bluuebunny/crossref_metadata_2025_split huggingface dataset:
Targeting precursor proteins to mitochondria
Authors: Lithgow Trevor , Schatz Gottfried 
Published: November 2023

Abstract
In order to grow and divide, eukaryotic cells need mitochondria. This statement is underscored most dramatically by the observation that deletion of any of the several genes essential for mitochondrial biogcnesis kills yeast cells (1) and that failure to direct at least one mitochondrion into the daughter cell after mitosis prevents the final steps of yeast cell division (2). Mitochondria cannot be made de novo (3). To assemble a new mitochondrion, a eukaryotic cell directs specific precursor proteins, synthesized in the cytosol, to the surface of its existing mitochondria, from where the precursors are directed into the organelle and sorted to one of the four submitochondrial compartments (Fig. 1). Assembly of new precursors into existing mitochondria then leads to the birth of new organelles through a poorly understood fission process. A long-standing question has been how eukaryotic cells target mitochondrial precursors specifically to that organelle.
HPLC of oligonucleotides and polynucleotides
Authors: Andrus Alex , Bloch Will 
Published: November 2023

Abstract
This chapter reviews applications of HPLC to two distinct classes of macromolecular analyte: single-stranded oligonucleotides and duplex polynucleotides. These two targets differ in several important respects apart from size and conformation, namely: origin; end-uses; position in a laboratory hygiene scheme; requirements for separation speed, throughput, and cost; goals of the separation; and maturity of the separation technology. Whereas optimized protocols are widely applied to oligonucleotide HPLC, polynucleotide HPLC is not yet popular enough to have evolved beyond the experimental stage of development. This contrast will be evident in what follows, which emphasizes refined procedures for oligonucleotide separations and tactical issues for polynucleotide analysis.
Modern molecular biology depends on nucleic acid separations at both ends of many procedures. Synthetic oligonucleotides should be purified and evaluated for quantity and purity before use as primers, probes, adapters, mutagenesis templates, anti-sense reagents, or ribozymes. In addition, qualitative or quantitative analysis of polynucleotide reaction products usually requires product resolution from excess reagents and side-products, either before or during the analysis. A multi-stage process, such as the sequencing or cloning of DNA amplified by the polymerase chain reaction (PCR), may require an intermediate purification so that preparative (e.g. amplification) reagents and side products do not interfere with downstream (e.g. sequencing) chemistry.
Glycosylphosphatidylinositols and the surface architecture of parasitic protozoa
Authors: Mcconville Malcolm J 
Published: November 2023

Abstract
A functionally diverse family of eukaryotic cell surface proteins are anchored to the plasma membrane via glycosyl-phosphatidylinositol (GPI) glycolipids (for recent reviews see Refs 1 and 2). These glycolipids constitute an alternative anchoring mechanism to the transmembrane polypeptide domain of type I membrane proteins. GPI-anchored proteins make up a variable proportion of the protein repertoire on the cell surface of complex metazoan organisms, but are usually less abundant than proteins with a transmembrane polypeptide domain. In contrast, GPI anchors are found on the most abundant surface proteins of many parasitic protozoa, including the African trypanosomes, Trypanosoma cruzi, Leishmania spp., Plasmodium spp. and Toxoplasma gondii (2) and are thus essential in maintaining the surface architecture of these organisms.
Expressing cloned genes in Escherichia coli
Authors: Bass Steve , Yang Maria 
Published: November 2023

Abstract
The expression of cloned genes in Escherichia coli for the production of recombinant proteins has provided a valuable system for studying protein structure and function. Production of recombinant proteins in L. coli is the basis or the multibillion dollar biotechnology industry. Proteins from E. coli or smaller eukaryotes like yeast arc usually easily over-produced in E coli. However. mammalian proteins are often more difficult. A combination of factors can conspire against the researcher to prevent high-level production or recombinant proteins.
Evolutionary theories of genomic imprinting
Authors: Hurst Laurence D 
Published: November 2023

Abstract
As deleterious recessive somatic mutations are hidden in diploids but not in haploids, and as most mutations are probably deleterious recessives, a multicellular organism is usually better off with two sets of chromosomes rather than one (for references and discussion see ref. 1). Why then do some genes (i.e. imprinted genes) behave as though they are haploid, with the same copy inactive in many tissues? A variety of hypotheses have been proposed to account for the evolution of imprinting. It is the function of this chapter to critically review and evaluate these. This is prefaced, however, with a brief word on how this chapter is different from the others in the book.
The instability of differentiation in hepatomas
Authors: Goss S J 
Published: November 2023

Abstract
In 1965, Henry Harris showed that viable heterokaryons could be made between HeLa cells and normal differentiated cells as distinctive as rat lymphocytes, hen erythrocytes, and rabbit macrophages; and in these heterokaryons he detected reactivation of RNA and DNA synthesis in the previously quiescent nuclei. Cell fusion was rapidly established as a powerful tool in the investigation of differentiation, as is clear from the intense activity in this field in the immediately following years (Ringertz and Savage 1976). In work with growing hybrid cells, it is liver differentiation that has been most extensively studied. Well-differentiated hepatomas are available that maintain a wide range of liver-specific traits even when they are grown rapidly in standard tissue culture media. Their phenotype is generally stable through repeated cloning (Deschatrette and Weiss 1974), and the usefulness of the system has been increased by the development of media and conditions that select either for or against liver-specific traits (Leffert and Paul 1972; Haggerty et al. 1975; Choo and Cotton 1977). When a hepatoma is fused to a non-liver cell, the simplest outcome is the cross-activation of liver-specific genes derived from the non-liver parent (Peterson and Weiss 1972; Darlington et al. 1974; Malawista and Weiss 1974; Brown and Weiss 1975).
Cancer suppression by the retinoblastoma gene
Authors: Lee Wen-Hwa , Goodrich, David , Lee Eva 
Published: November 2023

Abstract
A concentrated effort toward the molecular cloning of cancer suppressor genes had an important recent success: a candidate retinoblastoma (RB) gene was cloned, identified, and extensively characterized by us and others (Friend et al. 1986; Fung et al. 1987; Lee et al. 1987a). Our demonstration of the tumour suppression activity of this single gene when transferred into tumour cells containing inactivated endogenous RB genes constituted a critical milestone for the theory of cancer suppression (Huang et al. 1988). Since several other cancers are known to be familial, are associated with chromosomal deletions, or exhibit loss of heterozygosity, an extension of this concept to other genes and to other cancers is widely anticipated (Malkin et al. 1990).
Roles of the myc gene in cell proliferation and differentiation, as deduced from its role in tumorigenesis
Authors: Klein George 
Published: November 2023

Abstract
Myc, myb, fos, rel, and ski are oncogenes that code for DNA binding nuclear phosphoproteins that can trans-activate other genes and can stimulate DNA replication, directly or indirectly. They may contribute to tumour development and/or progression, after having been activated by structural and/or regulatory changes (Eisenmann 1989). The best known gene of this category, c-myc (Penn et al. 1990), is also most widely implicated in spontaneous tumour development in vivo. It is the cellular counterpart of v-myc, which has been isolated from four different acute avian leukaemia viruses. c-myc and v-myc can induce a broad spectrum of tumours, such as myelocytomatosis, endotheliomas, liver and kidney carcinomas, and erythroblastosis. One strain can also transform fibroblasts.
Human Linkage Mapping
Authors: Curran Julie L 
Published: November 2023

Abstract
The basic principles of linkage mapping have been used by scientists for many years, to establish whether a particular disease is associated with a specific chromosomal region. Linkage amongst simple Mendelian traits is often observable at the phcnotypic level. and the chromosomal rearrangements associated with some disorders could he located cytogenetically. But even in combination, these two approaches gave an extremely sparse map by today’s standards. One of the first loci to be mapped was that for zonal, pulverulent cataracts (CAEI) on lq21-q25, linked to the Duffy blood group (FY) at the same chromosomal location in 1969 (1).
Contig assembly by fingerprinting
Authors: Gregory Simon G , Soderlund, Carol A , Coulson Alan 
Published: November 2023

Abstract
A previous chapter in this series (1) described, primarily, the physical mapping of the 100 Mb Caenorhabditis elegans genome by fingerprinting of cosmid clones, and the linking of the contigs thus derived by YAC hybridization. At that time, the primary function of the map was to enhance the molecular genetics of the organism by facilitating the cloning of known genes, and to serve as an archive for genomic information. However, a clonal physical map—even with good alignment to the genetic map—carries only a tiny proportion of the information present in the genome. Consequently, the current objective of the C. elegans genome project (2) is to establish of the entire genomic sequence. The bacterial clone map, although incomplete by virtue of the uncloneability of regions of the genome in cosmid vectors (a factor which we shall discuss later in this chapter), has proved a sound basis for the systematic sequence analysis. The sevenfold cosmid coverage has a resolution sufficient to enable the selection of a subset of cosmids for sequencing such that, on average, each clone contributes 30 kb of unique sequence to the whole.
Separation by charge, size, and hydrophobicity
Authors: Millner Paul A 
Published: November 2023

Abstract
The material in this chapter, which covers perhaps the most generic forms of chromatography, is written very much from a pragmatic standpoint and results from experience of chromatography of every sort. Where appropriate, links have been drawn with later chapters, that cover more specific chromatographic approaches, although many of the procedures and precautions within this chapter are generic to all chromatographic procedures. General maxims for chromatographic work are to do the fewest chromatography runs possible and as quickly as possible. These aims stem not only from a desire for speed, but often from constraints imposed by the viability of the sample or target protein. The protocols presented are basic chromatographic procedures which cover the most common situations encountered.
Synthetic peptides as affinity ligands
Authors: Reynolds Gary J , Millner Paul A 
Published: November 2023

Abstract
There are many reasons for wanting to construct an immobilized peptide matrix. Purification of a specific antibody is one typical application. In this approach the antibody to be purified would either be raised against peptide conjugated to a carrier of some sort and the same peptide used to construct the matrix for purification. Alternatively, the antibody may have been raised against a whole protein and the peptide-matrix used to select antibodies binding to a specific epitope. The latter is the basis for many of today ‘s highly specific and powerful protein purification procedures using peptide affinity chromatography.
Cleavage using RNase to detect mutations
Authors: Nagase H , Nakamura Y 
Published: November 2023

Abstract
Recent advances in the human genome project will allow us to obtain information on the whole human genomic sequence and to map the positions of genes responsible for human hereditary diseases including both simple and complex genetic trait diseases. This information will provide a large number of candidate genes which will be used to identify the real responsible gene and assess its functions. For this purpose detection of mutations in genes is one of the most crucial techniques required. In order to screen for mutations, three methods have been frequently employed; detection of conformational changes between mutant and normal sequence, cleavage of mismatched heteroduplex, and direct sequencing. Conformational change detection, such as single-strand conformation polymorphism (SSCP) (1), denaturing gradient gel electrophoresis (DGGE) (2), and heteroduplex analysis (HA) (3), are most commonly utilized because of relatively easy handling and high sensitivity. However, there are some disadvantages in these methods. One main problem is a restriction of the fragment size for analysis; for SSCP this is less than 200 bp (4), for HA it is 200-300 bp, and for DGGE it is less than 600 bp (5). In SSCP and HA, sensitivity for special mismatches is condition-dependent, and DGGE require about 60 bp oligonucleotides with a G,C clamp to obtain high sensitivity (6). Chemical cleavage methods can analyse relatively long fragments, more than 1 kb. Among chemical cleavage methods, very early studies used SI and Mug bean nucleases.
Detection of mRNA in tissue sections with radiolabelled riboprobes
Authors: Simeone Antonio 
Published: November 2023

Abstract
Gene expression studies have been greatly advanced by techniques developed to visualize gene expression during embryonic development and tissue differentiation. Among these, in situ hybridization of sections with radiolabelled probes has a historical significance because the in situ expression of mRNA in invertebrate and vertebrate development was first described using this pro¬ cedure. Although alternative techniques are now available to identify gene transcripts in tissues and embryos (1, 2), in situ hybridization on sections with radiolabelled RNA probes is frequently found to be the more sensitive and reliable procedure. This chapter describes protocols for in situ hybridization with radiolabelled RNA probes on embryo and brain sections. In addition, a list is provided of diagnostic probes useful at specific stages of development for defining the expression domains of a given gene.
The amplified expression, identification, purification, assay, and properties of hexahistidine-tagged bacterial membrane transport proteins
Authors: Ward* Alison , Sanderson* Neil M , O’Reilly* John , Rutherford* Nicholas G , Poolmanꝉ Bert , Henderson* Peter J F 
Published: November 2023

Abstract
In bacteria, 3-15% of genes are predicted to encode proteins involved in membrane transport (1), a process which is vital for both the capture of nutrients and the excretion of waste products, toxins, and antibiotics. The energy required for these processes is derived from the electrochemical gradients of ions across the cell membrane (in bacteria the majority operate by proton-driven symport or antiport mechanisms but some utilize a sodium gradient) or from the hydrolysis of ATP (2). Whilst the 3-D structures of thousands of soluble proteins have been determined, to date only 21 membrane protein structures have been solved to atomic resolution (3, 4). The elucidation of the structures of membrane proteins using physical techniques is, therefore, a key area of research.
Spectroscopic and kinetic approaches for probing the mechanisms of solute transporters
Authors: Walmsley Adrian R 
Published: November 2023

Abstract
This chapter deals with the use of spectroscopic methods to monitor conformational changes in membrane transporters and kinetic approaches to time-resolve these changes. It outlines the use of fluorescence spectroscopy to monitor ligand-membrane protein interactions and the use of stopped-flow fluorescence spectroscopy to determine mechanisms of ligand binding and translocation. It also outlines the use of rapid-quench techniques for measuring the rate constants governing the translocation of the substrate across the membrane. To a large extent, these studies are illustrated by reference to the kinetics of substrate and inhibitor binding and translocation by various transporters studied in the author’s laboratory.
Detection and analysis of glucose transporters using photolabelling techniques
Authors: Gillingham Alison K , Koumanov Françoise , Hashimoto Makoto , Holman Geoffrey D 
Published: November 2023

Abstract
The use of photolabels to study biological systems has emerged as a powerful technique for tagging proteins, and the range of approaches and types of ligand that can be employed have been extensively reviewed (1). Although various affinity labelling approaches have been tried (for example using the highly reactive isothiocyanate group substituted into maltose (2) ), these have generally been unsuccessful in labelling glucose transporters. This difficulty can be largely attributed to the low affinity of glucose transporters for their substrate and the poor tolerance for groups substituted around the glucopyranose ring. Specificity studies (3) indicate that there is a close approach of the binding site around the sugar as it enters from the external solution. These properties of the glucose-transporter binding site have limited the range of approaches that can be used in photoaffinity labelling studies. Often photoreactive substituents are rather bulky and hydrophobic, and based on benzoic acid substituted with azides or diazirine substituents.
Mouse care and husbandry
Authors: Hetherington Colin M , Doe Brendan , Hay Donald 
Published: November 2023

Abstract
This chapter illustrates methods for the care and husbandry of mice. It out-lines various types of animal accommodation and methods of identification, record keeping, breeding systems, timed mating, superovulation and re-derivation. Mice may be housed under of a variety of conditions. The type of accommodation and husbandry will depend on the nature of the animal experimentation and financial considerations. The more sophisticated the methods used, the greater the cost of building, equipping and running the facility. A balance also must be struck between ease of access and working and the level of pathogen barrier used.
Radiolabelling of monoclonal antibodies
Authors: Mather Stephen J 
Published: November 2023

Abstract
Antibodies have been tagged with radionuclides since the 1940s when David Pressman, perhaps the first to recognize the potential of radiolabelled anti bodies in vivo, radiolabelled polyclonal antibody preparations with iodine-131, a radionuclide first produced by artificial means only a decade or so previously (1). Today the researcher may wish to radiolabel an antibody for a wide variety of different applications, many of which are described in other chapters in this book.
lmmunoassays
Authors: Peppard Jane V 
Published: November 2023

Abstract
This chapter describes methods by which monoclonal and also polyclonal antibodies may be used in order to quantitate antigens. Since the choice of format is extensive, the information is divided so that options for each stage of the assay, e.g. choices of label or solid support, which can be ‘mixed and matched’ according to requirements, are discussed individually. Finally, the most frequently used methods of immunoassay and their set-ups are described.
Immunofluorescent labelling of sections
Authors: North Alison J , Small J Victor 
Published: November 2023

Abstract
Over the past two decades, immunofluorescent labelling of cells and tissues has become one of the most powerful tools in cell biology. It is commonly performed in two ways: labelling of whole cells and labelling of sections. The problem with the first approach lies in rendering the intracellular targets accessible to the detecting antibodies. This necessitates some form of membrane permeabilization, generally using detergents or alcohols, or a direct physical approach such as micro-injection. Unfortunately such manipulations can lead to artefacts including ultrastructural changes and the loss or re distribution of the target antigen. Moreover, not all parts of the structure may be equally accessible to the antibody, even after extensive permeabilization. We have found this to be particularly pertinent in densely packed structures such as muscle (1). For this reason, the use of sectioned tissue, in which all cellular sub-structures are rendered unequivocally accessible at the section surface, can often prove to be the optimal procedure for consistent and reliable immunolocalization studies.
Green fluorescent protein in plants
Authors: Hawes Chris , Boevink Petra , Moore Ian 
Published: November 2023

Abstract
The introduction of the jellyfish green fluorescent protein (GFP) and its wavelength-shifted derivatives as reporter proteins for both gene expression and protein location has provided cell biologists with a powerful new tool with which to study cell structure and function in vivo. The protein has a number of intrinsic properties that make it attractive as a marker for cell biological studies. For instance, it is highly fluorescent and requires no co-factors or substrates other than oxygen to form the fluorochrome. It is relatively stable and pH insensitive, has a low level of cytotoxicity and in many instances does not interfere with the functioning of native proteins when expressed attached to them as chimeric constructs. Moreover, it has been genetically modified to alter its spectral and other properties such as folding rate, to make a family of tailor-made reporter proteins (1).
Ran
Authors: Macara Ian G , Brownawell Amy , Welch Katie 
Published: November 2023

Abstract
Ran is a very unusual GTPase. Unlike all other known members of the Ras superfamily of small GTP-binding proteins, Ran is not post-translationally modified. Moreover, its function depends critically on a spatial gradient of the GTP-bound form of Ran. This requirement sets it apart from other small GTPases that act in signal transduction pathways or in vesicle fusion and budding. Ran is concentrated in the nucleoplasm and plays a central role in active nucleocytoplasmic transport through the nuclear pore complex (NPC). It may have additional functions, but these remain to be identified. The vectoriality of transport and the accumulation of cargo in the nuclear or cytoplasmic compartments against a concentration gradient appear to be driven by the Ran-GTP gradient across the NPC. In this limited sense, therefore, nuclear transport behaves like a coupled transmembrane transport system, similar, for example, to the coupling of glucose accumulation to a sodium ion gradient. At a molecular level, however, Ran-mediated transport is unique, and is based on a complex sequence of protein-protein interactions that are controlled by the guanine-nucleotide-bound state of the GTPase. Despite the remarkably rapid progress being made in identifying the players in the nuclear transport machinery, we remain ignorant of many of the fundamental aspects of the transport mechanism.