Transcription of Superhelical DNA from Cell Nuclei

Abstract

Structures resembling nuclei may be released by gently lysing human or frog cells in solutions containing non-ionic detergents i n d 1.95 M NaC1. These structures, or nucleoids, sediment in sucrose gradients containing intercalating agents in the manner characteristic of DNA that is intact, supercoiled and circular. They are depleted of nuclear protein and contain no endogenous RNA polymerase activity. We describe conditions for the transcription in vitro of these nucleoids by the RNA polymerase of Escherichia coli. We compared the kinetics of RNA synthesis directed by nucleoids and by DNA prepared using conventional procedures : nucleoids direct RNA synthesis at three to four times the rate of an equivalent weight of pure DNA and under appropriate conditions the DNA of each nucleoid can direct the synthesis of twice its own weight of RNA. Most of the newlysynthesized RNA remains within the nucleoid. Experiments with the inhibitor, rifampicin, reveal that all RNA synthesis depends upon the initiation of new RNA chains and that nucleoids and pure DNA possess similar numbers of initiation sites. RNA synthesis directed by nucleoids has unusual kinetics : maximal rates are attained only after a lag of about ten minutes. Almost no lag is seen with pure DNA. The lag is due to the slow rate of formation of complexes of polymerase with nucleoid DNA that can initiate. Removal of supercoils by irradiation with y-rays or by the addition of the unwinding ligand, ethidium bromide, decreases the numbers of polymerase molecules able to initiate synthesis rapidly and increases the lag before maximal synthetic rates are achieved. Loss of supercoiling does not alter the maximum synthetic rate. Supercoiling in eukaryotic DNA clearly influences the initiation of RNA synthesis. These results are discussed with reference to the effects of supercoiling on the transcription of prokaryotic templates.Structures resembling nuclei may be released by lysing cells gently in solutions containing non-ionic detergents and high concentrations of salt. These structures, which we call nucleoids, sediment in sucrose gradients containing intercalating agents in a manner characteristic of DNA that is intact, supercoiled, and circular. These characteristic changes in sedimentation rate are abolished by irradiating the nucleoids with y-rays, a procedure known to break DNA. We interpret these results as showing that nucleoids contain DNA that is subject to the same kind of topological constraint restricting rotation of one strand of the double helix of DNA about the other as that found in intact and circular DNA [1,2]. When the DNA of Escherichia coli is isolated using the procedures of Stonington and Pettijohn [3] similar constraints are found [4].Supercoils are lost spontaneously from circles of DNA when only one phosphodiester bond is broken so that supercoiling is a sensitive index of DNA Enzymes. RNA polymerase (EC 2.7.7.6); ribonuclease A (EC 3.1.4.22).integrity. Since very low doses of y-rays sufficient to introduce few breaks into DNA remove the...

A general method for preparing chromatin containing intact DNA.

“…At various times samples were mixed with 3 volumes of 2.5% SDS and then spotted on to glass fibre discs (Whatman GF/C). After washing in 5% trichloroacetic acid, the discs were counted (Colman and Cook, 1977). During transcription the final concentrations were 50 mM Tris (pH 8.0), 25 mM KCI, 100 mM (NH4)2SO4, 2 mM MgCl2, 0.5 mM dithiothreitol, 1 mM EDTA, 5 mM spermidine, 5% glycerol, 50 MM S-adenosyl methionine, 500 AM ATP, 500 MM CTP, 500 AM GTP, 50 M UTP (supplemented with 10 ACi/mi [32P]UTP at -400 Ci/mmol).…”

Spectrofluorometric Measurement of the Binding of Ethidium to Superhelical DNA from Cell Nuclei

“…However, we cannot obtain concentrations of nucleoids great enough to provide an excess over the whole range of ethidium concentrations that we use. It would also be inappropriate to determine the amounts of the dye bound to nucleoids by reference to binding isotherms constructed using an excess of pure DNA since the nucleoids also contain RNA [23]. The fluorescence of ethidium bound to nucleoids is therefore expressed as the fluorescence of an equivalent concentration of free ethidium which is determined as follows.…”

A general method for preparing intact nuclear DNA.

“…Clearly, the pores in the gel permit rapid egress of soluble proteins. In contrastand in preparations containing few cells/beadessentially all the DNA and -50%7 of the cellular RNA is retained, just like free nucleoids (Colman and Cook, 1977). (This can be shown by prelabelling cells prior to lysis with [3H]thymidine or uridine and then determining the retention of label after lysis.)…”