Open AccessThe DNA‐RNA Hybridisation of [ 3 H]Methylated Mouse‐Globin mRNA in Conditions of RNA Excess
Author(s) -
Morrison Marcelle R.,
Paul John,
Williamson Robert
Publication year - 1972
Publication title -
european journal of biochemistry
Language(s) - English
Resource type - Journals
eISSN - 1432-1033
pISSN - 0014-2956
DOI - 10.1111/j.1432-1033.1972.tb01803.x
Subject(s) - rna , microbiology and biotechnology , dna , biology , nuclease protection assay , ribosomal rna , messenger rna , transcription (linguistics) , biochemistry , rna dependent rna polymerase , gene , linguistics , philosophy
Milligram quantities of mouse reticulocyte 9‐S RNA, which has been demonstrated to contain the mRNA for globin, have been isolated using the zonal ultracentrifuge. In vitro methylation of the 9‐S RNA with dimethylsulphate has allowed investigation of its hybridisation to mouse DNA. The RNA saturates between 0.084% and 0.15% of the DNA. The hybridisation is rapid, being complete in under 3 min. Repeated hybridisations of the RNA eliminate the possibility of the high saturation levels being due to the presence of contaminating traces of other RNA species. Similar saturation levels are found for mouse sperm DNA and total mouse embryo DNA. Slightly higher values are found for 14‐day mouse embryo liver DNA. There was no hybridisation with Escherichia coli DNA. The 9‐S RNA formed hybrids which had a T m 28 °C lower than that predicted from the 50.7% (G + C) content of the RNA. The size of the hybridised RNA was small after elution from RNAase treated filters, the largest fragments being approximately 60–120 nucleotides in length. Control experiments with [ 3 H]methylated 32 P‐labelled 5‐S ribosomal RNA showed that, although the saturation values obtained for hybridisation of methylated and non‐methylated RNA are identical, the methylated RNA‐DNA hybrid melts at a lower temperature. This difference in itself cannot, however, explain the low T m of the mRNA‐DNA hybrids.