Resultsmentioning

confidence: 99%

“…In summary, I have developed modifications to a polysome immunoadsorption procedure which had previously been applied only in certain animal systems (10,27). The specificity of the procedure was demonstrated by analysis of the selected mRNAs in an in vitro translation system, by hybridization of SBL or SBTI cDNA probes to mRNA gel blots, and by immunoselection of lectin polysomes from lectin-positive versus lectin-negative varieties.…”

Section: Resultsmentioning

confidence: 99%

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Isolation and Characterization of Messenger RNAs for Seed Lectin and Kunitz Trypsin Inhibitor in Soybeans

Vodkin¹

1981

Plant Physiol.

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The mRNAs for seed lectin and Kuntz tpin inbitor of In addition to substantial biochemical data for these two seed proteins, lectin and trypsin inhibitor have been defined by classical genetic analyses and soybean varieties which lack detectable SBL or SBTI protein have been found (21,22,24). It is the aim of this report and other studies in progress to determine what molecular defects cause the inherited absence of SBL or SBTI protein in certain soybean varieties. Such knowledge will help elucidate the normal functioning and expression of plant genes. As an initial step toward this objective, I have developed modifications to a polysome immunoadsorption procedure which have allowed the isolation of highly enriched mRNAs for lectin and Kunitz trypsin inhibitor. A preliminary report has been published (33). The selected mRNAs were analyzed by their activities in a rabbit reticulocyte cell free translation system and by hybridization of labeled SBL or SBTI cDNA to gel blots of total poly(A) RNA.

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Section: Resultsmentioning

confidence: 99%

Section: Resultsmentioning

confidence: 99%

Section: Resultsmentioning

confidence: 99%

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Isolation and Characterization of Messenger RNAs for Seed Lectin and Kunitz Trypsin Inhibitor in Soybeans

Vodkin¹

1981

Plant Physiol.

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“…Details of the procedures for the polyacrylamide gel electrophoresis in 98 % formamide and for the poly(A) determination by hybridization with [3H]poly(U) have been published [22,25]. Methods for the isolation of total poly(A)-containing mRNA from chicken oviduct and the procedure for the isolation of pure ovalbumin mRNA by adsorption of polysomes to matrix-bound antibodies have been described earlier [24,26].…”

Section: General Methodsmentioning

confidence: 99%

Size Distribution of Rat Liver Nuclear RNA Containing mRNA Sequences

Sippel

Groner

Hynes

et al. 1977

European Journal of Biochemistry

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Total rat liver poly(A)-containing polysomal mRNA was size-fractionated on polyacrylamide gels in 98 % formamide. Complementary DNA (cDNA) was prepared from the 8 -14-S mRNA fraction and separated into sequences representing abundant and non-abundant mRNAs. The cDNA complementary to the abundant small mRNA of the rat liver cell (approximately 20 species) was hybridized to nuclear RNA of different lengths to determine the size distribution of nuclear RNA molecules which contain these messenger sequences. It was found that :1. All abundant 8 -14 -S poly(A)-containing mRNAs have larger nuclear precursor molecules ; 20% of the different messenger sequences are found in nuclear RNA of several times their cytoplasmic length.2. 70 % of the mass of the examined nuclear messenger sequences is in RNA molecules of a size similar to their polysomal mRNA; 30% are in larger than 18-S RNA and 2% are between 37 S and 44 S.3 . The majority of small messenger-containing RNA molecules in the RNA prepared from isolated nuclei are of true nuclear origin, since their frequency distribution differs significantly from that of the polysomal 8 -14-S mRNA.To understand the regulation of gene expression in eucaryotic cells it is crucial to know more about nuclear processes involved in the biogenesis of mRNA. Several features of the synthesis and structure of hnRNA indicate its role in mRNA metabolism (for review see [l-51). Indirect evidence first led to the postulate that hnRNA contained precursor molecules to mRNA [6,7]. Hybridization-competition experiments later demonstrated base-sequence homology between polyribosomal mRNA and hnRNA from HeLa cells [8]. Furthermore, it was found that viral-specific RNAs in and polyoma-infected [lo] cells are transcribed as giant RNA molecules. However, owing to the enormous sequence complexity, the large size and the high turnover of hnRNA, a detailed describtion of the nuclear pathway of messenger sequences has not yet Abbreviations. Hepes. 4-(2-hydroxyethyl)-l-piperazineethanesulfonic acid; AMV, avian myeloblastosis virus; cDNA, DNA copy complementary to mRNA ; hnRNA, polydisperse high-molecularweight nuclear RNA; rot value, concentration of RNA (as mol phosphate/l) times time of hybridization (in s); rotllz value, rot value at which half the hybridizable cDNA is hybridized; vgt value, RNA derived from the amount of liver tissue (g), hybridized in IO-pI assay volume, times the hybridization time (h).Enzyme. S 1 nuclease, single-strand-specific endonuclease from Aspergillus oryzae (EC 3.1.4.21).been possible. The use of molecular hybridization with complementary DNA copies of specific mRNA molecules to identify messenger sequences in steadystate nuclear RNA has resulted in an increased amount of new information, but no general picture has emerged from these studies. For example, larger nuclear precursor RNA molecules for globin mRNA have been found, although with conflicting results as to their largest size [ l l -171. For the case of ovalbumin mRNA, no larger nuclear RNA containing the messenger se...

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“…Several laboratories have developed polysome immunoadsorption procedures (4,8,9,17,19,24) for isolation oflow abundance mRNAs. These procedures exploit the ability of antibodies, prepared against the native protein of the mRNA to be purified, to bind to the nascent polypeptide chains on the polysomes.…”

mentioning

confidence: 99%

Specific Polysome Immunoadsorption to Purify an Ammonium-Inducible Glutamate Dehydrogenase mRNA from Chlorella sorokiniana and Synthesis of Full Length Double-Stranded cDNA from the Purified mRNA

Bascomb¹,

Turner²,

Schmidt³

1986

Plant Physiol.

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A specific polysome immunoadsorption procedure, employing soluble rabbit anti-NADP-GDH IgG and sheep anti-rabbit IgG covalently-linked to an insoluble cellulose matrix, was used to immunoselect polysomes translating mRNA for a chloroplastic ammonium-inducible NADP-GDH in fully induced cells of Chlorella sorokiniana. The immunoselected polysomes were dissociated, and the NADP-GDH mRNA was recovered by oligo (dT)cellulose chromatography. The translatable NADP-GDH mRNA was estimated to be 0.07 and 90% of the total polysomal poly(A)+RNA before and after immunoselection of the polysomes, respectively. The immunoadsorption procedure resulted in an 83% recovery and 1,291-fold purification of translatable NADP-GDH mRNA. In vitro translation of the immunoselected poly(A)'RNA yielded a single radioactive protein (on sodium dodecyl sufate polyacrylamide gels) with a molecular weight of 58,500, i.e. size of the putative precursor-protein of the NADP-GDH subunit in the holoenzyme in fully induced cells. The purified NADP-GDH mRNA was used for synthesis of a high proportion of nearly full-length single-stranded cDNA and double-stranded cDNA molecules.

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Isolation of specific messenger RNA by adsorption of polysomes to matrix-bound antibody

Cited by 57 publications

References 26 publications

Isolation and Characterization of Messenger RNAs for Seed Lectin and Kunitz Trypsin Inhibitor in Soybeans

Isolation and Characterization of Messenger RNAs for Seed Lectin and Kunitz Trypsin Inhibitor in Soybeans

Size Distribution of Rat Liver Nuclear RNA Containing mRNA Sequences

Specific Polysome Immunoadsorption to Purify an Ammonium-Inducible Glutamate Dehydrogenase mRNA from Chlorella sorokiniana and Synthesis of Full Length Double-Stranded cDNA from the Purified mRNA

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