遗传学实验技术

A method for the photocrosslinking of proteins to DNA in purified complexes is described. It makes use of the juxtaposition of a limited number of photoreactive nucleotides with a limited number of radiolabeled nucleotides at a specific location in a DNA fragment. Protein–DNA complexes are ...

Protein–DNA interactions underpin life and play key roles in all cellular processes and functions including DNA transcription, packaging, replication, and repair. Identifying and examining the nature of these interactions is therefore a crucial prerequisite to understand the ...

Several methods have been developed to site-specifically incorporate photoreactive nucleotide analogs into DNA for the purpose of identifying the proteins and their domains that are in contact with particular regions of DNA. The synthesis of several deoxynucleotide analogs that ...

Electromobility shift assay is a simple, efficient, and rapid method for the study of specific DNA–protein interactions. It relies on the reduction in the electrophoretic mobility conferred to a DNA fragment by an interacting protein. The technique is suitable to qualitative, quantita ...

We present a single-molecule method for studying protein–DNA interactions based on fluorescence resonance energy transfer (FRET) and alternating-laser excitation (ALEX) of single diffusing molecules. An application of this method to the study of a bacterial transcription ini ...

Certain DNA-interacting proteins induce a pronounced bending in the double helix and cause topological stresses that are compensated by the formation of supercoils in DNA. Such supercoils, when forming on a circular plasmid, give rise to a series of topoisomers that run at different speeds d ...

So-called architectural DNA binding proteins such as those of the HMGB-box family induce DNA bending and kinking. However, these proteins often display only a weak sequence preference, making the analysis of their DNA binding characteristics difficult if not impossible in a standard el ...

Understanding the underlying structural and physico-chemical basis for the recognition of specific DNA sequences by regulatory proteins is a central goal of modern biochemical genetics. A method for the rapid identification of mutant molecules altered in the affinity and/or spec ...

Several nuclear mechanisms involve specific DNA-protein interactions. The electrophoretic mobility shift assay (EMSA, also known as the gel mobility shift or gel retardation assay), first described almost two decades ago (1,2), provides a simple, efficient and widely used method to s ...

In the phage display method, peptides (1) or protein domains (2,3) cloned as fusions to the coat proteins of filamentous bacteriophage are displayed on the capsid, which encloses the viral genome. Proteins of interest and their associated phage can be selected from a large pool of variants (a libra ...

Bending of DNA by proteins plays an important role in transcription initiation, DNA replication, and recombination. The degree of protein-induced DNA-bending can be simply and conveniently determined by combining gel electrophoresis of DNA-protein complexes with the use of speci ...

Site-specific protein-DNA photocrosslinking has proved to be the method of choice for analysis of the formation of nucleoprotein complexes such as those involved in transcription by mammalian RNA polymerase II (RNA Pol II). The method has two principal advantages. First, it yields struc ...

Atomic force microscopy (AFM; also called scanning force microscopy ) is a rather novel technique that offers unique advantages in the potential for the very high resolution of DNA and small ligands in the absence of stains, shadows, and labels (1,2). Furthermore, the scanning can be performed in a ...

Structural data on biological macromolecules provide invaluable insights into the interactions of proteins with nucleic acids. Data obtained at atomic resolution by X-ray diffraction or nuclear magnetic resonance (NMR) studies ultimately describes the exact folding of poly ...

In work carried out in collaboration with the laboratory of D. Reinberg (University of Medicine and Dentistry of New Jersey), we have developed a site-specific protein-DNA photocrosslinking procedure to define positions of proteins relative to DNA in protein-DNA and multiprotein-D ...

In biological systems, photoreactive derivatives have been widely applied to study specific interactions of receptor molecules with their ligands by photoaffinity labeling (1–3). While the receptors are generally proteins (e.g., enzymes, immunoglobulins, or hormone recept ...

Limited Proteolysis is a useful structural probe for investigating the globular nature of proteins by preferentially digesting the more accessible regions often found between domains. Generally, proteases require a small region of polypeptide chain possessing conformatio ...

The basic side chains of lysine residues often play essential roles in DNA-protein recognition. They are able to contribute to the overall affinity of an interaction through nonspecific charge-charge interactions with the phosphate backbone and contribute substantially to the sp ...

Gene targeting of mouse embryonic stem cells facilitates the deliberate engineering of the mouse genome. The gene targeting vector is introduced into cells by electroporation, and cell clones carrying the targeting vector are obtained by drug selection. Clones can be screened effect ...

Appropriate culture of murine embryonic stem cells is critical to enabling introduced mutations to be passed through the germ line. ES cells must be carefully cultured to ensure that pluripotency is maintained. The feeder cells and foetal calf serum used to culture the cells can significant ...