bluuebunny/crossref_metadata_2025_split huggingface dataset:
Baculovirus expression vectors
Authors: Stewart Lorna M D , Possee Robert D 
Published: November 2023

Abstract
Progress in molecular virology has given us sufficient understanding of genetic organization and regulation in certain viruses of eukaryotes to enable us to utilize these systems as expression vectors of foreign genes. Analysis of the baculovirus genome has identified genes which are redundant when the virus is grown in cell culture. Furthermore, the promoters associated with these genes are exceptionally active, leading to the production of considerable quantities of protein within virus-infected cells. Consequently, baculoviruses have been used as a vehicle for the production of a variety of foreign gene products. In most cases the yields are very high and provide material suitable for studies of protein structure-function, therapeutic applications, or as vaccines. Foreign genes have also been inserted into the baculovirus genome to enhance the effectiveness of these agents as virus insecticides.
Expression of growth factors in microbial systems
Authors: Dudgeon Timothy J , Clements John M , Hunter Michael G 
Published: November 2023

Abstract
This chapter describes the use of microbial systems for the expression of growth factors. The important parameters which influence the choice of expression system are discussed. Protocols for the construction and expression of a synthetic gene for PDGF-B are shown. We describe in detail the construction and use of a yeast expression system to secrete platelet-derived growth factor B-chain (PDGF-BB), and draw comparison to the different expression routes used for epidermal growth factor (EGF), and transforming growth factor alpha (TGF-a), highlighting the benefits and disadvantages of the various approaches. Table 1 summarizes some of the routes which have been used to express various growth factors. Many of the standard molecular cloning techniques are now widely practised and are not described here. Protocols for these techniques may be found elsewhere (1, 2).
Isolation and characterization of glycoproteins
Authors: Carlsson Sven R 
Published: November 2023

Abstract
Today, significant information on the structure of a protein can be obtained without the need of having the protein in a pure form. After the breakthrough of cDNA cloning, very few full-length protein sequences have been determined at the amino acid level. Instead, the primary structure is deduced from the sequence of nucleotides in the DNA encoding the protein. From the derived sequence, certain properties of the protein can be predicted. Such sequence-specific information include ER-translocation signals, transmembrane domains, and potential glycosylation sites for N-linked glycans. Furthermore, with refined algorithms for secondary structure and folding predictions, molecular modelling in three dimensions can be made from only primary sequence information, especially in combination with crystallographic or NMR data from homologous proteins.
Isolation and fractionation of N-linked oligosaccharides from glycoproteins
Authors: Kobata Akira , Endo Tamao 
Published: November 2023

Abstract
For many glycoproteins, the carbohydrate moieties endow their polypeptide portion with important physical properties such as conformational stability, protease resistance, charge, and water-binding capacity. Interest in the biological roles of the carbohydrate chains of glycoproteins has been intensified because of interesting examples of their importance as recognition markers for protein targeting, and cell-cell and cell-matrix interactions (1). Furthermore, the importance of carbohydrate chains has been increasingly recognized year by year, as evident from the recent data of recombinant glycoproteins produced in heterologous cell types (2). In order to elucidate the molecular basis of such physicochemical effects and recognition phenomena of the carbohydrate moiety of glycoproteins, information as to the exact structure of the relevant oligosaccharide moieties are indispensable.
Structural characterization of oligosaccharides from glycoproteins
Authors: Kobata A Kira , Takasaki Seiichi 
Published: November 2023

Abstract
The final goal of structural analysis of sugar chains is to determine sequences, linkages, and anomeric configurations of monosaccharides included in oligosaccharides. Various enzymatic and chemical methods have been developed and applied to the structural analysis. One of the useful approaches is to use exoglycosidases which release exposed monosaccharide units of sugar chains. Usually, each of these enzymes has the strict glycon specificity toward monosaccharide species and its anomeric configuration. For example, β-galactosidases can hydrolyse β-galactosides, but not any other β-glycosides. β-Galactosidases do not hydrolyse a-galactosides. There is no glycosidase acting on both α- and β-glycosides. Thus, an exoglycosidase can release a particular monosaccharide with either α- or β-configuration from the non-reducing termini of sugar chains. A typical exception is β-N- acetylhexosaminidases which cleave both β-N-acetylglucosaminyl linkages and β-N-acetylgalactosaminyl linkages.
Structural characterization of N-glycans obtained from metabolically-radiolabelled glycoproteins
Authors: Cummings Richard D 
Published: November 2023

Abstract
Within the last few years tremendous advances have been made in our understanding of the structure and function of glycoconjugates synthesized by mammalian cells. The oligosaccharide moieties of glycoproteins, glycolipids, and glycosaminoglycans are now known to promote specific recognition events important in cell-cell adhesion, cell-matrix adhesion, binding and uptake of extracellular glycoproteins, and intracellular targeting of macro-molecules to organelles. The excitement in this area of research has created a need to improve the analytical techniques for structural characterization of extremely small amounts of glycoconjugates. One highly sensitive and reliable approach involves the structural characterization of metabolically-radiolabelled oligosaccharides. In this technique living cells are incubated with radioactive sugar precursors such as 2-[3H]mannose, 6-[3H]glucosamine, and 6-[3H]galactose, and glycoconjugates synthesized by the cells are radiolabelled by the respective sugar nucleotide donors. The radiolabelled oligosaccharide moieties of glycoconjugates can be then be released, either enzymatically or chemically, separated, and analysed by a variety of techniques. All the techniques of structural analysis of glycoconjugates, except those involving spectroscopic instrumentation, are useful for characterizing metabolically-radiolabelled N-glycans.
Advances in antibody eng1•neer1• ng
Authors: Verhoeyen Martine E , Windust John H C 
Published: November 2023

Abstract
In 1894, Emil Behring reported the discovery of antibodies, then called antitoxins, and their therapeutic effects (1). About 80 years later, Kohler and Milstein established the techniques to reliably produce monoclonal antibodies (mAbs) by hybridoma cell lines (2). This event, together with the availability of increasingly sophisticated molecular biology and protein engineering techniques, opened up the exciting new science area of antibody engineering, and led to the creation of antibodies and antibody-derived molecules designed to suit possible applications not only in the medical but also industrial world.
Technology for real time fluorescent ratio imaging in living cells using fluorescent probes for ions
Authors: Mason W T , Hoyland J , Davison I , Carew M A , Jonassen J , Zorec R , Lledo P M , Shankar G , Horton M 
Published: November 2023

Abstract
In this article, we shall discuss the means for applying fluorescent probes and computer-controlled image acquisition and analysis to previously intractable problems. Image processing is at the heart of virtually all of these new approaches. This article will focus mainly on how image acquisition and analysis can be used to study ionic gradients in living cells with optical probes. Some specific applications from our laboratory using this technology will be used to illustrate the potential for such techniques.
Chemical synthesis of oligosaccharides
Authors: Khan Shaheer H , Hindsgaul Ole 
Published: November 2023

Abstract
That oligosaccharides can function as ligands in biological recognition, usually through their binding to protein receptor sites, is now beyond dispute. The active structures have been identified in cases ranging in diversity from bacterial and viral adhesion to cell-cell recognition in the development of whole organisms. But the potential range of activities seems almost unlimited as noted in a recent, comprehensive and unusually open-minded review article entitled 'Biological function of oligosaccharides: all of the theories are correct’ (1).
The Contribution of the molten Globule model
Authors: Christensen Henriette , Pain Roger H 
Published: November 2023

Abstract
One of the conclusions that have been drawn from the 'thermodynamic hypothesis' of Anfinsen (1) is that amino acid sequence determines protein conformation. While ribonuclease has a very different structure from lysozyme there are NAD binding domains in different dehydrogenases in which the structures are very similar despite the lack of sequence homology (2). It is evident that the code relating sequence to conformation is highly degenerate as, by extension, must be the information that determines how the polypeptide chain folds. If we assume that similar 3D structures are the end-products of similar pathways, it seems reasonable to ask whether we should expect to find similarities, based on the physicochemical and structural properties of the amino acid side-chains, between the ways in which both the pathway and the final conformation are determined.
DNA radiolabelling and detection
Authors: Carvajal-Valdecasas Jaime 
Published: November 2023

Abstract
The hybridization process, whereby a target nucleotide sequence can be detected by the complementary binding of a labelled nucleic acid (DNA or RNA probe) is one of the most fundamental techniques used in molecular biology. Based on the procedure originally described by Southern (1), electro phoretic separation of DNA according to molecular weight, usually after endonuclease restriction, is followed by transfer and immobilization on a solid support such as a cellulose membrane. The target sequence is detected by hybridization (2) with a radiolabelled probe and subsequently visualized using autoradiography. Alternatively, non-radioactive probes may be used, coupled with an appropriate detection method. In this chapter, I describe the techniques and methods commonly used for the generation of radiolabelled probes to be used for the detection of DNA fragments by Southern blotting or RNA fragments by Northern blotting, respectively, for colony or plaque hybridization, and for in situ hybridization. All the main methods are covered except the use of PCR which is described in Chapter 6. The preparation of non-radioactive DNA probes is covered in Chapter 4.
Antibody probes
Authors: Banting George 
Published: November 2023

Abstract
The use of antibodies as tools in the fields of molecular and cell biology is widespread. They are used in the affinity purification of diverse molecules, in immunolocalization studies, as specific inhibitors of intermolecular inter actions, as a method of targeted delivery of conjugated molecules, and in a wide range of other applications. The aim of this chapter is to provide practical guidance regarding the production, characterization, and use of antibodies. For information not given here, the reader is referred to other sources, particularly the guide by Harlow and Lane (1). The focus of this chapter will be antibodies which recognize protein anti gens, although it is clear that antibodies to other molecules exist and are of undoubted importance and use. The structures recognized by antibodies are known as epitopes. An epitope can be defined as that region of a protein which is recognized by the antigen-binding site of an antibody. It may result from the local folding of a linear sequence of amino acids (a continuous epitope) or it may arise due to the spatial proximity of specific residues in the secondary or tertiary structure of the protein; residues which are far apart in the primary amino acid sequence (a discontinuous epitope). Thus, anti protein antibodies recognize specific epitopes; they do not recognize specific proteins. More than one protein may possess the same epitope, and this ‘epitope sharing’ may occur irrespective of linear sequence homology.
Screening recombinant DNA libraries by hybridization and amplification
Authors: Mason Philip J , Vulliamy Tom J 
Published: November 2023

Abstract
The methods described in this chapter are all designed to isolate and purify a particular recombinant DNA molecule from a complex mixture of such molecules. Whether one is screening a library containing an entire mammalian genome or subcloning from a few dozen DNA fragments, the techniques are essentially the same. In these days of large-scale genome projects, much gene isolation is performed using gridded libraries in cosmid or yeast artificial chromosome (YAC) vectors and these techniques are described in the following chapter. However, before detailed analysis of such clones can be made they must be subcloned into manageable pieces; moreover, there is not a genome project running for every organism. This chapter is concerned with the day-to-day use of library screening for the typical laboratory. The most common use of these techniques nowadays is probably in screening genomic or cDNA libraries in phage X using a radiolabelled DNA probe, so we will describe this procedure in detail. Modifications required when using different cloning vectors and probes will also be mentioned.
In situ hybridization of nucleic acid probes to cellular rna
Authors: dÉcimo Didier , Georges-labouesse Elisabeth , DollÉ Pascal 
Published: November 2023

Abstract
Hybridization of a nucleic acid probe to endogenous mRNA present in cytological or histological preparations is the most precise method for studying gene expression since it permits detection of the target mRNA virtually at the resolution of a single cell. For this reason, in situ hybridization (ISH) is widely used in various research areas, especially in the field of developmental biology, as well as for diagnostic purposes in pathology. Several types of nucleic acid probes and labelling methods have been described over the past years. At present, it appears that complementary RNA (cRNA) probes (riboprobes) labelled with a low-energy p emitter offer the best sensitivity and are thus most widely employed. We describe here a complete ISH protocol using S-labelled cRNA probes, applied to sections of paraffin- embedded tissue.
Directed mutagenesis and mutant analysis
Authors: White Roger , Butler Alison , Parker Malcolm 
Published: November 2023

Abstract
The ability to introduce specific mutations into a cloned DNA sequence provides a powerful technique for studying both gene regulation and the structural and functional properties of the encoded proteins. In particular, the construction of defined mutations allows analysis of specific properties and the organization of functional domains within a protein, and the determination of the role of specific promoter or enhancer sequences in gene regulation. The aim of this chapter is to describe the approaches and methodology involved in the design and construction of specific mutations and to explain how these techniques may be applied to the modification of any DNA sequence. The basic principles and methods of mutagenesis will be discussed, this will then be followed by a description of methods for identifying mutated sequences and a list of some of the applications of mutagenesis and methods of mutant analysis.
Human gene mutational analysis
Authors: Roberts Roland G 
Published: November 2023

Abstract
Mutational analysis of human genes is a key procedure in both human molecular biology and molecular genetic diagnosis. At the most fundamental level, it is rapidly becoming the rate-determining step in the identification of disease genes; recent advances in large-scale physical mapping, contig assembly and gene-hunting have meant that the route from meiotic linkage data to a set of candidate cDNAs has become a relatively short one. The ability to pare this set down to a single gene locus is then dependent on an efficient method for screening the candidate sequences for disease-associated mutations. After identification of a disease gene, much can be inferred about the function of parts of the gene product by ascertaining the nature and distribution of naturally occurring mutations which cause the disease phenotype. Characterization of such mutations also allows direct prenatal and carrier diagnosis in relatives of affected individuals.
Quantification of DNA and RNA by PCR
Authors: Kang Jie , Kühn Jochen E , Schäfer Peter , Immelmann Andreas , Henco Karsten 
Published: November 2023

Abstract
The ability to quantify low copy numbers of a specific nucleic acid target in limited amounts of sample is important in both basic and applied medical research. The combination of PCR and TGGE (temperature gradient gel electrophoresis) provides an exact and reliable system for such quantification (1).
In vitro expression of proteins from PCR products
Authors: Lesley Scott A 
Published: November 2023

Abstract
The primary advantage of in vitro translation of gene products over the in vivo expression of cloned genes is that in vitro translation allows the use of a defined template to direct total protein synthesis. There is a direct correlation between the sequence of the DNA added as a template for transcription and translation, and the protein produced without any background synthesis of endogenous proteins. This is a particular advantage when one would like to specifically radiolabel a protein of interest. The advent of PCR as a common tool for manipulating DNA sequences presents additional opportunities to accelerate such studies, by providing template for in vitro translations without the usual cloning and in vivo expression. By utilizing in vitro translation, the gene product can be amplified, translated, and assayed in a single day.
PCR-based approaches to human genome mapping
Authors: Morrison J F J , Markham A F 
Published: November 2023

Abstract
Genetic maps are constructed by determining how frequently two markers are inherited together. These maps allow the ordering of DNA markers and give estimates as to the distances between these markers. Genetic maps differ from physical maps in that they can be used to site loci which are associated with disease. Physical maps, in contrast, specify nucleotide distances between markers along a chromosome. PCR is used to generate both types of map through the generation of sequence-tagged sites (STS) (1). Sequence-tagged sites are used to identify DNA segments in both genetic and physical maps, and usually comprise 200-500 bp of DNA sequence unique to that segment, sufficient to allow a PCR reaction to be performed. Thus these STSs can conveniently be identified by specific PCR primers which can be used to screen genomic DNA contained in yeast artificial chromosomes (YACs) or cosmids.
Linear amplification for the in vivo study of ligand/DNA interactions
Authors: Saluz Hans Peter , Jost Jean-Pierre 
Published: November 2023

Abstract
Genomic footprinting is a tool to study specific ligand/DNA interactions in vivo (1). The DNA in the region where proteins bind shows altered reactivity towards footprinting reagents and therefore usually escapes modification and/ or cleavage. The cleavage products of the DNA are visualized by either electroblotting and indirect end-labelling (1, 2), linear amplification (3, 4), or via primer extension with labelled primers (5). Protected areas appear as a ‘window’ in the sequence ladder, when compared with treated protein-free control DNA.
Production of foreign proteins at high level
Authors: Gérard Loison 
Published: November 2023

Abstract
The yeast Saccharomyces cerevisiae is one of the most commonly used host cell systems for the production of foreign proteins for research and for industrial or medical use. Although the state of knowledge on yeast molecular biology is increasing day by day, a large part of the yeast biotechnologist’s work remains based on empirical approaches rather than on assertive predictions. Insertion of a foreign coding sequence into a standard multiple-copy expression vector for yeast is not always sufficient to ensure a high level of accumulation of the foreign protein. Various strategies have been tested by different groups to overcome limitations in protein production; mostly these depend upon empirical mutagenesis-screening. Reviewing all these cases is beyond the scope of this chapter. Interested readers should consult the recent and comprehensive review of Romanos et al. (1), which is entirely devoted to foreign gene expression in S. cerevisiae as well as in other yeasts. I will rather limit myself to emphasizing some basic pieces of advice and outlining some successful strategies we have experienced ourselves.
Cell-free translation of natural and synthetic mRNAs in yeast lysates
Authors: Tuite Mick F , Zhu, Delin , Hartley Aland. 
Published: November 2023

Abstract
All three phases of the translational process—initiation, elongation, and termination—can be faithfully reconstituted in the test tube with defined natural and synthetic mRNA templates. The establishment of such in vitro translation systems has underpinned not only studies into the mechanism of protein synthesis in both prokaryotes and eukaryotes, but has also provided a key experimental tool for the elucidation of the genetic code. While the fundamental details of how one prepares a cell-free translation system and uses it to translate mRNA templates have not really changed since Pelham and Jackson first described the mRNA-dependent reticulocyte cell-free lysate (1), progress has recently been made in refining these systems for the study of post-translational processing of secretory proteins (2, 3) and for generating quantitative yields of translation products (4). There have also been impressive developments in the area of in vitro transcription of cloned DNA templates (5) to generate both natural and engineered mRNAs for cell-free translation studies. Cell-free systems have been described from a relatively limited number of eukaryotic cells with most researchers still focusing on the rabbit reticulocyte and wheat germ systems primarily due to their commercial availability and reproducibility rather than any innate biological relevance.
Historical introduction
Authors: Gershoni J M 
Published: November 2023

Abstract
In 1975 Southern published his method for the identification of specific DNA fragments which had been resolved by gel electrophoresis via transfer of the band pattern to nitrocellulose membrane filters (1). This technique immediately gained recognition, proving to be a milestone in molecular biology, and has since been referred to as Southern blotting. It was not long thereafter that a new technique was developed in which the macromolecule to be analysed was RNA, also immobilized to a filter matrix; this technique was coined Northern blotting (2).
Protein blotting and immunoblotting using nitrocellulose membranes
Authors: De Maio Antonio 
Published: November 2023

Abstract
During the last decade protein blotting has emerged as a powerful technique for the study of proteins or glycoproteins immobilized on a membrane filter, without the necessity for painful protein purification procedures. The detection of antibody epitopes in total cell homogenates has been without any doubt the preferred application. However, the use of protein blotting has been expanded to other applications such as the detection of receptors through the binding of their respective ligands, physical chemical analysis of protein-protein interactions, and the study of glycoconjugates (see Chapters 12-14 of this text).