Purification of Low-Abundance Messenger RNAs by Polysome Isolation with Polyclonal Antibodies
Current interest in the isolation and characterization of normal and mutant genes has focused attention on the need for recombinant DNA molecules complementary to low-abundance mRNAs. The low-abundance class of mRNAs (i.e., each constituting <0.5% of total cellular mRNA) comprises approximately 30% of the mRNA of the eukaryotic cell and codes for a majority of its enzymes. Starting with a total unfractionated mRNA, a cDNA library of at least 200,000 independent clones is required to obtain a complete representation of low-abundance mRNAs.1 A major problem in recovering a cDNA corresponding to a low-abundance mRNA from such a library is the laborious screening required to dotoct the rare cloned low-abundance sequences. A substantial initial enrichment of a given low-abundance mRNA would simplify dramatically the procedures involved in preparing or identifying recombinant DNA molecules containing complementary nucleotide sequences.
KeywordsSodium Dodecyl Sulfate Magnesium Acetate Vanadyl Sulfate Nascent Polypeptide Chain Bethesda Research Laboratory
Unable to display preview. Download preview PDF.
- 1.Williams, J. G., 1981, The preparation and screening of a cDNA clone bank, in: Genetic Engineering, Volume 1 (R. Williamson, ed.), Academic Press, New York, p. 2.Google Scholar
- 6.Lee, D. C., McKnight, G. S., and Palmiter, R. D., 1978, The action of estrogen and progesterone on the expression of the transferrin gene. A comparison of the response in chick liver and oviduct, J. Biol. Chem. 253:3497–3503.Google Scholar
- 27.Sul, H. S., Wise, L. S., Brown, M. L., and Rubin, C. S., 1984, Cloning of cDNA sequences for murine ATP-citrate lyase. Construction of recombinant plasmids using an immunopurified mRNA template and evidence for the nutritional regulation of ATP-citrate lyase mRNA content in mouse liver, J. Biol. Chem. 259:1201–1205.PubMedGoogle Scholar