Nuclear extracts

Proteins. Standard proteins for column calibration curves were obtained from Boehringer Mannheim. The methods outlined by Latham et al, (15) were followecLto prepare and label crude rat liver nuclear extract with -triiodothyronine. Normal con-... 

Figure 7. Purification of Tj nuclear receptors on the SW 3000 column. Crude liver nuclear extract was prepared and incubated with as outlined in the...

The factor Elf 1 was isolated as a factor that binds to the CNS-specific element I in the proximal regulatory region (Bray et al., 1988). Several lines of evidence pointed to Elfl as a strong candidate for a specific Ddc regulatory factor (Bray et al., 1989) First, mutations of element I that disrupt function also reduce the affinity of Elfl. Second, Elfl is the only element I binding activity detectable in an embryonic nuclear extract,... 

NA isolation and molecular characterization will be important to define the origin and functions of these proteins. At this time, infected cell nuclei offer the only source of these proteins, and NA have proved resistant to classic nuclear extraction methods (Yao and Jasmer, 1998). NA can be solubilized under conditions that co-extract nuclear lamins a/c and b (4 M urea, pH 8.0). Despite these similar physical properties, NA do not co-localize with lamins in the nucleoskeleton. However, both disulphide bonds and ionic interactions appear to contribute to nuclear complexes containing NA. In addition, NA can be cross-linked within host nuclei with protein cross-linking reagents. The foregoing properties represent current information available for the development of strategies to isolate and characterize these proteins and to investigate host proteins with which NA interact. 

Yao, C. and Jasmer, D.P. (1998) Nuclear antigens in Trichinella spiralis infected muscle cells nuclear extraction, compartmentalization and complex formation. Molecular and Biochemical Parasitology 92, 207—218. 

Finally, GTP is bound by many proteins, and diazirine-bearing thiol-cleavable GTP analogs (e.g., 23) have been proposed as probes to detect such proteins. By this method, H-Ras was successfully labeled in a nuclear extract of cultured human cells [70]. 

Fig. 12.3 The +294T/C polymorphic site in PPARD influences the binding of transcription factor Spl (24). Electromobihty shift assays (EMSAs) of nnclear extract derived from hnman monocytic U937 cells using double-stranded 25-mer oligonucleotides corresponding to the sequence from position -1-281 to -1-305 of the common T allele and the rare C allele. Arrow refers to the -I-294C allele-predominant factor. F denotes free DNA. Lane 1, without extract lanes 2 to 4,12, with 10 ag nuclear extract in the absence of competitor lanes 5 to 7,13 to 15, with 10 ag nuclear extract in the presence of 150-fold excess of unlabeled DNA as competitor. Competitors used were -I-294C probe (lanes 5, 13 indicated with C) -I-294T probe (lanes 6, 14 indicated with T) and a nonspecific probe (lanes 7, 15 indicated with X). Spl complex. Lanes 8 to 11, with lOng nuclear extract in the presence of antibodies directed against Spl, p50, p65, and c-Rel, respectively...

In regenerating rat liver only pre-existing H4 was phosphorylated at the peak of DNA synthesis [116,117]. The modification has a half-life of two hours and may be involved in replication of DNA [109,116,117]. The histidine kinases in yeast and Physarum polycephalum nuclear extracts are unable to phosphorylate H4 in... 

It has been suggested that DADS is actually a prodrug and the active HD AC inhibitor is its metabolite allyl mercaptan. This suggestion was reinforced when equal amounts (200 /tM) of allyl mercaptan and DADS were tested to inhibit HD AC activity in nuclear extracts of Caco-2 cells. 

Fig. 10. Chemical structure of apicidin (left), the potency of the different apicidins on HeLa nuclear extract.

Commonly, in vitro determination of HDAC activity is a manual assay utilizing a coupled two-step process, including enzymatic deacetylation of a substrate followed by reaction termination and readout [10]. Assays utilize nuclear extracts and substrates containing labeled (radioactive or fluorescent) acetylated histones. For the isotope-based assays, the enzymes are incubated with acetate-radiolabled histones prepared from chicken reticulocytes or chemically [ Hjacetylated peptide substrates, and the enzymatic activity is determined by liquid scintillation counting [11]. Alternatively, histones may be obtained from cells following treatment with [ H]acetyl-CoA [12]. The caveats of these approaches include the variability of prelabeled acetylated core histones within preparations, potential high costs, their labor-intensive nature and the presence of radioactive waste. 

Mixture of HDAC isoforms from HeLa cell nuclear extract. 

HeLa cell nuclear extracts were incubated at different concentrations with a radiolabeled acety-lated Histone 4 peptide fragment as substrate. HDAC activity was assessed measuring release of free acetyl groups. 

Simple experimental approaches to this problem recently started postulate that the repair of clustered DNA damage leads to conversion of nonlethal lesions, e.g., dihydrothymine, or mutagenic lesions, such as 8-OxoGuanine, into lethal double strand breaks. These early experiments have studied kinetics and influence of excision of base lesion within clustered DNA damage by E. coli and nuclear extracts [27,123-129]. 

Huebner, V.D. Matthews, H.R. Phosphorylation of histidine in proteins by a nuclear extract of Physarum polycephalum plasmodia. J. Biol. Chem., 260, 16106-16113 (1985)... 

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. 

We have used gel retardation assays to study proteins binding to the ARE. We used a probe consisting of the 42 base ARE. When the probe was multimerised in four or six tandem copies, protein, AREF (ARE Factor) from nuclear extracts of anaerobically induced suspension cultures was bound to the probe and showed a ladder of retarded bands (Fig. 2). This ladder of bands was completed specifically by the 4x ARE probe but not by a probe containing four copies of the octopine synthase enhancer element or pUC DNA even in 100-fold excess. Probes which contained one or two copies of the ARE did not show any binding. The need to multimerise the probe presumably results either from low affinity of the protein for the DNA or low abundance of AREF in nuclear extracts. 

Fig. 2. Binding of a nuclear extract to the ARE probe. A four copy radioactively labelled ARE probe was incubated with a nuclear extract from maize suspension culture cells. From the left the lanes represent free probe probe plus extract probe plus extract competed with a 10-fold excess of unlabelled ARE and, lastly, with a 50-fold excess of unlabelled ARE. The top bands are competed by unlabelled ARE but not by other unlabelled DNAs.

We identified a protein, designated GBF, in plant nuclear extracts, which specifically interacts with the G-box motif (Giuliano et al., 1988 Donald et al., 1990). 

David-Cordonnier M-H, Laval J, O Neill P (2000) Clustered DNA damage, influence on damage excision by XRS5 nuclear extracts and Escherichia coli Nth and Fpg proteins. J Biol Chem 275 11866-11873... 

David-Cordonnier M-H, Boiteux S, O Neill P (2001a) Efficiency of excision of 8-oxo-guanine within DNA clustered damage by XRS5 nuclear extracts and purified human OGGI protein. Biochemistry 40 11881-11818... 

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