bluuebunny/crossref_metadata_2025_split huggingface dataset:
Telomeres and mammalian genome analysis
Authors: Kipling David 
Published: November 2023

Abstract
This chapter reviews the cloning and sequence organization of human telomeres, their integration with the genetic map, and how their structure compares with those in the mouse. Evidence for true terminal deletions, clustering of VNTR (variable number of tandem repeats) loci in proterminal regions, terminal map expansions, and telomere–telomere exchanges in humans will be reviewed, as will the ability to manipulate chromosome structure experimentally by telomere-associated chromosome fragmentation. Some internal (TTAGGG)n arrays may be the remnants of ancestral telomere fusion events, which will be illustrated by the evolution of human chromosome 2. Telomeric sequences can be used to investigate a number of aspects of chromosome structure and behaviour, such as fragile sites, and in particular can reveal cryptic translocations in patients with apparently normal karyotypes. Finally, a potential link between the unusual genetic behaviour of (TTAGGG)n repeat arrays, as exemplified by those of the mouse, and the instability of short repeated sequences which underlie some human genetic diseases will be discussed.
Atninoacyl-tRNA synthetase-tRNA recognition
Authors: Arnez John G , Moras Dino 
Published: November 2023

Abstract
Aminoacylation is the first and key step in the translation of genetic messages into proteins. The reaction brings together amino acids and their cognate tRNAs and is catalysed by aminoacyl-tRNA synthetases (aaRSs). In the reaction the amino acid is esterified to one of the hydroxyl groups of the 3'-terminal adenosine; the energy in the overall reaction is supplied by the hydrolysis of ATP.
Interaction of 5S RNA -with TFIIIA
Authors: Pieler Tomas 
Published: November 2023

Abstract
Among the structural motifs which identify DNA- and RNA-binding proteins in eukaryotes, zinc finger modules are unique in defining the nucleic acid-binding domain in proteins with a demonstrated ability to form sequence-specific com plexes with either RNA or DNA or even with both classes of nucleic acids (1). Transcription factor IIIA (TFIIIA) is the founding member of the zinc finger protein superfamily, which is comprised of several hundred members in vertebrates (2, 3). TFIIIA is one of the most abundant proteins in immature Xenopus laevis oocytes, where it is found primarily in a complex with 5S ribosomal RNA (4). The key structural feature in TFIIIA comes from nine copies of a sequence motif that has been termed zinc finger, since two pairs of invariant cysteines and histidines mediate the three-dimensional (3-D) folding of two antiparallel !3-sheets and one a helical element, mainly by coordination of zinc (Figure 1). The zinc finger duster in TFIIIA is not only responsible for the specific recognition of 5S RNA, but also promotes sequence-specific binding to the internal control region of the 5S RNA gene, thereby defining the first step in the formation of an active transcription complex (5, 6).
Assaying inositol phospholipid turnover in plant cells
Authors: Brearley Charles A , Hanke David E 
Published: November 2023

Abstract
The inexorable growth in the complexity of inositol phospholipid and inositol phosphate science, principally in the animal field but also in yeast, has served to emphasize our lack of understanding of even the fundamentals in plant cells. The fragmented nature of plant inositol phosphate research is a primary problem (1). The lack of the plant equivalent of the avian or human erythrocyte, which features throughout this chapter as a model system in which virtually all the methods described here were developed or tested, holds back the efforts of many plant scientists. Thus, there is much to be gained from seeking a simple and, most importantly, rigorous description of the identities of the numerous inositol phosphates and inositol phospholipids present in plant tissues before proceeding to the rather more complicated task of investigating their function.
The purification and assay of inositide binding proteins
Authors: Theibert Anne B , Prestwich Glenn D , Jackson Trevor R , Hammonds-Odie Latanya P 
Published: November 2023

Abstract
A valuable strategy towards understanding the physiological roles of inositol polyphosphates (InsPns) and inositol lipids (PtdlnsPns) will be the identification, characterization, isolation, and molecular biological and genetic manipulation of their intracellular receptors. Several experimental approaches have been used to isolate InsPn and PtdlnsPn binding proteins, including conventional chromatography and reversible ligand binding, InsPn affinity column chromatography, and covalent modification of binding proteins with photoactivatable ligands. These approaches have led to the identification of an unexpected variety of InsPn and PtdlnsPn binding proteins, whose functions are only now being uncovered.
Mass assay of inositol 1,4,5-trisphosphate and phosphatidylinositol 4,5-bisphosphate
Authors: Challiss R A John 
Published: November 2023

Abstract
Elucidation of the phosphoinositide cycle (by which PtdIns(4,5) P 2 is hydrolysed, and Ins(l,4,5) P 3 and DAG are metabolized for re-incorporation into cellular the phosphoinositide pool), and studies which have highlighted the emerging roles of PtdIns(3,4,5) P 3 and higher inositol polyphosphates in cellular regulation, have been achieved largely by employing radioisotopic labelling strategies. Although radiolabelling methods continue to be invaluable for addressing many of the unresolved questions concerning phosphoinositides and cellular regulation, methods for the mass determination of key cycle intermediates can offer a number of clear advantages in terms of speed/throughput, reproducibility and cost. In this chapter methods for the mass assay of two critical phosphoinositide cycle intermediates are described.
The Ins(1,4,5)P
3 receptor
Authors: Mikoshiba Katsuhiko 
Published: November 2023

Abstract
Inositol 1,4,5-trisphosphate (Ins(l,4,5)P 3) is the second messenger derived from the hydrolysis of phosphatidylinositol bisphosphate via activation of phospholipase C. The Ins(l,4,5)P 3 binds to its specific receptor, which is an Ins(l,4,5)P 3-induced Ca2+-releasing channel located on the endoplasmic reticulum. In this way the Ins(l,4,5)P 3 signal is converted to a Ca2+ signal.
Affinity methods for isolating RNA binding proteins
Authors: Jackson Richard J 
Published: November 2023

Abstract
The need or wish to purify a particular RNA binding protein usually originates from the following type of scenario: At this stage it becomes important to purify and characterize the particular RNA binding protein(s). However, it is worth appreciating that purification is usually not an end in itself. Most commonly the aim is limited to obtaining sufficient protein in sufficient purity for amino acid sequencing, which then allows either the protein to be directly identified if it is already in the databases, or the isolation of cDNA clones leading ultimately to expression as recombinant protein. Thereafter all further work, including the raising of antibodies, exploits the recombinant protein if at all possible. The only reason, though it is an important one, why purified protein from the original source would still be required is to verify that the recombinant protein exhibits the same properties and binds to the RNA element with the same specificity and affinity.
A three-hybrid system to detect and analyse RNA:protein interactions in vivo
Authors: Zhang Bejlin , Kraemer Brian , Sengupta Dhruba , Fields Stanley , Wickens Marvin 
Published: November 2023

Abstract
RNA:protcin interactions arc critical in diverse cellular processes, such as splicing. translation, early development, and infection by RNA viruses. For example, the replication cycle of human immunodeficiency virus (HIV-1) relics on the interactions between reverse transcriptasc and the RNA genome to generate viral DNA, between Tat and TAR to produce mRNA, between Rev and RRE to export mRNA, and between viral proteins and the RNA genome to assemble viral particles. Few general methods are available to detect and analyse RNA:protein interactions in vivo. In vitro.
Prevention Of Unwanted Proteolysis
Authors: North Michael J , Beynon Robert J 
Published: November 2023

Abstract
To many biochemists and cell biologists, proteinases are not so much interesting enzymes or useful tools but persistent nuisances. while many proteins seem to be immune to the ‘ proteinase problem’ , the frequency with which artefacts arise indicates that a cautious approach is justified. Moreover, the availability of accurate mass from new mass spectrometric methods may well bring to light more examples of unwanted proteolytic degradation, especially terminal trimming that might have previously remained undetected through the low resolution of SDS-PAGE.
Neuronal promoter analysis by in vitro transcription using nuclear extracts from rat brain
Authors: Schwartz M L , Schlaepfer W W 
Published: November 2023

Abstract
We present a reliable method for making nuclear extracts from rat brain and for using these extracts in an in vitro transcription assay. Our nuclear extract technique allows for the isolation of nuclear proteins from post-mitotic neurones that presumably contain all of the factors required for the transcriptional regulation of neurone-specific genes. The use of brain as a source of nuclear extract is in contrast to the more commonly used cultured cell lines where expression of neurone-specific genes may be poor or lacking in tissue specificity. In vitro transcription using nuclear extracts from expressing (brain) and non-expressing (liver) tissues provide a direct assessment of promoter strength and level of tissue-specificity apart from the effects of higher order DNA structure (i.e. chromatin). In vitro transcription is also amenable to DNA competitor studies and to the addition of purified or partially purified protein factors and antibodies. In this way, true neurone-specific transcriptional activator or repressor sequences or factors can be characterized. The approach is rapid, reliable, and easily quantifiable. Finally, advancements in the field of nucleosome reconstitution have allowed the coupling of in vitro transcription to nucleosome assembly systems and provide a novel and promising approach to the understanding of neuronal-specific gene expression.
Cell culture
Authors: Blake K , Stacey A 
Published: November 2023

Abstract
The ability to culture cells in vitro has enabled enormous advances to be made in biology, particularly in virology. Many cell types can be routinely pro liferated and cryopreserved, allowing consistent, characterized, contaminant free cells to be used both as a research and diagnostic tool. However, despite the utility of cell culture, researchers must recognize that cells grown in vitro may not behave as cells in the intact organism. For example, the proliferation of cells in vitro is not necessarily conducive to full differentiation, where cell growth is normally absent. The potential loss of differentiated characteristics will inevitably affect properties and therefore the utility of cells. Cell behaviour will also be affected by the loss of cell-cell, and cell-matrix interactions, and loss of tissue architecture. The goal of attaining a greater range of in vivo characteristics is becoming more achievable with the increased availability of growth and differentiation factors, and the development of three-dimensional culture systems which allow cells to assume their correct polarities, shapes and interrelationships. The future perspective looks promising, and cell culture systems may be becoming available which enable in vitro growth and study of currently non-cultivable viruses (Chapter 2).
Antiviral drug testing
Authors: Oxford J S , Kelly L S , Davies S , Lambkin R 
Published: November 2023

Abstract
The clinical effectiveness of acyclovir, and more recently penciclovir, against herpes simplex type I and II infections (1) has led both to great expectations of further discoveries of antiviral compounds and to a change in the scientific direction of clinical diagnostic laboratories. Most such laboratories are at present used primarily to diagnose viral infections, either by culture or serology, or, more often, by detection of viruses using rapid molecular techniques such as PCR. The advent of the new generation antivirals such as protease inhibitors of HIV-1 (2), and neuraminidase inhibitors of influenza (3-5), together with new molecular methodologies, now requires clinical laboratories to quantify virus, usually by PCR or by molecular probing. Also, the therapeutic benefit of antiviral therapy has been enhanced by the use of PCR to analyse viral genes and search for drug-resistant mutations (6). Nowhere is this more important than with HIV, where, on the one hand, a choice of antivirals exists, and on the other hand the introduction of combination chemotherapy has led to a substantial increase in demand for scientific information on which to base clinical decisions.
Mutagenesis of RNA virus genomes
Authors: Barclay Wendy S , Almond Jeffrey W 
Published: November 2023

Abstract
Since the pioneering experiments of Burnett (1) and Cooper (2) in the 1950s and 1960s, the isolation of RNA virus mutants has been a cornerstone of studies aimed at determining the function of virus genomes and their encoded proteins. Not only have the phenotypes of individual mutants been informative about the replication and biology of the wild-type virus, but, collectively, the exploitation of mutants in complementation and recombination assays has provided useful information on the number of genes and the sizes of genomes. Nowadays the generation of mutants is a routine procedure exploited widely in structurefunction studies and for the development of designer viruses as vaccines, vectors, and reporter systems for studies on gene expression and genome replication.
Detection and analysis of host gene targets for oncogenic retroviruses
Authors: Neil J C , Terry A 
Published: November 2023

Abstract
Unlike the DNA viruses, where oncogenic representatives are found in disparate families, only two RNA virus families, the Retroviridae and the Flavoviridae, have so far been implicated in neoplasia. The mechanisms by which the latter operate are largely unknown and the prototypic example, hepatitis C virus, appears to be an indirect cause of cancer, for which the roles of specific gene products are as yet undetermined. In contrast, the Retroviridae have received much attention as causes of cancer. Animal retroviruses have been studied for many years, mainly as model carcinogens; their chief contribution has been through the identification of oncogenes that play a role in cancers of diverse aetiology. This field of work is the main focus of this chapter. However, it should be noted that these agents can be important causes of disease in their natural host species, and that since the discovery of human retroviruses there is much wider interest in retrovirus pathogenesis and methods of their control.
Direct assessment of conformational changes in G protein-coupled receptors, using fluorescence spectroscopy
Authors: Gether Ulrik , Kobilka Brian 
Published: November 2023

Abstract
G protein-coupled receptors (GPCRs) represent the largest class of transmembrane signalling molecules. The mechanism by which hormones and neurotransmitters activate intracellular signalling cascades through these homologous proteins is of great interest. While GPCRs all share a common seven-helix transmembrane structure, domains involved in ligand binding are nearly as diverse as the chemical structures of the known agonists. Small molecular weight ligands bind to sites within the hydrophobic core formed by the transmembrane (TM) a-helices, while binding sites for peptides and protein agonists also include the amino terminus, and extracellular hydrophilic loops joining the transmembrane domains (1). This diversity in ligand binding suggests that different classes of receptors may have unique mechanisms of activation. However, when one considers that all these receptors function by activating a highly homologous family of G proteins, it is reasonable to propose that agonist binding is linked to G protein activation through changes in the conformation of the transmembrane domains, and that what we learn about receptor activation from one class of receptors will apply to most, if not all, members of this large receptor family. It is generally assumed that binding of the agonist to the receptor induces a set of finely orchestrated changes in the tertiary structure of the receptor, that are recognized by the associated G protein a-subunit.
Probing the structure of receptor-binding sites by the substituted-cysteine accessibility method
Authors: Simpson Merrill M , Liapakis, George , Javitch Jonathan A 
Published: November 2023

Abstract
The substituted-cysteine accessibility method (SCAM) provides an approach to map systematically the residues on the water-accessible surface of a binding site, such as the binding-site crevice of a G protein-coupled receptor (GPCR) which binds ligand within the transmembrane portion of the receptor. Consecutive residues in putative membrane-spanning segments are mutated to cysteine, one at a time, and the mutant receptors are expressed in heterologous cells. The water-accessible residues are identified by assessing the reaction of charged, hydrophilic, sulfhydryl reagents with the engineered cysteines.
Post-translational acylation of signal transducing polypeptides: palmitoylation of G protein-coupled receptors
Authors: Bouvier Michel , Petäjä-Repo Ulla , Loisel, Thomas , Bélanger Charlène 
Published: November 2023

Abstract
In recent years, a number of proteins involved in signal transduction have been found to be acylated by fatty acids or prenyl derivatives. Three major classes of fatty acylation have been characterized. These are: (a) the Nterminal myristoylation of glycine residues through amide linkage; (b) the prenylation of cysteine residues by the formation of thioether links to farnesyl or geranylgeranyl moieties; and (c) the palmitoylation which occurs through the thioesterification of cysteine residues. In contrast to myristoylation and prenylation, which typically occur co-translationally, palmitoylation is a genuine post-translational modification (1). The chemical reactivity of sulfhydryls and thioester linkages have led several investigators to propose that protein palmitoylation may represent a reversible modification which could undergo dynamic regulation. In fact, rapid turnover of the covalently attached palmitate has been reported for several proteins, and this turnover is modulated by external stimuli (2-4).
G protein-coupled receptor phosphorylation and desensitization
Authors: Tobin Andrew B , Rae, Angela , Budd David C 
Published: November 2023

Abstract
Phosphorylation of G protein-coupled receptors (GPCRs) has been linked with receptor desensitization, primarily through work on the 13-adrenergic receptor(l-3). Extensive studies have demonstrated that agonist stimulation of the !3-adrenergic receptor results in phosphorylation of the receptor by two kinases; protein kinase A (PKA), and the receptor-specific kinase !3-adrenergic receptor kinase (GRK-2) (1-3). Each of these kinases phosphorylates distinct sites on the receptor. PKA sites are at RRSS motifs in the third intracellular loop and C-terminal tail (4), and GRK-2 sites are in an acid serine/threoninerich region of the C-terminal tail (5). Phosphorylation at both PKA and GRK-2 sites results in receptor desensitization (1). In the case of GRK-2- mediated desensitization the mechanism has been demonstrated to involve the intermediary protein !3-arrestin, which binds to the phosphorylated receptor and in doing so is able to ‘displace ‘ the Gas-protein, thereby uncoupling the receptor from adenylyl cyclase (6). Cloning studies have revealed that GRK-2 is a member of a protein kinase family called the G protein-coupled receptor kinase (GRK) family (7). There are at present six members of this family (GRK-1 to GRK-6); each is thought to be able to mediate agonist-stimulated phosphorylation of certain GPCRs (7).
Construction and analysis of receptor-G protein fusion proteins
Authors: Wise Alan 
Published: November 2023

Abstract
It is now well established that a multitude of diverse extracellular signals such as neurotransmitters, hormones, odorants, and light elicit intracellular responses via vectorial signal transduction mechanisms across the lipid bilayer, following binding to specific cell surface receptors possessing seven transmembrane-spanning domains. Agonist-occupation of these receptors leads to activation of effector proteins, and the concomitant mobilization or production of second messengers that initiate the desired physiological response within the cell. In all eukaryotic organisms the family of heterotrimeric G proteins plays a pivotal role, acting as intermediates between such cell surface receptors and cytoplasmic or membrane-bound effector molecules, leading to the regulation of humoral, neural, metabolic, and developmental functions. Examples of intracellular effectors which are regulated by G protein activation include adenylate cyclase, phosphoinositidase C, cyclic GMP phosphodiesterases, phospholipase A2, and a number of ion channels (1, 2).
Analysis of the polyphosphorylated inositol lipids of Saccharomyces cerevisiae
Authors: Dove Stephen K , Michell Robert H 
Published: November 2023

Abstract
The yeast Saccharomyces cerevisiae offers several advantages to the researcher interested in determining the functions of phosphoinositides in eukaryotes. The ease with which this yeast can be genetically manipulated, coupled with knowledge of the entire nucleotide sequence of its genome, means that certain approaches can be used which cannot yet be applied to other organisms. For instance, genes that encode proteins involved in the synthesis, degradation, or effector functions of phosphoinositides can be deleted or rendered partially defective, and the phenotypes of the mutants determined. In this way, the contribution of an enzyme or lipid to the operation of a signalling system can rapidly be gauged. By screening for suppression of the phenotypes of such mutants, genes whose actions lie upstream or downstream of a mutated gene can be identified and characterized. In addition, genetic complementation studies are used to test whether the function(s) of an inactivated yeast gene can be replaced by expression of an apparently orthologous gene from another organism (such as a mammal).
Detection, isolation, and quantitative assay of mitogenactivated protein kinases in intact cells and tissues
Authors: Anderson Neil G 
Published: November 2023

Abstract
The landmark discovery of the insulin-stimulated protein kinase, originally termed microtubule-associated protein 2 (MAP-2) kinase, by Sturgill and colleagues in 1987 (1), was of fundamental importance in initiating the huge advances since made in our understanding of how messages received by receptors on the cell surface are transduced to the cell interior to produce specific responses. Although originally isolated from insulin-stimulated 3T3- Ll adipocytes, MAP-2 kinase was subsequently shown to be present in all cells, to be activated in response to an enormous variety of extracellular stimuli, and to have been evolutionarily conserved from yeast to man (see ref. 2 for a review). Appreciation of the ubiquitous nature of MAP-2 kinase led to it being renamed mitogen-activated protein (MAP) kinase. Since then, a number of related protein kinases have been isolated. These all have similar structures and mechanisms of activation, but co-exist in cells as members of parallel signalling pathways stimulated by distinct types of extracellular stimulus. Three major classes of MAP kinase have, so far, been characterized in detail.