bluuebunny/crossref_metadata_2025_split huggingface dataset:
Linkage mapping of plant and animal genomes
Authors: Miller Ross 
Published: November 2023

Abstract
The human genome project has provided increasingly detailed physical (1-3) and genetic maps (4). A large number of other genomes are also being mapped: animal genomes under study include those of mouse (5), rat (6), sheep (7), pig (8-10), cow (11,12), dog (13), chicken (14), the pufferfish Fugu (15), zebrafish (16), honey bee (17), Drosophila (18), and the nematode C. elegans (19), whilst plant genomes being studied include rice (20), wheat (21), maize (22), Arabidopsis (23), spruce (24), tomato (25) and potato (25). These lists are intended merely to illustrate the breadth of the field rather than to be exhaustive.
Genomic libraries and the development of species-specific probes
Authors: Warner J P 
Published: November 2023

Abstract
The development of standard methods for reliable cloning and propagation of DNA fragments of various sizes has transformed our understanding of gene structure and regulation. The ability to generate representative libraries of sequences comprehensively covering the genome regardless of size, complex ity, or type of organism has enabled population geneticists to undertake fine resolution analysis of sequence variation. The purpose of this chapter is to describe general protocols for the construction and screening of genomic libraries and for the development of species-specific probes. Although the construction of libraries was traditionally regarded as technically demanding, modern high-quality enzymes, commercially prepared packaging extracts, competent cells, and complete cloning kits have made genomic cloning universally accessible. A high quality representative library of clones avail able for rapid re-screening whenever new sequences suitable for population analysis are described is ample reward for the time and effort invested in the initial library construction.
PCR protocols and population analysis by direct DNA sequencing and PCR-based DNA fingerprinting
Authors: Hoelzel A R , Green A 
Published: November 2023

Abstract
The polymerase chain reaction (PCR) has become a mainstay of molecular ecology and population genetic research in the brief time since its invention (1). By this technique, defined segments of DNA can be amplified to micro gram quantities from as little as a single template molecule. Although the procedure is in some ways deceptively simple, and the reaction can entail complex biochemical interactions, it is in most applications a fast, relatively inexpensive and easy way to generate ample material for further analyses. For this reason its application is now widespread and facilitating an ever greater diversity of research. From early applications to previously intractable or difficult procedures, such as the pre-natal diagnosis of sickle-cell anaemia, PCR has now been applied to a great variety of problems in molecular biology, including cDNA cloning (2) and mutagenesis (3). It is a natural for population genetic studies where large numbers of individuals need be screened at as fine a resolution as is practical and feasible. The combination of PCR amplifica tion and DNA sequencing has been especially powerful for these studies.
Automated DNA detection with fluorescence-based technologies
Authors: yid Victor A Da , menotti-raymond Marilyn 
Published: November 2023

Abstract
The use of fluorescence-based technologies in DNA detection systems has truly come of age. Automated DNA sequencing instruments, which were once relegated to core sequencing facilities at a few large institutions, are now found at thousands of laboratories throughout the world. The capabilities of these instruments have been expanded to encompass new technical develop ments such as microsatellites (1,2), random amplified polymorphic DNA (RAPD) markers (3,4), variable number tandem repeats (VNTRs; 5) and single-strand conformational polymorphism (SSCP; 6). The amenability of this technology to automation has made it especially useful in large-scale sequencing and genotyping projects; however, high sample throughput can be accomplished without expensive robotic systems. DNA storage, polymerase chain reaction (PCR) amplification, DNA sequence reactions, and sample loading can all be accomplished in a format compatible with a standard 96-well plate. This allows for the use of multi-channel pipettes to facilitate sample processing and multi-channel syringes for sample loading. This chapter will assess current applications of fluorescence-based DNA detection systems with emphasis on the two most widely used applications, DNA sequence analysis and microsatellite genotyping.
Radiolabelled Liposomes for Imaging and Biodistribution Studies
Authors: Goins Beth A , Phillips William T 
Published: November 2023

Abstract
The development of liposome-based agents for diagnostic and therapeutic applications requires an understanding of the blood clearance kinetics and tissue bio-distribution of the agents after administration into the body. Non-invasive biodistribution studies of liposomes labelled with gamma (photon)-emitting radionuclides performed with scintigraphic imaging is a powerful approach for studying liposome-based agents in vivo. This methodology permits determination of the distribution of liposomes at multiple time points in an individual animal or patient (1, 2). While invasive studies of liposomes labelled with beta-emitting radionuclides such as carbon-14 may be suitable for animal studies, human studies with non-invasive photon imaging is a significantly more efficient approach for liposome-based drug development.
Comprehensive mutation detection with denaturing gradient gel electrophoresis
Authors: Lerman L S , Beldjord Cherif 
Published: November 2023

Abstract
Since the basic laboratory procedures for denaturing gradient electrophoresis and the introduction of G,C clamps have not changed significantly from previous descriptions, and they depart only slightly from conventional gel electrophoresis, we provide guidance and references to published complete instructions. However, the computational preparations for mutation searching are critical to efficient application of the method. We provide a detailed introduction to the computational tools, their use in planning a search, and their role in interpreting results.
Mutation detection using T4 endonuclease VII
Authors: Youil Rima 
Published: November 2023

Abstract
The ideal method for analysis of genes implicated in human disease would be a simple, easy to perform procedure that utilizes standard technology available in most laboratories. It should offer high throughput capacity even when performed manually. The procedure should also lend itself to automation to allow for increased throughput, particularly for laboratories that routinely handle large numbers of samples. The ability of the scanning technique to localize the point of mutation would also be highly advantageous.
DNA detection and sequence distinction through oligonucleotide ligation
Authors: Landegren Ulf , Samiotaki Martina , Kwiatkowski Marek , Parik Juri , Nilsson Mats , Hagberg, Anette , Barbany Gisela 
Published: November 2023

Abstract
Synthetic oligonucleotides and DNA ligases are combined in a number of methods for gene analysis. Generally, two oligonucleotide probes are designed to hybridize immediately next to each other on a target DNA or RNA strand. Ends of the oligonucleotide probes, brought close by hybridization, can be covalently joined by a DNA ligase, provided that the probes are properly base paired to the target sequence at the junction. This joining act thus refiects the presence of the target sequence. Below, a method will be presented to use oligonucleotide ligation-assisted analysis to distinguish known variants of amplified DNA sequences.
Calcium and pH imaging in living cells
Authors: Parton Richard M , Read Nick D 
Published: November 2023

Abstract
Calcium and pH imaging, involving the use of ion-sensitive fluorescent dyes, have had a profound impact on our understanding of signal transduction in animal, plant, and fungal cells. This chapter provides an introduction to the techniques involved in using the light microscope to image and measure intracellular free calcium and pH within living cells and tissues. ‘Second messengers’ are intracellular mediators which provide important links in signal-response coupling within cells and act in both signal propagation and amplification (1). The most common and ubiquitous second messenger within eukaryotic cells appears to be Ca2+ (2-4). The role of pH has received far less attention as a second messenger. Nevertheless, a variety of cellular responses to stimuli involve changes in cytoplasmic pH (5-8). In addition, pH homeostasis is important in regulating numerous other aspects of cell metabolism (9-11).
Analysis of macrophage lytic functions
Authors: Varesio Luigi , Bosco Maria Carla , Carta Luca , Musso Tiziana 
Published: November 2023

Abstract
Mononuclear phagocytes, comprising circulating monocytes and tissue macrophages, are phylogenetically ancient cells deriving from the primordial amoeba that was enclosed into a complex pluricellular organism. During the course of evolution, macrophages retained some of the basic features of the amoeba, such as mobility and phagocytosis, and acquired new immunoregulatory properties as a result of the interaction with theemerging immune system. Engulfing bacteria and fighting with competitors are among the primordial functional activities of macrophages that pre-existed the development of the immune system and that are triggered by classical environmental stimuli such as bacteria, toxins, organic molecules, pH, osmotic changes, etc. The need to communicate with the immune system and with the neighbouring cells caused profound modifications in the number and nature of the signals recognized by macrophages, that learned to recognize distress signals derived from lymphocytes, such as lymphokines and antibodies, or from tissues, such as hormones, adhesion molecules, and matrix proteins.
Crystallization of membrane proteins
Authors: Howard Tina D , Mcauley-Hecht Katherine E , Cogdell Richard J 
Published: November 2023

Abstract
As yet, unfortunately, there are no high-resolution structures for any membrane transporters. This probably represents the major barrier to our understanding the precise molecular details of how this important class of membrane proteins actually work. An excellent example of the urgent need for structural information comes from the extensive studies on the lac-permease from Escherichia coli (1). Almost every amino acid in this transporter has been changed by site directed mutagenesis (H. R. Kaback, personal communication) and yet the exact details of how it works are still a mystery. It has been argued that the crystal structure of a transporter may not be such a ‘holy grail’, since it will only give a static picture and transport is, by definition, a dynamic process. However, the recent success in understanding how ATP is made by examining the structure of the mitochondrial F1-ATP synthetase (2, 3) illustrates how, when a structure can be ‘caught’ in several functional states, the dynamics of the reaction can be inferred. Since for many transporters there are specific inhibitors which can ‘lock’ the protein in different functional conformations, there is an excellent chance that when the structure of a transporter is determined it will be possible to deduce the mechanism.
Cryopreservation and rederivation of embryos and gametes
Authors: Glenister Peter H , Rall william F 
Published: November 2023

Abstract
This chapter describes optimized cryopreservation protocols for the long-term storage and maintenance of pre-implantation embryos and gametes from mouse models of human genetic disease, and of normal biological processes. It outlines the theory and practical application of cryopreservation procedures and discusses the quality control requirements to ensure the availability of mouse models for future study. Comprehensive reviews of the history, theory and application of germplasm cryopreservation are available elsewhere (1,2).
Raising antibodies
Authors: Kane* M M , Bankst J N 
Published: November 2023

Abstract
Antibodies are essential components of immunoassays, and the versatility of immunoassays results from the unique features of these proteins. Antibodies can recognize and bind to their antigen with high affinity and specificity, even when they are part of a complex mixture, enabling sensitive measurements of antigens or antibodies in a variety of matrices.
Phosphorylation. site analysis by mass spectrometry
Authors: Mitchelhill Ken I , Kemp Bruce E 
Published: November 2023

Abstract
The determination of phosphorylation sites on proteins is a vital part of their characterization, and an important step in understanding the structural basis of how phosphorylation modulates their functions. With the recent explosive growth in the use of mass spectrometry in the structural analysis of proteins and peptides, this approach has also become the method of choice for the elucidation of post-translational modifications, including phosphorylation. Many developments have taken place since early studies using fast-atom bombardment mass spectrometry (FABMS) in 1985 (1). Matrix-assisted laser desorption/ionization time-of-flight (MALDITOF), electrospray ionization (ESI) triple quadrupole mass spectrometry and its low flow rate version, nano-ESI mass spectrometry, are currently the most widely used forms of instrumentation in these studies. They are both sensitive and highly precise, and this chapter will be confined to these instrumentation types, with which we have practical experience. Hybrid instruments e.g. Q-TOF (quadrupole time-of-flight) are now available, or in advanced stages of development, from several manufacturers, as well as both electrospray and MALDITOF instruments with Fourier transform detection at the frontiers of precision. Other mass spectrometric analytical methodologies, including ion-trap methods, have been applied to phosphorylation site analysis. It is now essential for all laboratories involved in protein phosphorylation studies to have access to mass spectrometry if they are to contribute to understanding this pre-eminent regulatory mechanism.
Cloning and expression of cDNAs encoding protein kinase subunits
Authors: Carling David 
Published: November 2023

Abstract
Protein kinases form a large family of enzymes that play a key role in the control of a diverse variety of cellular processes, including the regulation of metabolic pathways, cell differentiation, and cell proliferation. Changes in the normal activity of protein kinases can lead to the development of a large number of different disease states. A critical step in our understanding of the precise regulatory role of protein phosphorylation within the cell, and the lesions that occur within this regulation, is the identification and characterization of all the protein kinases present in the organism. Whilst this may seem an ambitious goal at present, the rapid progress being made in several key areas of biology suggests that it will be achievable in the not too distant future. The completion of the Saccharomyces cerevisiae genome sequencing project has revealed that budding yeast contain 113 protein kinase genes, over half of which have known or predicted functions (1). Sequencing of the human genome is well underway, and it has been estimated that in humans there are at least 1000 genes encoding protein kinases (2), although in this case the percentage for which there is a known function is much smaller. Advances in gene cloning and manipulation have made analysis of mammalian genes, both in vitro and in vivo, a routine procedure. In order to elucidate the exact function of a specific protein kinase, isolation of the cDNAs encoding the protein kinase subunit(s) is an important and fundamental goal for the researcher. This chapter discusses the methods available for isolation of cDNAs encoding protein kinase subunits, including the new approach of ‘in silica’ cloning which has been made possible by the large-scale DNA sequencing projects currently being carried out around the world. Having isolated the cDNA, methods for expression of the protein kinase in heterologous systems are also described.
Crystallization of protein kinases and phosphatases
Authors: Kobe Bostjan , Gleichmann Thomas , Teh Trazel , Heierhorst Jörg , Kemp Bruce E 
Published: November 2023

Abstract
X-ray crystallography is the most powerful method for determination of macromolecular three-dimensional structures, and it has made enduring contributions to the understanding of the function of protein kinases and phosphatases. Determination of protein crystal structures requires single crystals that are suitable for high resolution X-ray diffraction analysis (Figure 1).Crystallization will only occur within a limited range of conditions that need to be empirically determined. Despite numerous successful crystallization experiments, our understanding of protein cry st allogenesis and macromolecular crystal growth processes remains limited. Crystallization of proteins can be broken down into two aspects: screening (based mainly on trial and error),where one attempts to obtain crystals; and optimization, where one strives to improve the diffraction quality of the crystals.
Kinetic analysis of nuclear receptor interactions
Authors: Cheskis B , Freedman L P 
Published: November 2023

Abstract
Steroid/nuclear receptors modulate transcription by virtue of their binding as part of a multiprotein complex to specific DNA sequences in promoter regions of target genes. This process directs a cascade of protein-protein and protein-DNA interactions affecting gene expression. Careful biochemical analysis or protein-protein and protein-DNA interactions is critical to the understanding of how genes are regulated by this family of ligand-modulated transcription factors. It is also a key to comprehending how small molecules. the cognate ligands for these receptors, affect receptor activity by directing the mode and amplitude of gene transcription.
Hormone-resistance syndromes
Authors: Gurnell M , Chatterjee V K K 
Published: November 2023

Abstract
The nuclear receptors comprise a superfamily of ligand-inducible transcription factor which exhibit a conserved structural organization with distinct functional domains. Members of this family include the glucocorticoid (GR), androgen (AR), oestrogen (ER). mineralocorticoid (MR), and progesterone (PR) receptors, together with those for thyroid hormone (TR), vitamin D (VDR). retinoids (RAK RXR). eicosanoids (PPAR), and an expanding number of ‘orphan· receptors whose ligand and function remain to be elucidated.
Bacteriology of the genital tract
Authors: Ison Catherine A , Davies Alun J , Hawkey Peter M 
Published: November 2023

Abstract
Whilst the diagnosis of genital infections by means of bacteriological examination is straightforward in those infections where an organism has a dear pathogenic role, such as is the case in gonorrhoea and syphilis, other infections are encountered where the contribution of diagnostic bacteriology is less dear. Culture of specimens from patients with genital infections may yield organisms such as Gardnerella spp., Listeria spp., and anaerobic bacteria, which, although capable of causing infections under suitable conditions, do not do so invariably. Unless an organism is generally recognized as a pathogen, its presence in large numbers does not provide evidence of pathogenicity.
Antimicrobial susceptibility testing
Authors: Brown Derek F J , Andrews Jenny , Winstanley Trevor , Alasdair P. MacGowan 
Published: November 2023

Abstract
In vitro antimicrobial susceptibility testing is undertaken in clinical laboratories to predict the likely in vivo response of the infecting organism to a selected range of antimicrobial agents. Susceptibility tests are designed to give a result interpreted as susceptible (sensitive. S), intermediate (moderately susceptible. moderately resistant, I), or resistant (R). A patient infected with a susceptible organism should respond to treatment with the manufacturer’s recommended dosage regimen. whereas one infected with a resistant organism is unlikely to respond. For an organism categorized as intermediate there is uncertainty whether or not the patient will respond to standard doses, but they will be more likely to respond to higher doses or if the agent is concentrated at the site of the infection, such as in the urinary tract. However, intermediate is a term which clinicians find unhelpful. and in practice the intermediate and resistant categories are combined and reported as resistant.
Quality control and quality assurance
Authors: Hyde W A , Curley P K 
Published: November 2023

Abstract
The late European Committee for Clinical Laboratory Standards produced a document on quality control for microbiology, which stated (1): “Internal quality control is the set of procedures undertaken by the staff of a laboratory for the continual evaluation of laboratory work and the emergent results, in order to decide whether they are reliable enough to be released to clinicians or epidemiologists.
Transition states in protein folding
Authors: Daggett Valerie , Fersht Alan R 
Published: November 2023

Abstract
Until the end of the 1980s the approach to protein folding concentrated on the gross characterization of folding intermediates and the refolding of well-known proteins such as bovine pancreatic trypsin inhibitor, ribonuclease A, and lysozyme. These studies utilized primarily circular dichroism and fluorescence, and chemical trap ping methods exploiting disulphide bridges. One of the major developments in the 1990s has been to focus on entire folding pathways at the level of individual residues and atoms. This became possible because of developments in three areas.
Protein folding coupled to disulphide-bond formation
Authors: Creighton Thomas E 
Published: November 2023

Abstract
Understanding in detail the mechanism of protein folding in vitro requires all the conformational states encountered during folding to be known, including the rate limiting transition state. On initiating refolding, different proteins adopt a wide variety of different conformations very rapidly, before folding to the native state. Some protein:-, remain unfolded, others adopt partly folded conformations, while many adopt a compact but disordered state that has come to be known as the ‘molten globule’ (MG) (1). These relatively stable partly folded and MG conformations are frequently assumed to be crucial for directing the folding process, and much effort has gone into characterizing them (Chapter 6). This is usually difficult to achieve, since they are present only transiently. Determining the kinetic roles of such intermediate species, as to whether they are on-pathway intermediates, kinetically trapped, or simply the initially preferred conformational state of the unfolded protein under refolding conditions, is especially difficult with a complex process like protein folding. There is little direct evidence about the kinetic roles of most folding intermediates that have been detected (2, 3).
Case study 1: The folding process of apomyoglobin
Authors: Wright Peter E , Baldwin Robert L 
Published: November 2023

Abstract
The folding of the globular protein apomyoglobin is characterized by well-populated intermediates which occur both transiently, as kinetic intermediates in folding, and at equilibrium near pH 4. If the structure of these intermediates could be determined, and if their relation to each other as part of a kinetic scheme could be rigorously established, then a significant contribution would have been made to understanding the folding process of this model protein. Further, characterization of these inter mediates as persistent conformational species on a folding pathway would constitute an important clement to bring to the ‘classical’ versus ‘new view” debate.