bluuebunny/crossref_metadata_2025 huggingface dataset:
Promoter potentiation and activation: chromatin structure and transcriptional induction of heat shock genes
Authors: Lis John , Wu Carl
Published: November 2023

The heat shock gene regulatory circuit is an attractive model for elucidating general principles that govern inducible gene transcription and for investigating the role of chromatin and nucleosome structure in transcription activation. The degree of transcriptional stimulation is robust: transcription of the major heat shock genes can be increased over 100-fold upon heat shock (1), enhancing the detection of specific regulatory components and allowing a quantitative analysis of the role of these components. The transcriptional response is mediated by proteins that are present in induced cells (2, 3) and is extremely rapid (4), thereby facilitating kinetic and mechanistic investigations of the activation of these genes. Comprehensive studies from many laboratories provide a strong foundation of information on the primary structure of these genes, the ds-regulatory elements and trans-acting protein factors, as well as the chromatin structure of the uninduced gene and structural changes occurring after heat shock (for reviews see 5, 6, 7).
Fractionation of cells by sedimentation methods
Authors: Patel D , Ford T C , Rickwood D
Published: November 2023

Cells can be separated and fractionated by a number of techniques using the different properties of the individual cell types. One of the most common methods used for the isolation and purification of cells is sedimentation, usually using centrifugation. Cells may be separated by size using simple dif ferential pelleting procedures or separated on the basis of size and/or density using density gradient centrifugation. Nearly all cell separation procedures involve the pelleting of cells as part of the procedure for harvesting or con centrating cells prior to most fractionation procedures. However, differential pelleting is seldom used for separating different cell types, because the required difference in size, about tenfold, is rarely seen and so this technique will not be described in detail. Instead this chapter will concentrate on cell separations using density gradients with cells sedimenting either under unit gravity or in a centrifugal field. Aspects of cell separations that involve elutria tion are described in Chapter 3 and will not be discussed here.
Separation and fractionation of cells by partitioning in aqueous two phase systems
Authors: Fisher D
Published: November 2023

In the technique of cell partitioning, cells are added to the aqueous two-phase systems that are formed when aqueous solutions of certain polymers (typically dextran and polyethylene glycol) are mixed above critical concentrations. The cells distribute (partition) between the top phase, the bottom phase, and the interface between them (frcqucnlly termed the bulk interface).
Separation of cells by flow cytometry
Authors: Ormerod M G
Published: November 2023

A flow cytometcr uses a flow system to deliver cells in single file past a point of measurement. Light is focused at this point and the fluorescence and the light scattered by the cells is collected and measured. Flow cytometers measure several parameters on single cells at rates hetween hundreds to thousands of cells per second. The method can be used to define and to enumerate accurately subpopulations. Once identified, such subpopulations can be physically sorted for further study.
Nucleic Acid Structures
Authors: Riesner Detlev
Published: November 2023

From the wide discipline of nucleic acid structure only selected topics have a direct bearing on antisense technology. Thus nucleic acid structure will not be discussed in this chapter as an independent field of research but as an essential factor for a particular field of application. The restrictions for selecting the topics of interest concern the structural elements as well as the solution conditions and the thermodynamic conditions. I shall mainly address RNA intramolecular structure and RNA:RNA double-strand formation. A few examples of DNA oligonucleotides directed against RNA targets will also be considered. Antisense technology is predominantly an in vivo technology, whereas studies on nucleic acid structure have been carried out in the past mainly in vitro. Therefore, in vitro conditions either have to be adapted to reflect the in vivo situation or the data have to be extrapolated. This requirement restricts the applicability of several experimental methods. Not only the solution conditions but also the thermodynamic approach has to resemble the requirements of antisense technology. Antisense RNA as well as target RNA act in structures which are formed right after synthesis in the cell. These structures, which might also exist as mixtures of conformers, deviate in many cases from the equilibrium structure. This complicates the experimental as well as the theoretical approach. Having these restrictions in mind, an outline will be given on the structures, on methods to predict them, and on experimental approaches for structure analysis.
Catalytic Antisense RNA Based on Hammerhead Ribozymes
Authors: Tabler Martin , Sczakiel Georg
Published: November 2023

Some plant viruses are accompanied by so-called satellite RNAs which cannot replicate autonomously but require a helper virus for replication (1). Some of these satellite RNAs, especially the circular satellite RNAs, which are also called virusoids, undergo Mg2 catalysed autocatalytic self-cleavage at a defined position of their RNA genome. A specific phosphodiester bond is chemically cleaved in an intramolecular trans-esterification reaction resulting in a 2’ ,3’ cyclic phosphate located 5’ to the cleavage site, whereas the 3’ cleavage product has a 5’ hydroxyl group. A similar type of reaction can be found in two plant viroids which, unlike the satellite RNAs, do not depend on the presence of a helper virus for replication, and in the RNA transcript of a satellite DNA 2 of newt. A feature common to all these RNAs is the presence of a so-called ‘hammerhead structure’ (2).” The hammerhead structure consists of several conserved nucleotides and three helices that can he drawn such that the resulting secondary structure resembles the head of a carpenter’s hammer. A generally accepted numbering system has been defined (4) which can be seen in Figure 1. Despite the generality of the hammerhead structure and the conservation of the nucleotides that are involved in catalysis, there is considerable flexibility with regard to loop size and length of the helices.
Construction and use of antibody gene repertoires
Authors: Pope A R , Embleton M J , Ugh R Merna
Published: November 2023

The display of antibody molecules on the surface of bacteriophage (1) pro vides a powerful method of selecting a specific antibody from a mixed popu lation of antibodies together with the gene encoding it. This ability to co-select proteins and their genes has been exploited to enable the isolation of high-affinity, antigen-specific antibodies derived from an immunized mouse (2) and from unimmunized humans (3). In addition, after selecting such an antibody, a mutant antibody library can be constructed and higher affinity antibody clones selected. Alternatively, a monoclonal antibody can be subcloned from a hybridoma cell line into the bacteriophage and after mutagenesis, antibodies of improved characteristics selected (4).
Rodent to human antibodies by CDR grafting
Authors: Bendig Mary M , Jones S Tarran
Published: November 2023

The concept that a functional human-like antibody could be created by graft ing the antigen binding complementarity determining regions (CDRs) from variable domains of rodent antibodies on to human variable domains was first demonstrated by Dr Greg Winter and his colleagues at the MRC Labo ratory for Molecular Biology in 1986 (1). Since then several different research groups have developed and improved the methods for designing and constructing humanized antibodies via CDR grafting (2). There are reports of at least 80 antibodies being humanized by CDR grafting and at least eight of these antibodies being evaluated in clinical trials (3, 4). This chapter describes the procedures currently used at the MRC Collaborative Centre to create a ‘reshaped human’ antibody from a mouse antibody via CDR grafting.
In vitro reconstitution and analysis of nucleosome positioning
Authors: Davey Colin , Pennings, Sari , Allan James
Published: November 2023

It is now established that positioned nuclcosomes play a role in controlling the genetic functions of DNA (1–3). This is a consequence of the ability of the core-histone octamer to: (a) sequester a DNA sequence, by incorporating it within the nucleosome; (b) ensure that a DNA sequence remains exposed within the linker DNA between nucleosomes; or (c) to bring distant DNA sequences into close proximity. The precise positioning of nucleosomes with respect to the underlying DNA sequence thus determines its capacity to interact with, or to promote interaction between, other DNA-binding factors. The specific disposition of nucleosomes over long stretches of DNA is also likely to determine the architecture and stability of the higher-order chromatin fibre, which may, in turn, dictate its functional properties.
Microbiological techniques for molecular biology: bacteria and phages
Authors: Bainbridge Brian W
Published: November 2023

Recombinant DNA technology depends on the manipulation of particular strains of bacteria and bacteriophages (phages). Expertise in isolating, checking, growing, and analysing these strains is crucial for the success of genetic engineering. Many experiments are now permitted under conditions of ‘good microbiological technique’ and anyone embarking on research in this area should check exactly what is meant by this phrase. Essentially, all of the basic techniques depend on the culturing of a particular microbe in the absence of other organisms (sterile or aseptic technique), the isolation of a genetically pure culture or clone derived from a single cell (single-colony or single-plaque isolation) and the character ization of the known genetic features of the strain.
Construction of a cDNA library
Authors: Bhattacharyya Madan K
Published: November 2023

This chapter primarily describes a protocol for cDNA library construction that has worked routinely in our laboratory. This protocol includes methods for RNA and poly(A+) RNA isolation, complementary DNA (cDNA) synthesis and cDNA cloning. In writing this protocol, construction of a cDNA library is considered for etiolated hypocotyls of dark-grown soybean seedlings as an example. This protocol is for unidirectional cloning of cDNA in the ,\-based vector Uni-ZAP XR (Stratagene). However, this general protocol, with suitable modifications, should be applicable for construction of cDNA libraries in any other cloning vector. Reverse transcriptase, the enzyme that synthesizes a cDNA molecule using RNA as a template, frequently fails fully to transcribe RNA molecules, especially if they are very long. Therefore, full-length molecules become only a minor portion of the total cDNA. A new technique, based on addition of a biotin group to the cap structure of mRNA, has been developed for the construction of cDNA libraries in which more than 90% of the clones are full length (ref. 1, and references there in). This technique, or others like it, should be considered if the transcripts to be cloned are especially long.
Nucleic acid labelling and detection
Authors: Mundy C R
Published: November 2023

The recent explosive progress in our understanding of genome structure and function has depended upon methods for detecting small amounts of nucleic acid. As described in Chapter 5, specific fragments of genomic DNA can be detected in complex mixtures by hybridization with labelled complementary DNA or RNA probes. A 1 kb fragment of a single-copy human gene is present at the level of only 300 fg/µ,g of total DNA. Thus, a very high level of sensitivity is required, and for several applications a high degree of spatial resolution is also important. The purpose of this chapter is to describe the available labels and labelling methods and to define as far as practicable the optimum combinations for a range of key applications.
Protein expression in Escherichia coli
Authors: Ward John M
Published: November 2023

There now exist a variety of host-vector systems for the synthesis of protein from recombinant DNA. Systems using bacteria such as Bacillus subtilis, lower eukary otes such as Saccharomyces cerevisiae and Pichia pastoris (1), and higher eukaryotes such as Spodoptera frugiperda (2) insect cells and Chinese hamster ovary cells all have their advantages and disadvantages for protein expression. However, by far the most common host is still Escherichia coli. These expression systems are used for a variety of purposes. Very often, pure recombinant protein is needed for functional analysis, such as the determination of kinetic parameters, substrate specificity and inhibitor sensitivity. The structural analysis of proteins by X-ray diffraction and NMR techniques demands multi-milligram amounts of very pure proteins. High-throughput screening by pharmaceutical companies for novel drug discovery can use significant amounts of a protein target. At the small scale of growth of a few tens of millilitres to a litre or two, there are several highly efficient systems which can be used for the rapid production of recombinant protein, and these will be discussed in this chapter. Larger-scale growth and expression systems in E. coli involve a somewhat different set of problems and an overview of these will be given at the end of the chapter.
Methodological tips for human T cell epitope mapping when using pin technology peptide arrays
Authors: Mutch David A , Westwood Olwyn M R
Published: November 2023

The conditions for efficient large scale mapping of T cell epitopes within proteins recognized by human T helper cells are discussed. Many of the inherent difficulties in using freshly isolated human peripheral blood mononuclear cells (PBMC) and T helper cells are also described, as well as how such problems may best be overcome. Observation and awareness of such difficulties are crucial to obtaining accurate and meaningful results for epitope mapping studies. Although these methods precisely locate and size T helper cell epitopes, they rely on the availability of high quality, large scale, low volume peptides presented to freshly isolated human peripheral blood lymphocytes or cultured clone cells, in a nontoxic, biologically compatible buffer system. MultiPin Technology type peptides developed at Chiron Mimotopes Pty Ltd. (see also Chapter 2) are used in the procedures described in this chapter.
Phage display libraries
Authors: Williams Samantha , Logt, Paul van der , Germaschewski Volker
Published: November 2023

Two independent developments were brought together in the late 1980s to create a technique which since then has been shown to prove extremely powerful for the determination of amino acid sequences recognized by antibodies. First, the construction of synthetic peptide libraries on solid matrices, for example plastic pins (1) offered the possibility to screen for peptides that were bound by an antibody of interest (see also Chapters 2 and 3). Although peptides were isolated on the basis of binding the antibody used for screening the library, in many cases the amino acid sequences of the peptides bore no resemblance to any part of the native sequence of the antigen against which the antibody was originally raised. Rather, these sequences mimicked native epitopes and Geysen consequently called them ‘mimotope libraries’.
Chromosome mechanics; the genetic manipulation of aneuploid stocks
Authors: Gubb David
Published: November 2023

The use of chromosomal aberrations for cytogenetic mapping has been critical in the development of Drosophila as a model genetic organism. In 1932 Muller (1) designed a series of genetic tests, using chromosomal deletions and duplications, which define five classes of mutation; broadly corresponding to lack of function (amorphic and hypomorphic) and gain of function (hypermorphic, neomorphic, and antimorphic) alleles. Although Muller’s terminology is ignored frequently, his basic insight, that duplications and deletions could be used to characterize the nature of mutant alleles, remains true. In recent years, a large number of genes have been mapped between the breakpoints of chromosomal deletions or associated with the breakpoints of translocations, transpositions, or inversions. As a result there is an increasingly fine-scale correlation between the genetic map and cytological positions in polytene larval salivary glands (2). New mutations can be mapped by recombination and then rapidly assigned to a cytogenetic region using the available chromosome aberrations. Deletions are particularly useful for this purpose as failure to complement a mutant phenotype localizes the mutation within the cytological boundaries of the deletion. Translocation and inversion breakpoints can give a precise localization, if one end-point is mutant for the target locus, but breakpoints more frequently fall between transcription units. In this situation, it is often possible to construct a synthetic deletion from the aberration breakpoint. Similarly, the cytogenetic map position of cloned sequences can be identified by in situ hybridization to wild-type polytene chromosomes and refined by probing chromosomal aberrations. An up to date catalogue of available aberrations is maintained by FlyBase (Chapter 1).
Identification and characterization of DNA helicases
Authors: Bean Daniel W , Matson Steven W
Published: November 2023

DNA helicases catalyse disruption of the hydrogen bonds between the two strands of duplex DNA to generate single-stranded DNA (ssDNA) products (1, 2). This reaction is generally referred to as an unwinding reaction. The ssDNA product(s) of the unwinding reaction is then available for use as a template for DNA replication or repair, or as a substrate in recombination. Enzymes that catalyse the unwinding of duplex RNA, RNA secondary structure, and DNA:RNA hybrids have also been described (3-7). These enzymes have important roles in mRNA biogenesis, transcription, and translation. Thus, helicase catalysed unwinding is not confined to the unwinding of duplex DNA. In fact, there are examples of enzymes that catalyse the un winding of more than one of these substrates (8-10). The well characterized T-antigen, for example, unwinds both duplex DNA and RNA:DNA hybrids (9). Presumably these substrate preferences are related to the biological function of the protein, although these relationships are not always immediately obvious. None the less, it is clear that all transactions involving either DNA or RNA require the (transient) existence of a single-stranded intermediate, and thus helicases play key roles in all aspects of nucleic acid metabolism (11).
Analysis of telomere replication activities: telomerase and telomere binding proteins
Authors: Harrington Lea A
Published: November 2023

This chapter outlines the types of assays currently available for the analysis of two major classes of proteins important for telomere replication: telomerase, and telomere binding proteins. Since many of the other activities important for telomere replication, including DNA polymerases and helicases, are not necessarily telomere-specific, they will not be discussed here. There have been many recent advances in the detection of telomerase activities in vitro. The finding that telomerase activity is detected in a wide variety of human cancers, and is present at very low levels or undetectable in many normal, somatic tissues, has considerably raised interest in the study of telomerase activity regulation (reviewed in ref. 1). In addition, the recent identification and cloning of telomere binding proteins and telomerase components from several organisms has opened the way for a detailed biochemical examination of telomerase structure and function in many organisms (reviewed in refs 2-4).
Mitotic changes in the nuclear envelope
Authors: Miller Brian R , Forbes Douglass J
Published: November 2023

In higher eukaryotes, the disassembly of the nuclear envelope is one of the most dramatic events to occur at mitosis. The nuclear envelope consists first and foremost of a double membrane system surrounding the chromosomes. Lying under the inner nuclear membrane is a filamentous network composed of the nuclear lamins and associated proteins. At multiple places in the envelope, the inner and outer nuclear membranes fuse at the nuclear pore complexes. Nuclear pores are large, multi­ protein structures responsible for the signal-mediated transport of molecules into and out of the nucleus (Fig. 1). The lamina, pores, and the membrane system itself are all disassembled at the beginning of mitosis and reassembled during telophase.
Inheritance of the cytoplasm during cell division
Authors: Shima David T , Warren Graham
Published: November 2023

The inheritance process for nuclear DNA consists of a biogenesis phase (replication) and a partitioning phase (mitosis/meiosis). The inheritance of the cytoplasm also has biogenesis and partitioning phases, in which cytoplasmic components must double in unit mass, then be appropriately divided between nascent daughter cells during cytokinesis, respectively. The need to inherit cytoplasmic constituents such as mitochondria and chloroplasts is obvious since their self-contained DNA is absolutely required for proper organelle and cellular function and therefore must be transmitted to progeny through each cell division (1). Similarly, the endoplasmic reticulum (ER), the major site of synthesis for membrane lipids and proteins, must be derived from pre-existing membranes since there is no apparent mechanism to assembly these cellular membranes de novo (2).
European Brewery Convention
Authors: Devreux A
Published: November 2023

Held every two years, the European Brewery Convention draws together experts from around the world to exchange the latest information about the art and science of their trade. This volume collects a wealth of current research, with topics ranging from hop varieties to brewery automation.
Einsatz eines Separators zur Kühltrubentfernung
Authors: Eils Hans-Georg , Jünemann Andreas
Published: November 2023

A separator for the removal of cold trial was tested in a full scale trial. The flow was 700 hl/h and the system was designed to replace the presently used process of cold !rub removal with sedimentation tanks. A 50 % reduction of cold trub was aimed for. To achieve this target the construction of the separator had to be optimised and trials with different parameters had to be carried out. In selecting the proper conditions the target could be met. The beer quality with the new system was the same as with cold !rub removal by means of sedimentation. Flavour stability was positively influenced by cold trub removal with the separator. Preliminary results of experiences in a new word cooling plant will be reported.
The in vivo 31P-NMR analysis of brewing yeast and its practical application to brewing
Authors: Furukubo Susumu , Matsumoto Takehiro , Yomo Hideko , Fukui Nobuyuki , Ashikari Toshihiko , Kakimi Yoshi
Published: November 2023

We have established a procedure to measure the state of intracellular phosphor-containing substances of a brewing yeast by the 31P-NMR analysis. Cytoplasmic pH value of intact cells can be precisely determined from the state of intracellular inorganic phosphate. Fermentation performance tests revealed that the yeasts with higher cytoplasmic pH value had grown and assimilated amino acids with higher degree. Cytoplasmic pH value was proved to be a good index to assess yeast vitality for the subsequent fermentation. By applying this method to the practical brewing, various conditions of yeast handling and fermentation process could be optimized.
Bitterness by HPLC, a practical solution for routine QA use in breweries
Authors: Cope R , Hegarty P K , White F H
Published: November 2023

HPLC of iso-a-acids has been possible for at least two decades but routine adoption of the technology has been slow because of the complexity of the technique. A practical solution to this problem is described which involves the automated use of commercially available Solid Phase Extraction Disc technology. This robotic HPLC method has been routinely adopted in seven Bass laboratories. Inter collaborative test results show significantly improved repeatability and reproducibility over the Brenner method, which is currently internationally accepted for bitterness measurement in beers. HPLC results also suggest that Brenner results do not accurately reflect the iso-a-acid content of many beers.
Planungsgrundsätze und Betriebserfahrungen beim Brauereineubau
Authors: Eβlinger H M
Published: November 2023

In the planning of the new brewery of Freiberger Brauhaus AG particular emphasis was laid on quality and economy. In addition to meeting completion date and cost targets, the requirements included a modular expandable brewery with the shortest pipe runs and highly automated operation. The investments in environmental protection and energy economy combined with technological advance and minimal depreciation were particularly stressed. Currently there are 10 staff in general areas, 9 in the workshop, 12 in production and 24 in paclaging who produce over 650,000 hi beer per annum, requiring 35 kWh primary energy, 9 kWh electricity and 3.5 hi water per hi of beer produced.
Practical aspects of fluorescence resonance energy transfer (FRET) and its applications in nucleic acid biochemistry
Authors: Stuhmeier Frank , Clegg Robert M
Published: November 2023

Fluorescence spectroscopic methods can be used to detect substance (which contain fluorescent chromophores) at very low concentratiom and are especially useful for studying biomolecular structures and their dynamics in solution (see (l-3) for introductions into fluorescence spectroscopy). FRET, which is a non-radiative exchange of excitation energy between two chromophores, is strongly dependent on the distance between the tv.ro chromophores. If two such molecules are attached to a single biopolymer at different and well-defined positions, l-’Rl.:I can be used to estimate the distance between the two chromophores, and these data can be used to study structural aspects of the macromolecule. The distances that can be estimated arc in the range of0.5 to= 10 nm, which are in the same range as many distances in macromolecules. Because well-defined biopolymers can be synthesized and labelled at defined sites, FRET has become an important tool for structural studies io biophysics.
Determination of DNA-ligand interactions by fluorescence correlation spectroscopy
Authors: Langowski J , Tewes M
Published: November 2023

The quantitative characterization of protein-DNA interactions is of fundamental importance for our understanding of the mechanism of transcription regulation. A number of methods are applied to measure the thermodynamics and kinetics of such interactions, but many of those commonly used, such as gel shift, nitrocellulose filter binding, or surface plasmon r’sonance arc restricted to systems that are bound to or confined within some kind of matrix.
Nucleotide incorporation by DNA dependent RNA and DNA polymerases
Authors: Sclavi Bianca , Roux Pascal , Bue Henri
Published: November 2023

The steady-state analysis of the’ incorporation of nucleotide substrates by nucleic acid polymerases has be’en a subject of intensive research for many years. It has provided interesting re’ Wards in its specific field, for example a heller understanding of the faithfi.11 or the incorrect incorporation of nucleotides into zin elongating template by a specific enzyme. It has also proved to be of great use for a more indirect purpose, namely the understanding of how, in a time-resolved manner, nucleic acid polymerases interact with their cognate templates as the copying process is initiated or proceeds at steady state. This topic the subject of this chapter. Such assays were initially used in a rather crude. analytical manner. For example run-off transcription experiments were inserted into purification protocols to assess the degree of purity of a preparation of RNA polymerase or the extent of activation generated by a given factor. We describe here how more quantitative assays, performed with purified enzymes and templates, provide significant information on the interactions occurring between the two partners. Such assays arc essential prerequisites for the establishment of a plausible kinetic scheme which will in turn be challenged by the specific molecular methods developed in other chapters of this volume.
and elongation phases.
Authors: Temiakov Dmitry , Karasavas Pamela E , McAllister William T
Published: November 2023

Although the DNA-dependent RNA polymerase (RNAP) encoded by bacteriophage T7 consists of a single subunit, this enzyme is able to carry out all of the steps in the transcription cycle as the more complex multi subunit RNAPs. Its relatively simple structure, together with the availability of structural data, makes the phage RNAP an attractive model for studies of transcription at the molecular kvd (for review, see refa. 1 and 2).
Analysing the substructure of mammalian nuclei, in vitro
Authors: Jackson Dean A
Published: November 2023

If we are to understand the importance and complexities of interactions within the nucleus, as they exist in vivo, it is vital to develop approaches that allow us to access nuclear contents in vitro in such a way that the multiple layers of their organization can be analysed. This can only be worthwhile, however, if every effort is made to maintain and protect the organization as it exists inside the cell. Chapters 6 and 7 have shown how these goals can be pursued whilst using FISH and immunofluorescence to study chromosome structure in whole cells and tissues. There are particular additional problems in studying complex underlying nuclear structures using classical biochemical approaches. This chapter details the principal methods through which attempts have been made to study nuclear substructures that are important for both nuclear structure and function.
Macromolecular interactions in hepatitis B virus replication and particle assembly
Authors: Nassal Michael
Published: November 2023

Hepatitis B virus (HBV) is the causative agent of acute and chronic hepatitis B in humans. Chronic infection in particular poses a serious problem to public health. According to the World Health Organization (WHO), about 5% of the world population are HBV carriers, with a greatly increased risk for developing liver cirrhosis and, eventually, primary liver cell carcinoma. It is estimated that more than one million deaths per year are due to liver disease associated with chronic HBV infection. While an effective prophylactic vaccination based on a recombinant virus envelope protein is available, there is as yet no generally effective therapy (1, 2). The only currently approved treatment is the high-dose systemic administration of interferon-a but virus elimination is achieved in less than one-third of the patients. Hence the search for novel targets to combat chronic HBV infection continues to be one driving force for efforts to understand in detail the molecular mechanisms underlying the viral infectious cycle.
Epstein–Barr virus proteins involved in cell immortalization
Authors: Rowe Martin , Eike Floettmann J
Published: November 2023

The discovery in 1964 of herpesvirus particles in tumour cell lines derived from patients with Burkitt’s lymphoma (1) was a crucial observation that subsequently led to the so-called Epstein-Barr virus (EBY) being the first virus to be implicated in the pathogenesis of a human cancer. This gammaherpesvirus has since been associated with several other malignant diseases, including nasopharyngeal carcinoma, B cell lymphomas in immunosuppressed patients, certain T cell lymphomas, and Hodgkin’s disease (2). Absolute proof of the involvement of EBY in these malignancies was hampered by the unexpected finding that EBY was in fact a ubiquitous virus, being carried as a persistent and largely asymptomatic infection by more than 95% of adults in all populations world-wide (2). However, in support of its potential oncogenic properties, EBY was found to be a powerful transforming agent for normal resting human B lymphocytes in vitro, resulting in the regular outgrowth of EBY-immortalized lymphoblastoid cell lines (LCL) (3). Viral gene expression in LCL is restricted to about nine of the approximately 100 genes encoded by EBY and, since LCL are largely non-permissive for virus production, the EBY genes expressed in LCL were defined as latent genes. Six of the latent genes encode the nuclear antigens, EBNA-1,- 2, -3A,-3B, -3C., and -LP, while three encode the latent membrane proteins, LMP-1, -2A, and -2B; the most abundantly expressed latent transcripts in LCL are non-polyadenylated RNA, the EBERs, that do not encode proteins.
Interactions between viral and cellular proteins during DNA virus replication
Authors: Botting Catherine H , Hay ronald T
Published: November 2023

Initiation of viral DNA synthesis is preceded by assembly of a pre initiation complex at the viral origin of DNA replication. This nucleoprotein complex is composed of interacting viral and cellular proteins, which together recognize the viral origin of DNA replication and impart great specificity to the process. The initiation event is then triggered, and the pre-initiation complex dissociates as elongation proceeds. Thus the protein-protein and protein-DNA interactions involved are dynamic, with association and dissociation being induced by conformational changes, which may be triggered by one or more binding changes distal to the interaction under study. The methods described below have been used to study protein-protein and protein-DNA interactions in adenovirus DNA replication. It is therefore of value to describe briefly this replication system, to give context to the methods.
Synthesis and screening of positional scanning synthetic combinatorial libraries
Authors: Pinilla Clemencia , Appel Jon R , Blondelle Sylvie E , Dooley Colette T , Eichler Jutta , Nefzi Adel , Ostresh John M , Martinꝉ Roland , Wilson Darcy B , Houghten Richard A
Published: November 2023

Combinatorial library synthesis and screening methods, which enable the rapid identification of highly active compounds, have revolutionized basic research and drug discovery. A number of different combinatorial approaches based on the principles of solid phase synthesis have been used to generate enormous molecular diversities, including peptides, peptidomimetics, and small organic molecules. The advantage of the various combinatorial approaches compared to traditional drug synthesis and screening methods is the very large number of compounds that can be simultaneously synthesized and rapidly screened in biological assays.
Analytical methods in combinatorial chemistry
Authors: Yan Bing , Chu╪ Yen-ho , Shapiroꝉ Michael , Richmond§ Ramsay , Chinꝉ Jefferson , Liuꝉ Lina , Yuꝉ Lina
Published: November 2023

This chapter describes an array of techniques successfully applied to the optimization of solid phase organic synthesis (SPOS) (1, 2), quality control (QC) of combinatorial libraries, and in the selection of active lead compounds. Since low quality of the final library is largely attributable to poorly developed chemistry at the optimization stage, investing more time and effort in reaction optimization is crucial in combinatorial synthesis. QC data gives confidence on both the molecular diversity generated and the subsequent screening results. While the screening of pure compounds is a well-developed routine operation, the screening of compound mixtures is more difficult and this will be covered at the end of this chapter. Various analytical techniques are described below in the order encountered in a combinatorial synthesis process.
An introduction to differential display and related techniques
Authors: Robertson Harold A , Lesliet Ronald A
Published: November 2023

It is worth acknowledging, as there is no way around it, that differential display has been, and remains, a controversial technique. Any technique that engenders articles with the word ‘dismay ‘ in the title (1) must be controversial! More than a thousand articles, however, have now been published describing results achieved with the use of the differential display technique and hundreds of genes have been ‘discovered ‘. This book has been written with the firm belief that much of the scepticism that differential display has engendered is based upon a frequent poor understanding of the technique and the premature publication of many studies. Such studies have often been undertaken by groups who were early to recognize the power of this technique in biological applications but who had not yet achieved the necessary technical expertise to apply the technique successfully. Thus, in the heady days after the introduction of the technique by Liang and Pardee in 1992 (2), many biologists with divergent areas of expertise (neurobiology, psychology, plant sciences, pharmacology) rushed into applying the technique in their particular systems without, perhaps, a sufficiently solid background in modern molecular biology. This was a testi monial to the apparent simplicity of the technique and to the potential breadth of biological systems to which the technique might be applied.
A modified approach for the efficient display of 3 ‘ end restriction fragments of cDNAs
Authors: Subrahmanyamt Y V B K , Yamaga Shigeru , Newburger Peter E , Weissman Sherman M
Published: November 2023

One of the remarkable developments in science in the last few years has been the progressive improvement in technology and paradigms for obtaining the sequence of very large amounts of genomic DNA. In parallel with this develop ment, sequences identifying specific mRNAs have accumulated from random sequencing of segments of cDNA clones as well as from genomic sequences. The development of approaches for rapidly and economically screening the expression patterns ofmRNAs in different tissues or cell types and as a function of different physiological or pharmacological perturbations promises to generate bodies of data of the same order of magnitude and biological significance as the sequences themselves.
Cloning of differentially expressed brain cDNAs
Authors: Denovan-Wright Eileen M , Gilby Krista L , Howlett Susan E , Robertson Harold A
Published: November 2023

The ability of cells to alter gene expression in response to stimuli or during development and ageing underlies the plasticity of the central nervous system. Although there are multiple levels of control of gene expression, induction or repression of specific genes is an important first step in the regulation of the function of the CNS. Therefore, molecular screening and selection techniques based on comparing mRNA populations can reveal important differences in gene expression between and among brain tissues.

Differential display is a PCR-based screening method (for an overview see 1) that is especially useful to study changes in steady-state levels of mRNA in heterogeneous tissue such as brain for several reasons (1). First, this screening technique can be used to identify novel brain mRNAs or previously described mRNAs whose relative expression levels are altered as a result of a physiological change, a disorder, a disease state, or the administration of pharmacological agents. Secondly, it is possible to simultaneously compare multiple tissues, treatments, and time points (up to 60 comparisons per experiment and primer set) using small amounts of tissue from a number of individual animals for the original isolation of RNA. These features distinguish differential display from methods that require prior information about specific gene expression and from methods that are used to screen or select cDNAs from only two states. Moreover, differential display can be easily established in any laboratory that has basic molecular biology equipment and supplies.
Pleckstrin homology domains: phosphoinositide-regulated membrane tethers
Authors: Lemmon Mark A , Ferguson Kathryn M
Published: November 2023

Pleckstrin homology (PH) domains are small β-sandwich protein modules of approximately 120 amino acids that were first noted as a twice-repeated pattern in pleckstrin, the major protein kinase C substrate in platelets (1). In simultaneous communications to Nature(2) and Cell(3) in 1993, it was pointed out that a variety of proteins involved in cellular signalling also contain PH domains. Subsequent reports extended the number of putative PH domains to more than 100 (4, 5), and this number continues to grow. Confirmed or putative PH domains have been found in the following classes of intracellular protein (Fig. 1):
Inositol phosphatases: constructive destruction of phosphoinositides and inositol phosphates
Authors: Woscholski Rudiger , Parker Peter J
Published: November 2023

Phosphoinositide metabolism and signalling rely on the integrated action of several enzyme activities that act upon the inositol moiety. Phosphorylation and phosphate removal, performed by various kinases and phosphatases respectively, as well as the release of the inositol headgroup by phospholipases, are the crucial players that control the flux of inositol lipid metabolism (Fig. 1). While the pathway of the soluble inositol polyphosphates and their recycling to phosphoinositides is well established (1), phosphoinositide metabolism itself is still relatively unexplored. This has been made evident by the recent discovery of novel phosphoinositides such as phosphatidylinositol 3,5-bisphosphate (PtdIns(3,5)P2), which defined a completely new set of pathways and controlling enzymes (2–4). In particular, the influence of certain lipid kinases, such as PtdIns and PtdIns(3)P 3and 5-kinases, on the level of PtdIns(3,5)P2has recently been explored. This particular lipid might be as important for cellular signalling as those already established for the phosphoinositide 3-kinase products—PtdIns(3)P, PtdIns(3,4)P2, and PtdIns(3,4,5)P3—(see Chapter 2) (5–7) and diacylglycerol (DAG) derived from phospholipase C (PLC) (Chapter 1).
Antisense oligonucleotides: tools for elucidating gene function
Authors: Wolf John J , Michael Flanagan W
Published: November 2023

The goal of antisense technology is to design potent, sequence-specific inhibitors that can be used to elucidate the molecular basis of diseases, validate drug targets, and treat human diseases. Currently, one oligonucleotide (ON) has been approved by the Food and Drug Administration for the treatment of cytomegalovirus-induced retinitis in AIDS patients, and several additional antisense ONs are in various phases of clinical testing for the treatment of inflammatory diseases and cancer (1).
Confocal scanning microscopy in microarray detection
Authors: Schermer Mack J
Published: November 2023

All microarrays require fluorescence scanning to extract their experimental results. The confocal laser scanner delivers the highest image and data quality, a significant performance advantage. This chapter will describe the scanning process from an instrument point of view. It is divided into three parts. Sections 1 and 2 will describe the relevant characteristics of microarrays and all types of microarray scanners. Sections 3– 7 will describe the design options and critical characteristics of confocal scanners. Finally, Section 8 will describe one commercial confocal implementation, the ScanArray®. This section will describe the aspects of microarrays that are most relevant from the point of view of a scanning instrument. Microarrays consist of small samples of DNA or other biological matter arranged on a flat surface. The DNA or other material is tagged with a fluorescent probe so that a fluorescence measurement will reveal the concentration of the sample, with a typical fluorescence dynamic range of between about 400:1 and 4000:1 limited by background fluorescence. The flat substrate is generally made of chemically treated glass, and is often in the form of a 25 mm × 75 mm microscope slide. Microarrays are described at length in several other chapters; this chapter will confine itself to the aspects of microarrays related to fluorescence detection.
The cytoskeleton in the transduction of signal and regulation of cellular function
Authors: Carraway Coralie A Carothers
Published: November 2023

Cells are constantly bombarded with signals from their environments, and their responses to these cues determine all aspects of their function. Each type of cell responds to these environmental stimuli in a sensitive selective, and temporally ordered manner. Each signal is perceived and integrated into a carefully regulated cellular response which reflects a hierarchy imposed by each cell. Responses to stimuli can range from relatively simple ones, such as activation of a housekeeping metabolic pathway, to global reorganizations which encompass multiple metabolic pathways and elicit massive morpho logical alterations. In many cases there is at least minimal involvement of portions of the intracellular cytoskeletal network. In some instances, such as mitogenesis, complete disassembly and reassembly of all the cytoskeletal net works is required. In these cases morphology becomes destiny, and the pathways which link cell activation to cytoskeletal changes are key to the transmission of signals for the pleiotypic effects necessary for appropriate cellular response.
Signalling complexes: association of signalling proteins with the cytoskeleton
Authors: Carraway Coralie A Carothers , Carraway Kermjt I
Published: November 2023

A plethora of studies in the areas of membranes, cytoskeleton and. more recently. signal transduction have contributed to both concepts and technologies for the study of interactions of signalling proteins with the cytoskeleton (for overviews. see refs 1 and 2). These studies have implicated association of signalling proteins with the membrane and with the cytoskeleton in targeting these proteins to appropriate cellular loc,11ions for their participation in signal transduction events. Appropriate cellular localization is requisite for activation processes (3) and can occur reversibly. to turn off signal. The approaches used historically are diverse, ranging from tissue and cell localization by microscopy, to protein sequence analyses for the identification of domains for structure-function determinations to genetic perturbations. The recent explosion in genomics/proteomics and informatics is beginning to contribute in a major way to design and interpretation of cxpcrimenb based on sequence similarities, structural and functional domains. Further, these areas arc expected to play signilicant roles in crossfertilization of concepts among major areas of technology and of hiological investigation.
The study of mRNA-cytoskeleton interactions and mRNA sorting in mammalian cells
Authors: Bermano Giovanna , Hesketh John E
Published: November 2023

It is vital for a wide range of cell functions that newly synthesized proteins are delivered to the correct place in the cell at the appropriate time. There is abundant evidence for targeting motifs in proteins themselves which play a role in protein delivery (e.g. nuclear import signals), and there is increasing evidence that, for a least some proteins, the localization and targeting of their mRNAs plays a significant role in determining the site of protein synthesis within cells (1-3). Thus, in Drosophila embryos, the localization of certain mRNAs functions to ensure correct development by determining the site of synthesis, and in this way, the distribution of proteins critical in development (4). mRNA localization has also been demonstrated to occur in mammalian somatic cells (2), and again its role appears to be to determine or restrict protein distribution. This is likely to be important where there are closely similar isoforms and protein targeting alone could perhaps not segregate isoforms (e.g. actin, creatine kinase, metallothionein) (5-7), where the mRNA and protein are unstable (e.g. c-myc) (8) or where inappropriate deposition of the protein might cause disruption of cell organization (e.g. myelin basic protein, vimentin) (9,10).