bluuebunny/crossref_metadata_2025 huggingface dataset:
Production of monoclonal antibodies against proteins expressed in E. coli
Authors: Picksley Steven M , Lane David P
Published: November 2023

The production of monoclonal antibodies against the product of a genetically identified open reading frame (ORF) is often a major starting point in its characterization. The procedure is more complex than that described in Chapter 2 for the production of polyclonal antibodies and should be seen as a complementary technique. Ideally, both procedures should be employed to exploit the full potential of immunochemical analysis. For while the major benefits of monoclonal antibodies, namely their unlimited supply and defined specificity, come into their own during the advanced stages of characterization, polyclonal antibodies do allow simple detection and immunodepletion studies. The preparation of monoclonal antibodies is, however, preferred for the quantitative assay of the ORF product and for rapid purification of the product in a biologically active form. Moreover, the fine mapping of the antibody binding sites (epitopes) on the ORF product, when correlated with their effect on biological activity, provides a valuable insight into possible structure-function relationships.
Production of transgenic rodents by microinjection of cloned DNA into fertilized one-cell eggs
Authors: Waller Sarah Jane , Ho Mei-Yin , Murphy David
Published: November 2023

Since the pioneering demonstration in the early 1980s that the introduction of recombinant growth hormone DNA into the pronuclei of single cell mouse embryos could result in inheritable alterations in the growth of these mice (1), the techniques of mammalian transgenesis have led to significant ad vances in our understanding of the regulation of numerous complex biological processes. The power and appeal of transgenesis lies in its ability to allow the study of almost any protein within a truly physiological environment, that of the whole organism. Thi& permits detailed studies of the developmental and physiological regulation of the protein as well as its function in context with the normal processes occurring in the animal.
Databases, computer networks, and molecular biology
Authors: Fuchs R , Cameron g. N
Published: November 2023

Since M. Dayhoff established the first public macromolecular sequence data collection in the mid-1960s databases have become indispensable research tools in molecular biology (1). The number of databases of interest to biologists has grown steadily since then, and in 1993 the ‘database of databases’ LiMB (Listing of Molecular Biological Databases) presented more than a 100 databases in this area (2). Regular consultation of databases for nucleotide or protein sequence information, mapping data, or the description of the functional role of a particular protein in the living cell is now essential to the work of any molecular biologist. All biological databases today are available, often exclusively, in electronic form. Thus a certain amount of computer literacy is crucial for a modern biologist, even though computing has so far not been a key element of biological training and education. A biologist who mastered the principles of using a computer to support him in his paperwork may still face apparently insurmountable obstacles when he tries to find a small piece of relevant information in the mass of information provided by current databases.
Complement genetics
Authors: Schneider Peter M , Rittner Christian
Published: November 2023

With the advent of recombinant DNA methodology about 20 years ago, tremendous progress has been made in the definition of the genetic basis of the complement system. Most genes coding for complement components and related proteins have been cloned, partially or completely sequenced, their chromosomal location determined, and a variety of mutations leading to partial or complete genetic deficiency described. Nevertheless, genetic typing of complement polymorphisms is still being carried out routinely for almost all components at the protein level. Protein typing technology using electrophoretic as well as serological methods has been proven to be a reliable tool for the definition of polymorphic variants. The analysis of complement polymorphisms has been applied to a wide range of problems, such as association with diseases, structure–function relationship, phylogenetic as well as ethnological studies, and paternity testing. The analysis of complement polymorphisms at the DNA level has provided an even more complex picture by revealing further variants, which subdivide alleles defined at the protein level. DNA sequence analysis of protein allotypes of a number of complement components has demonstrated that most sequence variations are not confined to short stretches of genomic DNA, but are rather scattered throughout the entire gene. Therefore it appears to be technically difficult to replace protein typing methods completely by direct DNA typing using PCR methodology. In contrast, PCR typing has been established successfully for the direct genetic typing of HLA class II genes. A combination of typing methods both at the protein and DNA level has to be applied to obtain complete genetic typing results for complex polymorphic complement components as well as for the detection of complement deficiency.
Map-based cloning in Arabidopsis
Authors: Putterill Joanna , Coupland George
Published: November 2023

Novel plant genes are often first identified as mutations that disrupt or modify plant processes. However, in most species it is still extremely difficult to isolate the affected genes if nothing is known of their sequence or the biochemical properties of the proteins they encode. Insertional mutagenesis using transposons or T-DNA has proved a successful method of isolating such genes in maize, Antirrhinum, Arabidopsis, and Petunia (1-5), as long as appropriate alleles caused by insertion of one of these elements are available or can be identified (see also Chapter 7). However, the isolation of such alleles is always laborious and is often the rate-limiting step in this approach. More recently map-based cloning has proved to be a powerful alternative to insertional mutagenesis in tomato (6) and Arabidopsis (see below). This involves isolating the gene after first accurately locating a mutation on the chromosomes relative to previously isolated DNA markers. The approach therefore overcomes the major difficulty of T-DNA or transposon tagging, in that a rare allele caused by insertion of one of these elements is not required.
Comparative polymerase chain reaction
Authors: Kossodo Sylvie De
Published: November 2023

Comparative PCR may be used to compare the expression of cytokines in a series of diverse settings: for example, in different tissues or cell lines; in the same tissues or cell lines; before and after specific treatments; during kinetic studies; in uninfected versus infected organs etc. In each case the small amount of tissue, low cell-numbers or low copy-number would limit the utilization of Northern blots or the RNAse-protection assays described in Chapter 3. In contrast to the techniques described in Chapter 4, the method detailed here does not necessitate the construction of a multispecific control fragment. Comparison is achieved by subjecting samples to amplification, in sub-saturating conditions, of the constitutively expressed glyceraldehyde phosphate dehydrogenase (GAPDH) gene and the specific cytokine, either in parallel, or preferably, in the same tube, in the presence of a radiolabelled deoxynucleotide. The relative product amounts are quantitated by measuring the incorporated radioactivity of the specific bands from dried gels. This can be done by phosphor-imager analysis 01, after exposure to film, by conventional densitometry. The values obtained by measuring the incorporated radioactivity in the cytokine amplification products are normalized to the values obtained in the GAPDH amplification products.
Measurement of cytokine induction of PC-specific phospholipase C and sphingomyelinases
Authors: Schötze Stefan , Kronke Martin
Published: November 2023

Cytokines play a vital role as mediators of intercellular communication within the immune system. One of the hallmarks of cytokine function is the marked redundancy of biological activities. The overlapping biological functions of TNF/IL-1 and GM-CSF/IL-3 are just two examples. The redundancy of cytokine activities may be based on the utilization of shared key components of intracellular signalling pathways. The principal architecture of major signalling pathways used by cytokines consists of series of proteins including G-proteins, second messenger generating enzymes, protein kinases, and phosphatases. Receptor-mediated hydrolysis of cellular phospholipids by phospholipases of distinct specificities (PLA2, PLC, PLD, SMase) is a ubiquitous event of central importance in cellular signalling. Activation of these enzymes results in generation of early second messenger molecules which further transduce receptor signals to cytoplasmic target proteins. Since multiple intracellular targets exist for a given second messenger, the cytokine signal may branch off to more complex and diversified signals.
Assays for chemotaxis
Authors: Damme Jovan , Gs Rene Con In
Published: November 2023

In view of the recent developments of cytokine research, interest in chemotaxis assays has received a real revival. Indeed, within a short time a number of novel chemotactic cytokines have been identified. These low molecular weight proteins are different from other cytokines such as interleukin-1 (IL-1) and tumour necrosis factor (TNF), previously reported to be chemotactic. They belong to a novel supergene family and include proteins exerting their chemotactic activity specifically on granulocytes or monocytes (1-4). Some members of this family of inflammatory proteins were already biochemically characterized, but their chemotactic effect has only recently been demonstrated. The lack of sensitive and specific bioassays for measuring chemotaxis, as well as the complexity of these tests, can in part explain the late discovery of this family of chemotactic cytokines. However, the assays that were used to detect their activity during purification were basically identical to those already used for a long time, such as the Boyden chamber assay (5) and the agarose method (6) described in this chapter. Since many substances can exert chemotactic activity, their identification rather had to wait for techniques to produce sufficient amounts and to purify them to homogeneity.
Assessment of DNA damage in mammalian cells by DNA filter elution methodology
Authors: Bertrand Richard , Pommier Yves
Published: November 2023

A variety of DNA lesions can be measured in mammalian cells by the DNA filter elution methodology. All DNA elution methods were developed in Dr Kurt W. Kohn’s laboratory (Laboratory of Molecular Pharmacology, NCI, NIH, Bethesda, MD). DNA elution is commonly used to study the effects and mechanisms of action of chemotherapeutic drugs and carcinogens (1-3). The basic DNA elution filter methods were originally designed to assay DNA damage in intact cells or tissues from living animals (1). More recently, the DNA elution filter assays were adapted to study drug mechanisms in isolated nuclei (4-8) and in a reconstituted cell-free system (9-12). Altogether, the various elution methods are currently applied to study the DNA effects of a variety of anticancer agents (topoisomerase inhibitors, alkylating and crosslinking drugs) in cells in culture and to analyse the DNA fragmentation associated with programmed cell death (apoptosis) (9-12).
Immunomethods: magnetic, column, and panning techniques
Authors: Dyer P A , Brown P , Edward R
Published: November 2023

Cell separation techniques mediated by antibody-antigen interactions are now widely used in both research and clinical laboratories. This has largely come about with the advent of monoclonal antibodies and their exquisite specificity for defined antigen cpitopes. Cell surface molecules to which mono clonal antibodies have been developed arc identified with the internationally recognized nomenclature of cluster of differentiation (CD) antigens. Each CD antigen is assigned a unique number following Leucocyte Typing Workshops (1). Some commonly occurring CD antigens, such as CD4 and CD8 present on T lymphocytes, have been recognized for many years while others, largely those with higher CD numbers, have been defined only recently.
Analysis of human antibody sequences
Authors: Walter Gerald , Tomlinson Ian M
Published: November 2023

Diverse repertoires of antibody genes are generated during B-cell develop ment by the combinatorial rearrangement of relatively small numbers of basic building blocks: VH (variable), D (diversity), and JH (joining) segm nts for the heavy (H) chain variable domain and VL and h segments for the light( L) chain variable domain (for review see ref. 1). The antigen-binding site comprises three loops from the heavy chain variable domain (Hl, H2, and H3) and three from the light chain variable domain (Ll, L2, and L3), which are supported by a conserved f3-sheet scaffold (2).
Converting rodent into human antibodies by guided selection
Authors: HOOGENBOOM HENNIE R , ALLEN DEBORAH J , ROBERTS ANDREW J
Published: November 2023

Hybridoma technology, developed 18 years ago, has given rise to many rodent antibodies with exquisite specificity. Unfortunately, the therapeutic potential of rodent antibodies in humans has not yet been fully realized owing, to a great extent, to the immune responses they elicit, which can hamper or abolish their activity. Obtaining human antibodies directly by hybridoma technology has been relatively difficult, particularly in the case of those antibodies with specificity for ‘self’ antigens. Efforts to convert rodent antibodies into less immunogenic, more human structures have led to the construction of humanized antibodies (1), where the regions of the murine antibody responsible for antigen binding are transferred on to a human back bone (or framework) (Chapter 7). As a consequence, a substantial portion of the antibody (the six complementarity-determining regions or CDRs) remains non-human, which, in some cases can still lead to an anti-idiotype response, compromising the therapeutic benefit of the antibody treatment.
Extraction and separation of core histones and non-histone chromosomal proteins
Authors: Thorne Alan W , Cary Peter D , Crane-Robinson Colyn
Published: November 2023

The participation of the nucleosome in DNA processing events such as transcriptional activation is now well established (see Editor’s Introduction). It has been shown. for example, that certain transcription factors can access their recognition sites within a nucleosome alone or with the help of the SWI/SNF complex. The involvement of core histone modifications.
In vivo footprint and chromatin analysis by LMPCR
Authors: Riggs Arthur D , Singer-Sam Judith , Pfeifer Gero P
Published: November 2023

In vitro footprinting, as introduced by Galas and Schmitz (1 ). has become a common procedure which uses cloned DNA and purified DNA binding proteins or nuclear extracts. In vivo footprinting, performed by genomic sequencing after treatment of nuclei or intact cells with dimethyl sulfate (DMS). was introduced by Church et al. (2, 3) and improved by Becker et al. (4) and Saluz and Jost (5). However, for mammalian single-copy genes, the technique required the use of high levels of radioactivity and remained difficult until the advent of ligation-mediated PCR (LMPCR) (6, 7). LMPCR takes advantage of the sensitivity of PCR and the specificity of nested PCR. This enables a high-resolution, nucleotide-level analysis of chromatin, and only a microgram or less of total genomic DNA is needed. LMPCR was introduced by Mueller and Wold (6) for OMS footprinting and extended by Pfeifer and his colleagues for use in DNA sequencing (7), methylation analyses (7, 8). DNase I footprinting (9, 10), nucleosome analyses (9), and UV photofoot-printing (11, 12). In essence, LMPCR is a procedure for detecting the presence of rare breaks in the phosphodiester backbone of DNA. Generally, the breaks or nicks are the result of enzyme or chemical agents that directly, or indirectly after additional treatment, provide a 5’ phosphate.
Recovery of DNA from electrophoresis gels
Authors: Towner Paul
Published: November 2023

During molecular biology projects it is frequently necessary to isolate DNA fragments for further manipulations. The DNA could be from many sources but would typically be from PCRs or restriction enzyme digestion of recombinant plasmids. The simplest way to separate DNA is by agarose gel electrophoresis in the presence of ethidium bromide, the required DNA bands being identified using a UV transilluminator. The quality of agarose is now so good that concentrations as high as 8% (w/v) can be cast and used to separate DNA fragments down to 30 bp with very good resolution, allowing virtually all double-stranded DNA fragments to be isolated.
CTL epitopes
Authors: Elliott Tim , Haurum John S
Published: November 2023

Cytotoxic T lymphocytes (CTL) are primarily responsible for the clearance of acute virus infections. Thus, for example in mice, recovery from influenza A infection correlates with the induction of CTL before a neutralizing antibody response emerges (1). In humans, a rise in the frequency of influenza-specific CTL correlates with a reduction in virus titres in nasal washes (2, 3), and similar observations have been made for RSV (4), HSV (5), measles virus (6), and hepatitis A virus (7). CTL may also be important in controlling chronic viral infections such as EBV (8), CMV (9), and HIV (10). Some intracellular bacterial infections such as Listeria monocytogenes (11) as well as parasitic infections such as Theileria parva (12) and Plasmodium yoelii (13, 14) also appear to be susceptible to attack by CTL. The protective effect of CTL has been amply demonstrated in several cases by adoptive transfer experiments (3–5, 12, 15–19). In addition to providing protection against infectious agents, CTL are thought to provide some degree of protection against spontaneous tumours, by virtue of their ability to detect subtle quantitative and qualitative antigenic differences in transformed cells (reviewed in ref. 20).
Poles apart? Spindle pole bodies and centrosomes differ in ultrastructure yet their function and their function and regulation are conserved
Authors: Hagan Iain M , Gull Keith , Glover David M
Published: November 2023

One of the most overpowering images in biology has to be the simplicity symmetry of the apparatus that forms in order to segregate the eukaryotic genome before cell division, the spindle. Nothing illustrates more graphically the complexity of structure that can be generated by regulating the function of biological polymers. The essential bipolarity of the spindle requires the function of specialized microtubule organizing centres (MTOCs) at its poles, termed centrosomes in animal cells and spindle pole bodies (SPBs) in fungi. These specialized organelles differ markedly in structure from one class of organisms to another.
Eco-controlling - a tool to implement value-based environmental management
Authors: Storm Andreas , Wittke Anja
Published: November 2023

Managerial eco-controlling is based on the basic process of financial controlling.[1] Eco-Controlling envisages a strategic approach to environmental issues and proposes a systematic management procedure with various steps from target and strategy formulation to data management, decision support, control, implementation and communication [Schaltegger & Sturm 1995]. The concept is specifically developed to link environmental strategy with the financial and strategic targets of top management. The system therefore focuses on the improvement of eco-efficiency.
New mash filtration technique for the processing of powder grist
Authors: Lotz Markus , Schneider Jan , Weisser Horst , Krottenthaler Martin , Back Werner
Published: November 2023

Lauter tun and mash filter separate with a natural filter layer consisting of husks. Therefore the malt grain cannot be ground too fine and it is not possible to disclaim the use of husks. A new filtration technique with a dynamic disc filter was adapted to mash filtration to exploit the advantages of fine grinding and husk separation. The wort clarification was done with membranes. The fine grinding of malt in combination with the new filtration technique leads to shorter mashing and filtration times. The filter performance is comparable to the one of traditional mash filters. The extract yield could be improved. At the same time the B-glucan content was reduced. Poorly solved malts can be processed without problems. The beers were compared with traditionally brewed beers sensorial and valued better. Therefore the new filtration technique opens totally new perspectives for the brewing industry.
Continuous proportional fed-batch propagation of brewers’ yeast
Authors: Naudts D , Ae1ts G , IserentantB D
Published: November 2023

The effective control of brewers yeast propagation and the optimation of the quality of the propagated yeast remain important aspects in beer production and ask for further research. During our research is was found: - how in a continuous, proportional fed-batch propagation the medium composition and the yeast growth can be controlled; - how, by defining medium composition, oxygen concentration, temperature and culture conditions during the late exponential growth phase, yeast quality can be controlled. The results of this research gave us the possibility to define the propagation parameters in function of the final fermentation requirements.
Studies of yeast behaviour in fully automated test plant
Authors: Skands Birthe , Riis Preben , Thomsen Henrik , Hansen Jan Ruud
Published: November 2023

With the purpose of carrying out systematic studies of yeast behaviour in practical fermentation a fully automated 2 L test plant has been developed. Fermentations in the test plant are carried out in 16 EBC test tubes arranged in 8 pairs allowing the process parameters i.e. wort composition, wort aeration, fermentation temperature profile, time and pressure to be individually controlled. The studies have concentrated on mapping the impact of changes in word composition on yeast behaviour during fermentation.
Effects of cone cropping and serial re-pitch on the distribution of cell ages in brewery yeast
Authors: Deans K , Pinder A , Catley B J , Hodgson J A
Published: November 2023

Breweries that employ yeast cropping procedures for serial re-pitch into fermentations consider which portion of the crop to retain and how many fermentations to pitch with the same yeast culture. Cell age distribution analysis in yeast samples by fluorescence microscopy, fluorescence spectrophotometry and tlow cytometry revealed changes in age profile upon serial re-pitch of yeast through subsequent brewery fermentations. Analysis of samples from fractionation of a lager yeast cone showed a gradient of cell ages across !he depth of the cone and differences in fermentation performance. Yeast quality, re-use and crop selection can be monitored by cell age analysis.
Zeolites as catalysts for the cold and direct conversion of acetolactate into acetoin
Authors: Andries M , Derdelinckx G , Ione K G , Delvaux F , van Beveren P C , Masschelein C A
Published: November 2023

Our objective in initiating this research being the conversion of acetolactate directly into acetoin, the catalytic activities of twelve synthetic zeolites were tested. Experiments were performed between 2 and 20°c using a fluid bed in upward flow and phosphate buffer solutions at pH 4.2 containing 400 ppb acetolactate. The catalyst with the highest performance demonstrates conversion yields between 60 and 80 % al a flow rate of 75 1/kg.h. The highest values are obtained at 5°C. Only acetoin is formed. Conversion patterns are similar for beer. The kinetics and specificity of these transformations are discussed with a view to the optimisation of the decarboxylation reaction.
Product integrity during the packaging process
Authors: Bergh Herman J van de
Published: November 2023

Product integrity will be explained in terms of the highest possible quality in combination with the lowest possible risks for the consumer. The Packaging Process will be related to the present product and packaging assortment as well as the future developments. To reach the objectives different systems and procedures in the packaging process are used, judged on efficiency and functionality. Four subjects will be discussed to present the outline of Heineken’s approach. The subjects are: co-makership, HACCP in the packaging process, functional design of packages and efficiency of the packaging process.
Use of DNA microcircles in protein-DNA binding studies
Authors: Payet Dominique
Published: November 2023

In many protein-mediated DNA transactions such as site-specific recombination and transcription initiation, the DNA is tightly bent or otherwise distorted in order to bring DNA sequences that are separated by many double-helical turns into close spatial proximity. For example, in the core nucleosome 145 bp of DNA arc wrapped around the histone octamer so that the entering and exiting duplexes are dose to each other (1). Similarly DNA looping can be induced by proteins bound at separate distant sites. One example is the tetrameric lac repressor that interacts simultaneously with two sites and can form a tightly bent repression loop of 90 bp(2).
Bandshift, gel retardation or electrophoretic mobility shift assays
Authors: Fairall Louise , Buttinelli Memmo , Panetta Gianna
Published: November 2023

In this chapter we describe how to pe1iOnn a band shift experiment also known as the gel retardation assay or electrophorctic mobility shift assay, and some of the uses of the bandshift assay. 171c basis of the bandshift assay is that protein DNA complexes remain intc1ct when gently fractionated by gel electrophoresis and migrate as distinct b<mds, but more slowly than the free DNA fragment. The assay is simple and quick, and the use of radioactive binding-site DNA makes it highly sensitive. Originally, this method was primarily used in the detection of DNA-binding proteins in crude cell extracts, but as more and more proteins have become available in a pure form it is largely used to study the binding activity of purified DNA-binding proteins. It can be used for both highly sequence-specific (1, 2) and non-specific proteins, such as histones (3, 4). Bandshift assays can be used quantitatively to estimate dissociation constants for protein-DNA complexes. Bandshift experiments can also be used to visualize protein-protein interactions between a DNA-binding protein and other non-DNA-binding proteins. The binding of a second protein to a protein-UNA complex to form a triple complex is visualized by a further retardation of mobility that is called a supershift. Supershift experiments can also be used to assay thC’ binding of a second DNA-binding protein to the DNA or the binding of a second molecule of DNA to the protein.
Protein-DNA crosslinking with formaldehyde in vitro
Authors: Brodolin Konstantin
Published: November 2023

Formaldehyde is a highly reactive reagent which produces protein-DNA and protein-protein crosslinks b’tween macromolecules in close contact. TI1e distance between the’ crosslinking groups of protein and DNA should be around 2 A, suggesting an interaction at the range of van der Waals radii (1). Therefore, the formaldehyde crosslinking procedure provides a tool for identification of proteins and protein domains closely positioned to DNA. Formaldehyde crosslinking had been widely used for studies of protein-DNA interactions in chromatin in vivo and has been described in detail recently (2). In this chapter, the form is on applying formaldehyde crosslinking to studies of specific protein DNA interactions in vitro. This approach is principally different from the one mentioned above since relatively mild crosslinking conditions will be described that allow preferential formation of protein crosslinks of RNA polymerase from Escherichia coli with single-stranded (locally melted) regions of DNA in open complexes. In this context, crosslinking provides a simple way to identify domains on RNA polymerase bound to promoter ONA in open complexes as well as the identification of those subunit domains contacting the melted DNA. It can also be used for monitoring conformational changes in nucleoprotein complexes induced by different factors (temperature, salt conditions, pH) (3, 4). ln general, this approach can be applied to the study of any protein contacting dist011ed or melted regions of the DNA double helix.
Radiolytic cleavage of DNA. Mapping of the protein interaction sites
Authors: Charlier Michel , Spotheim-Maurizot Melanie
Published: November 2023

In aerobic conditions, DNA lesions induced by ionizing radiations such as y-, |3and X-rays result mainly from attack by the oxidative hydroxyl radicals (OH*) arising from the radiolysis of water (see Figure 1). The OH* radicals either abstract H atoms from the deoxyribose moiety, or add to the bases (see Figure 2A).
In vivo UV-laser footprinting
Authors: Boccard Frederic , Dethiollaz Sylvie , Engelhorn, Manuel , Geiselrnann Johannes
Published: November 2023

This chapter describes the use of UV-laser footprinting for detecting protein DNA interactions within a living bacterial cell. The principle of UV-laser footprinting is described in Chapter 14, and we concentrate on protocols that specifically address the extension of the technique to in vivo experiments. DNA-binding proteins exert their function when they associate with a specific DNA target. The biological activity of such c1 protein is therefore a direct function of the saturation of the specific DNA-binding site with the protein. The question that we want to answer, using UV-laser footprinting in vivo, is: ‘to what extend is a specific site on the DNA saturated by the protein that binds this sequence?’
Kinetics of DNA interactions surface plasmon resonance spectroscopy
Authors: Persson Bjorn
Published: November 2023

The procedures for biomolecular interaction analysis described in this chapter are based on biosensor technology, which offers an opportunity to study the interaction between an immobilized partner, the ligand, and an analyte molecule in solution. The interaction is monitored continuously and the binding events are studied in real time, with no need for labelling of any component. The technology is generally applicable to the study of any molecular structures which express affinity for one another, including proteins, nucleic acids, carbohydrates, lipids, low molecular weight substances, but also large particles such as vesicles, bacteria and cells. The wealth of information provided by this technology includes qualitative data such as binding specificity, but also quantitative aspect of binding like stoichiomet1y, affinity, concentration and kinetics and are amenable to detailed analysis (for general technical reviews see refs. 1, 2).
Mapping protein/DNA interactions in vivo using ligation-mediated polymerase chain reaction
Authors: Macleod Donald
Published: November 2023

This chapter describes the procedures currently used to identify sites of protein interaction on specific DNA sequences. One of these methods, ligationmediated PCR (LMPCR), can be used for in vivo footprinting analysis and is discussed in detail. Using the mouse aprt gene as an example, I show how this method can be used to build up a profile of gene structure in vivo,by mapping transcription factor binding sites and the position of nucleosomes.
Studying the progression of vertebrate chromosomes through mitosis by immunofluorescence and FISH
Authors: Sullivan Beth A , Warburton Peter E
Published: November 2023

Mitosis is a dynamic process in which replicated chromosomes, that have aligned at the metaphase plate, move along spindle microtubules (MTs) and become equally distributed into daughter cells. This chapter describes several methods used to study chromosome structure and segregation in mitosis, particularly during anaphase and telophase. Incorporating FISH and immunofluorescence, the protocols include the treatment of cells with drugs that reversibly affect spindle dynamics and cell cleavage, as well as immunofluorescence techniques on unblocked cells or unfixed cells. Advantages to each approach are discussed with respect to cell type and growth characteristics. The techniques are generally suitable for basic fluorescence microscopy, however confocal microscopy and imaging using charge-coupled devices (CCDs) have also been used to analyse cells prepared according to the protocols in this chapter.
Chromosome assembly in vitro using Xenopus egg extracts
Authors: Swedlow Jason R
Published: November 2023

The replication, transcription, and segregation of DNA in eukaryotes occurs within the context of chromatin and chromosomes. Chromatin shifts from condensed to decondensed states during transcriptional activation and during the transition from mitosis to interphase. Two extremes, the 10000-fold linear compaction of the DNA in heterochromatin or the mitotic chromosome, and the completely unfolded DNA present at replication forks and highly transcribed loci, define the limits of a range of structures that can occur simultaneously in a single cell. To mediate these changes, a host of proteins are available that promote efficient packaging by neutralizing the high negative charge of the DNA-phosphate backbone and forming specific nucleoprotein structures (reviewed in ref. 2). The DNA is wrapped twice around a disc-shaped octamer formed by the histones H2A, H2B, H3, and H4. Numerous non-histone proteins also serve to set up the structure of chromatin and chromosomes and mediate the various processes that utilize an organism’s DNA.
p53 and the integrated response to DNA dainage
Authors: Smith Paul J , Jones Christopher J
Published: November 2023

In multicellular organisms the process of DNA repair is one facet of the cellular response to DNA damage and as such must be integrated with other pathways to effect physiologically and genetically satisfactory outcomes for the individual cell, affected tissue system, and organism. Integration has occurred through evolution, linking pathways for stress signalling, adaptive responses, recovery mechanisms, cell-cycle delay, transcription and replication, and not least the molecular guillotine of programmed cell death, apoptosis. The programmed senescence shown in many cell lineages appears to borrow signalling pathways used to herald the presence of DNA damage and change proliferative capacity. It is becoming increasingly clear that the fidelity of these integrated responses is affected by the accrual of genetic lesions which constitute the multistep progression typical of neoplasia. Currently, it is believed that malignant transformation requires several such mutations, co-operating in multistage pathways that differ from one tumour type to another. Some estimates suggest that as many as ten such mutations could be involved (1), whereas statistical analysis of radiation-induced malignancies is compatible with a smaller number of ‘rate-limiting’ events, typically about three (2).
Molecular piracy by KSHV: a strategy of live and let live
Authors: Jayachandra Sukhanya , Chang Yuan , Moore Patrick S
Published: November 2023

Viruses have developed elaborate strategies to subvert the host cell metabolism and evade host immune surveillance in order to ensure their survival. One strategy makes use of molecular piracy in which homologues to cellular proteins involved in critical pathways are encoded by the virus. Molecular piracy is used most effectively by members of the herpesvirus and poxvirus families. For example, a common characteristic of the herpesvisuses is that they encode not only viral structural proteins but also enzymes involved in nucleic acid metabolism that play important roles in the synthesis of genomic viral DNA (1).
Cellular sites and mechanisms of human cytomegalovirus latency
Authors: Sinclair John
Published: November 2023

Human cytomegalovirus (HCMV) is a member of the herpesvirus family whose seroprevalence generally depends on socio-economic status but can vary from 50 – 90% depending on the population. Like all herpesviruses, after primary infection, HCMV maintains a latent infection throughout the lifetime of the infected host. Normally, primary infection of immunocompetent individuals with HCMV rarely causes disease but problems arise when infection or reactivation occurs in immuno-supressed individuals, particularly transplant patients and patients with AIDS (1, 2).
Adenovirus proteins that regulate apoptosis
Authors: Wold William S M , Chinnadurai G
Published: November 2023

Viruses and the hosts they infect are involved in an evolutionary battle. The host has physical barriers that protect itself from viruses. Infected cells can commit suicide to preclude virus replication. Virus-infected cells are destroyed by the innate and adaptive arms of the immune system, and virions are eliminated by specific antibodies. Viruses, in tum, have mechanisms that allow the virus to infect cells and survive attack by the host. Virus proteins inhibit intrinsic cellular apoptosis, and block killing of infected cells by cytokines and cells of the immune system. These viral proteins are interesting tools to understand virus pathogenesis, cellular apoptosis, and the immune response. They also provide novel insights into many key regulatory aspects of molecular and cellular biology. In this article we describe human adenovirus (Ad) proteins that counteract host antiviral responses. Aspects of this topic have been reviewed elsewhere (1–5). There are 51 serotypes of human Ads, which fall into six subgroups, A–F. We will limit our discussion to the commonly studied serotypes 5 (Ad5) and Ad2, members of subgroup C.
Extraction, purification, and characterization of virus DNA
Authors: Halliburton Ian W
Published: November 2023

Since viruses will only replicate within living cells, special methods have to be employed for growth in vitro. Four main systems are used for growth of viruses in the laboratory: Laboratory animals: Before other techniques were available, viruses were mainly isolated by inoculation of laboratory animals. The obvious choice of experimental animal is the natural host, but for human infections this is obviously not generally possible, and animals such as mice, rabbits, ferrets, and monkeys have been commonly used. The cost of upkeep of animals is, however, high, and there tends to be a large variation in response between individual animals even if inbred, so large numbers are therefore needed. To a growing extent, ethical considerations are also important, but animals are still required for the isolation of some viruses which will not grow in tissue culture, and animals still have to be used to study virus-host interactions or symptoms of a virus infection in its natural host.
Analysis of transcriptional control in DNA virus infections
Authors: Thomas S K , Latchman D S
Published: November 2023

Transcriptional control in DNA viruses is highly regulated, showing a high degree of both tissue-specificity and temporal control of gene expression. During the replication of DNA viruses, transcription occurs first from the immediate-early (if applicable) and early genes, followed by the late genes, with these two phases of transcription being separated by DNA replication. In general, genes expressed in the early phase encode regulatory and other nonstructural viral proteins, while most of the genes expressed in the late phase encode virion components. Although this highly regulated cascade is controlled by virus-encoded factors, it is now well established that expression is also controlled by host-encoded factors. For example, although the herpes simplex virus (HSV) virion protein Vmw65 (VP16) is essential for the transactivation of the immediate-early genes, it has to be complexed with the cellular transcription factors Oct-1 and host cell factor (HCF) in order to function (1). The presence or absence of these necessary cellular transcription factors can obviously determine the outcome of infection, thus (at least in part) determining the cellular tropism of the virus. An understanding of the control of these interactions is therefore clearly important both for an understanding of the cellular tropism of the virus, and also to aid the development of antiviral agents.
Maintenance of C. elegans
Authors: Stiernagle Theresa
Published: November 2023

Wild-type and mutant stocks of Caenorhabditis elegans are available from the Caenorhabditis Genetics Center (CGC). C. elegans is easily grown in the laboratory, with stocks routinely maintained on agar-filled Petri plates. However, they can be grown in liquid culture when larger quantities of worms are needed.
Conventional genetics
Authors: Hodgkin Jonathan
Published: November 2023

Caenorhabditis elegans was originally chosen as an experimental organism partly because of the advantageous features of its genetic system (1). Genetic analysis is a particularly powerful approach for investigating complex biological topics. One gene, once thoroughly characterized, can become the starting point for further exploration. With a strain bearing a mutation in the gene, functional interactions between genes/gene products can be examined by generating double mutants through simple crosses with previously charac terized mutants. Genetic screens can be performed for mutations with similar phenotypes or for mutations that suppress or enhance a phenotype. The power of the genetic system in C. elegans means that these experiments can move very quickly, and novel genes thereby identified can be mapped and cloned. Normally, populations of this animal consist almost entirely of diploid self-fertilizing hermaphrodites, which each produce a limited number of sperm and a larger number of oocytes. Both types of gametes result from con ventional meioses, with about one crossover event per c. The advantages of self-fertilization are several: first, mutants with very severe behavioural or anatomical phenotypes can nevertheless grow and reproduce as homozygous stocks, because the animal needs little more than a functional gut and pharynx in order to ingest and digest bacterial food. Second, self-fertilization means that a hermaphrodite carrying a recessive mutation will automatically produce animals homozygous for that mutation, as one quarter of its progeny, according to standard Mendelian principles. As a result, screening for mutants is made much easier, because there is no need to set up specific crosses in order to generate homozygotes. Diploidy also means that lethal or sterile mutations can be maintained, by using heterozygous animals. Finally, self-fertilization means that both recessive and dominant modifier mutations can be easily detected, if screens for suppressors or enhancers of a given phenotype are carried out. Again, there is no need to set up specific crosses; the screening can be carried out on whole populations.
High-throughput combinatorial synthesis of discrete compounds in multimilligram quantities: non-chemical encoding and directed sorting
Authors: Xiao* Xiao-yi , Nicolaouꝉ K C
Published: November 2023

Over the past few years the pharmaceutical community’s need to generate and screen chemical libraries with large diversity has fuelled the rapid development of combinatorial chemistry (1, 2). Research areas such as high-throughput solution (3, 4) and solid phase (5-7) synthesis, the development of novel solid supports and linkers (Chapters 6 and 7) (8, 9), as well as new powerful encoding strategies (10-14) (Chapter 1) have received exceptional attention due to their central role in the development of this field. The chemical encoding strategies (15, 16), coupled with the split and pool technique (17) (Chapters 1-3) greatly increase the efficiency of combinatorial synthesis and screening. However, these strategies produce each member of the library in very small amounts (nanograms to micrograms), making subsequent multiple screenings and characterization procedures quite difficult. Although traditional parallel synthesis (18, 19) in combination with the remarkable recent progress in laboratory automation (19) produces larger quantities of material, it still lacks the efficiency of the split and pool technique particularly for the preparation of very large libraries. We have recently developed two new combinatorial synthesis strategies employing non-chemical encoding and directed sorting in a batch synthesis fashion (10, 11) that combine both the advantages of split and pool and parallel synthesis. These strategies are capable of producing large combinatorial libraries and delivering milligram quantities of each library member. This chapter discusses the development of these two non-chemical encoding techniques: radiofrequency (Rf) tagging (10) and bar coding (11) in combination with a directed sorting strategy, and their application to the synthesis of combinatorial libraries of a variety of chemical structures.
Combinatorial synthesis on multivalent oligomeric supports
Authors: Kim Ronald M , Chang Jiang
Published: November 2023

This chapter describes a strategy for liquid phase combinatorial chemistry which we term combinatorial synthesis on multivalent oligomeric supports (COSMOS). In this approach, compounds are synthesized on multivalent soluble supports through iterative cycles of solution phase synthesis and size-based product purification. Final products may be cleaved from the support, and isolated from the oligomer by size. A brief comparison of solid phase and liquid phase synthesis will be presented, followed by an introduction to COSMOS methodology. The role of the support in COSMOS, and methods for rapid product purification will be highlighted. Detailed procedures for the preparation of soluble supports, and an illustrative synthesis using the COSMOS approach will be described. Emphasis will be placed on protocols for isolating support-bound and cleaved products. Parallel sample processing and automation of the COSMOS methodology will be discussed.
Differential display analysis of memory-associated genes
Authors: Huang A-Min , Lee Eminy H Y
Published: November 2023

Memory formation is a complicated process, especially in mammals. In the form of long-term memory formation, it is well known that it requiresde novaRNA and protein synthesis because protein and mRNA synthesis inhibitors impair long-term memory formation (1, 2). In the invertebrate system, extensive efforts have been made to identify specific genes associated with memory formation. For example, by using quantitative 2D gel analysis, Castellucciet al.(3) have identified several proteins that are related to the process of long-term sensitiza tion, a form of long-term memory studied in the invertebrateAplysia.Also, screening ofDrosophilamutants has yielded about ten genes that are related to the process of olfactory learning and memory (4). Although these methods are effective, two disadvantages exist by using these approaches for studies in the mammalian system. First, they take a long time to identify and characterize the genes of interest. Secondly, gene expression associated with learning and memory in mammals is much more complicated which may not be well detected by using the traditional methods.