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Procedure for the preparation of milligram quantities of adenovirus messenger ribonucleic acid
Abstract:
Late after adenovirus 2 infection (18 hr), nearly all newly synthesized polysomal messenger ribonucleic acid (mRNA) is viral specified. Large amounts of adenovirus mRNA have been purified by utilizing membrane filtration at high ionic strength. With this procedure, molecules that contain polyadenylic acid [poly (A)] tracts are bound selectively, and ribosomal RNA can be separated from the viral mRNA which contains poly(A). Polysomal RNA synthesized 18 hr after infection was labeled in the presence of 0.02 mug of actinomycin D per ml and extracted at pH 9.0. This RNA annealed 40% to 3 mug of adenovirus 2 deoxyribonucleic acid; the RNA selected by membrane filtration bound 80% under the same conditions. The RNA eluted from membrane filters was 80 to 90% greater than 18S and contained species migrating as 31, 27, and 24S. Binding of polysomal RNA to individual membrane filters was linear, using as much as 300 mug of RNA per membrane. A 1.1-mg amount of viral RNA was prepared from 17.7 mg of polysomal RNA that had been purified by extraction at pH 9.0.
Insights
Researchers purified adenovirus messenger RNA (mRNA) using polyadenylic acid [poly(A)] tract binding. This method efficiently separated viral mRNA from host ribosomal RNA, yielding substantial amounts of pure viral mRNA for further study.
Area of Science:
- Molecular Biology
- Virology
Background:
- Adenovirus infection leads to the synthesis of viral messenger RNA (mRNA).
- Separating viral mRNA from host cell components is crucial for studying viral gene expression.
Purpose of the Study:
- To develop a method for purifying large quantities of adenovirus mRNA.
- To characterize the purified viral mRNA.
Main Methods:
- Utilized high ionic strength membrane filtration to selectively bind polyadenylic acid [poly(A)] tracts present in viral mRNA.
- Separated viral mRNA from ribosomal RNA based on the presence of poly(A).
- Labeled RNA in the presence of actinomycin D and extracted at pH 9.0.
Main Results:
- Achieved selective binding of polyadenylic acid [poly(A)]-containing viral mRNA.
- Demonstrated efficient separation of viral mRNA from ribosomal RNA.
- Purified viral mRNA was predominantly larger than 18S, with species observed at 31, 27, and 24S.
- Obtained 1.1 mg of viral RNA from 17.7 mg of polysomal RNA.
Conclusions:
- High ionic strength membrane filtration is an effective method for purifying adenovirus mRNA.
- The purified viral mRNA consists of distinct size species and is suitable for further molecular analysis.