Identification of tissue-specific DNA-protein binding sites by means of two-dimensional electrophoretic mobility shift assay display

We developed a technique of differential electrophoretic mobility shift assay (EMSA) display allowing identification of tissue-specific protein-binding sites within long genomic sequences. Using this approach, we identified 10 cell type-specific protein-binding sites (protein target sites [PTSs]) within a 137-kb human chromosome 19 region. In general, tissue-specific binding of proteins from different nuclear extracts by individual PTSs did not follow the all-or-nothing principle. Most often, PTS-protein complexes were formed in all cases, but they were different for different nuclear extracts used.     

Thermal denaturation profiles and gel mobility shift analysis of oligodeoxynucleotide triplexes.

Oligodeoxypurine- and oligodeoxypyrimidine-containing strands were mixed under conditions conducive to the formation of triple stranded assemblies. The mixtures were characterized both by their UV absorbance change with increasing temperature and by their mobility in non-denaturing polyacrylamide gels. Duplexes 34 bp long containing 15 central purines on one strand and 15 complementary pyrimidines on the other strand yielded new melting transitions and showed different gel mobilities upon combination with oligopyrimidine 15-mers. The dependence of the thermal denaturation profiles on pH, salt concentration, GC content, strand orientation, base mismatches, and strand length was investigated.     

A mobility shift detection method for DNA methylation analysis using phosphate affinity polyacrylamide gel electrophoresis

We describe a procedure for DNA methylation analysis using the bisulfite-mediated cytosine-to-uracil conversion of a target DNA followed by methylation-specific polymerase chain reaction (MSP) and phosphate affinity polyacrylamide gel electrophoresis (PAGE). The MSP was performed using a 1:1 mixture of 5′-phosphorylated methylation-specific and 5′-OH non-methylation-specific primers. The PAGE using an immobilized phosphate-binding tag molecule (i.e., a polyacrylamide-bound dizinc(II) complex, Zn²⁺-Phos-tag), which selectively captures the 5′-phosphorylated DNA fragment, enabled the mobility shift detection of the methylation-specific product as a slower migration band. Using this novel procedure, we demonstrated the detection of a methylated cytosine base in a pUC19 plasmid.     

Specific binding of human immunodeficiency virus type 1 gag polyprotein and nucleocapsid protein to viral RNAs detected by RNA mobility shift assays.

Packaging of retroviral genomic RNA during virion assembly is thought to be mediated by specific interactions between the gag polyprotein and RNA sequences (often termed the psi or E region) near the 5' end of the genome. For many retroviruses, including human immunodeficiency virus type 1 (HIV-1), the portions of the gag protein and the RNA that are required for this interaction remain poorly defined. We have used an RNA gel mobility shift assay to measure the in vitro binding of purified glutathione S-transferase-HIV-1 gag fusion proteins to RNA riboprobes. Both the complete gag polyprotein and the nucleocapsid (NC) protein alone were found to bind specifically to an HIV-1 riboprobe. Either Cys-His box of NC could be removed without eliminating specific binding to the psi riboprobe, but portions of gag containing only the MA and CA proteins without NC did not bind to RNA. There were at least two binding sites in HIV-1 genomic RNA that bound to the gag polyprotein: one entirely 5' to gag and one entirely within gag. The HIV-1 NC protein bound to riboprobes containing other retroviral psi sequences almost as well as to the HIV-1 psi riboprobe.     

Ligand binding analysis and screening by chemical denaturation shift

The identification of small molecule ligands is an important first step in drug development, especially drugs that target proteins with no intrinsic activity. Toward this goal, it is important to have access to technologies that are able to measure binding affinities for a large number of potential ligands in a fast and accurate way. Because ligand binding stabilizes the protein structure in a manner dependent on concentration and binding affinity, the magnitude of the protein stabilization effect elicited by binding can be used to identify and characterize ligands. For example, the shift in protein denaturation temperature (T_m shift) has become a popular approach to identify potential ligands. However, T_m shifts cannot be readily transformed into binding affinities, and the ligand rank order obtained at denaturation temperatures (?60 °C) does not necessarily coincide with the rank order at physiological temperature. An alternative approach is the use of chemical denaturation, which can be implemented at any temperature. Chemical denaturation shifts allow accurate determination of binding affinities with a surprisingly wide dynamic range (high micromolar to sub nanomolar) and in situations where binding changes the cooperativity of the unfolding transition. In this article, we develop the basic analytical equations and provide several experimental examples.     

A rellable mapping method for sequence determination of oligodeoxyribonucleotides by mobility shift analysis

The method for sequence analysis of large oligodeoxyribonucleotides based on the characteristic mobility shifts of their sequential partial degradation products on two-dimensional homochromatography has been perfected using a large number of synthetic oligodeoxyribonucleotides of defined sequences as standards. Flat bed electrophoresis with careful temperature control gave entirely reproducible mobilities in the first dimension. Using this information, an accurate formula has been derived for calculating the relative electrophoretic mobilities of oligodeoxyribonucleotides of any composition. This formula is used to calculate the mobility shifts between two consecutive oligodeoxyribonucleotides in a series of partial products of an unknown oligomer distributed in the two-dimensional homochromatogram which differ by one nucleotide in length. This is compared with the observed mobility shift value to identify the added nucleotide. This provides a direct and rapid method for obtaining the unambiguous sequence of an entire oligodeoxyribonucleotide up to 15 nucleotides in length.     

Defining the sequence recognized with BmFTZ-F1, a sequence specific DNA binding factor in the silkworm, Bombyx mori, as revealed by direct sequencing of bound oligonucleotides and gel mobility shift competition analysis.

BmFTZ-F1 is a Bombyx mori homologue of FTZ-F1, a positive regulator of the fushi tarazu gene of Drosophila melanogaster. In order to determine the sequence recognized with this factor, we made three sets of oligonucleotide mixture which contain 4 possible nucleotides at different positions within the previously proposed 12-bp binding consensus sequence. Oligonucleotides which bound to purified BmFTZ-F1 were separated by a gel mobility shift procedure and a binding sequence was determined by direct sequencing through Maxam-Gilbert method. By this analysis, 7 positions showed clear sequence preference and 5 positions showed weak or no sequence preference. The importance of each nucleotide at each position was confirmed by a gel mobility shift competition analysis and results were presented as a quantitative difference in the binding affinity. From these analyses, we conclude that the best binding sequence of BmFTZ-F1 is 5'-PyCAAGGPyCPu-3'. This method may be useful for the determination of a binding sequence of other sequence specific DNA binding factor.     

Detection and analysis of neutral endopeptidase from tissues with substrate gel electrophoresis

Neutral endopeptidase from human or bovine tissues retains enzymatic activity following electrophoresis and immobilization in polyacrylamide gels. Infiltration of the gel with a fluorogenic substrate permits identification of the active enzyme by fluorescence associated with a distinct protein band. This technique both separates and identifies the enzymatically active species from a crude cell membrane fraction or from partially purified extracts that contain contaminating proteins. Enzymatic activity is quantitated by photographing the fluorescent bands and scanning the negatives with a laser densitometer. Because as little as 25 ng of enzyme can be detected by this method, it could be used where the amount of material is limited.     

A simple method for predicting serum protein binding of compounds from IC(50) shift analysis for in vitro assays

The shift in apparent IC(50) that attends addition of serum proteins to in vitro cellular, enzymatic, and receptor binding assays can be used to determine the dissociation constant for compound-serum protein complexes. We show here that a simple linear relationship exists between the apparent IC(50) in the presence of serum protein and the inverse of the apparent K(d) for the compound-serum protein complex. Using a series of cell-active kinase inhibitors we demonstrate that the K(d) value derived in this way can be used to predict the extent of protein binding in serum for various compounds. This method should provide a simple means of assessing the relative serum protein binding propensity of compounds early in the compound optimization phase of drug discovery campaigns.     

An electrophoretic mobility shift assay for the identification and kinetic analysis of acetyl transferase inhibitors

Histone acetyl transferases are important regulators of cellular homeostasis. This study describes a sensitive acetyl transferase electrophoretic mobility shift assay applicable both for kinetic analysis of acetyl transferase inhibitors and for high-throughput testing. Application of the assay for human GCN5L2 enabled dissection of inhibitor competition with respect to acetyl coenzyme A. Furthermore, we demonstrated that the assay can detect time-dependent inhibition of human GCN5L2 by reactive inhibitors.     

Design of a fluorescent electrophoretic mobility shift assay improved for the quantitative and multiple analysis of protein-DNA complexes

We describe a protocol for the fluorescent electrophoretic mobility shift assay improved for the quantitative analysis of protein-DNA complexes. Fluorescent-labeled oligonucleotide probes incubated with nuclear proteins were followed by electrophoresis. The signals for protein-DNA complexes were measured and normalized with fluorescent-labeled marker using fragment analysis software. This assay proved reliable measurement and multiple detection of DNA binding proteins.     

Trp repressor-operator binding: NMR and electrophoretic mobility shift studies of the effect of DNA sequence and corepressor binding on two Trp repressor-operator complexes

In Trp repressor-DNA complexes, most interactions either occur with phosphate groups or are water-mediated hydrogen bonds to bases. To examine the factors involved in DNA selectivity, we have studied Trp repressor binding to two operator sequences, trpR(S)() and trpO(M)(), with L-tryptophan or 5-methyltryptophan as corepressor. These operators contain all the consensus bases but differ at base pairs contacted by their phosphate groups. In electrophoretic mobility shift assays (EMSAs) the trpR(S)() sequence gives solely 1:1 protein-DNA complexes with either corepressor. The trpO(M )()sequence binds more weakly than trpR(S)(). It gives dissociating 2:1 complexes in EMSAs with L-tryptophan, but both 1:1 and 2:1 complexes are observed with 5-methyltryptophan or if glycerol is present in the gel. The backbone resonances of the TrpR-L-tryptophan-DNA complexes were assigned using triple-resonance experiments and selectively (15)N labeled protein. On changing the DNA sequence, the largest differences in the NMR spectra are at residues 78-81, at the turn of the helix-turn-helix motif and the tip of the recognition helix. I79 and A80 interact with the conserved bases of the operators, while G78 and T81 interact with phosphate groups at bases that differ between the two sequences. Changing the corepressor from L-tryptophan to 5-methyltryptophan causes effects at residues 52, 60, 61, and 85, which do not interact with the DNA. The spectra suggest that there is mutual induced fit between protein and DNA so that sequence changes at bases contacted only by the phosphate groups affect the environment of the protein at residues that bind to conserved bases elsewhere in the DNA.