bluuebunny/crossref_metadata_2025 huggingface dataset:
Some Varieties of Pope’s Classicism
Authors: Hopkins David
Published: November 2023

Educated eighteenth-century English culture was permeated at every level by the art, history, mythology, philosophy, and literature of ancient Greece and Rome. School and university curricula were dominated by the study of classical (and particularly Latin) texts, in ways that had changed little since the Renaissance. Schoolboys and undergraduates were drilled not only in classical poetry, philosophy, and oratory but also in the historical, geographical, medical, math- ematical, and legal lore of the ancient world. Figures from republican Rome attained the status of cult heroes among the English ruling classes. Eminent politicians and land-owning grandees had themselves sculpted in the manner and garb of virtuous Romans. Young aristo- crats and gentlemen on the Grand Tour visited the monuments of Rome, and later Herculaneum and Pompeii, and brought back with them physical relics of the classical past. (Travel to Greece became more common as the century progressed.) Parliamentary speakers modelled their orations on those of Cicero and Demosthenes.
Neural specializations for speech and pitch: moving beyond the dichotomies
Authors: Zatorre Robert J , Gandour Jackson T
Published: November 2023

The idea that speech processing relies on unique, encapsulated, domain-specific mechanisms has been around for some time. Another well-known idea, often espoused as being in opposition to the first proposal, is that processing of speech sounds entails general-purpose neural mechanisms sensitive to the acoustic features that are present in speech. Here, we suggest that these dichotomous views need not be mutually exclusive.
Language processing in the natural world
Authors: Tanenhaus Michael K , Brown-Schmidt Sarah
Published: November 2023

The authors argue that a more complete understanding of how people produce and comprehend language will require investigating real-time spoken-language processing in natural tasks, including those that require goal-oriented unscripted conversation. One promising methodology for such studies is monitoring eye movements as speakers and listeners perform natural tasks. Three lines of research that adopt this approach are reviewed: (i) spoken word recognition in continuous speech, (ii) reference resolution in real-world contexts, and (iii) real-time language processing in interactive conversation.
More on sets and functions
Authors: Sutherland Wilson A
Published: November 2023

In the previous chapter we assumed familiarity with a certain amount of notation and terminology about sets and functions; but some readers may not yet be as much at ease with the concepts in the present chapter. In topology the idea of the inverse image of a set under a map is much used, so it is good to be familiar with it. If you are at ease with Definitions 3.1 and 3.2 below, then you could safely skip the rest of this chapter. (If in doubt, skip it now but come back to it later if necessary.)
Metric spaces
Authors: Sutherland Wilson A
Published: November 2023

The motivation for metric spaces comes from studying continuity. We begin by rephrasing Definition 4.29 using more English and no Greek: a real-valued function of a real variable is continuous at a if we can make the distance f (x) f (a) between f (x) and f (a) as small as we please by choosing x so that the distance x a between x and a is sufficiently small.
More concepts in metric spaces
Authors: Sutherland Wilson A
Published: November 2023

We now explore some more concepts in metric spaces which generalize notions from real analysis and which in turn will be generalized to topological spaces later.
Topological spaces
Authors: Sutherland Wilson A
Published: November 2023

In this chapter, we make our final leap into generality: we introduce topological spaces as our ultimate framework for studying continuity. At the end of the last chapter we saw that the open sets in a metric space are the most important elements when defining continuity. In the light of the remarks there, the following is a plausible definition.
Continuity in topological spaces; bases
Authors: Sutherland Wilson A
Published: November 2023

Now we come to the main point of topological spaces as far as analysis is concerned: the definition of continuity. The second part of this chapter concerns bases, which are useful in considering continuity.
Subspaces and product spaces
Authors: Sutherland Wilson A
Published: November 2023

In this chapter we consider two ways of getting new spaces from old— subspaces and product spaces.
Connected spaces
Authors: Sutherland Wilson A
Published: November 2023

Intuitively a connected space is one which does not fall apart into two or more pieces. To make this mathematical we need a precise definition of ‘fall apart’. We should probably agree that in the real line the sub-space [0, 1] is connected while the subspace [0, 1] [2, 3] is not. But what about the subspace Q of R, or the subspace of the plane consisting of the graph of y = sin(1/x) for x > 0 together with the line segment on the y-axis joining the points (0, 1) and (0, 1) (see Figure 12.1)? It is not so easy to decide intuitively whether these should be considered to be connected.
Mathematics teaching, teachers and students at St Andrews University, 1765–1858
Authors: Craik Alex D D , Roberts Alonso D
Published: November 2023

St Andrews University in Scotland has had a chequered six-hundred-year history. The mid-eighteenth to mid-nineteenth century was a period of slow recovery from near-ruin and dereliction. Yet among the professors and assistants were some notable scholars and teachers, and several former students rose to national prominence. We outline the university environment, the teaching of mathematics at St Andrews, and the careers of its main teachers and most notable students. Nicolas Vilant was Professor of Mathematics for over forty years, from 1765 to 1807; but, incapacitated by ill-health, he mostly employed a series of assistants to teach in his stead.
The Cambridge Ritualists, 1876–1924: A Study of Commensurability in the History of Scholarship
Authors: Lubenow W C
Published: November 2023

If we are to be true and worthy heretics we need not only new heads, but new hearts, and, most of all, that new emotional imagination, joint offspring of head and heart which is begotten of enlarged sympathies and a more sensitive habit of feeling. About the moral problem this is nothing mysterious; it is simply the old, old question of how best to live together. We no longer believe in an unchanging moral law imposed from without. We know that a harder incumbency is upon us; we must work out our law from within. Greek studies were important in the nineteenth century because they contained properties of stabilization and subversion and served as an agent of liberalism.
Organ-specific autoantibodies
Authors: Bird A G
Published: November 2023

Autoimmune diseases are conditions in which structural or functional damage to tissues or organs result from immunological mechanisms. Either humoral or cellular mechanisms or both can be responsible for the pathology seen in these diseases. The ease of detection of antibodies reacting with a range of tissue antigens has led to the description of a large range of autoantibody specificities. Some react against common cell constituents such as nuclear components or mitochondrial antigens and are thus detectable in different tissue substrates in contrast to those with restricted tissue specificity. The presence of autoantibodies does not necessarily imply an underlying disease process, especially in elderly subjects. Cellular immune responses are, for technical reasons, more difficult to identify and have received less attention. This is partly because of the need to separate and culture lymphocytes in order to identify specific T-cell responses, the relative scarcity of appropriate target tissues and the severe MHC restriction imposed on the study of T-lymphocyte responses.
Detection of immune complexes and antibody mediated target cell lysis
Authors: Oldstone Michael B A
Published: November 2023

Experience with a large number of viral infections in animals and humans indicates that immune complexes form and can be detected both in the circulation, as well as in target tissues, where they localize. Presumably, the interaction of viral antibody with virus or viral antigens in the circulation during acute and chronic virus infection not only serves as a marker for the presence of virus but also produces immune complexes that commonly cause nephritis and arteritis. Immune complex deposits are the source of tissue damage that is immunopathologically similar in humans and animals and is likely to be responsible for several of the signs and symptoms that accompany acute viral infections.
Synthetic peptides as antigens
Authors: Dyrberg Thomas , Kofods Hans
Published: November 2023

Antibodies have always had a prominent place in the armamentarium available to research. In general, antibodies to complex antigens obtained from immunized animals or through hybridoma technology are characterized by a high degree of specificity; however, the exact position of the binding site on the antigen is only rarely known. Further, the production of such antibodies requires that the antigen is available, either in a purified state or as part of a heterogeneous mixture. One approach to circumvent some of these problems has been to generate antibodies to defined epitopes of proteins, using synthetic peptides, representing sequences of the proteins in question, as antigens (1-3).
Isolation of potassium currents
Authors: Lancaster B
Published: November 2023

Potassium movement across the membrane can occur in the form of up to seven different currents. The challenge for the experimenter then is not to isolate potassium from other ionic currents, but to separate these variants of K current from each other. To start with a framework we will consider the following broad categories of K current that have been identified-although be warned that complications to this scheme will appear later.
Phosphorothioate-based site-directed mutagenesis for singles-tranded vectors
Authors: Sayers Jon R , Eckstein Fritz
Published: November 2023

Oligonucleotide-directed mutagenesis allows the molecular biologist to alter a defined nucleotide sequence within a gene in a precise manner. This alteration, a mutation, may be a single mismatch, i.e. a transition or transversion, or it may involve the insertion or deletion of one or more bases. The effect of a mutation may manifest itself in the production of a new protein on expression of the modified gene. Most often the proposed active site of an enzyme is manipulated in this way. However, protein engineering is not the only application for site-directed mutagenesis (1. 2). The technique also has applications in the study of molecular recognition. Alterations in promoter sequences (3), repressor binding sites, and other regulatory sequences can be systematically introduced and the effect of the changes studied. The basic approach to oligonucleotide-directed mutagenesis results in low mutational efficiency for the variety of reasons discussed in Chapter 1. As a consequence large numbers of putative mutant clones must be screened in order to obtain a clone with the required sequence. This involves a lot of effort and often the use of large amounts of radioactivity for hybridization screening or the sequencing of large numbers of clones. Thus, several strategies have been developed which aim to increase the efficiency of the basic site-directed mutagenesis procedure. Our method provides one such solution, while others are presented in Chapters 1 and 2 of this volume.
Recombination and mutagenesis of DNA sequences using PCR
Authors: Horton Robert McArn , Pease Larry Readington
Published: November 2023

The polymerase chain reaction makes possible a new method for engineering genes and is useful for both mutagenesis and recombination of sequences. This approach has significant advantages over current techniques because it is fast, straightforward, and not limited by the need for restriction sites. The development of recombinant DNA technology can be divided into three generations. The first generation approaches take advantage of natural recombination mechanisms in vivo to map and study genes. Into this category would fall classical genetic linkage analysis, and the use of mechanisms for homologous recombination between phages, for example, to generate sets of random recombinants between related genes. The second generation began with the use of restriction endonucleases and DNA ligase to specifically take genes apart, and put them back together in new combinations in vitro. It was the advent of this technology that made the field of molecular genetics what it is today.
Electron microscopical enzyme histochemistry
Authors: Van Noorden Cornelis J F , Hulstaert Caesar E
Published: November 2023

Good preservation of the ultrastructure of tissues and cells is a prerequisite for an adequate localization of enzyme activity at the electron microscopical level. For light microscopical purposes, tissue preservation is not as critical because the resolution of the microscope generally excludes enzyme localization within organelles or other cytoplasmic structures. Morphology is usually preserved by chemical fixation but for the localization of enzyme activity cells and tissues cannot be fixed in a similar way as for morphological ultrastruc- tural investigations, because the majority of enzymes are inactivated rapidly by fixation.
Three-dimensional electron microscopy of tissues and cells
Authors: James Nigel
Published: November 2023

The three-dimensional (3D) visualization and analysis of structures is of such considerable importance to investigators in many biomedical and other disciplines that it requires no justification. A fundamental difficulty in many subjects is that the 30 structures of interest are either opaque or too small to be seen without microscopical facilities. The smallest of structures requires the use of the electron microscope, which immediately places several important constraints upon the investigator. First, only extremely small portions of a structure can be analysed. Second, only dead and biased representations of the biological structures can be studied. Third, the most likely specimen preparation to be of use is a section of sufficient thinness to allow either light or a beam of electrons to penetrate, to yield a visible image or generate a photographic negative. Such a section is variously either relatively ‘thick’ or ‘thin’ according to the dimensions of the test objects under study, which is either limited in the total amount of information it contains or possesses inherent artefacts limiting adequate visualization when examined by two dimensional (2D) microscopical techniques in which all images are essentially projections.
Liquids
Authors: Eylers John P
Published: November 2023

The starting point for any analysis of circulatory systems is a simple model of a Newtonian fluid (i.e. a fluid whose viscosity is independent of the rate at which it is sheared) flowing through a rigid walled pipe of uniform diameter (Figure la). Under conditions of constant flow rate and laminar flow rate is directly proportional to the pressure drop per unit length and the fourth power of the radius while inversely proportional to the viscosity. Consequently small changes in radius are of much greater significance than small variations in pressure head or viscosity.
Bone, nacre, and other ceramics
Authors: Jackson Andrew P
Published: November 2023

Biological ceramics, like all ceramics, are stiff and relatively brittle materials. These properties pose several problems to mechanical evaluation. For ex ample:
Adhesives
Authors: Naldrett M J
Published: November 2023

The simplest and most general definition of an adhesive is ‘a material which when applied to the surfaces of two solids can join them together such that they resist separation’.
X-ray Analysis
Authors: Sawyer L , Turner M A
Published: November 2023

This chapter covers the preliminary characterization of the crystals in order to determine if they are suitable for a full structure determination. Probably more frustrating than the failure to produce crystals at all, is the growth of beautiful crystals which do not diffract, which have very large unit cell dimensions, or which decay very rapidly in the X-ray beam.
Overview of structural mechanics
Authors: Biewener Andrew a.
Published: November 2023

Organisms must contend with the physical characteristics of their environment as they carry out a wide range of physiological and mechanical functions. Particular structural solutions must be evolved to meet particular biological demands, both within and external to theorganism. The application of engineering principles to living (and fossil) organisms, and to the materials of which they are built, represents an important interdisciplinary approach that can facilitate and help to open new insights into our understanding of organismal design and function. An understanding of this relationship is also important in terms of how organisms respond and adapt to changes in their physical environment. Although biomechanics has seen a recent growth in a number of fields within the biological sciences, the approach has had a long history (1, 2).
Compartment models
Authors: Wiegert Richard g.
Published: November 2023

Models are abstractions of reality. They must possess at least some of the attributes of the system of interest, but none possess all or even a large fraction of such attributes. Models are of many kinds: a painting of a landscape, for example, is a model of reality in the sense that it suggests something of the ecosystem it represents. Such models are static— they cannot be used to simulate the changes in a system with time. Dynamic models can simulate changes with time; they were introduced in Chapter 8 by way of simple, single-compartment models.
The spinal cord as an in vitro preparation
Authors: Bagust Jeffery
Published: November 2023

1. Introduction

The last decade has seen an explosive increase in the use of isolated mammalian nervous system preparations for the study of the biophysics, physiology, and pharmacology of the central nervous system (CNS) (1, 2, 3). They offer to the mammalian brain researcher advantages that previously were only available to those working on isolated invertebrate nervous systems:

absence of anaesthetic agents

control over the composition of the medium surrounding the tissue, allowing known concentrations of test compounds to be added

improved mechanical stability; movements caused by respiration and blood pressure are eliminated

control over activity entering the system; inputs from other brain areas and the peripheral nervous system are removed

improved visibility of structures within the preparation allows electrodes to be guided accurately by visual means to the recording site

experimental convenience; once isolated the tissue is classified as a preparation and is not subject to the legislation that controls the use of living animals in experiments.
Introduction
Authors: Singh Juswinder , Thornton Janet M
Published: November 2023

All things are made of atoms, and everything that living things do can be understood in terms of the jigglings and wigglings of atoms

Richard P. Feynman

The structure, function, and stability of a protein is dependent upon the interactions between its amino acid groups. Although proteins are simple polymers, they adopt complex three-dimensional folds, which are a consequence, in part, of the packing geometries adopted by the protein side chains. Since these twenty groups differ in their size, shape, and chemistry they vary in their packing arrangements with each other. Determining the types of packing geometry adopted by these pairs is crucial to understanding the nature of the forces which stabilize protein structures.
Nuclear organization of pre¬ mRNA splicing factors and substrates
Authors: huang Sui , Spector david l.
Published: November 2023

Pre-mRNA transcripts must be processed and transported to the cytoplasm where the mature mRNAs are translated into proteins. For most RNA polymerase II tran-scripts, this processing includes addition of a 7-methyl-guanosine cap structure at the 5’ end of the nascent RNA transcripts; hnRNP assembly; removal of non-coding intron regions and ligation of exons; 3’ end cleavage and polyadenylation; and the ex¬ change of hnRNP proteins for mRNP proteins. Splicing occurs in a multicomponent complex termed a spliceosome. Many of the detailed biochemical steps involved in the prc-mRNA splicing reaction have been extensively studied in vitro and are well understood (for reviews sec Chapters 3-8). However, the integration of the splicing reaction with other related cellular functions, such as transcription and RNA trans-port, is best studied in intact cells. The cellular approach to understanding the organization of nuclear function has lagged somewhat behind the in vitro approach. However, during the past few years, major inroads have been made toward our understanding of where in the nucleus pre-mRNA splicing occurs and how the organization of both splicing factors and substrates are interrelated to the transcrip¬ tional activity of the cell. In this chapter, the nuclear organization of both snRNP and non-snRNP RNA processing factors, as well as pre-mRNA substrates, arc examined. In addition, their organization throughout the cell cycle, and the relationship of their nuclear distribution to the transcriptional activity of the cell, are discussed.
Studying cell movements in vivo by time-lapse video microscopy
Authors: Thorogood Peter v. , Amanze dee
Published: November 2023

Cell movement plays an important role during embryonic development (1). Much of what is presently known about cell movement is based on the behaviour of cultured cells. Despite refinements of culture conditions, however, extrapolation from events in vitro to behaviour in the embryo is difficult. Direct observation of living cells in vivo, when this can be done, is clearly preferable. Naturally transparent tissues such as the cornea (2), nematode embryos (3) (Chapter 2) and teleost fish embryos (4) (Chapters 4 and 9) (Figure /) lend themselves particularly well to in vivo analyses of cell movement. Techniques such as Nomarski DIC (Chapter 8), confocal scanning microscopy, and the labelling of living cells with fluorescent dyes coupled with fluorescence microscopy (Chapter 9), have begun to give us a much better appreciation of how cells normally behave in vivo.
Culture of Avian Neural Crest Cells
Authors: Dupin Elisabeth , Arin Nicole M Le Dou
Published: November 2023

As the unique source of very diverse cell types in the adult (such as peripheral glial and neuronal cells, mesenchymal cells, melanocytes, and endocrine cells), the neural crest (NC) of the vertebrate embryo has been the subject of considerable research effort aimed at elucidating several basic embryological problems related to cell commitment and differentiation, and to the segregation of cell lineages during morphogenesis. Although NC derivatives and migration pathways of crest cells have been mapped with great precision in birds, much remains to be known about the mechanisms underlying cell determination and phenotypic diversification in NC development. Attempts to understand the molecular basis of these fundamental processes have led many laboratories to undertake in vitro experiments where the differentiation of defined populations of NC cells can be analysed in controlled environmental conditions.
cDNA library construction
Authors: Kakizuka Akira , Yu Ruth T , Evans, Ronald M , Umesono Kazuhiko
Published: November 2023

DNA libraries allow the identification, isolation, and characterization of genes expressed in particular cell types or at specific developmental stages. They provide information on primary structures of mRNAs for comparative studies, and can yield molecular markers for use in descriptive or experimental developmental studies.
DNA-protein interaction at high resolution
Authors: Luisi Ben
Published: November 2023

Forty years on since the elucidation of a fiber-diffraction model for double-stranded DNA, structural scrutiny of the molecule continues to provide insights into key biological processes. Over the course of the last decade, and, in particular, during the last few years, there has been an explosive growth in high resolution structure determinations of DNA, DNA-binding proteins, and their complexes. These studies have provided a wealth of detailed stereochemical information, revealing how the DNA molecule´s sequence is read and how its structure is manipulated. Surprisingly, the mechanical properties of DNA have been implicated in these recognition processes, and this realization has revitalized an interest in the structure of the molecule itself. Herein, some of the principal lessons about DNA structure and mechanics, and their bearing on protein-DNA interactions, will be described.
|Enzymes that supercoil DNA
Authors: Wang James C
Published: November 2023

Supercoiled DNA has been known for three decades. It became clear in the sixties that a double-stranded DNA ring with intact strands could be viewed as two multiply intertwined and topologically linked single-stranded rings (1, 2). The degree of topological linkage of the complementary strands in such a ring is specified by a quantity Lk termed the linking number. For discussions in this chapter, Lk for a DNA ring is defined as the minimal number of times one strand must be passed through a transient break in the other in order to unlink the two complementary strands in a right-handed, double-stranded DNA ring (see ref. 3 for alternative definitions of Lk).
|Recognition of DNA damage and repair
Authors: Sancar Aziz
Published: November 2023

DNA is a highly reactive molecule. In particular, the bases and the phosphodiester bond are modified at a genetically unacceptable rate by common cellular metabolites (1). External agents ranging from ultraviolet and ionizing radiation to polycyclic aromatic hydrocarbons and methylating agents add to the cellular burden of maintaining genetic integrity. In addition, mismatches and 1-3 nucleotide loops are produced during replication and recombination reactions. Although mismatches, strictly speaking, are not considered as DNA damage, physiologically they have some of the same consequences and biochemically they are processed by similar mechanisms (2) as DNA lesions. Both non-physiological DNA modifications and base mismatches occur with such a high frequency that these structures might be considered among the most common targets for high-specificity DNA binding proteins which do not rely on DNA sequence.
|Structure-recognition by DNA junction resolving enzymes
Authors: LILLEY David M J
Published: November 2023

The junction resolving enzymes are a widespread class of nucleases that are characterised by a selectivity for branched DNA. Their role lies in recombination and repair events, in the recognition and manipulation of branched DNA intermediates of various kinds. In general, such enzymes exhibit relatively little specificity for base sequence, but exquisite selectivity for DNA structure. This involves recognition of DNA structure on a relatively large scale, and understanding the mechanism of this aspect of molecular recognition provides a fascinating challenge.
Making nucleic acid probes
Authors: Alphey Luke , Parry Huw D
Published: November 2023

The ability to make specific labelled nucleic acid probes has been central to the rapid expansion of molecular biology. The methods are part of the basic repertoire of a molecular biology laboratory. Rather than attempting to detail all of the methods historically used to label nucleic acids, we describe easy, rapid, and reproducible protocols for routine work. In the following chapter we will describe a set of frequently used hybridization protocols that utilize probes labelled as we detail here.
Screening ‘A expression libraries with antibody and protein probes
Authors: Margolis Benjamin , Young Richard A
Published: November 2023

The λgtll vector-host system and related gene expression systems permit the isolation of specific recombinant DNA sequences by virtue of the antigenicity or activity of the expressed gene product. The expressed protein, or domain of the protein, can be detected by using antibodies or through binding to a specific substrate such as a DNA fragment or a polypeptide. The design of the λgtll,, λZAP, and λEXlox systems have been described in detail elsewhere (1-4). We describe here features of the recombinant DNA libraries and the antibody and the protein probes that are important for successful screening. We also describe our favourite screening protocols using antibody and protein probes.
Expression of foreign proteins in E. coli using plasmid vectors and purification of specific polyclonal antibodies
Authors: Williams Jim A , Langeland James A , Thalley Bruce S , Skeath, James B , Carroll Sean B
Published: November 2023

For several decades antibodies have been used to identify, localize, quantitate, and analyse proteins. Before the advent of gene cloning, only natural sources were available for the isolation of proteins to which antibodies were raised. Low protein yields and impurities often compromised the quality and quantity of antibodies that could be produced. In addition, the vast majority of proteins that play critical roles in regulating cellular processes were completely unknown. As powerful genetic approaches have been devised to isolate the genes involved in a vast array of biological processes in diverse organisms, most proteins are now first identified at the gene level. The analysis of the structure, expression, regulation, and function of proteins identified in this manner usually requires the production of specific antibodies that can be used to detect the protein in different cells, tissues, organisms, and other biological samples.
The baculovirus expression system
Authors: Gunvanti Patel , Jones Nicholas C
Published: November 2023

Detailed biochemical and structural analysis of proteins encoded by cloned mammalian genes requires efficient expression systems for their high level expression and purification. Although very many proteins have been successfully over-produced in bacteria, there are certain inherent disadvantages which often makes this approach unsuitable. Foreign proteins over-expressed in bacteria often accumulate as insoluble occlusion bodies which require harsh denaturing agents to purify the recombinant protein. In addition, proteins produced in bacteria are not post-translationally modified. Since in many cases protein modification is crucial to activity, their over-production and purification from bacteria will be of limited use in functional studies. This chapter describes the helper-independent baculovirus expression system which offers a convenient and efficient method of over-producing posttranslationally modified proteins in a eukaryotic cell and may therefore be the method of choice under many circumstances. This system takes advantage of unique features in the life cycle of baculoviruses, including the production of two classes of viral progeny, and the presence of the strong polyhedrin promoter which is non-essential both for viral replication and the production of infectious extracellular virus. We describe the key features of this expression system including the latest modifications which will facilitate the isolation of recombinant baculoviruses expressing foreign proteins. Descriptions of general DNA manipulations, protein analysis, tissue culture procedures, and handling of baculoviruses can be found in other laboratory manuals (1-5).
High efficiency gene transfer into mammalian cells
Authors: Chisholm Vanessa
Published: November 2023

Over the past decade, recombinant or hcterologous gene expression in mammalian cells has emerged as an essential tool for studying multiple aspects of cukaryotic biology. The development of highly efficient vector based gene transfcction methods has provided researchers with almost un limited possibilities for the use of engineered protein products derived from mammalian cell culture. What has become apparent is that highly individual ized scientific questions can be routinely addressed using a relatively distinct battery of these types of expression protocols. The choice of technique and how it can be effectively used depends primarily on the specific requirements of the experiment. An extensive series of expression vector designs have been developed. Some arc more suited to introducing and analysing gene products transiently while others are better for the stable and continued expression of heterologous DNA in cells. Vectors have been constructed to exploit certain cell types. Likewise, transfection methodscan becategorized into those more effect ive for short-term expression analysis and those more suited to select stably expressing cell lines. The identification of recombinant products has evolved from time-consuming radioactive assays to more straightforwardimmunoassays and luminescent techniques. What may appear to be a most formidable field of players to choose from is best approached by becoming familiar with the basic characteristics essential for the efficient transfer and subsequent expression of genes in mammalian cells. In general terms these include:
Construction and characterization of vaccinia virus recombinants
Authors: Mackett Mike
Published: November 2023

The use of cowpox to vaccinate against smallpox was popularized by Jenner at the end of the 18th century and ultimately led to the eradication of smallpox. Although cowpox was used as a vaccine for a short period, the closely related poxvirus vaccinia has been in use for this purpose since the 1850s. Cowpox and vaccinia virus have played an important role in the history of human medicine since they represent the first widely used human vaccine and the only vaccine whose use has resulted in the eradication of a human disease. Surprisingly, interest in poxviruscs in general, and in vaccinia virus in particular, increased substantially after the disappearance of smallpox. This interest has arisen largely from the development of vaccinia virus as an expression vector with the potential use of vaccinia virus recombinants as live vaccines against pathogens other than the smallpox virus.
Chromosome-specific gridded cosmid libraries: construction, handling, and use in parallel and integrated mapping
Authors: Nizetic Dean , Lehrach Hans
Published: November 2023

The analysis of the information in human genomic DNA is based on the cloning and molecular characterization of the genes together with the genetic analysis of the phenotypes a-;sociated with the genetic loci under study. Libraries of cloned genomic DNA serve as essential intermediates to relate the information from the genetic analysis of phenotypic variation to the molecular analysis of the genes (I). Such libraries, can be either constructed in a yeast or bacterial vector-host system. The yeast system. using yeast artificial chromosomes (YACs) (2) as vectors, is well suited for rapid coverage of long genomic regions and entire chromosomes (3) (see also Chapter 4). This offers easier integration of the data with the genetic and physical maps of the genomic DNA, for example in the comparison of the maps of cloned DNA to long-range restriction maps of genomic DNA constructed using restriction endonucleases that cut at infrequent intervals. The yeast system is however not the best choice for the application of methods to identify transcribed regions (genes), to analyse the transcription unit and exon-intron organization, and to obtain the sequence of the genes. The longer the YAC clones arc, the more frequently they suffer from deletions, rearrangements, co-cloning, and chimerism, which makes the establishment of a highresolution map difficult. Moreover, irrespective of the YAC length, the isolation of pure YAC DNA from the genomic DNA of the host is difficult to achieve in most cases (sec Chapter 4 for a discussion of these problems). The bacterial (E. coli)-based vector systems (phage, cosmid, Pl, F-) (4-6) are much more suitable for the characterization of transcribed sequences (7-10), to study the molecular organization of the genes (11), and to provide material for DNA sequencing. The isolation of the cloned DNA in a pure state, free from the DNA of the host, is much more straightforward. Cosmid libraries are probably the simplest to use. They can be constructed with very high efficiency, allowing libraries to be made from limited amounts of DNA, and have been in use for longer than the Pl, PAC, and other new bacterial vector systems.
Amplification of DNA microdissected from mitotic and polytene chromosomes
Authors: Saunders Robert D C
Published: November 2023

Techniques permitting access to defined chromosomal regions are invaluable for the analysis of complex genomes. Microcloning is one such technique, which is particularly powerful when applied to dipteran polytene chromosomes. Microcloning was first described by Scalenghe et al. (1), who microdissected approximately JOO kb from the white region of the Drosophila melanogaster X chromosome, and recovered DNA fragments cloned in a phage lambda insertion vector, essentially by conventional cloning methods in miniature. Despite the obvious technical problems in transferring this technique to the study of mitotic chromosomes, microcloning has been applied to the analysis of specific chromosome regions of a variety of organisms.
Long-range restriction mapping
Authors: Bickmore Wendy
Published: November 2023

Mapping large and complex genomes relies on techniques that permit the analysis and manipulation of long stretches of DNA, hundreds to thousands of kilobases in length. This has been achieved largely through the developments of pulsed field gel electrophoresis (PFGE)-allowing the resolution of large DNA fragments, and yeast artificial chromosome vectors (YACs) for cloning large DNA fragments. Being able to resolve very large fragments of DNA also necessitates being able to restrict DNA into suitably long length . This can be accomplished with restriction enzymes with large, and therefore rare recognition sequences. In vertebrates, large restriction fragments can also be generated using restriction enzymes with CpG dinucleotides in their recognition sequences (this also applies to plant DNAs and includes enzymes with CpXpGs in their recognition sites). The principle cleavage sites for CpG recognizing enzymes in vertebrate genomic DNA, as well as providing convenient landmarks for the construction of long-range restriction maps, also mark out the position of many of the genes-critically important in organism where only a few per cent of the DNA has a coding capacity. This chapter aims to demonstrate how to create and interpret long-range restriction maps. primarily, of mammalian genomes.
Studies of cellular energetics using 31P Nmr
Authors: Brindle Kevin M , Fulton Alexandra M , Simon-peter Williams
Published: November 2023

Nuclear magnetic resonance (NMR) has become an established technique for the non-invasive study of metabolism in systems ranging from microbial cells to the human brain. It is a tremendously versatile technique in terms of the nuclei that can be monitored and thus the type of problems that can be addressed. For example flux of material in a metabolic pathway can be monitored by feeding the system a C-labelled substrate and then monitoring the subsequent isotope redistribution using C NMR (1). Ion fluxes and gradients can be monitored using Na and K NMR or by using Rb NMR in studies where Rb+ is used as a replacement for K+ (2). The intracellular concentrations of cations such as Ca2+ and Mg2+ can be monitored using 19F NMR in tissues which have been loaded with fluorine labelled chelating agents which bind these ions (3).H NMR can be used to monitor the levels of a variety of cellular metabolites and, in imaging studies, to determine the spatial distribution of water and hence the structure of the system under study. Although all these nuclei can be used to a greater or lesser extent to study cellular bioenergetics we will restrict our discussion here to the nucleus which has made the greatest contribution in this area, the naturally abundant isotope of phosphorus, P.
Physical mapping: YACs, BACs, cosmids, and nucleotide sequences
Authors: Bancroft Ian
Published: November 2023

The most detailed description of a genome is its nucleotide sequence, which represents a complete genetic blueprint. Within a short time of writing, the complete nucleotide sequence of the genome of Arabidopsis will have been determined and will become a key resource for plant scientists studying processes in Arabidopsis and many other plants. We discuss the systematic approaches that have been used to generate Arabidopsis nucleotide sequences and the opportunities that the sequence data offer for mapping.
Web-based bioinformatic tools for Arabidopsis researchers
Authors: Rhee Seung Y , Flanders David J
Published: November 2023

The introduction briefly defines the term ‘bioinformatics’ and describes sources of bio-informatic data. The body of the chapter is divided into four sections, followed by a short glossary: (a) How to get connected to the Internet and how to use Web browsers. (b) A flowchart describing an experimental scenario that uses bio-informatic tools.
Determination of the cell proliferative fraction in human tumours
Authors: Silvestrini Rosella , Daidone Maria Grazia , Costa Aurora
Published: November 2023

During the last two decades, increasing effort has been made to obtain as much information as possible on the cell proliferative fraction of individual clinical tumours in order to increase the knowledge on tumour biology and to provide clinicians with data on consecutive series of patients. As a consequence, an impressive number of reports has supported the relevance of cell kinetics as a tool to investigate growth pattern, biological heterogeneity and clinical progression of human tumours (1-4).
Chromosome analysis in cell culture
Authors: Mohamed Anwar N , Wolman Sandra R
Published: November 2023

The study of chromosome morphology and number (cytogenetics) describes and defines the cellular vehicles of inheritance. Structural features of the entire set of chromosomes within a cell can only be visualized in the late prophase and metaphase stages of cell division (mitosis); normally the mitotic process occupies a relatively brief (30-40 min) fraction of the entire cell cycle. Thus, only actively replicating tissues are amenable to cytogenetic investigation. The few tissues with sufficient spontaneous cell division to permit direct chromosome preparation, without culture in vitro, are bone marrow, trophoblast, testis, and some examples of malignant tumours. In order to obtain metaphase chromosome preparations from other tissues, it is necessary to induce cell division. Therefore, the cultivation of these tissues in vitro has become a critical component of cytogenetic investigation. It is essential to remember that this is an indirect approach to the tissues intended for analysis and that selection for growth in culture may result in cells that are not fully representative of the original population. Despite this drawback, almost all the cytogenetic information available on human tumours has been derived from cells grown in culture for various time periods. The methods described herein were developed for culture and cytogenetic analysis of human cells; most are applicable to other mammalian cells from the same tissue sources with minor modifications.
Growth and activation of human leukaemic cells in vitro and their growth in the SCID mouse model
Authors: Visonneau Sophie , Cesano Alessandra , Santoli Daniela
Published: November 2023

Despite their aggressive growth in the patient’s body, leukaemic cells usually fail to proliferate autonomously in tissue culture conditions (1). The dependence of human leukaemic cells on specific haematopoietic growth factors for in vitro growth was first demonstrated in well-or partially-differentiated acute T lymphoblastic leukaemias (T-ALL) which could be maintained in conditioned media containing interleukin 2 (IL-2) (2). Many of these cell lines have subsequently become growth factor-independent due to autocrine production of IL-2. The growth factors required by acute myelogenous leukaemias (AML) for in vitro growth have been defined more recently. Initially, it was observed that a small fraction of AML cells could form colonies in semi-solid medium supplemented with conditioned media from normal myeloid cells (3-5), or from phytohaemagglutinin-stimulated lymphocytes (6). It was later found that IL-3 and granulocyte-macrophage colony-stimulating factor (GM-CSF) represented the active factors in these preparations (7, 8).
Senescence and immortalization of human cells
Authors: Hubbard Karen , Ozer Harvey L
Published: November 2023

Normal vertebrate diploid cells in culture have a finite lifespan (1). The maximum number of population doublings is dependent on cell type and species. It has also been shown to vary as a function of age of the donor organism, resulting in its utilization as a model for the proliferative aspects of cellular ageing. Although cells from all species eventually cease to proliferate and become ‘senescent’, the capability to achieve infinite or continuous cell growth (i.e. became ‘immortal’) by mechanisms which overcome cellular senescence is strikingly dependent on the animal species. Rodent cells readily become ‘spontaneously’ immortal such that there are multiple established fibroblastoid cell lines; for example, mouse 3T3 cells. In marked contrast normal chicken and human fibroblasts (HF) rarely, if ever, become immortal. The observation that many human tumours are immortal cell lines when introduced into tissue culture suggests that overcoming senescence is a stage in carcinogenesis. One experimental manipulation which has been successful in facilitating the immortalization of HF has been through the introduction of genes from DNA tumour viruses into otherwise normal cells. In particular, the gene encoding the large T antigen (or A gene) from SV40 has been especially useful in this regard. Microinjection of large T antigen will reactivate DNA synthesis in senescent cells, although mitosis is not induced. Stable introduction of the A gene (as by viral infection or DNA-mediated gene transfer) prior to senescence will extend the limited lifespan for multiple generations; however, such cells typically undergo cell death, termed ‘crisis’ as distinct from senescence. These SV40 transformed cells can also be considered pre-immortal cells since they have an increased likelihood of becoming immortal, albeit with a varied and, most often, low frequency. Indeed many continuous cell lines have been developed in vitro. It should be emphasized that SV40-immortalized cells are still dependent on large T antigen function. We (2) and others (3) have developed a two-stage model.
DNA structure: implications for chromatin structure and function
Authors: Wolffe Alan P , Drew Horace R
Published: November 2023

DNA has a simple yet intricate structure that serves as the template for the assembly of a chromosome. Each chromosome has one DNA molecule, a long unbranched double helix consisting of two antiparallel polynucleotide chains. The structure is stabilized by base-stacking interactions and hydrogen bonding between the bases: G (guanine) being paired to C (cytosine) and A (adenine) to T (thymine). Physical studies using X-ray diffraction indicate that, under conditions of physiological ionic strength, DNA is a somewhat regular helix with a diameter of 2 nm, making a complete turn every 3.0 to 3.4 nm. This particular DNA structure (B form) has approximately 10.5 bp/tum of the helix, with a variation due to base sequence of 9.9 to 11.1 bp/tum. Every base pair is rotated approximately 34° around the axis of the helix relative to the next base pair, resulting in a twisting of the two polynucleotide strands around each other. A double helix is formed that has a minor groove (very approximately 1.2 nm across) and a major groove (very approximately 2.2 nm across). The geometry of the major and minor grooves of DNA is crucial in determining the interactions of proteins with the DNA backbone sugars and phosphates. The double helix is usually right handed.