Single positioned nucleosomes have been extensively employed as simple model experimental systems for analysis of various intranuclear processes. Here we describe an experimental system containing positioned mononucleosomes allowing transcription by various RNA polymerases. Each DNA template contains a pair of fluorescent labels (Cy3 and Cy5) allowing measuring relative distances between the neighboring coils of nucleosomal DNA using Forster resonance energy transfer (FRET). The single-particle FRET (spFRET) approach for analysis of DNA uncoiling from the histone octamer during transcription through chromatin is described in detail.
`Phage display has become a powerful tool for screening large libraries of peptides or proteins for the purposes of identifying high-affinity ligands for molecules of interest. In this article, we describe the construction and screening of libraries of 10(8) different peptides expressed at the N-terminus of mature protein III of bacteriophage M13. We discuss the assembly and cloning of double-stranded oligonucleotides into restriction enzyme-digested M13 RF DNA, electroporation of bacteria, harvesting of phage recombinants, isolation of binding phage with target molecules immobilized in microtiter plate wells, and confirmation of binding isolates. (C) 1997 Wiley-Liss, Inc.
Success in iterative synthesis/screening strategies for drug discovery from combinatorial libraries is sensitive to the composition of the libraries. Well-defined library subsets, generally with equal representation of composite compounds, are extremely important for finding the most active compounds for a given disease-target assay. We present methods for creating libraries of well-defined composition, with natural nucleosides, nonnatural nucleoside analogs, and novel nonnucleoside monomers linked together via phosphoramidite chemistry. A variety of methods are used to ensure nearly equimolar incorporation of monomers into oligomers, whether or not they have similar reactivities. Simple methods have been developed to characterize novel nucleoside-analog and nonnucleoside monomers quickly, which are necessary for defining libraries made with these elements. We also describe considerations for library design that affect the ability to identify active compounds. (C) 1996 Wiley-Liss, Inc.
Split synthesis can be applied to the synthesis of nonpeptidic combinatorial libraries based on the one-bead-one-compound principle. Examples of the synthesis and screening of libraries based on (1) building a rigid scaffold in the randomization process, (2) randomization of a flexible scaffold structure, and (3) building of heterogenic organic molecules are presented in this article. (C) 1997 Wiley-Liss, Inc.
In the last 5 years, combinatorial chemistry has emerged as a powerful tool for drug discovery within the pharmaceutical industry. Large, diverse, nonoligomeric libraries of low molecular weight (250-600) compounds are of significant interest for lead discovery and optimization. These collections should be adapted to a variety of screening formats with high efficiency and throughput. We will describe the process of preparation and screening of small-molecule combinatorial libraries encompassed within Pharmacopeia's Eclips(R) technology. Eclips(R) libraries are synthesized on PEG-grafted polystyrene beads (Rapp Polymer, Tubingen, Germany), which are encoded in a binary strategy with releasable, electrophoric tags. An example, based on the benzopyran template, has been designed and synthesized. Optimization of solution and solid-phase reactions for each combinatorial step will be discussed, along with parallel optimization of the controlled elution and extraction of library compounds from the beads. The engineering and informatics that support the preparation, screening, and structure decoding of a combinatorial library is critical to the success of a drug discovery effort. We will describe novel bead and fluid handling instruments used to prepare our libraries for screening in 96-well formats. We will also outline the design of an integrated database for barcode-based tracking of plates and compounds and a chemical-data-management system that can handle large combinatorial collections of structures. (C) 1997 Wiley-Liss, Inc.
The generation of peralkylated peptidomimetic combinatorial libraries can be accomplished by applying a "libraries-from-libraries" concept. The chemical modification of existing resin-bound tri- and tetrapeptide libraries through amide N-alkylation creates libraries having different physical, chemical, and biological properties. The synthetic methods used to produce these peralkylated compounds with different alkylating reagents, such as methyl iodide, allyl bromide, and benzyl bromide, are described. (C) 1996 Wiley-Liss, Inc.
`Phage display has become a powerful tool for screening large libraries of peptides or proteins for the purposes of identifying high-affinity ligands for molecules of interest. In this article, we describe the construction and screening of libraries of 10(8) different peptides expressed at the N-terminus of mature protein III of bacteriophage M13. We discuss the assembly and cloning of double-stranded oligonucleotides into restriction enzyme-digested M13 RF DNA, electroporation of bacteria, harvesting of phage recombinants, isolation of binding phage with target molecules immobilized in microtiter plate wells, and confirmation of binding isolates. (C) 1997 Wiley-Liss, Inc.
The multiple simultaneous synthesis of nonpeptide arrays of small organic molecules for biological screening is efficiently demonstrated on solid support for a bis-urea-substituted phenolic library, A versatile and economical apparatus is described for array synthesis, which utilizes a 96-well plate modified for filtration, Various techniques for solid support and array synthesis are described, including the washing of resins after each chemical step, the loading of resins, reagents, and solvents into plates, the agitation of plates to effect chemical reactions, and processing solution libraries derived from solid supports. (C) 1997 Wiley-Liss, Inc.
The utility of nonsupport-bound synthetic combinatorial libraries (SCLs; i.e., those that are not attached to a solid support) is due to their ability to be readily used in virtually any assay system that involves not only soluble receptors but also membrane-bound receptors and whole-cell organisms. This last application is illustrated here by the screening of soluble, nonsupport-bound SCLs composed of peptides or peptidomimetics for their ability to inhibit bacterial and viral cell growth. The same SCLs have also been tested in the presence of erythrocyte cells for their ability to inhibit known cytolytic compounds. (C) 1997 Wiley-Liss, Inc.
The four general methods to generate and screen a huge combinatorial peptide library (>107 peptides) are: biological libraries such as filamentous phage (), plasmid (), or polysome () libraries; the "one-bead one-compound" synthetic combinatonal library method or the "Selectlde process" (, , ); synthetic peptide library methods that require deconvolution, such as an iterative approach (,), positional scanning (); orthogonal partition approach (), or recurse deconvolution (); and synthetic library using affinity column selection method (,).
The isolation of good-quality high molecular weight DNA is probably the most important step in preparing yeast or bacterial artificial chromosome libraries and in physical mapping studies, This report describes a novel methodology for extraction of plant nuclei involving etiolated tissue, plasmolysis, mechanical disruption, and a high concentration sucrose buffer, which gives improved yields of DNA of size greater than 5.7 Mb, employs a simple cheap buffer, allows easy processing of large amounts of tissue, and reduces contamination from plastid DNA to a minimum, The technique is also applicable to different plant species including bean and tomato. (C) 1996 Wiley-Liss, Inc.
Introduction of foreign DNA into mammalian cells by electroporation is a popular method of choice, and it is usually performed by the use of commercially available pulse generators and electroporation cuvettes. Although these systems provide suitable and optimal conditions for most applications, large numbers of electroporation experiments are time consuming to perform, require many cuvettes, and are thus costly. Here, we describe reusable single, 8, and 96 well coaxial electrodes for electroporation of DNA into COS-7 cells in flat-bottomed 96 well tissue culture plates. This method is easy and rapid to perform, and there is no need for cuvettes, thus reducing labor and supply costs. Since electroporation in 96 well plates requires lower amounts of plasmid DNA, and the absolute cell number is not critical, cell and DNA requirements are not a constraint to large-scale experimentation. Electroporated cells can be cultured further in the microplate, or if high numbers of cells are electroporated, cultures can be set up in larger culture vessels, e.g., in 75 cm(2) tissue culture flasks. (C) 1996 Wiley-Liss, Inc.
DNA-protein covalent cross-linking in combination with two-dimensional gel electrophoresis and hybridization allows mapping of DNA-protein contacts in vivo and determination of the protein's approximate molecular weight (Mw), Here we present a strategy and detailed protocol for UV cross-linking, purification, two-dimensional gel electrophoresis, and "protein image" hybridization of cross-linked complexes, This method permits direct study of DNA-protein contacts without constructing artificial in vitro or in vivo systems. (C) 1995 Wiley-Liss, Inc.
The technique of Southwestern blotting is an easy and reliable way of identifying a DNA-binding protein in crude or purified cellular material after separation by gel electrophoresis and transfer onto a suitable membrane, Resolved proteins are detected with a labeled DNA probe that can be either a known and characterized cis-acting element or a sequence designed to search for a DNA-binding protein with desired properties, The advantage of this method is that knowledge of the approximate molecular mass of the DNA-binding protein allows an appropriate isolation strategy to be devised and can be helpful in assessing the purity and biochemical activity during the isolation steps. (C) 1995 Wiley-Liss, Inc.
We developed an original strategy to perform solid-phase sequencing of an amplified bacterial gene, Design of sequencing primers was based on the consensus sequences of related genes in different bacterial species, Ambiguities were resolved using inosine-containing primers instead of degenerate primers as previously used. The data reported herein show that such deoxyinosine-containing primers can be used for direct, solid-phase automated sequencing, for up to 3 deoxyinosine residues for a 20-mer primer. (C) 1995 Wiley-Liss, Inc.
We have developed a simplified method for transformation of bacterial cells with DNA fragments without needing to use previously prepared competent cells. The transformation procedure is easy to carry out, reduces time and effort, and provides transformation efficiencies which are adequate for many purposes. (C) 1995 Wiley-Liss, Inc.
Rapid amplification of cDNA ends (RACE) is widely used in molecular biology to amplify either end of a target cDNA from a cDNA population. This report details a variant of RACE, based on hexadecyltrimethylammonium bromide (CTAB) precipitation, that has a number of advantages over standard methods; it is effective when starting from small amounts of tissue, it requires no expensive reagents or equipment, and it is comparatively quick. The value and versatility of this method are demonstrated by amplification of cDNAs corresponding to the 5' ends of four distinct mRNAs. Following amplification, each of the RACE reactions contained a major band of approximately the size expected for the particular full-length amplification product. The correct identity of each product was confirmed by Southern blotting. (C) 1995 Wiley-Liss, Inc.
The isolation of coding sequences by exon frapping is known to be efficient on pools of cosmids. On yeast artificial chromosomes (YACs), however, this procedure is far less efficient for isolating human transcripts because yeast host DNA unavoidably contaminates gel-purified YACs. In the present report we demonstrate that the yeast background problem can be overcome efficiently by carrying out Alu long-range polymerase chain reaction (PCR) on unpurified YACs and then subjecting the PCR products to exon trapping, Using the YAC Alu long-range exon-trapped products as probes on enriched cDNA libraries, four of the five genes known to lie on the YACs used as starting material were recovered, Moreover, three new partial genes were isolated and mapped. Our data show that exon trap probes generated from YAC Alu long-range PCR products are sufficiently complex to isolate and map novel cDNAs efficiently and specifically. (C) 1996 Wiley-Liss, Inc.
Using a yeast genomic library mutagenized by transposon insertion, a bank of yeast strains containing random lacZ insertions throughout the genome can be generated. Such strains can be analyzed for gene expression, localization of beta-galactosidase-fusion proteins, and disruption phenotypes. The library can be hence be utilized for a variety of purposes, including insertional mutagenesis, analysis of differential gene expression, and identification of downstream targets of a protein of interest. We describe techniques for generating and utilizing such a bank and protocols we have developed to allow these analyses to be performed rapidly on a large number of individual strains. Also included are modifications of existing protocols for yeast transformation and DNA isolation to allow their use on large numbers of samples. (C) 1996 Wiley-Liss, Inc.
