bluuebunny/crossref_metadata_2025 huggingface dataset:
NG 108-15 neuroblastoma × glioma hybrid cell line as a model neuronal system
Authors: Docherty R J , Robbins, J , Brown D A
Published: November 2023

Clonal cell lines are cultured tumour cells, i.e. they divide and proliferate spontaneously when given appropriate conditions in tissue culture. This property of cell lines gives them several distinct advantages over most other preparations available to biologists, especially neurobiologists, not least that they provide versatile model systems which involve a minimal sacrifice of life. Cell lines are homogeneous preparations of cells grown in a controlled environment where as few or as many cells may be prepared as are required for a particular purpose. Hence they have equal utility for investigators from different disciplines and provide a good preparation for multidisciplinary approaches to many of the problems facing biologists today.
Modelling the non-linear conductances of excitable membranes
Authors: Borg-Graham Lyle J
Published: November 2023

Modelling the electrical response of single neurons is a tool that can be used by the electrophysiologist both (a) to understand the mechanisms underlying cell behaviour and (b) to integrate data from different protocols into a consistent theoretical description of a cell in toto. Models of single neurons have appeared in many contexts, including squid axon (1, 2), molluscan neuron (3), amphibian spinal neurons (4), bullfrog sympathetic ganglion cells (5), rabbit nerve (6), cerebellar Purkinje cells (7, 8), and hippocampal pyramidal cells (9, 10).
Aequorin Measurements of Cytoplasmic Free Calcium
Authors: Cobboldand P H , Lee J, A C
Published: November 2023

1. Introduction The photoproteins aequorin and obelin generate blue-green light upon binding Ca2+ ions. Aequorin has been used over the past twenty years to measure cytoplasmic free Ca2+ (Cat) in single cells by injecting the cell with purified protein and then simply measuring the light emitted from the cell with a photomultiplier. The technique has been used in giant cells [giant barnacle muscle fibre (1), amoeba (2)] down to single mammalian cells [oocytes (3), and cells from heart (4), liver (5), and adrenal gland (6)]. Populations of mammalian cells have been loaded with obelin by liposome fusion, or with aequorin by various reversible permeabilization procedures such as hypo-osmotic shock, ATP-EGTA, scrape-loading, or spin-loading (for review see ref. 7).Photoproteins have several advantages over fluorescent indicators, includ ing the need for much simpler recording instrumentation and freedom from factors such as autofluorescence, intracellular compartmentalization, rapid leakage of the probe from the cell and buffering of calcium by the probe. Relative disadvantages are the need (at present) to microinject single cells, and lack of sufficient signal to make meaningful Cat images in mammalian cells. Recently it has been possible to compare data obtained with aequorin and fura-2 in single hepatocytes (5, 8, 9), when similar cellular Cat responses were found during hormone-stimulation; a gratifying result in view of the complexities of the intracellular environment and possible sources of spurious probe behaviour.
Metallochromic Indicators
Authors: Thomas M V
Published: November 2023

1. Introduction Metallochromic indicators were used quite extensively for intracellular Ca measurement before the introduction of the fluorescent indicators, and they can still be useful alternatives under some circumstances. In any case the two indicator families have a great deal in common, because they measure Ca in exactly the same way, i.e. by undergoing an optically detectable change as a result of binding Ca ions.
Quantification of the Affinity of Drugs Acting at the Calcium Channel
Authors: Kenny Barry A , Kilpatrick Andrew T , Spedding Michael
Published: November 2023

Functional calcium channels Calcium plays a vital role as an intracellular second messenger and is responsible for a variety of stimulus-response events. Intracellular calcium in resting cells is maintained at very low levels (less than 10-7 M) much of it being bound and sequestered at a variety of sites including endoplasmic and sarcoplasmic reticulum and intracellular Ca2+ binding proteins. Intracellular Ca2+ can rise as a result of increased permeability of the plasma membrane by the selective opening of Ca2+ channels allowing Ca2+ to pass into the cellular cytoplasm along its electrochemical gradient.
Sodium-Calcium Exchange Activity in Plasma Membrane Vesicles
Authors: Reeves John P
Published: November 2023

1. Introduction The Na+-Ca2+ exchange system is a carrier-mediated transport process in which the movement of Ca2+ions across the membrane is directly coupled to the movement of Na+ ions in the opposite direction. It is found in the plasma membrane of many types of cells, particularly excitable cells such as nerve and cardiac muscle. The exchange system is electrogenic with a stoichiometry in most cells of 3 Na+ per Ca2+ (1); in retinal rods, however, K+ movements are coupled to the exchange process and it can function with a stoichiometry of 4 Na+ per 1 Ca2+ + 1 K+ (2, 3).
Site-directed in vitro mutagenesis using uracil-containing DNA and phagemid vectors
Authors: Yuckenberg Patricia D , Witney Frank , Geisselsoder Janet , McClary John
Published: November 2023

Oligonucleotide directed in vitro mutagenesis is a widely used procedure for the study of the structure and function of DNA and the protein for which it codes, for studies on regulation of gene expression and DNA replication, and for modification of food and therapeutic proteins. A wide variety of techniques is available for performing in vitro mutagenesis (reviewed in ref. 1 and this book). One strategy is to clone the segment of DNA to be mutated into a vector whose DNA exists in both single and double-stranded forms. An oligonucleotide complementary to the region to be altered, except for a limited internal mismatch, is hybridized to a single-strand copy of the DNA. A complementary strand is then synthesized by DNA polymerase using the oligonucleotide as primer. Ligase is used to seal the new strand to the 5’-end of the oligonucleotide. The double-stranded DNA, homologous except for the intended mutation, is then transformed into E. coli, resulting in two classes of progeny; the parental and those carrying the oligonucleotide directed mutation. Since there are both parental and mutant progeny, no more than half of the progeny will be mutant. In practice, a much lower fraction is usually obtained. Recently, a number of methods have been developed which are more efficient. These methods create an asymmetry between the two strands of the heteroduplex and permit selection against the wild-type strand (see also Chapters 1 and 3).
Tensile fibres: strings and straps
Authors: Ker R F
Published: November 2023

This chapter is about the strings and straps of animals. By ‘strings’ I mean passive (i.e. non-muscular) tissues that are long and thin; they are flexible but, none the less, can transmit longitudinal tensile loads. ‘Straps’ are relatively broader but otherwise the same.
An Introduction to the Crystallogenesis Of Biological Macromolecules
Authors: Giege R , Ducruix A
Published: November 2023

The word ‘crystal’ is derived from the Greek root ‘krustallos’ meaning ·clear ice’. Like Ice, crystals are chemically well defined, and many of them are of transparent and glittering appearance. like quartz, which was for a long time the archetype. Often they are beautiful geometrical solids with regular faces and sharp edge which probably explains why crystallinity, even in the figurative meaning, is taken as a symbol of perfection and purity. From the physical point of view, crystals are regular three-dimensional arrays of atoms. ions, molecule or molecular assemblies. Ideal crystals nm be imagined as infinite and perfect arrays in which the building hlock (the a ymmetric units) arc arranged according to well-defined symmetries (forming the 230 space groups) into unit cells that are repeated in the three-dimensions by translations. Experimental crystals. however, have finite dimensions. An implicit consequence is that a macroscopic fragment from a crystal is still a crystal, because the orderly arrangement of molecules within such a fragment still extends at long distances. The practical consequence is that crystal fragments can be used as seeds (see Chapter 5) In laboratory-grown crystals the periodicity is never perfect, due to different kinds of local disorders or long-range imperfections like dislocations.
Methods of crystallization
Authors: Ducruix A , Giege R
Published: November 2023

There are many methods to crystallize biological macromolecules (for recent reviews see references 1-3). all of which aim at bringing the solution of biological macromolecules to a supersaturation state (see Chapters 9 and 10). Although vapour phase equilibrium and dialysis techniques are the two methods most favoured by crystallographers and biochemists, batch and interface diffusion methods will also be described.
Crystallization in Gels and Related Methods
Authors: Robert M C , Provost, K , Lefaucheux F
Published: November 2023

Gel growth is a particular case of, solution growth and consequently is ruled by the same parameters; however, some of the parameters can be varied much more conveniently by gelling the growth medium. After a short description of gel structures allowing identification of the parameters involved, typical techniques used for macromolecule crystal growth are reviewed in order to explain how the techniques are tailored for gel growth. Practical advice for the preparation of different gel-growth experiments is given together with comments about the expected results. To some extent, gel growth has features common with growth under reduced gravity or hypergravity. These related techniques are considered as well.
Automating Crystallization Experiments
Authors: Ward. K B , Perozzo. M A , Zuk W M
Published: November 2023

This book on the practical aspects of preparing single crystals of macro molecules suitable for diffraction analysis describes in detail the various activities required to find conditions suitable for optimum crystallization. These often involve the repetitive manipulation of small (1-10 µI) volumes of reagents to produce a solution which must then be inspected visually at intermittent times throughout a period ranging from several days to several months. This experimental protocol is ideally suited for the use of laboratory automation equipment. Conse4uently, several research groups have developed equipment in which one or more laborious, repetitive, and tedious operations have been automated.
Preparation of Selenomethionyl Protein Crystals
Authors: Doublie S , Carter C W
Published: November 2023

For many years multiple isomorphous replaccement (MIR) has been the method of choice for phase determination. In most cases MIR is reliable, but occasionally it fails: heavy-atom binding may not occur, or if it does it may lead to loss of isomorphism. Use of Multiwavelength Anomalous Dispersion (MAD) has been introduced to circumvent these two problems (I, 2). On the one hand, the MAD method exploits the presence of anomalous scatterers, such as Cu or Fe in metalloproteins, or any of the heavy-metal derivatizing agents. On the other hand, all measurement relevant to determining a single phase can be made on the same crystal o isomorphism is exact, and electron density maps are of very high quality (3, 4). Thus, the MAD method will likely become a preferred method for phase determination.
Finite element analysis biomechanics
Authors: Beaupre Gary s. , Carter dennis r.
Published: November 2023

Most bioengineers would agree that finite element analysis (FEA) has had a profound impact in the field of biomechanics. Finite clement analysis provides analysts with a technique to quantitatively analyse complex biological systems for which other solution strategies are either impractical or completely intractable. For example, in the field of orthopaedic biomechanics, FEA has increased our understanding of successful design strategics for total joint replacements to such an extent that the majority of research laboratories, as well as most manufacturers of orthopaedic implants, now comider FEA to be an indispensable analysis and design tool. In addition, FEA is becoming established as a powerful approach to implement and test new theories for the role of mechanical stresses in the development, maintenance, and repair of many tissue and organ systems.
Hydrodynamics of animal movement
Authors: DcMont M E , Hokkanen J E I
Published: November 2023

All organisms, whether plant or animal, terrestrial or aquatic, sessile or freely moving, large or small, experience now of fluid over and through external and internal structures. This flow can be an unwanted consequence of some other process that induces the flow, or generated purposefully for functional mechanisms. The forces generated by this flow, for all situations, have important implications to the life of the organism, and influence the physiology and ecology of the organism, as well as many other biological processes. Clearly, knowledge of these fluid processes will have important implications in understanding the relationship of an organism to its environment. This chapter introduces some basic theory of fluid dynamics and experimental techniques, important in understanding one particular aspect of biology — how animals move through water.
Functional analysis of uvomorulin by site-directed mutagenesis and gene transfer into eukaryotic cells
Authors: Stappert Jorg , Kemler Rolf
Published: November 2023

The generation and expression of suitable mutants of a gene is a powerful technique for altering the primary structure of the coded protein to study structure-function relationships. New methods for the isolation and cloning of genes and, above all, the chemical synthesis of DNA allow genes to be altered specifically by introducing deletions or insertions or by replacing individual bases in the DNA. In this way virtually any change desired may be made to the protein primary structure involving the 20 amino acids. The biological activity of these mutants can then be tested in vitro and in vivo. The availability of full-length CDNAs coding for cell adhesion molecules (CAMs) has opened new possibilities for detailed studies on the molecular mechan isms of cell adhesion.
Integrin mutagenesis
Authors: Reszka A , Horwitz A
Published: November 2023

Integrins are cell-surface receptors involved in cellular adhesion to the extra cellular matrix (ECM), to other cell receptors, and to blood molecules (1-4). In the cell’s interior, integrins are connected to the cytoskeleton through a supramolecular linkage. By virtue of these interactions, integrins are implicated in a vast number of functions (Table J).
Biochemical approaches for analysing de nova assembly of epithelial junctional components
Authors: Pasdar Manijeh
Published: November 2023

The epithelial intercellular junctional complex is composed of four major components: tight junctions (1-4), adherens junctions (5-8), desmosomes (1, 9-12), and cell adhesion molecules (13-22). These components rapidly appear on the plasma membrane of adjacent cells upon induction of cell-cell contact. A common feature of these junctional components is their apparent structural similarity (Figure IA). Each junctional component consists of an extracellular or membrane core domain and an intracellular or cytoplasmic plaque domain to which cytoskeletal elements are attached. In all identified cases the extracellular domain consists of glycosylated integral membrane proteins and the cytoplasmic domain comprises non-glycosylated proteins (Figure 1B). These integral membrane and cytoplasmic proteins presumably go through different biosynthetic pathways (Figure 2). The glycoproteins of the membrane core domain are synthesized and core glycosylated in the endoplasmic reticulum. These proteins are then transported to the Golgi complex, where they become complex glycosylated. Subsequently, the glycoproteins are transported via transport vesicles to the plasma membrane. The protein components of the cytoplasmic domain however, are synthesized on free ribosomes and are then transported to the plasma membrane. Consequently, the assembly of a multi-subunit junctional complex requires the coordinated expression and processing of the constituent proteins and their final association in the fully assembled structure. Therefore, a comparative analysis of the biosynthetic pathways of the protein constituents of a multi subunit complex should provide valuable insights into mechanisms regulating its assembly.
Recording membrane currents of peripheral neurones in short-term culture
Authors: Stansfeld Catherine , Mathie Alistair
Published: November 2023

In comparison with central neurones, ganglion cells are attractive because they are accessible collections of relatively uniform cell types, easily identified visually. An added advantage is that they are generally large—dorsal root ganglion (DRG) cells being 10-60 μm and sympathetic superior cervical ganglion (SCG) cells being 15—40 μm—thus, they are readily accessible to electrophysiological techniques. Quite apart from questions concerning the physiology of the sensory system, the unipolar DRG and nodose ganglion cells can be viewed as model spherical neurones to which can be applied more general questions concerning pre- or post-synaptic membrane processes. They have, for example, been used extensively to examine biochemical processes governing calcium channel function. Thus, they are an invaluable electrophysiological tool from which we can gain insights into the mechanisms at work in less accessible neuronal membranes. On the other hand, SCG cells are normally multipolar, and so lack the ideal compactness afforded by the sensory ganglion cells; however, they temporarily adopt this ideal spherical form when dispersed from the ganglion. At this stage their membrane currents can be studied without the complications of dendrites. In this chapter, we concentrate on a short-term preparation or isolated cells-to be studied within 24 h-and have chosen sympathetic SCG cells as our example. The purpose of this preparation is to achieve rapid cell dissociation for electrophysiology in which high cell yield is not important (4000 cells per day is usually enough for most electro physiologists). Our emphasis is on how to optimize conditions for good control of the membrane voltage, and how to make use of this in interpreting the membrane currents seen.
Transport mechanisms in ocular epithelia
Authors: Jacob T J c. , Stelling J W , Gooch Amanda , Zhang Jin Jun
Published: November 2023

1. Introduction

The epithelia of the lens and ciliary body of the eye represent very different structures both from structural and functional viewpoints. The reasons for studying them are, however, related. Two diseases of the eye, cataract and glaucoma, result from the malfunctioning of these respective epithelia.

All cpithelia arc involved in transport and, although there are some broad categories, each individual epithelium tends to possess specific faculties that arc directly related to its particular function. In the case of the lens, this function is to maintain the ionic and solute balance in the tissue to prevent swelling and opacification, and in the case of the ciliary epithelium, it is to secrete aqueous humour. Although these two epithelia have distinct functions, they nevertheless have a number of channels, exchange mechanisms, and pumps in common, and this chapter deals with the techniques used in their identification.
Intracellular staining of enteric neurones
Authors: Lees Gordon M
Published: November 2023

It is now widely accepted that the enteric nervous system (ENS) provides neuroscientists with many challenges, one of the most important problems being the identification of neuronal pathways involved in intestinal reflexes, particularly intrinsic reflex responses, such as peristalsis. The intrinsic neurones of the gastrointestinal tract lie in ganglionated plexuses between the (outer) longitudinal muscle and the circular muscle layers (myenteric plexus) and in the submucosa (inner and outer submucous plexuses) (Figure J). Morphologically, elcctro physiologically, immunohistochcmically, and pharmacologically these enteric neurones show an amazing diversity, both between and within the plexuses (1-10).
Making of the atlas
Authors: Singh Juswinder , Thornton Janet M
Published: November 2023

This section describes how the information in the atlas was derived. It describes the protein dataset used, the definition of the side chain interactions and their geometries, and the use of local coordinate systems to generate three-dimensional distributions of interacting pairs. The suite of programs used for the calculation and analysis of the interacting pairs is called SIRIUS (Singh and Thornton 1990), and a flowchart of the approach is shown in Fig. 1.
Cyclin-dependent kinases: an entbarrassntent of riches?
Authors: Pines Jonathon , Hunter Tony
Published: November 2023

One of the most exciting advances in our understanding of the cell cycle has been the identification in multicellular organisms of a family of protein kinases that are involved in the regulation of different checkpoints. These protein kinases are related both in their primary structure, and because they bind and are activated by the cyclin family of proteins. The latter property has led to this family of protein kinases being called the ‘cyclin-dependent kinases’, or cdks. The cyclins are a family of structurally related proteins that typically vary in amount during the cell cycle in a programmed manner (the cyclins are reviewed in Chapter 5). The first two cyclins to be isolated, cyclins A and B, were called mitotic cyclins, because they were specifically destroyed in mitosis, implicating them in the regulation of the G2/M transition. Subsequently, another class of cyclins was discovered in yeast that have roles in the Gl/S transition, and are called Gl cyclins. More than ten different cyclins have been identified in humans and in budding yeast, and the different cyclin/cdk complexes are involved in regulating several different check –points throughout the cell cycle. Furthermore, it now appears that some cyclin –cdk complexes regulate processes quite separate from the cell cycle. The paradigm for the cyclin-cdk interaction is that between cdc2 and cyclin B (reviewed in Chapter 5). Therefore, in this article we will only outline the salient points.
The packaging of pre-Mrna
Authors: Mcafee James g. , huang mae , soltaninassab syrus , Rech janee. , iyengar, sunita , Lestourgeon wallace m.
Published: November 2023

In eukaryotes, pre-mRNA is bound by protein either simultaneously with the initiation of transcription or very soon thereafter (1-3), and it is not possible to isolate pre-mRNA molecules free from a unique set of abundant nuclear proteins unless protein denaturants are used in the purification scheme. The packaging of DNA into nucleosomes, and their association in the 30 nm chromatin fibre, greatly foreshortens the DNA substrate and provides some protection of the genome from nuclease activity (4). However, the major pre-mRNA binding proteins possess well- characterized, helix-unwinding activities (5-8) and, in comparison with nucleosome packaging, provide less protection from nuclease activity (9,10). It seems then, that the pre-mRNA packaging mechanism functions primarily to maintain the transcript in a single-stranded state. This generally fits with the short-lived nature of pre- mRNA, and with the fact that nascent transcripts must be accessed by factors involved in intron removal, polyadenylation, and transport. Unlike rRNA, tRNA, and snRNA, pre-mRNAs are usually transcribed from single-copy genes and the pool of total nuclear pre-mRNA reflects great length heterogeneity (hence the term, heterogeneous nuclear RNA or hnRNA).
Spliceosome assembly
Authors: Reed Robin , Palandjian Leon
Published: November 2023

Pre-mRNA splicing takes place within a large, highly dynamic complex designated the spliceosome. Spliceosomes are 50-60S, have a calculated molecular weight on the order of 3-5 × 106 kDa, and are estimated to be 40-60 nm in diameter (see 1, 2 for reviews). Among the best characterized of the spliceosomal components are the small nuclear RNAs (snRNAs) Ul, U2, U4, U5, and U6, which are thought to play central roles not only in spliceosome assembly, but also in the two catalytic steps of the splicing reaction (reviewed in 1, 3, 4; see Chapter 5). Less is known about the protein components of the splicing machinery. Genetic studies in yeast have identified over 50 protein-coding genes essential for splicing (reviewed in 2; see Chapter 7), and an equal number of proteins have been detected in highly purified mammalian spliceosomes (5, 6). In addition to obtaining direct evidence for RNA- mediated catalysis, major challenges of the splicing field include cloning, characterizing, and determining the functions of the protein components of the spliceosome and achieving a detailed understanding of spliceosome assembly.
snRNP structure and function
Authors: Will Cindy l. , lÜhrmann reinhard
Published: November 2023

In contrast to self-splicing RNA molecules which contain extensive, highly con¬ served structural elements sufficient for intron excision and exon ligation, nuclear pre-mRNA molecules possess short, conserved sequences which are limited to the 5’ and 3’ splice site and branchpoint regions (see Chapters 1, 4, and 8). While these sequences are necessary for the conversion of the pre-mRNA to mRNA, they are not sufficient. Nuclear pre-mRNA splicing is thus dependent upon the activity of a number of trans-acting splicing factors, which in addition to protein factors, include the four small nuclear ribonucleoprotein particles (snRNPs) Ul, U2, US, and U4/U6. These evolutionarily conserved RNA-protein complexes interact stepwise with nuclear pre-mRNA molecules to form the large multicomponent RNP complex (the spliceosome) in which splicing occurs (see Chapter 4). Ul, U2, US, and U4/U6 snRNPs play a central role in the recognition and alignment of splice sites. In addition, snRNPs may directly contribute to the catalytic activity of spliceosomes: snRNA constituents have been proposed to form the catalytic centre responsible for excision-ligation events.
Mammalian pre-mRNA splicing factors
Authors: Caceres Javier f. , Krainer adrian r.
Published: November 2023

Since the discovery of pre-mRNA splicing in 1977, considerable progress has been made in dissecting the czs-elements and frzms-acting factors involved in carrying out this complex reaction. Multiple protein and nucleoprotein components are necessary to catalyse pre-mRNA splicing, which occurs within a particle termed the spliceosome. The components of the splicing machinery, and/or factors involved in the assembly of the spliceosome, include the small nuclear ribonucleoprotein particles (snRNPs) Ul, U2, U4/U6, and U5; the polypeptides that associate with hnRNA to form hnRNP particles (hnRNP proteins); and a large number of non- snRNP essential splicing factors (for earlier reviews see 1-6).
Alternative pre-mRNA splicing
Authors: wang Yung-chih , selvakumar, meenakshi , Helfman david
Published: November 2023

The formation of a mature mRNA from a primary transcript generally requires excision of intervening sequences (introns) with the subsequent joining together (splicing) of exons. The general splicing reaction of metazoan intrans has been well characterized and involves a two-step process, which occurs in a large ribonuclear protein complex, termed the spliceosome (see Chapter 4; reviewed in 1, 2). In the first step, a 5’ exon is cleaved and two intermediates are formed: a 5’ exon with a 3’ hydroxyl end, and an RNA molecule containing the downstream intron sequence and 3’ exon, in which the 5’ terminal guanosine of the intron is covalently linked via a 2’-5’-phosphodiester bond to a residue termed the branchpoint, usually an adenosine, located 18-40 nucleotides upstream of the 3’ splice site. In the second step, cleavage at the 3’ splice site results in the release of a lariat intron and con-comitantly the two exons are ligated together.
Analysis of Gene Expression by Reverse Transcriptase-Polymerase Chain Reaction
Authors: Koopman Peter
Published: November 2023

A molecular genetic approach to development often involves isolating genes controlling cellular developmental processes, analysing their structure and regulation, and gaining clues about their function by examining their expression. Two constraints commonly encountered in studying gene expression in embryos are scarcity of tissue and low levels of expression. When the gene of interest is expressed in small groups of cells, or when transcripts are present in vanishingly low amounts, it is difficult to perform Northern blotting, RNAase protection assays, or in situ hybridization. In these cases the exquisite sensitivity of the polymerase chain reaction (PCR) can provide a solution. PCR has been used to detect transcripts present at down to one copy per thousand cells (1), and in samples as small as a mouse blastocyst (2-4), or even a single cell (5, 6).
Oligonucleotide-directed mutagenesis with single-stranded cloning vectors
Authors: Kolmar Harald , Fritz Hans-Joachim
Published: November 2023

Directed mutagenesis with synthetic oligonucleotides was proposed in 1971 by Hutchison and Edgell (1) and first developed to a practical procedure by Zoller and Smith (2, 3). According to the original protocol, the gene of interest is first cloned in a vector derived from phage M13 (single-stranded circular DNA genome). An oligonucleotide is synthesized that is complementary to the target site of mutagenesis with the exception of one or more non complementary nucleotide central residues. The oligonucleotide is annealed to the single-stranded, recombinant vector DNA, and covalently closed double-stranded heteroduplex DNA (hd DNA) containing a single site of non-homology is synthesized in an enzyme catalysed reaction. This hd DNA is transfected into E. coli where it replicates in vivo, to segregate DNA clones carrying the predetermined mutation carried on the synthetic oligonucleotide. Some of the first spectacular successes of protein engineering were achieved with this method (see for example ref. 4). It was soon realized, however, that the procedure is bound to produce only low yields of the desired mutants because cellular processes of DNA mismatch repair are directed against the DNA strand synthesized in vitro (5). Marker yield is indeed an important aspect, since it drastically influences the work needed to identify a clone with the correct structure, which-in the absence of a phenotype-has to be accomplished solely by biochemical means such as nucleotide sequence analysis (5).
Expression of cloned genes in yeast
Authors: Romanos Michael A , Scorer Carol A , Clare Andjeffrey J
Published: November 2023

Saccharomyces cerevisiae has long been a model organism for biochemical and genetic studies, and the expression of cloned genes in yeast was examined as soon as transformation procedures became available. The particular advantages it offers over other expression systems are mainly due to the fact that it is both a micro-organism and a eukaryote. Its genetics are more advanced than any other eukaryote, so that it can be genetically manipulated almost as easily and rapidly as Escherichia coli. As a eukaryote, it is more likely to provide a suitable environment for the folding of foreign eukaryotic proteins than E. coli, and can be more readily used for secretion of proteins. Yeast can be grown rapidly on simple media, and to high cell density. Additionally, as a food organism, yeast is regarded as highly acceptable for the production of pharmaceutical proteins.
Interaction trap cloning with yeast
Authors: Russell L , Finley JR , Brent Roger
Published: November 2023

The interaction trap is a two-hybrid system for cloning cDNAs encoding proteins that interact with a protein whose coding sequences are known. The method uses the transcription of yeast reporter genes as a synthetic phenotype to detect protein-protein interactions. It can also be used to study interactions between known proteins.
Expression using a defective herpes simplex virus (HSV-1) vector system
Authors: Lim Filip , Starr Philip , Song Song , Hartley Dean , Lang Phung , Wang yaming , Geller Alfred I
Published: November 2023

Genetic intervention in the brain through the use of vector systems capable of transferring recombinant genes into mammalian neurones is becoming an increasingly useful strategy for investigating the molecular basis of various brain functions ranging from regulating homeostasis to information processing and higher cognitive functions (1, 2). Genes have been isolated and characterized which encode molecules such as signal transduction enzymes, transcription factors, and components of the neurotransmitter release machinery that may play critical roles in regulating neuronal physiology. Relatively little is known about the function of these molecules in mediating alterations in neuronal physiology and the resulting changes in complex behaviours. Genetic intervention in the brain has the theoretical potential to reveal precise information about specific brain functions through the use of mutations in neuronal genes, and then subsequent expression of the altered gene products in their normal cellular milieu.
Adenovirus vectors
Authors: Gerard Robert D , Meidell Roberts
Published: November 2023

The introduction of foreign genetic material into somatic cells in culture and in intact organisms is an important investigational technique and holds prom ise as a therapeutic tool.
Cosmid clones and their application to genome studies
Authors: Ivens Alasdair c. , Little Peter F R
Published: November 2023

A 1000-fold range of DNA sizes may be cloned in the current range of cloning vectors: the ideal vector for genome mapping studies would be one that is easy to use while containing as much DNA as possible. Cosmids now occupy the middle ground: they have a significant capacity, thus reducing the number of steps required to clone an entire gene or region, combined with very simple methods for isolating inserted DNA in pure form. As a consequence, positional cloning strategies frequently involve the use of cosmids as a final cloning vector for reducing a yeast artificial chromosome (YAC) clone to manageably sized DNA fragments (1). Cosmids are units that are very likely to contain an entire gene, are easily mapped with respect to restriction sites, and are amenable to the application of a number of other functional assays, e.g. exon trapping (2, 3), genomic sequencing (4-6), and fingerprinting to generate contig maps (7, 8). As a result, the detailed information that can be obtained from a cosmid clone makes it the ideal medium for genome analysis. Indeed, several genome mapping studies (e.g, Caenorhabditis elegans (9), Escherichia coli (10)), have relied on physical DNA maps built around cosmid clones.
Isolation and characterization of photosynthetic reaction centres from eukaryotic organisms
Authors: Evans Michael c.w. , Hallahan Beverly j. , Hanley Jonathan A , Heathcote Peter , Gumel Nicola J , Saul Purton
Published: November 2023

Photosynthetic reaction centres are the site of solar energy conversion in biological systems. The basic organization of photosynthetic systems is the same in all chlorophyll-containing organisms. Light is absorbed by a large array of chlorophyll molecules and the energy transferred by random processes through the array to a reaction centre chlorophyll, which undergoes photochemical oxidation transferring an electron to an acceptor molecule. The chlorophyll array is highly organized with the pigments bound to a range of proteins which modulate the chlorophyll energy levels and provide an organization which facilitates energy transfer to the reaction centre. The light harvesting protein complexes surround the reaction centre forming a photosynthetic unit. The photosynthetic units are in tum bound in a specific membrane system, the thylakoid. In eukaryotic organisms the thylakoids are in tum limited to a specialized organelle, the chloroplast. The high chlorophyll to reaction centre concentration makes optical analysis of the reaction centre extremely difficult and also limits the concentration of reaction centres which can be obtained for spectroscopic analysis. The presence of two types of reaction centre in oxygen evolving organisms further complicates analysis. The successful development of models of reaction centre structure and mechanism has depended on the development of techniques to isolate and characterize the reaction centres.
Haemolytic assays for whole complement activity and individual components
Authors: Whaley Keith , North Jonathan
Published: November 2023

The ability of the intact classical or alternative pathways of complement to lyse erythrocytes is used in functional assays of entire pathways or of single components. Functional assays have the advantage over immunochemical assays of individual components, as the latter, depending on the choice of detecting antibody, can measure non-functional proteins and breakdown products generated by complement activation in addition to the native proteins. Reproducible haemolytic assays depend on performing the tests in conditions similar to those in vivo in respect of isotonicity and pH, as deviations from this can lead to spontaneous lysis of the target cell.
Purification of complement components
Authors: Daha Mohamed R
Published: November 2023

Purified complement components are required for many purposes, including assessment of their biological activities, use as standards and reagents in assays, and raising of antibodies. In particular, the role of complement in vivo requires further extensive research in the near future. It is thought that transgenic and knockout models of complement may provide valuable additional information, and in these studies the substitution of missing complement components with purified reagents may prove to be essential.
Biosynthesis of complement components
Authors: Circolo Antonella , Nutter Thomas B , Strunk Robert C
Published: November 2023

Complement protein biosynthesis has been studied since the 1890s when Metchnikov suggested that leucocytes were a source of complement (1). Ehrlich and Morgenroth (2) were the first to suggest that the liver was a site of complement production when they demonstrated that complement, but not antibody, was depressed in sera of animals poisoned with phosphorus. In the late 1960s, Alper and co-workers (3) confirmed that the liver was a major site of serum complement synthesis by studying a patient following liver transplantation; the shift of C3 from recipient to donor type indicated that the liver synthesized at least 95% of the C3 found in serum.
Arabidopsis mutant characterization; microscopy, mapping, and gene expression analysis
Authors: Kalantidis Kriton , Greg Briarty L , Wilson Zoe A
Published: November 2023

Mutants provide a valuable resource for the study of developmental biology, since the effects of genes can be identified and their role in development assessed in homozygous and heterozygous states. One problem with gene identification by mutagenesis can be the cloning of the specific gene (see Chapters 7 and 8). This chapter will cover a range of approaches which can be used to characterize mutants of Arabidopsis thaliana. These include the initial mapping of the mutation and determination of allelism between other mutations with similar phenotypes, both by classical linkage analysis and by molecular approaches. Different approaches are presented to allow the detailed analysis of phenotype by microscopy. The final part of the chapter gives protocols to permit the analysis of gene expression by Northern and in situ hybridization.
Tissue culture, transformation, and transient gene expression in Arabidopsis
Authors: Lindsey Keith , Wei Wenbin
Published: November 2023

Tissue culture and transformation technologies have revolutionized studies on plant biology. They allow investigations, in a way previously only dreamed of, of the relationships between, plant morphology on the one hand and gene expression, metabolism, and cell differentiation on the other. Disorganized growth, such as occurs during callus formation, can be experimentally induced and the developmental, biochemical, and genetic consequences studied (1). Furthermore, and of fundamental significance, disorganized tissues can be induced to reorganize and to regenerate intact organs and whole plants. This observation has been crucial in the development of the concept of the totipotency of plant cells, and has been equally fundamental to the establishment of reproducible techniques for the genetic engineering of plants. Thus, it is possible to introduce foreign genes into cultured plant cells, protoplasts, or tissues, and regenerate from them stably transformed individuals that will transmit the introduced transgenes to their progeny. It is beyond the scope of this chapter to discuss the commercial or agronomic implications of plant genetic engineering, but applications that are of a more basic scientific nature, and to which Arabidopsis thaliana lends itself ideally as an experimental organism, can briefly be considered here.
Cell cycle analysis by flow cytometry
Authors: Crissman Harry A
Published: November 2023

Mechanisms that control and regulate the cell cycle are currently being intensively studied in numerous laboratories. Central to these studies is the basic understanding of those regulatory mechanisms that appear to be lost or dysfunctional when cells become transformed from the normal to the malignant state. This information can lead to a better understanding of the nature of cancer and potentially result in the design of more effective chemotherapeutic agents or even to the intervention of those processes that lead to the development of cancer. Studies in these areas will necessarily rely on the constant assessment of cell cycle distribution and cycle progression.
Analysis of flow cytometric DNA histograms
Authors: Rabinovitch Peters.
Published: November 2023

Analysis of the DNA content of cells and their distribution within G1, S and G2/M phases of the cell cycle is one of the earliest applications of flow cytometry. Because of its simplicity, DNA content analysis remains a commonly used and powerful technique. Chapters 2 and 8 within this volume describe additional variations and enhancements of the original flow cytometric techniques; however, mathematical analysis of single parameter DNA content histograms remains a common element in many of these methods. Recent interest in the clinical application of DNA ploidy and cell cycle measurements from DNA histograms has stimulated improvements in the methods and models used to derive these measurements, and guidelines for the implementation of DNA cytometry have been established to help ensure greater accuracy in implementation of these techniques. This chapter describes a protocol consistent with these guidelines for DNA content histogram analysis and discusses some of the principles in methods of analysis.
Morphological and biochemical criteria of apoptosis
Authors: Ramachandra Sumant , Studzinski George P
Published: November 2023

When cells receive mixed signals for growth they usually die. For instance, when the developmental programme requires cell division but external growth signals are lacking, or when a growth-related gene such as c-myc is highly expressed but the cellular environment has insufficient nutrient content, or a xenobiotic is present, the cell dies by a process termed apoptosis. Although there are differences in the phenomena observed during this sequence of events, depending on the cell type, and agent or circumstance which initiates the cell’s demise, there are morphological and biochemical similarities which suggest that these are variants of the same biological process designed to control the size of cell populations.
Cell synchronization as a basis for investigating control of proliferation in mammalian cells
Authors: Stein Gary S , Stein Janet L , Lian Jane B , Last T J , Owen Thomas , Mccabe Laura
Published: November 2023

From a historical perspective, synchronized cells have provided the basis for defining regulatory mechanisms operative during specific periods of the cell cycle; for example, those at key transition points that separate Gi, S-phase, G2 and mitosis as well as those operative when quiescent cells are induced to proliferate. Cell synchrony studies have also contributed to establishing modifications in gene expression which are functionally related to initiation and execution of DNA replication during S-phase and to the onset and completion of mitotic division. More recently this experimental approach has increased our understanding of key modifications in gene expression and the interrelated cellular signalling pathways that transduce, integrate and amplify information that support each stage of the cascade of regulatory events requisite for progression through the cell cycle. This chapter is restricted to methods that have proven to be effective for synchronizing continuously dividing normal diploid and tumour cells as well as for induction of quiescent cells to embark upon a progression of events that leads to cell division. Protocols that can be applied for monitoring cell synchrony will be addressed. In addition, we will evaluate the extent to which cell synchrony can be achieved in a manner that is compatible with physiological regulation that is operative under in vivo conditions.
Analysis of the effects of purified growth factors on normal human fibroblasts
Authors: Phillips Paul D , Cristofalo Vincent J
Published: November 2023

Normal human diploid fibroblast-like cells are widely used for studies of in vitro cell ageing and in studies of growth factor action and cell proliferation. Because of their special growth characteristics the manipulation of these cells requires special attention and understanding to ensure the vigorous and optimum growth of the cultures.

Cells from normal human tissues, explanted and grown in tissue culture, have the following characteristics: (i) the cultures possess a finite and predictable proliferative capacity most accurately measured as cumulative number of population doublings (chapter 12); (ii) the replicative life span of a cell line is inversely proportional to the age of the donor; (iii) normal human fibroblast-like cells have never been shown to transform spontaneously in culture, i.e. they have never been shown spontaneously to acquire the indefinite proliferative capacity characteristic of tumour derived or otherwise transformed cells; and (iv) normal human cells can be transformed into cultures with an indefinite proliferative capacity by infection with DNA tumour viruses such as simian virus 40 (SV40) (1, 2, and Chapter 12).
Epithelial cell culture
Authors: Shaw Andrew J
Published: November 2023

Our understanding of the functions of epithelia, and their development and maintenance, has been rapidly advanced by the use of cell cultures which faithfully display the morphological, biochemical, and functional characteristics of these tissues. This book gives detailed step-by-step descriptions of the methods used to mimic epithelial and endothelial tissue iin vitror, and the techniques used to characterize these cell culture models, together with hints and tips for success, troubleshooting guides, and key background information. Topics covered include: modelling epithelial tissues in vitro; identification and localization of cytokeratins; assessment of cell polarity; xenobiotic metabolism; culture and characterization of human endothelial cells; studying transport processes in absorptive epithelia; a model of glandular epithelium for studying secretion; a model of the blood-testis barrier for studying testicular toxicity; reconstruction of human skin epidermis in vitro. Epithelial Cell Culture: A Practical Approach is a timely and comprehensive practical guide for all researchers who are studying, or intend to study, epithelia-related tissues using modern cell culture techniques. As such, this book will have a very wide appeal to cell biologists, developmental biologists, biochemists, pharmaceutical scientists, pharmacologists, and toxicologists.