bluuebunny/crossref_metadata_2025 huggingface dataset:
Time series analysis
Authors: Dunstan F D J
Published: November 2023

In many biological and medical situations a variable is observed sequentially over a period of time. The resulting set of observations, ordered with respect to time, is called a time series. For example, if the temperature in a certain place is measured at noon on each day for a year, the resulting set of values is a time series of length 365 (provided the year is not a leap year). Similarly, if the acidity level of a lake is measured monthly for five years, a time series of length 60 results. While, in many branches of statistics, when a set of observations is made they are assumed to be independent for subsequent analysis, one of the key features of a time series is that the observations are almost invariably dependent. The analysis of the series acknowledges this dependency in modelling the series and, in some applications, exploits this dependency to forecast future values. In the past, time series analysis has been developed largely in the areas of economics and engineering, but recently there has been a growing realization that it is a subject with important applications in biology and medicine and there is increasing interest in its use in these fields.
Electrophysiology of Xenopus oocytes: an expression system in molecular neurobiology*
Authors: Fraser S P , Moon C , Djamgoz M B A
Published: November 2023

Oocytes are unfertilized egg cells which can reach more than 1 mm diameter at later stages of development in lower vertebrates and thereby enable intracellular recording with multiple electrodes to be performed with ease (1). Furthermore, all the biochemical steps, from translation of messenger ribonucleic acid (mRNA) to insertion of protein into the plasma membrane, which normally occur rapidly upon fertilization, may also be induced by injection of ‘foreign’ mRNAs (2, 3). If the resulting protein is capable of generating an electrophysiological signal, as in the case of neurotransmitter/ hormone receptors, ion channels, and pumps, then electrophysiological recording can be used to assay the induced characteristic. Oocytes of the clawed frog Xenopus laevis in particular have been found to be highly efficient in this respect. They have become indispensible in studies aiming al elucidation of the molecular basis of function in proteins, especially those of the nervous system. The aim of this chapter is to give an account of the electrophysiology of Xenopus oocytes with an emphasis on practical aspects. An earlier account of this topic (‘Functional expression in the Xenopus oocyte of mRNAs for receptors and ion channels’) was given in 1987 by Barnard and Bilbe in Neurochemistry: A Practical Approach.
Quantal analysis of synaptic transmission
Authors: Walmsley Bruce
Published: November 2023

1. Introduction

Our understanding of the mechanisms underlying synaptic transmission owes much to the early mcasurcmenls and analysis of evoked and spontaneous synaptic potentials at neuromuscular junctions (1).
Iontophoresis in the mammalian central nervous system
Authors: Roberts M H T , Gould T
Published: November 2023

The microiontophoretic application of drugs onto neurones of the central nervous system can be one of the more seriously misused of pharmacological techniques. It remains, however, almost the only way of applying relatively few molecules rapidly into the vicinity of central synapses. The technique gets closer to mimicking synaptically released neurotransmitter than any other, but its limitations must be thoroughly understood or very misleading data will emerge. This chapter will outline the strengths of the technique, as well as the methodological constraints, and will give practical examples of how it may sensibly be used. It will also discuss common mistakes which should be avoided.
The cdc2 kinase: structure, activation, and its role at tnitosis in vertebrate cells
Authors: Basi Gabriele , Draetta Guilio
Published: November 2023

In the current view of eukaryotic cell cycle control, the existence of factors which positively regulate the progression through different phases of the cell cycle is assumed. In the last few years much progress has been made towards the molecular characterization of a factor which promotes entry into mitosis called MPF (M-phase promoting factor). The first evidence for the existence of such a factor dates back to studies in the slime mould Physarum polycephalum(1). P. polycephalumis a plasmodial organism (myxomycete) in which nuclear division is naturally synchronous, and therefore it is particularly suitable for studying the regulatory mechanisms controlling entry into mitosis. Experiments in which plasmodia at different stages of the cell cycle were fused, revealed the existence of a positive regulator of mitotic initiation, the levels of which fluctuated during the cell cycle, reaching a peak just prior tomitosis. The results also indicated that protein synthesis was required for the appearance of the mitotic factor and suggested that a translocation of this factor from the cytoplasm to the nucleus occurred shortly before the qnset of mitosis. More than twenty years after this initial observation, MPF was characterized and it was confirmed that the view proposed for the regulation of mitosis in P. polycephalumis essentially correct for all eukaryotes.
Capping, methylation, and 3’-end formation of pre-Mrna
Authors: Wahle Elmar , Keller Walter
Published: November 2023

This chapter deals with three covalent modifications of mRNA precursors that do not change the RNA’s coding sequence: capping of the 5’ end; cleavage at the 3’ end followed, in nearly all cases, by the addition of a poly(A) tail; and methylation of internal bases. Both terminal modifications-cap and poly(A) tail-arc thought to be involved in the same set of reactions, splicing, RNA transport, translation initiation, and RNA stabilization or regulation of mRNA decay. Interestingly, there are indications of a direct or indirect interaction between 5’ end and 3’ end (1, 2). The function of internal methylations is unknown.
Trans-splicing
Authors: Nilsen Timothy w.
Published: November 2023

Intermolecular (trans-) splicing is by definition an RNA-processing reaction which precisely joins exons from separately transcribed RNAs. As discussed elsewhere in this volume, there are four types of cis-splicing reactions: excision of group I introns (in many cases by autocatalysis) (see Chapter 1); excision of group II introns (in some cases by autocatalysis) (see Chapter 1); snRNP-mediated splicing of nuclear pre-mRNAs (see Chapters 4-7); and enzymatic removal of introns from pre-tRNAs (reviewed in 1, 2).
mRNA editing
Authors: simpson Larry , Thiemann otavio h.
Published: November 2023

RNA editing is a term that was coined by Benne et al. (1) to describe the apparently post-transcriptional insertion of four Us at three sites in the coding region of the cytochrome oxidase subunit II (COII) mRNA in two kinetoplastid species, Crithidia fasciculata and Trypanosoma brucei. This modification overcame a -1 frameshift in the COII gene that had been shown to be evolutionarily conserved in these two species and also in Leishmania tarentolae (2). As additional examples of RNA sequence modifications were reported in a variety of organisms (2-10), RNA editing’ came to be defined in a broad sense as any process that results in the production of an RNA molecule which differs in nucleotide sequence in coding regions from the DNA template, with the exception of classical ds-splicing (11). However, even this definition was rendered obsolete by the discovery of specific sequence changes in rRNAs and tRNAs in several organisms, in one case involving the insertion of cytidine residues (12, 13) and in some cases the substitution of uridine for cytidine (14) or guanosine for adenosine residues (15). In this review we will only discuss the trypanosome type of RNA editing, which has been termed insertion-deletion editing by Bass (16), to distinguish it from the substitution editing found in other cells.
Fate maps and cell lineage analysis
Authors: Altaba Ariel Ruiz i , Warga, Rachel m. , Stern Claudio D
Published: November 2023

Knowledge about the fates of cells during development is of fundamental importance to our understanding of developmental mechanisms (1). In organisms like the nematode Caenorhabditis elegans, cell number is sufficiently reduced and the embryo is small and transparent enough to make lineage analysis by direct observation of cell divisions relatively straightforward. However, in systems where cell number is greater, special techniques have to be introduced to follow cell fates.
Microinjection Into Drosophila Embryos
Authors: Ingham Philip , Forbes Zan Dy
Published: November 2023

Microinjection of Drosophila embryos is relatively easy to perform and has several important applications. The most common of these is injection of DNA for the production of transgenic flies; the same general approach can be used for injection of RNA, oligonucleotides, metabolites, lineage tracers, cytoplasm, and antibodies, and for transplantion of nuclei or cells between embryos.
Cloning Genes Using the Polymerase Chain Reaction
Authors: Holland Peter W H
Published: November 2023

The polymerase chain reaction (PCR) is an in vitro method for amplifying regions of DNA, utilizing information based on nucleotide sequences flanking the region of interest (1-3). PCR is used in the study of DNA sequence variation, genomic organization or gene expression (the latter using cDNA as a template, reverse transcribed from RNA; Chapter 24). PCR can also be used to amplify DNA from previously uncharacterized genes, if predictions can be made regarding the sequence of part of the gene(s).
Monosaccharides
Authors: Chaplin M F
Published: November 2023

Monosaccharides exhibit a great variety of structural types with vastly different chemical and physical properties. In contrast to the several methods available for the extensive analysis of the other major class of biological molecules, the amino acids, there is no single method which is suitable for the quantitative or qualitative analysis of all monosaccharides. The method of choice will depend on a number of factors including the accuracy required and resources available. There are two techniques available for the quantitative analysis of mixtures of monosaccharides, high performance liquid chromatography (HPLC) and gas-liquid chromatography (GLC). If only a qualitative analysis is needed, either paper chromatography (PC) or thin-layer chromatography (TLC) may be used. Single monosaccharides may be identified by means of mass spectrometry (MS), infra-red spectroscopy (IR), or proton or carbon-13 nuclear magnetic resonance (1H-NMR, 13C-NMR), and quantified by use of specific colorimetric or enzymatic assays.
Neutral polysaccharides
Authors: Pazur John H
Published: November 2023

Polysaccharides are polymers of monosaccharide units joined together by glycosidic bonds which are formed by the elimination of the elements of water between the hemiacetal hydroxyl group of one residue and a primary or secondary hydroxyl group of an adjacent residue (1). Polysaccharides may consist of a small or a large number of residues and in structure may be linear, branched, or occasionally cyclic polymers. These polymers may be homopoly saccharides composed of a single type of residue or may be heteropoly saccharides composed of two or more different types of residues. Originally polysaccharides were named to reflect biological sources or properties of the polymer and many such names have remained in common usage. At the present time, it is the recommendation of the Joint Commission on Biochemical Nomenclature that polysaccharide nomenclature follow the general principles established for organic nomenclature (2). Glycan is the name recommended for a polysaccharide by the Commission.
Glycoproteins
Authors: Montreuil Jean , Bouquelet Stephane , Debray Henri , Lemoine Jerome , Michalski Jean-Claude , Spik Enevieve , Strecker Gerard
Published: November 2023

Glycoproteins (for recent reviews, see refs 1--4), which together with glycolipids constitute the class of glycoconjugates, result from the covalent association of carbohydrate moieties (glycans) with proteins. Glycosylation of proteins represents one of the most important post-translational events because of the universality of the phenomenon. Most proteins are glycosylated, the glycoproteins being widely distributed in animals, plants, micro organisms, and viruses. In addition, the development of recombinant proteins of therapeutic interest represents a formidable challenge since the structure of recombinant glycoproteins must conform with that of the natural ones (5). It has been established that glycans perform important biological roles including.
|DNA bending by sequence and proteins
Authors: Travers Andrew A
Published: November 2023

A fundamental problem confronting any living organism is how to package an immense length of DNA in an organised manner into a small volume. One solution is to compact the DNA by the tight bending of the duplex in the form of a superhelix which can itself then be folded into a more compact structure. Because the DNA double helix possesses axial stiffness, the bending necessary for this packaging is normally mediated by proteins which may themselves then promote further assembly into higher-order nucleoprotein structures. However, in addition to this primary structural role, DNA bending is also an essential component in the formation of the nucleoprotein complexes required for site-specific recombination and for the initiation of transcription and DNA replication.
Bioenergetics
Authors: Brown Guy C , Cooper Chris E
Published: November 2023

Bioenergetics is a very broad subject which covers a wide range of systems. The movement of electrons and ions across and through membranes is important in areas such as energy transduction, cell biology, and cell signalling. This book describes the core of bioenergetic techniques that are necessary to analyse membrane-translocating events.
Oxygen, proton and phosphate fluxes, and stoichiometries
Authors: Hinkle Peter C
Published: November 2023

The measurement of coupling ratios should never be considered a routine undertaking, for history shows that practically every example went through several stages of eliminating systematic errors before the current relative consensus was reached. For example, traditionally the P/O ratio of mitochondria with succinate was thought to ‘approach’ 2.0, but currently it is considered to be 1.5 (1). This is partly due to the fact that measurements of oxygen and protons are rather prone to error. Errors may also result from the frame of mind of the experimenter. Since ratios are dimensionless and reflect the mechanism of the coupling process they are hard to view with objective detachment. I will not review the sad history of errors in ratio determinations, which have happened to most workers in the field, including myself, but will describe examples of what seem to be the most reliable methods currently used to measure coupling ratios and some of the sources of error which should be avoided.
Membrane permeability and transport
Authors: Voegele Ralf T , Marshall Emmalee V , Wood Janet M
Published: November 2023

The limited permeability of biological membranes to large and/or hydrophilic solutes allows cells to modulate their own composition and to base their energy metabolism on transmembrane electrochemical potential gradients. Our current understanding of those phenomena, which were first placed within a comprehensive theoretical framework by Peter Mitchell (1), is based on measurements of membrane structure, of membrane permeability, and of transmembrane solute fluxes mediated by enzymes denoted as transporters. Photosynthetic and respiratory electron transport chains and ion-motive adenosine triphosphatases (ATPases) mediate creation of the membrane potential (Δψ), formation of transmembrane electrochemical potential gradients (Δμ1), and coupling of those gradients to the synthesis and hydrolysis of ATP. The membrane potential, solute gradients, ATP, and phosphoenolpyruvate (PEP) serve as the energy supplies powering many other transport reactions. Methodologies for the estimation of Δψ and of the protonmotive force (ΔμH+) are described in Chapter 3 of this volume. This chapter describes methodologies for the measurement of passive and transporter-mediated transmembrane solute fluxes. It does not describe methods specifically designed for the study of membrane channels. Detailed descriptions of theories, procedures for the preparation of cells, organelles, and membrane vesicles, and of methods for transport measurements can be found in other books in the Practical approach series and in the Methods in enzymology series of books.
Reconstitution of bioenergetic proteins and the uses of proteoliposomes
Authors: Wrlgglesworth john M
Published: November 2023

A variety of membrane proteins are involved in bioenergetic processes such as ATP synthesis, ATP hydrolysis coupled to ion and metabolite translocation, and redox reactions coupled to proton and sodium ion pumping. A study of their mechanism of action is often aided by purification and reconstitution into artificial bilayer systems, the most widely used of which is the liposome (1, 2). The advantage of reconstituted systems is that vectorial reactions of the protein can be recreated under defined conditions. The disadvantages are largely methodological. Problems can arise in the choice of reconstitution method, the lipid composition of the bilayer for optimum activity, and the yield and homogeneity of the final preparation. In addition, the properties of the vesicles themselves can impose constraints on the function of the protein. For example, the small vesicle size produced by some methods of preparation means that the electrogenic movement of just a few charged molecules can generate a significant membrane potential.
Quantitative biological assays for individual cytokines
Authors: Wadhwa Meenu , Bird Christopher , Page Lisa , Mire-Sluis Anthony , Thorpe Robin
Published: November 2023

Bioassays for IL-α and β commonly utilize the ability of this cytokine to induce IL-2 production by T cell-lines such as EL4.6.1 and LBRM 33, or in primary cultures of thymocytes (LAF assay). Alternatively, IL-1 can be assayed by measuring its ability to stimulate the proliferation of lines such as the subclone DlOS of the murine T-helper cell line D10.64.1 (8). A subclone, NOB-1, of the murine thymoma line EL4.6.l provides a simple and sensitive bioassay for IL-1 (see Figure 1). This line produces IL-2 in response to both human and murine IL-1, which is measured using the CTLL-2 cell-line based assay (see Section 2.1).
Modelling epithelial tissues in vitro
Authors: Shaw Andrew J
Published: November 2023

The human body is composed of four basic types of tissue: epithelial, connective, muscular, and nervous. Epithelia cover the surfaces, and line the cavities and tubes of the body. These tissues are supported on a basement membrane and consist of close-knit polyhedral cells bound laterally to each other by cell junctions.
Identification and localization of cytokeratins
Authors: Mackenzie Ian C , Gao Zhirong
Published: November 2023

The patterns of polarization, stratification, and maturation that characterize epithelial differentiation in vivo are usually lost when epithelial cells are grown on plastic substrates using standard cell culture methods. However, epithelia cultured on organic matrices containing mesenchymal cells often redevelop regionally specific patterns of growth and structure, and such culture techniques thus provide unique opportunities for the in vitro investigation of many aspects of epithelial differentiation and function. The demonstrable effects of mesenchyme on epithelial differentiation in organotypic cultures illustrate the importance of tissue interactions in the maintenance of normal epithelial structure. Organotypic cultures provide valuable systems for the study of the actions and molecular nature of these mesenchymal influences.
Xenobiotic metabolism in epithelial cell cultures
Authors: Guillouzo Andre , Chesne Christophe
Published: November 2023

Individuals are exposed to a variety of xenobiotics which are present in their environment (i.e. air, water, etc.), are administered (i.e. medicines), or are ingested (i.e. beverages and foods). Many of these compounds are lipophilic and must be converted to more hydrophilic metabolites in order to be eliminated. Most, if not all epithelial cells, contain some capacity to metabolize foreign compounds, however hepatocytes contain the most diverse and active xenobiotic metabolizing system. This explains why the hepatic enzyme profile has been the most extensively analysed and why it is critical to determine hepatic biotransformation of new compounds that could reach the systemic circulation. To gain this information, in vitro liver models and particularly cultured liver cells are increasingly used.
A model of the blood—testis barrier for studying testicular toxicity
Authors: Steinberger Anna , Jakubowiak Andrzej
Published: November 2023

Although diverse classes of chemicals have been found to produce spermatogenic damage in experimental animals, their primary target site(s) and/or mechanisms of action are, in most cases, not known (1). Certain compounds, e.g. diester phthalates, need to be metabolized to monoesters in the liver or gut before they can exert a toxic effect on the testis (2). A toxicant may affect the male gametes indirectly by interfering with the production of androgens by Leydig cells, or with various functions of Sertoli cells, e.g. metabolism, vectorial secretion, and the formation of specialized tight junctions. As Sertoli cells are frequently affected by exposure to testicular toxicants, they have been most widely utilized in studies of testicular toxicity in vitro.
Chromatin replication and assembly
Authors: Sogo José M , Laskey Ronald A
Published: November 2023

Chromosome replication is one of the most complex problems facing the cell. Not only must it produce exactly two accurate copies of the parental DNA, but it must copy patterns of chromosomal proteins. In this chapter we start by introducing the mechanism of eukaryotic DNA replication and some of the proteins involved. Next we consider eukaryotic origins of replication focusing on the contrast between the well-defined simple replication origins of lower eukaryotes or viruses and the paradoxically complex origins of metazoan cells. We then discuss how old nucleosomes are distributed and new nucleosomes assembled at replication forks. Finally we consider the termination of replication and the problems posed when two adjacent replication forks meet.
Initiation of expression: remodelling genes
Authors: Hager Gordon , Smith Catharine , Svaren, John , Hörz Wolfram
Published: November 2023

The periodic nucleosomal organization of DNA in eukaryotic cells has been appreciated for more than twenty years (1). However, despite the ubiquity of nucleosomes, the exact role which nucleosome structure plays in transcriptional regulation has been, and remains, a subject of vigorous debate. The omnipresent nature of nucleosome structure itself, which was initially an advantage in chromatin studies, has made it difficult to target studies in vivo to this problem.
Epigenetic regulation in Drosophila: a conspiracy of silence
Authors: Eissenberg Joel C , Elgin Sarah C R , Paro Renato
Published: November 2023

Understanding the mechanisms of epigenetic regulation, by which cell-specific gene silencing is established and stably maintained, is a major unresolved challenge. Gene expression in eukaryotes relies upon the accessibility of the DNA template to RNA polymerase and a variety of accessory proteins. It is clear that the accessibility of regulatory elements can be controlled by the chromatin structure at a given locus: in spite of the availability in a given cell of all of the requisite DNA-binding proteins, a gene can remain silent, and this silenced state can be propagated faithfully through mitosis. This might be accomplished by alternative modes of packaging DNA into a nucleosome array, but almost certainly involves other types of packaging as well.
Domains and boundaries
Authors: Schedl Paul , Grosveld Frank
Published: November 2023

The very large genomes found in many higher eukaryotes must be condensed into a relatively small volume inside the nucleus. This is accomplished by packaging the DNA into a complex, multi-level nucleoprotein structure, chromatin. At the first level of organization the DNA is assembled into a beads-on-a-string nucleosome array, the 10 nm fibre visualized by electron microscopy (see Chapter 1). In the second level of organization, the 10 nm fibre is coiled into a 30 nm fibre. While this transition is thought to be facilitated by Hl binding (1), the precise organization of the nucleosomes is not completely resolved. In one widely accepted model (2, 3) they are coiled into a fairly homogeneous solenoid-like structure, six nucleosomes per turn. Recently, some electron microscopy studies have suggested that the principle building block of the 30 nm fibre is a di-nucleosomal unit that appears to zigzag across the fibre axis, giving a much more irregular and potentially dynamic structure (4). Whichever model is correct, the 10 nm and 30 nm fibres most certainly coexist in the interphase nucleus, with specific regions of the chromosome undergoing transitions from one state to the other, presumably as a consequence of transcriptional activity or DNA replication and recombination (see 5-9). Additional levels of organization above the 30 nm fibre are required to fit the DNA inside the interphase nucleus and to achieve the even higher levels of DNA compaction observed in mitotic chromosomes. This higher order folding of the 30 nm fibre is very poorly understood, and there is still a great deal of controversy about the principles governing the 3-D orgnization of chromatin inside the nucleus.
Sources of microorganisms and their preservation
Authors: Smith David
Published: November 2023

It is essential when working with microorganisms that the strains employed arc representative of the species and possess the full range of properties typical of the organism. These properties must not be allowed to deteriorate or change throughout the duration of the study or use of the microorganism. It is therefore imperative that the source of the strain is reputable. If the strain is collected specifically for the study, it must be fully characterized and described so that variance from the original characteristics can be noticed. The organism must also be stored in such a way that it remains both viable and stable.
Analysis of Microbial Growth Data
Authors: Razin Michael J , Wood Ann P , Paget-Brown David
Published: November 2023

In this chapter we describe some of the ways in which microbial growth kinetic data can be analysed. Most of our attention is focused on dynamic models containing the Monod growth function because it is this function that is most commonly employed to describe the kinetics of microbial growth. But first, in order to minimize ambiguity. we define some of the terms we employ.
Centrifugal elutriation
Authors: Wilton Joanne C , Strain Alastair J
Published: November 2023

Centrifugal elutriation provides a versatile means of separating cells using continuous flow centrifugation. Elutriation is defined as separation by washing and straining or by decanting. Centrifugation provides a means of finely regu lating this process. It is a rapid, non-invasive method for the preparation of specific subpopulations of cells from mixed cell types utilizing a balance of two opposing forces; centrifugal force and a fluid counterflow. First described by Lindberg in 1932 (1), its use remained limited until the 1970s when Beckman Instruments first marketed separation chambers and rotors specifically designed for the process. The technique has since become widely used for the separation of cells from an extremely diverse range of sources.
In Vitro Rnas
Authors: Weber Ute , Gross Hans J
Published: November 2023

RNA can be synthesized in vitro in two different ways which complement each other. Chemical synthesis yields oligoribonucleotides of a maximal length of 40 nucleotides, in contrast to the 100 to 150 nucleotides possible for oligodeoxyribonucleotides (Chapter 3). Nevertheless, the chemical approach does not impose any restrictions on the nucleotide sequence, including the use of modified building blocks.
In Vitro Applications of Antisense RNA and DNA
Authors: Munroe Stephen H
Published: November 2023

Despite these diverse applications, there have been relatively few studies with antisense nucleic acids in cell-free systems in comparison with the large number of investigations carried out in intact cells. It is anticipated that in vitro analysis will play an increasingly prominent role in future investigations as new antisense systems are discovered and efforts are directed towards understanding their mechanisms. In this chapter I will discuss methods which are common to many investigations involving the use of complementary RNAs and DNAs to block gene expression and probe RNA structure and interactions in cell-free systems. Some of these methods, and a number of related procedures, arc further discussed in other compilations of methods (10) and in another volume in this series (11).
Shotgun Antisense Mutagenesis
Authors: Spann Timothy P , Brock Debra A , Gomer Richard H
Published: November 2023

The use of antisense RNA and DNA to block gene expression has proven to be a valuable technique for the examination of gene function in a number of different systems. Typically, a known CDNA fragment is directionally cloned into an expression vector so that cells transfected with this construct will synthesize antisense RNA transcripts. The antisense transcript will cause the loss or reduction of the corresponding gene product. The phenotypes of transformed cells are then assayed and possible functions of the gene are inferred. If, instead of transforming cells with one antisense construct, a population of cells is transformed with an antisense CDNA library, a complex pool of transformed cells is produced. This type of protocol (shotgun antisense) will yield many different clones; in each clone, the expression of a different gene product will he blocked by an antisense transcript. Mutants generated by shotgun antisense can be screened in the same manner as mutants generated by any other means. However, when a mutant is selected for further study, PCR can be used to immediately isolate the antisense CDNA, which can then be directly cloned and sequenced. This allows one to examine the sequence of the protein encoded by the repressed gene a few days after identifying a mutant with an interesting phenotype (Figure I). To confirm that the mutant phenotype was caused by the antisense CDNA construct, the PCR product isolated from the mutant of interest is cloned into the antisensc vector and used to transform cells. A matching phenotype indicates that the CDNA caused the phenotype. Alternatively, the CDNA can be used in a gene disruption construct, which in turn can be used to make a genomic knock-out of the corresponding gene. We have developed a shotgun antisense procedure for the simple eukaryote Dictyostelium discoideum (1).
Replication origins in metazoan chromosomes
Authors: Depamphilis Melvin L
Published: November 2023

Central to understanding how animal cells regulate DNA replication is under­standing the nature of DNA sites where replication begins. Based on analyses of the chromosomes of bacteria, bacteriophage, plasmids, yeast, animal viruses, and mito­chondria, initiation of DNA replication begins with the binding of specific trans-act­ing proteins to specific cis-acting DNA sequences referred to as origins of replication (1-3). This event is followed by DNA unwinding at or near the protein­binding site in order to expose the two single-stranded, complementary DNA tem­plates to DNA polymerases, their accessory proteins, and proteins that provide nucleotide primers for DNA synthesis. DNA synthesis is then initiated on one or both templates by a process that generally, but not always, results in replication bubble and fork structures such as those found in the chromosomes of prokaryotic and eukaryotic cells (Fig. 1).
Chromosome replication in Xenopus egg extracts
Authors: Blow J Julian
Published: November 2023

Chromosomal DNA replication must be coordinated with other events of the cell division cycle. This means that:

DNA replication should only occur when the cell is legitimately committed to undergoing cell division;

the entire genome must be fully replicated in each S phase, as any section of DNA left unreplicated at mitosis would lead to chromosome breakage;

no DNA should be replicated more than once in a single cell cycle, as this is likely to lead to an irreversible amplification of a section of the genome;

S phase and M phase must occur in strict alternation to maintain the ploidy of the cell.
Regulation of S phase in the fission yeast Schizosaccharomyces pombe [CT]
Authors: Forsburg Susan L
Published: November 2023

The fission yeast Schizosaccharomyces pombe provides an excellent model system for studies of cell cycle regulation (1, 2). Its cell cycle has a short Gl phase and is largely regulated at the G2/M transition in rapidly growing cells, in contrast to the cell cycle of the very distantly related budding yeast Saccharomyces cerevisiae. This has made study of the regulation of the Gl /S phase transition in fission yeast technically difficult. However, considerable advances have been made recently, informed in part by comparisons with the models established in S. cerevisiae (3, 4) (see Chapter 7). Just as analysis of the G2/M phase transition shows important differences as well as broad similarities in the two yeasts, work on the control of the Gl/S phase transition already has identified conserved as well as variable regulatory elements. Such comparisons between different cell types promise to extend our understanding of the control of cell cycle entry and DNA replication in all eukaryotes. The web of interactions in S. pombe that connects genetically defined regulators to biochemically characterized components of the replication apparatus is still being dissected.
Choosing and manipulating effector functions
Authors: Sandlie Inger , Michaelsen Terje E
Published: November 2023

The aim of antibody therapy is often target-cell destruction. The target cells may be microorganisms, infected cells, cancer cells, autoimmune T cells, or cells responsible for organ graft rejection and graft versus host disease. In all these cases it is crucial that the effector mechanisms of target-cell specific antibodies are elicited. Antibodies protect against bacterial infections by inducing bactericidal activity, i.e. complement-mediated bacteriolysis, or by inducing opsonophagocytosis mediated by neutrophils, monocytes, or macro phages. When experiments are carried out using antibodies against human target cells and with human serum as the source of complement, only a few of the antibody molecules induce good lysis. This is due to the presence of cell surface molecules such as ‘homologous restriction factor’, ‘decay accelerating factor’, CD59, and ‘membrane co-factor protein’ (collectively called comple ment regulatory proteins or homologous complement restriction factors).
Expression of immunoglobulin genes in mammalian cells
Authors: Bebbington Christopher
Published: November 2023

Expression systems for antibodies in mammalian cells have recently been re viewed (1). This chapter describes detailed procedures for some of the most efficient, currently available expression systems for use in Chinese hamster ovary cells and mouse myeloma cell lines. These are the two cell types that have been most widely used for antibody production. Both can be grown readily in a variety of bioreactors including large-scale suspension fermenta tion systems, and have been used to produce antibodies for clinical use in humans.
Preparation and uses of Fab’ fragments from Escherichia coli
Authors: Carter Paul , Rodrigues Maria L , Park John W , Zapata Gerardo
Published: November 2023

Antibody (Ab) engineering has been revolutionized by the advent of methods to express functional Fv (1) and Fab (2) from Escherichia coli by co-secretion of corresponding light and heavy chain fragments (see also Chapter 10). The low Ab fragment titres obtained using shake-flask cultures (typically< 5 mg/ litre) are useful for many research applications. However, such titres are inconveniently low for biophysical or X-ray crystallographic studies and unsuitable for biotechnology or human therapy where gram or even kilogram quantities of material may be required. This prompted us to develop a system for very high-level expression (up to 2 g/litre) of Ab fragments in E. coli grown to high cell-density in the fermenter (3). This high-level production of soluble and functional Ab fragments (reviewed in ref. 4) has facilitated bio physical (5, 6), X-ray crystallographic (7, 8), and pharmacokinetic (9) studies as well as preclinical efficacy studies in Rhesus monkeys.
Analysis of nuclear scaffold attachment regions
Authors: Gasser Susan M , Vassetzky Yegor S
Published: November 2023

The eukaryotic nucleus is a complex, highly organized structure. Separation into soluble and insoluble subfractions is one of the few ways to analyse nuclear organization. As long as 20 years ago, it was observed that the removal of histones by high salt extraction resulted in a halo of tortionally constrained loops of naked DNA, emanating from a residual nuclear structure (1–5). The halo size changed on addition of ethidium bromide, demonstrating that the individual loops could become supercoiled, and are thus topologically independent domains.
Nucleic acid electrophoresis in agarose gels
Authors: Robinson Douglas H , Lafleche Gayle J
Published: November 2023

Electrophoresis is defined as the movement of ions and charged macromolecules through a medium when an electric current is applied. Agarose and polyacrylamide are the primary stabilizing media used in the electrophoresis of macromolecules. Macromolecules are separated through the matrix based on size, charge distribution and structure. In general, nucleic acids migrate through a gel based on size, with little influence from base composition or sequence, whereas proteins separate through the matrix based on size, structure and charge because their charge density is not directly proportional to size.
Construction of recombinant DNA molecules
Authors: Powell Richard , Gannon Frank
Published: November 2023

Although the central role of DNA in all living forms was well accepted by the 1950s it was very difficult to study it directly at that time (1, 2). The information obtained about DNA came mostly from the interpretation of classical genetic experiments. Most of these were performed using microorganisms whose relative simplicity and ease of manipulation made them the organisms of choice. The difficulty of obtaining a detailed understanding of a bacterial virus or phage such as X in which there are a mere 50 000 nucleotides (approximately) made the wish to understand the structure and function of higher organisms in which there are almost 105 times more nucleotides seem unattainable. Then, in the mid-1970s, a technological revolution occurred which has allowed this great chasm in accessible information to be bridged. As in all revolutions, developments in many unrelated areas came together in a way which magnified the individual components into a powerful new ensemble.
Survey of cloning vectors for Escherichia coli
Authors: Brown T A
Published: November 2023

The vector plays the central role in a DNA cloning experiment and must be care fully chosen so that its properties are appropriate for the objectives of the pro ject. The features of the vector must then be taken into account when designing the experimental strategy that will be used to generate and identify recombinant clones. An understanding of the different types of vector that are available, and the properties of each one, is therefore a prerequisite for successful molecular biology.
Strategies for research in molecular biology
Authors: Brown T A
Published: November 2023

The techniques described in Volume I of this book form the foundation to re search work in molecular biology and, once they have been mastered, any of the more specialized procedures can be approached with confidence. However, successful use of these specialized procedures requires more than just the ability to string together basic manipulations. As in any scientific endeavour the experi mental programme has to be planned in accordance with the questions being asked and the goals being sought. There are two or three different way of doing just about anything in molecular biology, and choosing the best experimental approach requires an understanding of the requirements, scope and limitations of the different procedures, as well as a clear conception of what the overall research programme is intended to achieve. As with the procedures described in Volume I, the newcomer to molecular biology should approach the chapters in Volume II with the dual aim of understanding both the technical and conceptual aspects of the methodology.
Construction of genomic libraries in A and cosmid vectors
Authors: Frischauf Anna-Maria
Published: November 2023

A genomic DNA library is a set of cloned fragments of genomic DNA. For most organisms no prior information about the genome is required for library construction (though special methods for DNA isolation may be required). The first genomic libraries of complex genomes were constructed in phage X. vectors cloned in Escherichia coli. This was followed by the introduction of cosmid vectors, plasmids which also use the phage X. packaging system (see Volume I, Chapter 9, Section 3.6). Packaging of recombinant DNA molecules into phage X. heads provides an excellent selection for a minimum insert size. It also specifies a maximum insert size of about 22 kb for a X. vector and 40 kb for a cosmid. More recently, several additional systems for the production of genomic libraries have become available. Pl vectors, bacterial artificial chromosomes (BACs) and Pl arti ficial chromosomes (PACs) (1) are also propagated in E.coli, while yeast artificial chromosomes (YACs) are grown in Saccharomyces cerevisiae (2, 3). All the new vec tors offer significant increases in the size of inserts that can be accommodatedgreater than 100 kb for Pl, up to 300 kb for BACs and PACs, and more than 1 Mb for YACs. These large-insert vectors have the advantage that libraries containing fewer clones are needed in order to cover an entire genome. This greatly facilitates the construction of ordered clone maps and the physical linking of genetic markers. There are, however, some disadvantages inherent to propagating clones of very large DNA fragments in either yeast or E.coli. Not only is there a greater tendency for the DNA to undergo deletions or rearrangements, but the copy number of the cloned DNA also has to be quite low, leading to a relatively low yield of the rather complex insert DNA. Another important practical problem with such libraries is that very high-quality, high-molecular-weight insert DNA, of consistent size, must be prepared prior to cloning. Library construction is therefore laborious and technically demanding.
Immobilization of nucleic acids and hybridization analysis
Authors: Dyson N J
Published: November 2023

Nucleic acid hybridization is an important component of many molecular biology techniques. Filter hybridization methods exploit the specificity of molecular hybridization for the detection of rare sequences in a complex mixture. The applications of filter hybridization are widespread and are a fundamental part of any analysis of gene structure or gene expression.

In filter hybridization, denatured nucleic acids are immobilized on an inert support so that reannealing of sequences within the sample is prevented but the nucleic acids are still available for hybridization. A suitably-labelled probe is added and the filter is incubated under conditions that promote hybridization between the immobilized target and the nucleic acid probe. The filter is washed until only specifically hybridized probe remains and the label is detected. The intensity of the hybridization signal is an indication of the abundance of target sequences within the sample and, in several types of analysis, the position of the signal on the filter gives further qualitative information.

The first part of this chapter contains methods for the immobilization of nucleic acids on nylon and nitrocellulose filters. Most of the remainder of the chapter deals with the hybridization analysis of these filters. Parameters that affect both the hybrid stability and rates of hybridization are briefly described. Specific protocols for the basic techniques are given together with indications of how these can be adapted for related applications.
Transcript mapping
Authors: Smith Colin P
Published: November 2023

A detailed study of the structure and expression of any gene will normally include the determination of the end-points of the mature transcript derived from it. This type of analysis is generally conducted in order to identify the positions of DNA sequences potentially involved in the initiation or termination of transcription, or to identify the position of processing sites within the transcript. A knowledge of the ‘transcript map ‘ can also be very useful for designing subsequent genetic manipulations, including, for example, the construction of a transcriptional fusion of the test gene ‘s promoter to a reporter gene, or the cloning of the gene of interest into an expression vector.

Several methods are available for directly quantifying the amount of a specific transcript. These include northern hybridization analysis, slot-blotting and reverse transcription coupled with PCR (RT-PCR). However, these methods do not allow the mapping of transcript end-points (the subject of this chapter).
Mutagenesis
Authors: Grigliatti T A
Published: November 2023

Revising this chapter engendered the thought that perhaps the days of large scale screens for mutations had waned. But the genome projects have described many genes (ORFs), or expressed sequences, for which no known function exists, and this provides impetus for the isolation of mutations in specific genes or regions of the genome. Until homologous gene replacement becomes available to make full use of in vitro mutagenesis, there will be a need to induce mutations in specific genes in order to analyse or confirm the function of protein domains in vivo. In addition, as the basic steps for essential functions in developmental and physiological pathways are being defined, screens for more specific phenotypes such as aberrant chromosome pairing, aberrant migration of specific cell types, aberrant organelle assembly, or aberrant behavioural characteristics are being undertaken. The successful identification of mutants in a particular process has always depended on the specificity of the screen. Therefore, considerable attention needs to be paid in the screening protocol to:
Enhancer traps
Authors: O’Kane C J
Published: November 2023

Enhancer trapping is widely used in Drosophila for generation of cell type markers that are often exquisitely specific, for identification of novel genes on the basis of expression patterns, and more recently, for the manipulation of defined cell types by allowing targeted expression of transgenes. While its application must of course be dictated by rigorous scientific considerations, there can be few experimental exercises that rival the fun of seeing new expression patterns that are natural works of art, and occasionally discovering a pattern that labels exactly those cells you always wanted to work with.