Determination of cellular RNA concentrations by electron microscopy of R loop-containing DNA.
R loop hybridizations and electron microscopy have been used to determine cellular RNA concentrations for cloned genes. In plasmid DNA sequence excess, all the complementary RNA is driven into R loop structures that can be assayed by electron microscopy. To determine the concentration of a particular poly(A)+ RNA, plasmid DNA crosslinked once every 2000-5000 base pairs with trioxsalen and UV light is hybridized in DNA sequence excess to various known amounts of total poly(A)+ RNA, and the R loops are stabilized by treatment with glyoxal. If necessary, excess nonhybridized RNA is removed by Sepharose 2B chromatography, which enables the visualization of less abundant transcripts. Reconstruction experiments demonstrated that electron microscopic determination of the fraction of plasmid DNA molecules containing specific RNA loops gives accurate values of specific RNA weight fractions or concentrations in the total poly(A)+ RNA populations. These methods were also used to determine the concentrations of five RNA species complementary to sequences on TRT3, a recombinant DNA plasmid containing yeast histone 2A and 2B genes and three other nonhistone genes. The methods described allow one to visualize the R loop structures for both abundant and nonabundant transcripts and to estimate concentrations of these RNA species simply by determining the fraction of DNA containing R loops.
D000818:Animals; D001483:Base Sequence; D002645:Chickens; D003001:Cloning, Molecular; D004262:DNA Restriction Enzymes; D004271:DNA, Fungal / Q000235:genetics; D004274:DNA, Recombinant / Q000032:analysis*; D008854:Microscopy, Electron; D009690:Nucleic Acid Conformation; D009693:Nucleic Acid Hybridization; D010047:Ovalbumin / Q000235:genetics; D010957:Plasmids; D012331:RNA, Fungal / Q000235:genetics; D012333:RNA, Messenger / Q000032:analysis* / Q000235:genetics
D004271:DNA, Fungal; D004274:DNA, Recombinant; D012331:RNA, Fungal; D012333:RNA, Messenger; D010047:Ovalbumin; D004262:DNA Restriction Enzymes