bluuebunny/crossref_metadata_2025_split huggingface dataset:
Methods for studying mouse natural killer cells
Authors: Welsh Raymond M 
Published: November 2023

Abstract
Natural killer (NK) cells are bone marrow-derived, thymus-independent, large granular lymphocytes (LGL) which, without specificity or antigenic memory, lyse on contact a variety of target cells (1, 2). The prototypic NK cells lack T cell receptor or CD3 mRNA or proteins (3). NK cells lyse a variety of tumour cells and certain normal cells such as thymocytes. Infection of target cells with any of a variety of viruses contributes to enhanced NK cell-mediated lysis of those targets. The cytotoxic capacity of NK cells can be greatly augmented by interferon (α, β, or γ) and by interleukin-2 (IL-2), resulting in enhanced kinetics of lysis of highly sensitive targets and a greatly expanded target range encompassing most cultured cell lines. Interferon and IL-2 also contribute to the proliferation of NK cells. Virus infection, as a result of stimulating very high levels of IFN (α and β), causes marked activation and proliferation of NK cells in vivo. Studies in the mouse have characteristically shown a peak in NK cell activity and number early in infection, followed by a peak in cytotoxic T cells later in infection (2). In keeping with the general orientation of this book, most of the following discussion will focus on NK cells in the mouse, but many of the techniques and general principles discussed can also be applied to studies in man and other animals.
Culture of adult mammalian peripheral neurons
Authors: Lindsay Ronald M , Evison Caroline J , Winter Janet 
Published: November 2023

Abstract
The vast heterogeneity of both the neuronal and glial elements which constitute the nervous system has proved to be a major barrier to studies to elucidate the precise physiological function and underlying biochemistry of individual subclasses of neurons and glia. As in many other systems, tissue culture has been widely used in attempts to simplify such studies. In the nervous system, tissue culture has been particularly useful in studying factors that regulate the development of the peripheral nervous system (PNS). Explant and dissociated cultures of peripheral neurons, for example, have been invaluable in the discovery and explanation of the key role played by growth and survival factors (e.g. nerve growth factor — NGF (1, 2), brain-derived neurotrophic factor — BDNF (3, 4), and ciliary neurotrophic factor-CNTF (5)), substrate adhesion factors (e.g. the extracellular matrix molecules such as laminin, collagen, and fibronectin), and cell-cell adhesion molecules (N-CAM, NG-CAM) in the development of the PNS (6).
Photolabile calcium buffers to selectively activate calciumdependent processes
Authors: Gurney Alison M 
Published: November 2023

Abstract
Optical probes provide a particularly powerful means for manipulating the concentration of free calcium inside or outside neurons. Photosensitive calcium buffers are molecules that either release or sequester calcium upon irradiation with appropriate wavelengths. The experimental usefulness of these probes lies in the speed of the photochemical reactions, as well as the ease with which the intensity, duration, and area of activating light can be varied. Thus the light could be directed at a whole population of cells, or be focused into a spot small enough to irradiate a single cell or part of a cell. The approach therefore allows the possibility of studying how a neuron responds to changes in the calcium concentration imposed throughout the cell, or localized to one part of the cell. The probes currently available include nitr-5 (1) and DM-nitrophen (2), which evolved from two separate series of molecules that release calcium upon photolysis. Both of these agents can be obtained from Calbiochem Corp., PO Box 12087, San Diego, California 92112-4180, USA, or Novabiochem (UK) Ltd, 3 Heathcoat Building, Highfields Science Park, University Boulevard, Nottingham NG7 2QJ. Agents that mop up calcium after photolysis have also been described (3, 4), but are not yet available commercially. All of these probes are calcium chelators that incorporate a photolabile group, which upon photolysis results in molecules with altered affinities for calcium. This chapter discusses practical aspects in the use of the currently available probes, nitr-5 and DM-nitrophen, for studying calcium-dependent phenomena in neurons.
Mutagenesis facilitated by the removal or introduction of unique restriction sites
Authors: Carter Paul 
Published: November 2023

Abstract
Over the last decade, a variety of efficient and reliable methods have been developed for the construction of site-directed mutations in DNA using synthetic oligonucleotides (reviewed in refs 1-3). This has revolutionized the study of gene regulation and protein -structure and function. All of these mutagenesis methods fall into one of three broad strategies. One approach uses an oligonucleotide complimentary to part of a single-stranded (ss) DNA template but containing an internal mismatch(es) to direct the desired mutation (see Section 1.1, and Chapters 2 to 5 and 9). A second strategy is to replace the region of interest with a synthetic mutant fragment generated by annealing complimentary oligonudeotides (‘cassette mutagenesis’ (ref. 4 and Chapter 10)), or by hybridization and ligation of a number of oligonucleotides (reviewed in ref. 5). The third and most recent strategy, has been to harness the power of the polymerase chain reaction (PCR) (6) to generate a mutant fragment starting from a double-stranded (ds) DNA template using mismatched oligonucleotides (refs 7 and 8, and Chapter 11). In this chapter an efficient method of site-directed mutagenesis using mismatched oligonucleotides (first strategy above) is discussed.
Cassette mutagenesis
Authors: Richards John H 
Published: November 2023

Abstract
Cassette mutagenesis provides a powerful and versatile approach for the generation of mutations and allows a wide range of studies of the relationship between base sequence and function in nucleic acids or between structure and function in proteins. The central aspect of this approach is the replacement of a region of DNA with new sequences that can be of any length, any sequence or any mixture of sequences. Thus, in studying the basis for protein function one can introduce not only changes of a few amino acids (an important aspect of primer-extension in vitro mutagenesis) but can also create chimeric proteins in which entire domains of a protein have been replaced with a region of totally different amino acid sequence.
Expression cloning: transient expression and rescue from mammalian cells
Authors: Aruffo Alejandro 
Published: November 2023

Abstract
Cloning strategies based on transient expression in mammalian cells have been used effectively to isolate CDNAs encoding secreted, cell surface, and intracellular proteins. The first application of transient expression in mam malian cells in cloning was the isolation of CDNAs encoding the lymphokine GM-CSF (granulocyte macrophage colony stimulating factor) (1, 2). This was followed by the use of transient expression in mammalian cells in conjunction with antibody panning (3) to isolate CDNAs encoding cell surface proteins (4, 5). Transient expression in mammalian cells has also been used to isolate cell surface receptors based on the ability of the transfected cells to bind to either fluorescein-conjugated (6) or radiolabelled (7) ligands or ligand immobilized on a solid phase (8, 9) or ligand expressed on a cell surface (10). Recently, transient expression in mammalian cells has also been used to isolate CDNAs encoding intracellular proteins (11, 12).
Biochemical methods for studying supramolecular complexes involving cell adhesion molecules, integral membrane proteins, and the cytoskeleton
Authors: Nelson W James , Wilson, Rachel , Mays Robert W 
Published: November 2023

Abstract
During development of complex tissue patterns, cells undergo dramatic structural and functional reorganization (1). The inductive process(es) leading to cellular differentiation often involves specific cell-cell and cell-substratum interactions (2, 3). These interactions result in new programmes of gene expression and the spatial redistribution of plasma membrane and cytoplasmic proteins in line with the specialized structural and functional requirements of the fully differentiated cell. Understanding the mechanisms involved in the regulation of protein distributions during cellular differentiation is one of the most interesting and experimentally challenging areas of cell and developmental biology.
Looking for protein-protein interactions
Authors: Midgley Carol A , Lane David P 
Published: November 2023

Abstract
The specific interactions of protein molecules to form homo- and heterooligomers underlie many key events in biology, from the assembly of viruses to the formation of multisubunit protein machines of great complexity. Traditionally the study of such interactions has demanded the availability of large amounts of purified components, and the members of a complex have been identified through their extensive copurification. These techniques are restricted to the analysis of tight associations between relatively abundant proteins, and cannot easily be used to study the biological regulation of a protein complex in different cellular environments.
Pre-mRNA splicing factors in the yeast Saccharomyces cerevisiae
Authors: Hodges Peter e. , plumpton, mary , Beggs jean D 
Published: November 2023

Abstract
The budding yeast Saccharomyces cerevisiae provides a model system to study pre-mRNA splicing using a combination of classical genetics, molecular biology, and biochemistry {see 1-4 for other recent reviews). Attention must be paid to those aspects of yeast splicing that are not general to all eukaryotes. Initial discoveries left some doubt about the universality of the yeast mechanism. For instance, the yeast splicing machinery has more stringent requirements for branchpoint and splice site sequences (4), yeast Ul and U2 snRNAs arc unusually large (5-7), and initial genetic screens for yeast splicing mutants failed to identify genes encoding any of the snRNP proteins known from mammals (see below). However, the universal nature of spliceosome function is now accepted; many splicing factor homologues are recognized as conserved between organisms, and the mechanisms of spliceo-some assembly, deduced from mammalian and yeast splicing systems, are identical. This review summarizes the discoveries found in yeast splicing, compares splicing in yeast and in other eukaryotes, and enumerates the advantages of working with yeast as a model system.
Drosophila Embryos
Authors: Forbes Zandy , Ingham Philip 
Published: November 2023

Abstract
Adult fruitflies of the species Drosophila melanogaster are easily maintained in the laboratory. At 25 °C, the generation time of Drosophila is 9 days and embryogenesis is completed in 22 h. Traditionally, large numbers of flies are grown in half-pint milk bottles with a solid nutrient medium in the base; alternative containers include plastic fruit juice bottles or urine sample containers. Stocks of flies can be maintained at 18 °C in smaller tubes with the same medium. For 25 bottles or 120 tubes containing nutrient medium, boil 8 g agar in 1 litre tap water for 3 min, add 80 g corn (maize) meal, 18 g dried yeast, and 10 g soya flour (premixed with some of the water), plus 80 g malt extract and 40 g molasses. Boil for 20 min, cool to 50-80 °C, add 6.6 ml acid mix (a mix of 500 ml propionic acid and 32 ml phosphoric acid), and pour.
Zebrafish embryos
Authors: Warga Rachel m. 
Published: November 2023

Abstract
Zebrafish (Brachydanio rerio) embryos have gained considerable popularity in recent years because they offer several advantages for developmental studies. They are cheap and easy to breed, can be obtained all year round and the embryos are transparent. The latter are easy to manipulate and develop quite rapidly: from egg to hatching takes roughly 3 days.
Xenopus
Authors: Altaba Ariel Ruiz I 
Published: November 2023

Abstract
Amphibian embryos have a prominent place among the species used to study vertebrate embryonic development. There is a long and distinguished history of experimental manipulations that were carried out in salamanders and newts. Perhaps the best known example is the 1924 organizer transplant experiment of Hilde Mangold (1), then a student in Hans Spemann’s lab. This experiment was not an isolated event, rather it was the culmination of a long search for the basis of the formation of embryonic tissues and their patterning in several laboratories, including that of Spemann. To understand the roots of present day molecular embryology, the reader is invited to read the fascinating book by Viktor Hamburger (2) written 50 years after the appearance of Spemann’s magistral work (3).
Induction Assays in Xenopus
Authors: Taba Ariel Ruiz I Al 
Published: November 2023

Abstract
The study of inductive interactions requires the identification of tissues that produce the inductive signal and tissues that are competent to respond (1). The identification of the signal as well as the study of the molecular mechanisms underlying induction requires the development of an assay system with which to test different preparations and factors. Amphibian embryos have been used to identify and to define a series of embryonic inductions. Two of these, the induction of mesoderm and the induction of neural tissue, are crucial in the formation of the vertebrate body plan and were first described in amphibian embryos (2-4). In the first case, vegetal cells signal overlying animal cap cells at the equator of the blastula-stage embryo to change fate from ectodermal to mesodermal. In the second case, mesodermal cells signal to adjacent animal cap cells to change fate from epidermal to neural during gastrulation.
Embryonic Stem Cells and Gene Targeting
Authors: Robertson Elizabeth J , Martin Gail R 
Published: November 2023

Abstract
Embryonic stem cells (ES cells), in conjunction with protocols for targeted gene disruption, have made it possible to introduce mutations into specific genetic loci in mammalian embryos. ES cells are permanent tissue culture lines of pluripotential stem cells derived from the inner cell mass of the normal mouse embryo (1,2). They can be readily manipulated in culture and genetically altered cell clones then used to generate chimaeric animals following their microinjection into preimplantation stage carrier embryos. As ES cells reproducibly populate both the somatic cell lineages and the germ line, they afford a means of introducing mutations into the mouse genome. The discovery that mammalian cells can undergo a process of homologous recombination (site-specific recombination between exogenously introduced cloned DNA and the chromosomal cognate gene), has facilitated the generation of mice carrying a range of targeted genetic alterations, from large deletions to single nucleotide changes (3).
Purification of over-produced proteins from E. coli cells
Authors: Grisshammer Reinhard , Nagai Kiyoshi 
Published: November 2023

Abstract
Many biologically interesting proteins are present naturally in small quantities and are not amenable for structural and biochemical characterizations. The molecular mechanisms of gene expression in Escherichia coli became well understood in the late 1970s, allowing the over-production of foreign proteins from their cloned genes. A large proportion of new protein structures reported in 1990 and 1991 were derived from heterologously over-produced proteins (1) and this unequivocally demonstrates that the technique of overproduction has now become an essential tool for the study of protein structure and function. We mainly focus in this chapter on the purification of overproduced proteins from E. coli cells. It is a well-known fact that overproduced proteins are not always soluble and active in E. coli and often aggregate as insoluble inclusion bodies. As re-folding of proteins is difficult, it is desirable to produce proteins in a soluble and active form. We first describe how inclusion body formation can sometimes be avoided. We then detail purification methods for both soluble and insoluble proteins. Each protein has its own ‘personality’ and the methods described in this chapter should be regarded as a collection of experiences which may direct readers to the successful purification of their own proteins.
Library construction in Pl phage vectors
Authors: Sternberg N 
Published: November 2023

Abstract
The Pl cloning system was developed as an alternative to YAC and cosmid technology for the cloning of high molecular weight DNA fragments. It permits the recovery of inserts in E. coli that are up to 95 kb in size (more than twice the size of inserts that can be recovered in cosmids) with an efficiency that is intermediate between ;... cosmid and YAC cloning; greater than 105 clones can be generated using the Pl system with 1-2 µg of vector arms and 2-4 µg of Sau3Al digested, size selected genomic DNA insert (1). While the size of cloned insert recovered with the Pl system are significantly smaller than arc those recovered with YACs, the former offen, several advantages: libraries representative of multiple copies of the genome arc more easily produced, larger quantities of a particular cloned DNA are more easily isolated, the cloning process may be more faithful (2, 3), and physical dissection of cloned inserts may be more easily accomplished. Indeed, the ability to analyse cloned DNA before and after attempting amplification with the Pilytic replicon permits one to assess whether amplification of the insert by a high copy number replicon can indeed occur, and if so, whether DNA integrity is maintained.
Genetic mapping with microsatellites
Authors: Naom Isam S , Mathew, Christopher G , Town Margaret-Mary 
Published: November 2023

Abstract
Simple tandemly repeated sequences, or microsatcllites, are ubiquitous in the genomes of a wide range of organisms, and the number of repeats within many of them is highly variable in the population of a particular species. The advent of the polymerase chain reaction (1) provided the means for rapid analysis of the repeat number, and several groups showed that these sequences were likely to provide a rich source of very informative markers for genetic mapping (2-4).
Transposon and T-DNA mutagenesis
Authors: Aarts Mark G M , Koncz Csaba , Pereira Andy 
Published: November 2023

Abstract
Insertional mutagenesis with a known mobile DNA insert can generate mutations that are marked by a molecular tag. When the insertion disrupts a gene, causing a mutant phenotype, the tagged mutant gene can easily be cloned using the DNA insert as a molecular probe. DNA sequences of the cloned mutant gene can then be used to isolate the corresponding wild-type allele. This procedure, known as gene tagging, facilitates the analysis of both mutant and gene.
Analysis of cell death by flow cytometry
Authors: Darzynkiewicz Zbigniew , Li Xun , Gong Jianping , Hara Shinsuke , Traganos Frank 
Published: November 2023

Abstract
Flow cytometry allows one rapidly and accurately to measure individual cells in large cell populations. Because several cell attributes can be measured simultaneously and the data are recorded in a mode that allows one to relate all the measured attributes to a particular cell (‘list mode’), their multivariate analysis provides information on the relationship between these attributes. In contrast to biochemical analysis in bulk, cellular heterogeneity can be estimated and cell populations with distinct characteristics can be discriminated. If needed, cells with particular features, can be electronically sorted. These advantages have all contributed to the fact that flow cytometry has become a methodology of choice in a variety of functional and structural assays of the cell, including cell viability.
A model of glandular epithelium for studying secretion
Authors: Adessi Gérard L , Beck Laurent , Mahfoudi Abderrahim 
Published: November 2023

Abstract
The endometrium is a complex tissue consisting of the endometrial epithelium supported by a mesenchymal tissue called the endometrial stroma. The endometrial epithelium comprises the luminal epithelium, which faces the uterine lumen, and the glandular epithelium which invaginates into the endometrial stroma. The endometrium is a classic target tissue for ovarian steroids. It becomes a proliferative tissue under the action of oestrogens and, after ovulation, is changed by progesterone into a secretory tissue which accommodates ovum implantation. The cyclic morphological and functional differentiation of the whole endometrium under the effect of hormones represents the response of individual cell types within a complex signalling network.
Elements of chromatin structure: histones, nucleosomes, and fibres
Authors: Holde Kensal Van , Zlatanova Jordanka , Arents, Gina , Moudrianakis Evangelos 
Published: November 2023

Abstract
The subject of this book is chromatin, its structure, and how this structure functions in regulation of the eukaryotic genome. The biochemists’ operational definition of chromatin is probably the best we can do at present: chromatin is the DNA-protein complex extracted from lysed interphase nuclei. Just which of the multitudinous substances present in a nucleus will constitute a part of the extracted material will depend in part on the techniques each researcher uses. Furthermore, the composition and properties of chromatin vary from one cell type to another, during development of a specific cell type, and at different stages in the cell cycle. Nevertheless, we can provide at least a rough description of the features common to chromatin of most eukaryotic cells (1-3).
Metabolic Flux Analysis
Authors: Holms William H 
Published: November 2023

Abstract
Flux analysis is a method of organizing data which shows the quantitative relationships among all the metabolic events going on in a fermentation. It is a simple description of what is actually happening in the fermentation and does not, in itself, consider the theory of how fermentations are limited or controlled. However, once the analysis is complete, it defines the ballpark within which the game is actually being played and the interpretation of this can often suggest what might be the factors limiting the process engineer’s aspirations for his/hers defined goals. By definition, the biomass always does the best it can with what is available, unless human intervention has deliberately deranged its genetic machinery or put it into a deliberately misleading environment.
2-5A-Antisense Chimeras for Targeted Degradation of RNA
Authors: Silverman R H , Maran A , Maitra R K , Waller C F , Lesiak K , Khamnei S , Li G , Xiao W , Torrence P F 
Published: November 2023

Abstract
The 2-SA-antisense approach under development in our laboratories seeks to substantially enhance the efficiency of antisense oligodeoxyribonucleotides (oligos) in vivo by harnessing the activity of a ubiquitous intracellular endoribonuclease called 2-SA-dependent RNase (RNase L) (l-3). In this strategy, an activator of RNase L, the 2’,5’-linked tetraadenylate pA(2’p5’ Ah, is conjugated through linkers to antisense oligos. As a result, the cell’s ability to control RNA decay is reprogrammed to degrade RNA molecules of choice. We have used this strategy to demonstrate selective and catalytic destruction of target mRNA molecules in assays containing homogeneous RNase L, in crude cell-free systems and in intact human cells. Here we present our protocols to synthesize and analyse the activity of2-5A-antisense species for the ablation of target RNA molecules. Current work, to be described elsewhere, aims to further enhance the stability, activity. and mode of synthesis of2-5A-antisense.