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Structure of eukaryotic chromosomes and chromatin part 3 analysis of messenger rna populations



Structure of eukaryotic chromosomes and chromatin part 3 analysis of messenger rna populations



Philosophical Transactions of the Royal Society of London B Biological Sciences 283(997): 373-374



When studying transcription, whether in living cells or in cell-free systems, it often becomes important to know something of the composition of the RNA produced, in terms of the nucleic acid sequences that it contains. Nucleic acid reassociation methods very often offer the best approach. Those that employ copy DNA (cDNA), synthesized enzymically on an RNA template, are among the most accurate and sensitive. According to the procedure most commonly used, the cDNA, which is radioactively labeled to a high specific activity, is annealed with a sufficient excess of RNA to make the reassociation reaction effectively 1st-order with respect to the RNA concentration. The incorporation of the labeled cDNA into hybrid DNA-RNA duplexes can readily be measured, for example by the use of a single-strand specific endonuclease, or by chromatography on hydroxyapatite. Inferences can be drawn from both the rate and the extent of duplex formation. Experiments were based on this approach. A series of experiments was designed to analyze gene expression during the development of Drosophila melanogaster. Another series was carried out to determine whether the appearance of heat-shock sequences in Drosophila is due to a quantitative or a qualitative change in transcription.

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