Sergei G. Bavykin

Portable system for microbial sample preparation and oligonucleotide microarray analysis.

ABSTRACT We have developed a three-component system for microbial identification that consists of (i) a universal syringe-operated silica minicolumn for successive DNA and RNA isolation, fractionation, fragmentation, fluorescent labeling, and removal of excess free label and short oligonucleotides; (ii) microarrays of immobilized oligonucleotide probes for 16S rRNA identification; and (iii) a portable battery-powered device for imaging the hybridization of fluorescently labeled RNA fragments with the arrays. The minicolumn combines a guanidine thiocyanate method of nucleic acid isolation with a newly developed hydroxyl radical-based technique for DNA and RNA labeling and fragmentation. DNA and RNA can also be fractionated through differential binding of double- and single-stranded forms of nucleic acids to the silica. The procedure involves sequential washing of the column with different solutions. No vacuum filtration steps, phenol extraction, or centrifugation is required. After hybridization, the overall fluorescence pattern is captured as a digital image or as a Polaroid photo. This three-component system was used to discriminateEscherichia coli, Bacillus subtilis, Bacillus thuringiensis, and human HL60 cells. The procedure is rapid: beginning with whole cells, it takes approximately 25 min to obtain labeled DNA and RNA samples and an additional 25 min to hybridize and acquire the microarray image using a stationary image analysis system or the portable imager.

[20] Mapping DNA-protein interactions by cross-linking

Publisher Summary DNA- protein interactions attract much attention for they are involved in the structural organization of genomic DNA as well as in directing and regulating DNA function. This chapter describes here an experimental procedure for induction and analysis of DNA-protein cross-links. The chapter present the protocols for cross-linking proteins and DNA by dimethyl sulfate (DMS) methylation depurination- reduction and by UV light. Then, methods for recovery of the cross-linked complexes by depleting them of the uncross-linked DNA and proteins are explained. Methods for analysis of cross-links by gel electrophoresis diagonal techniques, DNA hybridization, immunological reactions, and peptide chemistry are described in this chapter. To characterize components of the cross-linked complex, hybridization with cloned DNA probes should be used for recognizing DNA sequences, whereas gel electrophoresis and/or specific antibodies are useful for identification of proteins. Gel electrophoresis is the method of choice for proteins that are present in the complex in a sufficient amount, for example, for histones. Thus, two-dimensional gel electrophoresis and hybridization with various probes, as described below, was used to demonstrate the reversible displacement of histones from transcribed gene regions, as well as an essential similarity in the arrangement of histones on DNA in core nucleosomes within transcribed and silent chromatin regions.

The method of crosslinking histones to DNA partly depurinated at neutral pH

Abstract The procedure of either reversible or stable “zero-length” crosslinking of histones through their lysine residues directly to DNA partly depurinated under mild conditions at neutral pH is described. DNA in chromatin was methylated with dimethyl sulfate. Methylated purine bases in DNA can be readily excised under mild conditions at neutral pH. Aldehyde groups formed in the depurinated residues of DNA react with the ϵ-amino groups of lysines in histones and crosslink histones to DNA through the Schiff bases. The Schiff bases catalyze the β-elimination reactions causing quantitative single-stranded scissions of DNA at the points of crosslinking in such a way that only the 5′-terminal fragments of DNA formed upon the breakage become attached to histones. These covalent DNA-protein crosslinks are reversible and can be split at pH 3 or stabilized by reduction of the Schiff bases with NaBH 4 .

Nucleosome Structural Transition during Chromatin Unfolding Is Caused by Conformational Changes in Nucleosomal DNA

We have recently reported that certain core histone-DNA contacts are altered in nucleosomes during chromatin unfolding (Usachenko, S. I., Gavin I. M., and Bavykin, S. G. (1996) J. Biol. Chem. 271, 3831–3836). In this work, we demonstrate that these alterations are caused by a conformational change in the nucleosomal DNA. Using zero-length protein-DNA cross-linking, we have mapped histone-DNA contacts in isolated core particles at ionic conditions affecting DNA stiffness, which may change the nucleosomal DNA conformation. We found that the alterations in histone-DNA contacts induced by an increase in DNA stiffness in isolated core particles are identical to those observed in nucleosomes during chromatin unfolding. The change in the pattern of micrococcal nuclease digestion of linker histone-depleted chromatin at ionic conditions affecting chromatin compaction also suggests that the stretching of the linker DNA may alter the nucleosomal DNA conformation, resulting in a structural transition in the nucleosome which may play a role in rendering the nucleosome competent for transcription and/or replication.

DNA-protein cross-linking applications for chromatin studies in vitro and in vivo.

Publisher Summary Covalent protein–nucleic acid cross-linking is a powerful tool for the analysis of protein–DNA and protein–RNA interactions in various biological systems. It allows a researcher to freeze protein–nucleic acid contacts within the context of a nucleoprotein complex and analyze cross-linked species by a variety of methods such as two-dimensional (2D) denaturing gel electrophoresis This chapter describes some of the methodical advances in the field of protein–nucleic acid covalent cross-linking applications. The innovations discussed include (1) a fast, relatively noninvasive method of cross-linking by radical-producing chemicals, such as bleomycin–iron and phenanthroline–copper complexes, (2) applications of chemical and photochemical cross-linking techniques to nucleosome-containing complexes following in vitro nucleosome reconstitution on specific DNA, and (3) a time-resolved cross-linking technique for studies of postreplicative chromatin assembly based on the immunoenrichment of newly replicated DNA. The chapter describes 5S rRNA gene nucleosome interactions with linker histones and transcription factor TFIIIA.