微生物学技术

Eucaryotes use a common theme for packaging genomic DNA: 146 bp of DNA are wrapped around an octameric protein core consisting of two molecules each of the histones H2A, H2B, H3, and H4. Although this nucleosomal arrangement is ubiquitous throughout the genome, different chromosomal portions ...

The method described here is based on a technique developed to analyze the chromatin structure of the SV40 origin of replication, and also alterations in nucleosomal structures in the hsp70 promoter of Drosophila after heat shock (1,2). It relies on the ability of formaldehyde to crosslink pro ...

Protein-nucleic acid complexes play a crucial role in the events involved in gene expression and regulation. Direct and powerful approaches in studying this regulation are footprinting and protein-DNA crosslinking. Protein-DNA crosslinking detects the presence of a protein on a g ...

The protein-DNA complexes that make up the chromosome serve not only to package the genomic DNA within the confines of the nucleus but to directly participate in the efficient and controlled utilization of the DNA for nuclear processes such as replication, transcription, recombination, a ...

In vivo UV crosslinking permits direct analysis of protein-DNA interactions in intact cells. This technique has been used to study DNA binding by a wide variety of proteins including RNA Polymerase II, Topoisomerase I, and sequence specific transcription factors such as Even-Skipped, Ze ...

The position of a nucleosome describes the arrangement of its core 147 base pairs of DNA relative to the core histone octamer (1). The DNA superhelix spiraling around the histone octamer has an outer, solvent exposed face and an inner, histone-associated face (2). Fixing of a particular exposed hel ...

Researchers often think of nucleosomes as inert and static structures. But the laws of physical chemistry—which apply to any molecular complex—remind us that this picture cannot be correct. Indeed, many early studies revealed that nucleosomes undergo a complex set of assembly/disass ...

DNA supercoiling in eukaryotic viruses was first described by Vinograd and co-workers more than 30 years ago (see ref. 1 for a review), but its origin was recognized to result from DNA complexation with host histones to form minichromosomes only 10 years later (2). Because they are in contact with endo ...

The assembly of DNA into the eukaryotic nucleus via an ascending hierarchy of intermediate chromatin structures has two major functional consequences: 1. The intermediate chromatin structures provide a filing system that greatly facilitates the search by RNA polymerase for regul ...

In the past five years, there have been numerous molecular and genetic investigations of the mechanisms by which chromatin condenses into interphase chromosomal fibers (1,2). In addition to traditional molecular techniques such as electron microscopy (3–5) and analytical ultrace ...

The ability of analytical ultracentrifugation to elucidate chromatin structure/function relationships originates directly from its capacity to accurately measure key structural properties of complex macromolecular assemblies in solution. Figure 1 schematically ...

Phage antibody (Ab) library selections on peptides or proteins are usually carried out using antigens (Ags) directly coated onto a plastic surface (e.g., Petri dishes, microtiter plate wells, and immunotubes). This straightforward method is easy to perform and has been shown to be successf ...

The generation of high-affinity antibodies (Abs) against hapten targets (molecular weight below 1000 Dalton) presents particular problems not encountered with larger antigens (Ags). By their nature, haptens are invisible to the host immune system unless presented as an epitope con ...

The use of combinatorial libraries displayed on the surface of filamentous bacteriophage offers an efficient route to obtain a diverse set of monoclonal antibodies (MAbs) with desired specificity (1,2). However, following selection of such libraries, certain epitope specificit ...

Cloning and expression of functional antibody fragments (Fabs) in bacteria and on the surface of filamentous phage has revolutionized the processes by which antibodies (Abs) are discovered and engineered. Cloning, expression, and screening efficiencies of phage systems permit t ...

Antibody (Ab) phage display is a recently developed recombinant DNA technology for making human monoclonal antibodies (MAbs) from immune sources, such as bone marrow, lymph node, or peripheral blood lymphocytes from patients with various diseases, or from healthy individuals (1,2). M ...

Repertoires of antibody (Ab) V genes derived from nonimmunized human donors (1) or made synthetically (2 3) have been cloned for display on filamentous bacteriophage as either scFvs or Fabs fused to the minor phage coat protein (pIII) (4). Phage Ab repertoires can be subjected to multiple rounds of p ...

Cell surfaces provide a rich source of potential antigen (Ag) targets for therapeutic and research reagent antibodies (Abs). However, in some circumstances, access to these targets may be difficult since it is technically challenging to purify individual Ags while retaining their nat ...

As described in other chapters, the selection of phage-displayed immunoglobulin (Ig) fragments with desired specificity can be accomplished through successive rounds of panning on purified antigen (Ag). However, for many applications, the target Ag may not be able to be purified becau ...

The generation of new drugs has long involved the screening of hundreds of thousands of components with well defined in vitro tests, seeking compounds to mimic as closely as possible the desired in vivo activity of the new drug. New library methodologies offer many alternative routes that are at le ...