Nuclear extracts fractionation

Nuclear extracts can be fractionated by chromatography on DEAE-cellulose to give three peaks of RNA polymerase activity (the use of column chromatography is explained in chapter 6). These three peaks correspond to three different RNA polymerases (I, II, and III), which differ in relative amount, cellular location, type of RNA synthesized, subunit structure, response to salt and divalent cation concentrations, and sensitivity to the mushroom-derived toxin a-amanitin. The three polymerases and some of their properties are summarized in table 28.4. 

RNA of these particles possesses other properties characteristic of information RNA or D-RNA. It hybridizes with homologous DNA to the same extent as nuclear D-RNA isolated by the hot phenol fractionation procedure. There is a strong cross-competition for binding sites on DNA between D-RNA isolated from the SOS particles and D-RNA isolated by hot phenol fractionation. This means that the RNAs prepared by the two procedures contain a similar population of molecules (Samarina et al., 1965a, 1967a). RNA prepared for nuclear extracts or from SOS particles effectively stimulates amino acid incorporation in a cell-free system (Jacob and Busch, 1967 Samarina, 1969). It is labeled strongly after short intervals, and its specific activity is of the same order as the specific activity of nonextractable RNA of the cell nucleus. The specific activity of this RNA increases with the increase of the number of extractions. 

The nuclear matrix-associated poIy(ADP-ribose) represent short polymers while complex polymers are released from the nucleus by the salt extraction. Molecular sieve chromatography analysis revealed that the nuclear matrix-associated ADP-ribose polymers, which represented 50% of the total residues are short polymers, while the polymers extracted by salt contain both short and complex chains (Fig. 2). These results demonstrate that there are considerably more polymer molecules in the nuclear matrix as compared to the salt extractable fraction. Therefore, it is very likely that in the nuclear matrix there are more molecules of protein modified by these... 

Our laboratory has been involved in the identification and characterization of the transcription factors for RNA polymerase I (Pol I)-directed transcription of ribosomal RNA genes (rDNA). To this end, we initially isolated and partially purified a transcriptionally active protein complex which contains RNA polymerase I and the essential Pol I transcription factors (1). Such a fraction was obtained from whole ceU extracts (1) or from nuclear extracts (2). Subsequently, we demonstrated that a fraction obtained during chromatography of the cell extract on a heparin sepharose coliunn could prevent nonrandom transcription of cloned rat rDNA in an in vitro system (3). The major protein in this fraction exhibited characteristics of purified poly(ADP-ribose) polymerase. The present report summarizes the properties of this protein, and describes experiments showing the dramatic appearance of accurately initiated transcript in an unfiractionated whole ceU extract or nuclear extract from a tissue foUowing addition of the protein factor. 

We then tested whether HS-C protein can promote specific and accurate transcription of rat rDNA in an unfractionated extract. Previous studies in our laboratory (2) have shown that unfractionated nuclear extracts from either rat hepatoma or rat liver caimot support rDNA transcription. If the lack of transcription in such extracts is predominantly due to nicks in the template caused by the action of DNase, addition of fraction HS-C should prevent random transcription and yield a distinct band corresponding to the expected size of the transcript. TTiat this is indeed the case was proven by... 

The present studies have demonstrated that a protein factor which exhibits properties of poly(ADP-ribose) polymerase can prevent nonspecific transcription of rat rDNA in either whole ceU extracts or nuclear extracts derived from tissues. This factor is analogous to that described for RNA polymerase Il-directed transcription (7). Highly purified fractions do not require this factor for accurate transcription of rDNA (Mealey and Jacob, unpublished data) probably due to removal of DNA-nicking enzyme(s) following purification. This observation does not preclude the importance of poly(ADP-ribose) polymerase in vivo in rDNA transcription following DNA damage. Clearly, rDNA can be nicked under these conditions and the requirement for poly(ADP-ribose) polymerase to prevent binding of RNA polymerase I to these nicks will be essential. 

The appearance of a distinct band corresponding to the accurate transcript of rat rDNA in an unfractionated hepatoma nuclear extract in response to exogenous HS-C is of considerable interest. This observation raises the possibility of using such a system to study rDNA transcription in response to a variety of physiological stimuli that are known to alter rRNA synthesis. Unfractionated samples have certain advantages over the fractionated samples since purification procedures could result in differential recovery of the transcription factors from control and test samples. 

To isolate RARs, we routinely incubate cultured cells with radiolabeled retinoic-acid isomers, prepare nuclei, extract the ligand-bound receptors from the purified nuclei and fractionate them using either sucrose density-gradient centrifugation or high-performance size-exclusion chromatography (HPSEC). It is also possible to prepare nuclear extracts from untreated cells and incubate this material with radiolabeled retinoic acid prior to fractionation (3). How-... 

RARs are believed to interact with retinoic-acid response elements as heterodimers with RXRs However, isolated RAR-retinoic-acid complexes consistently show a molecular weight of45,000 when fractionated by HPSEC, which suggests that it is the monomeric form that is being extracted (2,7) A similar result has been obtained when a nuclear extract is prepared prior to incubation with retinoic acid (3)... 

Isopycnic centrifugation. If a post-nuclear extract of hypo-tonically shocked rat TDL is fractionated by means of isopycnic centrifugation in an aqueous sucrose density gradient, all of the sedimentable acid hydrolases, including a small part of cathepsin D, band around a modal density of 1.18 (Fig.l). In agreement with the results presented in Table I, most of the cathepsin D activity is recovered in a soluble, unsedimentable form. The other lysosomal acid hydrolases contribute much less unsedimentable activity. 

This chapter contains two protocols, one for making a Drosophila embryonic nuclear extract called soluble nuclear fraction (SNF), and one for RNA polymerase II transcription in vitro. SNF transcribes DNA and chromatin templates and functions with several Drosophila and mammalian promoters containing different sets of core promoter elements. SNF also functions with several activating proteins. This highly active extract is suitable for many in vitro transcription experiments. 

Protocol 31.1 presents the preparation of embryonic nuclear extracts, a method from the Kadonaga laboratory (Kamakaka et al. 1991 Kamakaka and Kadonaga 1994) with minor modifications. This extract is called SNF, for soluble nuclear fraction, to distinguish it... 

Yttrium and lanthanum are both obtained from lanthanide minerals and the method of extraction depends on the particular mineral involved. Digestions with hydrochloric acid, sulfuric acid, or caustic soda are all used to extract the mixture of metal salts. Prior to the Second World War the separation of these mixtures was effected by fractional crystallizations, sometimes numbered in their thousands. However, during the period 1940-45 the main interest in separating these elements was in order to purify and characterize them more fully. The realization that they are also major constituents of the products of nuclear fission effected a dramatic sharpening of interest in the USA. As a result, ion-exchange techniques were developed and, together with selective complexation and solvent extraction, these have now completely supplanted the older methods of separation (p. 1228). In cases where the free metals are required, reduction of the trifluorides with metallic calcium can be used. 

Abscisin II is a plant hormone which accelerates (in interaction with other factors) the abscission of young fruit of cotton. It can accelerate leaf senescence and abscission, inhibit flowering, and induce dormancy. It has no activity as an auxin or a gibberellin but counteracts the action of these hormones. Abscisin II was isolated from the acid fraction of an acetone extract by chromatographic procedures guided by an abscission bioassay. Its structure was determined from elemental analysis, mass spectrum, and infrared, ultraviolet, and nuclear magnetic resonance spectra. Comparisons of these with relevant spectra of isophorone and sorbic acid derivatives confirmed that abscisin II is 3-methyl-5-(1-hydroxy-4-oxo-2, 6, 6-trimethyl-2-cyclohexen-l-yl)-c s, trans-2, 4-pen-tadienoic acid. This carbon skeleton is shown to be unique among the known sesquiterpenes. 

Uniformly labeled 2,4-dichlorophenol- C (purchased from New England Nuclear Corp, Boston, Mass.) was used in the tracer preparation. This provided a label at all carbon positions in the dibenzo-dioxin structure. 2,7-Dichlorodibenzo-p-dioxin- C after initial cleanup by fractional sublimation, contained approximately 5% of an impurity, detected by thin layer chromatography (TLC) which gave mass peaks at 288, 290, 292, and 294 in the mass spectrometer, consistent with a trichloro-hydroxydiphenyl oxide. This is probably the initial condensation product of the Ullman reaction and is most likely 2-(2,4-dichlorophenoxy)-4-chlorophenol. It was removed easily by extractions with aqueous... 

Denmark 1.5 days after the explosion. Air samples collected at Roskilde, Denmark on April 27-28, contained a mean air concentration of 241Am of 5.2 pBq/m3 (0.14 fCi/m3). In May 1986, the mean concentration was 11 pBq/m3 (0.30 fCi/m3) (Aarkrog 1988). Whereas debris from nuclear weapons testing is injected into the stratosphere, debris from Chernobyl was injected into the troposphere. As the mean residence time in the troposphere is 20-40 days, it would appear that the fallout would have decreased to very low levels by the end of 1986. However, from the levels of other radioactive elements, this was not the case. Sequential extraction studies were performed on aerosols collected in Lithuania after dust storms in September 1992 carried radioactive aerosols to the region from contaminated areas of the Ukraine and Belarus. The fraction distribution of241 Am in the aerosol samples was approximately (fraction, percent) organically-bound, 18% oxide-bound, 10% acid-soluble, 36% and residual, 32% (Lujaniene et al. 1999). Very little americium was found in the more readily extractable exchangeable and water soluble and specifically adsorbed fractions. 

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