Purification of specific adenovirus 2 RNAs by preparative hybridization and selective thermal elution

Abstract

A method is described for the preparative isolation of highly purified adenovirus RNA species. Cytoplasnic RNAs frcn cells infected with adenovirus 2 were selected by hybridization to viral DNA fragments bound to nitrocellulose membranes. A series of washes at elevated temperatures (50-700) determined conditions at which the true hybrids were stable but non-specific RNA was removed. This tenWerature has been found to correlate with the base composition of the DNA fragment. After wash at this predetemined tesxerature, the specific RNA was eluted at 85 . The purity of the eluted RNA was greater than 95% as determined by size, sequence specificity, and template activity in an in vitro protein synthesizing system. The method described should be generally useful for purification of specific RNAs. JlN CONCharacterization of individual mRNA species has become increasingly important in the study of eukaryotic gene expression and has increased the understanding of the extensive processing mechanisms involved in mRNA synthesis. In light of recent findings that demonstrate the presence of multiple inserts within the coding sequences of a gene (1-4) and show that a specific gene sequence may express different products by synthesizing overlapping mRNAs(5-8), it is often necessary to identify functional genes by purifying the mRNA coding for the gene product. Several methods have been described for the preparative isolation of specific RNA molecules by hybridization to DNA immobilized on a matrix (9-12), or by hybridization to DNA in liquid and purification of the DNA-RNA hybrid (13). The nitrocellulose membrane technique has several advantages: DNA can be bound to membranes by rapid and efficient methods (14) C) Information Retrieval Limited 1 Falconberg Court London Wl V 5FG England

Purification and mapping of specific mRNAs by hybridization-selection and cell-free translation.

“…Two steps in this procedure are essential in the elimination of nonspecifically bound RNAs: (i) after the transfer of the DNA to the filter but prior to baking, the membrane is purged of any adhering agarose by gently massaging the surface of the filter; (ii) after selection, the filters are exhaustively washed with buffers carefully maintained at a predetermined temperature (in these experiments, the critical temperature was 600C). It should be noted that the thermal stability of a DNA-RNA hybrid is a function of the G+C content of the DNA and therefore care should be taken to ensure that certain hybridized mRNAs are not selectively eluted during washing (9). The Applications of hybridization-selection include: (i) selection of mRNAs by small DNA fragments, which permits refined genomic mapping of these RNAs; (ii) elucidation of specific mRNAs whose cell-free products are not obvious in the complex pattern of products directed by total RNA; (iii) discrimination of comigrating cell-free products originating from the translation of different mRNAs; (iv) preparative purification of specific mRNAs; (v) correlation of precise size and genomic organization of these transcripts with their coding and noncoding sequences by SI nuclease analysis and hybrid-arrested translation on selected mRNAs (unpublished data); and (v)…”

“…DNA, either in liquid or bound to immobilized supports, has been used to hybridize and thereby sequester specific mRNAs from the plethora of cellular RNA species. These methods have included isolation of RNA-DNA hybrids by: selective binding to hydroxylapatite (1); selective exclusion through agarose (2) or Sepharose 4B (3); the use of DNA covalently bound to cellulose (4,5) or Sepharose (6); DNA bound directly to nitrocellulose (7)(8)(9); DNA enzymatically synthesized and covalently bound to oligo(dT)-cellulose (10).…”

A viable simian virus 40 variant that carries a newly generated sequence reiteration in place of the normal duplicated enhancer element.

“…To prepare RNAs, CV-1P cells were infected at a multiplicity of 10 plaque-forming units per cell and harvested at various times after infection. Cytoplasmic, poly(A)+ RNA was prepared (19) and subjected to RNA blot analysis (20) using probe DNAs specific for either the early or late SV40 transcription unit. RESULTS…”

Localization of the adenovirus E1Aa protein, a positive-acting transcriptional factor, in infected cells infected cells.

“…Cytoplasmic polyadenylic acid-containing RNA was selected by hybridization to nitrocellulose filters bearing an ElA plasmid (0 to 4.5 map units), using previously described procedures (28,49). The selected ElA-specific mRNA was translated in rabbit reticulocyte extracts (Bethesda Research Laboratories).…”