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Nucleosomes by Paul M. Wassarman and Roger D. Kornberg (Eds.)

By Paul M. Wassarman and Roger D. Kornberg (Eds.)

The severely acclaimed laboratory ordinary, Methods in Enzymology, is likely one of the so much hugely revered courses within the box of biochemistry. considering the fact that 1955, each one quantity has been eagerly awaited, usually consulted, and praised by way of researchers and reviewers alike. The sequence comprises a lot fabric nonetheless suitable this present day - really an important ebook for researchers in all fields of existence sciences

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The SV40 genome offers a compact, complete system containing an origin of replication and two sets of genes with their extensively studied METHODS IN ENZYMOLOGY, VOL. 170 Copyright© 1989by AcademicPress,Inc. All flightsof ttl~roduetionin any form reserved. 14 CHROMATIN AND NUCLEOSOMES: PREPARATIVE TECHNIQUES [9_] NaHSO3, and 1 m M PMSF, for 19 hr at 35,000 rpm in a Beckman SW41 rotor at 4*. 5% agarose gel. Peaks of nucleosome monomers, dimers, and trimers are generally in a single fraction (typically fractions 10, 6, and 3, respectively) and are dialyzed and concentrated as described above for H l-depleted chromatin.

If this lower band is collected and recentrifuged on another Percoll gradient, an upper band again forms, suggesting that nuclei previously trapped in the debris of the lower band have been released. Nuclei in the upper band seem undamaged, judging by the following criteria: they are not sticky; they support run-on transcription reactions; and they contain undegraded DNA and histones and intact chromatin, as assessed by digestion with micrococcal nuclease. 1 × I, and centrifuged for 5 min at 7500 rpm in the JA-10 or GSA rotor.

The spheroplasts are cultured for 1 hr with slow shaking at 30 ° in growth medium containing l M sorbitol prior to lysis or nuclear preparation. Using this approach and estimates of mRNA decay rates, early results suggest that at least 50% (and perhaps all) of HSP26 genes present as amplified episomal elements are transcribed at a rate comparable to that of the single-copy genomic gene after heat shock. We emphasize the importance of understanding the chromatin structure and functional DNA anatomy of the plasmid prior to making constructions to be used for study of protein-DNA interactions.

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