bluuebunny/crossref_metadata_2025_split huggingface dataset:
Chromosome analysis in cell culture
Authors: Mohamed Anwar N , Wolman Sandra R 
Published: November 2023

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

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

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

Abstract
DNA has a simple yet intricate structure that serves as the template for the assembly of a chromosome. Each chromosome has one DNA molecule, a long unbranched double helix consisting of two antiparallel polynucleotide chains. The structure is stabilized by base-stacking interactions and hydrogen bonding between the bases: G (guanine) being paired to C (cytosine) and A (adenine) to T (thymine). Physical studies using X-ray diffraction indicate that, under conditions of physiological ionic strength, DNA is a somewhat regular helix with a diameter of 2 nm, making a complete turn every 3.0 to 3.4 nm. This particular DNA structure (B form) has approximately 10.5 bp/tum of the helix, with a variation due to base sequence of 9.9 to 11.1 bp/tum. Every base pair is rotated approximately 34° around the axis of the helix relative to the next base pair, resulting in a twisting of the two polynucleotide strands around each other. A double helix is formed that has a minor groove (very approximately 1.2 nm across) and a major groove (very approximately 2.2 nm across). The geometry of the major and minor grooves of DNA is crucial in determining the interactions of proteins with the DNA backbone sugars and phosphates. The double helix is usually right handed.
Promoter potentiation and activation: chromatin structure and transcriptional induction of heat shock genes
Authors: Lis John , Wu Carl 
Published: November 2023

Abstract
The heat shock gene regulatory circuit is an attractive model for elucidating general principles that govern inducible gene transcription and for investigating the role of chromatin and nucleosome structure in transcription activation. The degree of transcriptional stimulation is robust: transcription of the major heat shock genes can be increased over 100-fold upon heat shock (1), enhancing the detection of specific regulatory components and allowing a quantitative analysis of the role of these components. The transcriptional response is mediated by proteins that are present in induced cells (2, 3) and is extremely rapid (4), thereby facilitating kinetic and mechanistic investigations of the activation of these genes. Comprehensive studies from many laboratories provide a strong foundation of information on the primary structure of these genes, the ds-regulatory elements and trans-acting protein factors, as well as the chromatin structure of the uninduced gene and structural changes occurring after heat shock (for reviews see 5, 6, 7).
Fractionation of cells by sedimentation methods
Authors: Patel D , Ford T C , Rickwood D 
Published: November 2023

Abstract
Cells can be separated and fractionated by a number of techniques using the different properties of the individual cell types. One of the most common methods used for the isolation and purification of cells is sedimentation, usually using centrifugation. Cells may be separated by size using simple dif ferential pelleting procedures or separated on the basis of size and/or density using density gradient centrifugation. Nearly all cell separation procedures involve the pelleting of cells as part of the procedure for harvesting or con centrating cells prior to most fractionation procedures. However, differential pelleting is seldom used for separating different cell types, because the required difference in size, about tenfold, is rarely seen and so this technique will not be described in detail. Instead this chapter will concentrate on cell separations using density gradients with cells sedimenting either under unit gravity or in a centrifugal field. Aspects of cell separations that involve elutria tion are described in Chapter 3 and will not be discussed here.
Separation and fractionation of cells by partitioning in aqueous two phase systems
Authors: Fisher D 
Published: November 2023

Abstract
In the technique of cell partitioning, cells are added to the aqueous two-phase systems that are formed when aqueous solutions of certain polymers (typically dextran and polyethylene glycol) are mixed above critical concentrations. The cells distribute (partition) between the top phase, the bottom phase, and the interface between them (frcqucnlly termed the bulk interface).
Separation of cells by flow cytometry
Authors: Ormerod M G 
Published: November 2023

Abstract
A flow cytometcr uses a flow system to deliver cells in single file past a point of measurement. Light is focused at this point and the fluorescence and the light scattered by the cells is collected and measured. Flow cytometers measure several parameters on single cells at rates hetween hundreds to thousands of cells per second. The method can be used to define and to enumerate accurately subpopulations. Once identified, such subpopulations can be physically sorted for further study.
Nucleic Acid Structures
Authors: Riesner Detlev 
Published: November 2023

Abstract
From the wide discipline of nucleic acid structure only selected topics have a direct bearing on antisense technology. Thus nucleic acid structure will not be discussed in this chapter as an independent field of research but as an essential factor for a particular field of application. The restrictions for selecting the topics of interest concern the structural elements as well as the solution conditions and the thermodynamic conditions. I shall mainly address RNA intramolecular structure and RNA:RNA double-strand formation. A few examples of DNA oligonucleotides directed against RNA targets will also be considered. Antisense technology is predominantly an in vivo technology, whereas studies on nucleic acid structure have been carried out in the past mainly in vitro. Therefore, in vitro conditions either have to be adapted to reflect the in vivo situation or the data have to be extrapolated. This requirement restricts the applicability of several experimental methods. Not only the solution conditions but also the thermodynamic approach has to resemble the requirements of antisense technology. Antisense RNA as well as target RNA act in structures which are formed right after synthesis in the cell. These structures, which might also exist as mixtures of conformers, deviate in many cases from the equilibrium structure. This complicates the experimental as well as the theoretical approach. Having these restrictions in mind, an outline will be given on the structures, on methods to predict them, and on experimental approaches for structure analysis.
Catalytic Antisense RNA Based on Hammerhead Ribozymes
Authors: Tabler Martin , Sczakiel Georg 
Published: November 2023

Abstract
Some plant viruses are accompanied by so-called satellite RNAs which cannot replicate autonomously but require a helper virus for replication (1). Some of these satellite RNAs, especially the circular satellite RNAs, which are also called virusoids, undergo Mg2 catalysed autocatalytic self-cleavage at a defined position of their RNA genome. A specific phosphodiester bond is chemically cleaved in an intramolecular trans-esterification reaction resulting in a 2’ ,3’ cyclic phosphate located 5’ to the cleavage site, whereas the 3’ cleavage product has a 5’ hydroxyl group. A similar type of reaction can be found in two plant viroids which, unlike the satellite RNAs, do not depend on the presence of a helper virus for replication, and in the RNA transcript of a satellite DNA 2 of newt. A feature common to all these RNAs is the presence of a so-called ‘hammerhead structure’ (2).” The hammerhead structure consists of several conserved nucleotides and three helices that can he drawn such that the resulting secondary structure resembles the head of a carpenter’s hammer. A generally accepted numbering system has been defined (4) which can be seen in Figure 1. Despite the generality of the hammerhead structure and the conservation of the nucleotides that are involved in catalysis, there is considerable flexibility with regard to loop size and length of the helices.
Construction and use of antibody gene repertoires
Authors: Pope A R , Embleton M J , Ugh R Merna 
Published: November 2023

Abstract
The display of antibody molecules on the surface of bacteriophage (1) pro vides a powerful method of selecting a specific antibody from a mixed popu lation of antibodies together with the gene encoding it. This ability to co-select proteins and their genes has been exploited to enable the isolation of high-affinity, antigen-specific antibodies derived from an immunized mouse (2) and from unimmunized humans (3). In addition, after selecting such an antibody, a mutant antibody library can be constructed and higher affinity antibody clones selected. Alternatively, a monoclonal antibody can be subcloned from a hybridoma cell line into the bacteriophage and after mutagenesis, antibodies of improved characteristics selected (4).
Rodent to human antibodies by CDR grafting
Authors: Bendig Mary M , Jones S Tarran 
Published: November 2023

Abstract
The concept that a functional human-like antibody could be created by graft ing the antigen binding complementarity determining regions (CDRs) from variable domains of rodent antibodies on to human variable domains was first demonstrated by Dr Greg Winter and his colleagues at the MRC Labo ratory for Molecular Biology in 1986 (1). Since then several different research groups have developed and improved the methods for designing and constructing humanized antibodies via CDR grafting (2). There are reports of at least 80 antibodies being humanized by CDR grafting and at least eight of these antibodies being evaluated in clinical trials (3, 4). This chapter describes the procedures currently used at the MRC Collaborative Centre to create a ‘reshaped human’ antibody from a mouse antibody via CDR grafting.
In vitro reconstitution and analysis of nucleosome positioning
Authors: Davey Colin , Pennings, Sari , Allan James 
Published: November 2023

Abstract
It is now established that positioned nuclcosomes play a role in controlling the genetic functions of DNA (1–3). This is a consequence of the ability of the core-histone octamer to: (a) sequester a DNA sequence, by incorporating it within the nucleosome; (b) ensure that a DNA sequence remains exposed within the linker DNA between nucleosomes; or (c) to bring distant DNA sequences into close proximity. The precise positioning of nucleosomes with respect to the underlying DNA sequence thus determines its capacity to interact with, or to promote interaction between, other DNA-binding factors. The specific disposition of nucleosomes over long stretches of DNA is also likely to determine the architecture and stability of the higher-order chromatin fibre, which may, in turn, dictate its functional properties.
Microbiological techniques for molecular biology: bacteria and phages
Authors: Bainbridge Brian W 
Published: November 2023

Abstract
Recombinant DNA technology depends on the manipulation of particular strains of bacteria and bacteriophages (phages). Expertise in isolating, checking, growing, and analysing these strains is crucial for the success of genetic engineering. Many experiments are now permitted under conditions of ‘good microbiological technique’ and anyone embarking on research in this area should check exactly what is meant by this phrase. Essentially, all of the basic techniques depend on the culturing of a particular microbe in the absence of other organisms (sterile or aseptic technique), the isolation of a genetically pure culture or clone derived from a single cell (single-colony or single-plaque isolation) and the character ization of the known genetic features of the strain.
Construction of a cDNA library
Authors: Bhattacharyya Madan K 
Published: November 2023

Abstract
This chapter primarily describes a protocol for cDNA library construction that has worked routinely in our laboratory. This protocol includes methods for RNA and poly(A+) RNA isolation, complementary DNA (cDNA) synthesis and cDNA cloning. In writing this protocol, construction of a cDNA library is considered for etiolated hypocotyls of dark-grown soybean seedlings as an example. This protocol is for unidirectional cloning of cDNA in the ,\-based vector Uni-ZAP XR (Stratagene). However, this general protocol, with suitable modifications, should be applicable for construction of cDNA libraries in any other cloning vector. Reverse transcriptase, the enzyme that synthesizes a cDNA molecule using RNA as a template, frequently fails fully to transcribe RNA molecules, especially if they are very long. Therefore, full-length molecules become only a minor portion of the total cDNA. A new technique, based on addition of a biotin group to the cap structure of mRNA, has been developed for the construction of cDNA libraries in which more than 90% of the clones are full length (ref. 1, and references there in). This technique, or others like it, should be considered if the transcripts to be cloned are especially long.
Nucleic acid labelling and detection
Authors: Mundy C R 
Published: November 2023

Abstract
The recent explosive progress in our understanding of genome structure and function has depended upon methods for detecting small amounts of nucleic acid. As described in Chapter 5, specific fragments of genomic DNA can be detected in complex mixtures by hybridization with labelled complementary DNA or RNA probes. A 1 kb fragment of a single-copy human gene is present at the level of only 300 fg/µ,g of total DNA. Thus, a very high level of sensitivity is required, and for several applications a high degree of spatial resolution is also important. The purpose of this chapter is to describe the available labels and labelling methods and to define as far as practicable the optimum combinations for a range of key applications.
Protein expression in Escherichia coli
Authors: Ward John M 
Published: November 2023

Abstract
There now exist a variety of host-vector systems for the synthesis of protein from recombinant DNA. Systems using bacteria such as Bacillus subtilis, lower eukary otes such as Saccharomyces cerevisiae and Pichia pastoris (1), and higher eukaryotes such as Spodoptera frugiperda (2) insect cells and Chinese hamster ovary cells all have their advantages and disadvantages for protein expression. However, by far the most common host is still Escherichia coli. These expression systems are used for a variety of purposes. Very often, pure recombinant protein is needed for functional analysis, such as the determination of kinetic parameters, substrate specificity and inhibitor sensitivity. The structural analysis of proteins by X-ray diffraction and NMR techniques demands multi-milligram amounts of very pure proteins. High-throughput screening by pharmaceutical companies for novel drug discovery can use significant amounts of a protein target. At the small scale of growth of a few tens of millilitres to a litre or two, there are several highly efficient systems which can be used for the rapid production of recombinant protein, and these will be discussed in this chapter. Larger-scale growth and expression systems in E. coli involve a somewhat different set of problems and an overview of these will be given at the end of the chapter.
Methodological tips for human T cell epitope mapping when using pin technology peptide arrays
Authors: Mutch David A , Westwood Olwyn M R 
Published: November 2023

Abstract
The conditions for efficient large scale mapping of T cell epitopes within proteins recognized by human T helper cells are discussed. Many of the inherent difficulties in using freshly isolated human peripheral blood mononuclear cells (PBMC) and T helper cells are also described, as well as how such problems may best be overcome. Observation and awareness of such difficulties are crucial to obtaining accurate and meaningful results for epitope mapping studies. Although these methods precisely locate and size T helper cell epitopes, they rely on the availability of high quality, large scale, low volume peptides presented to freshly isolated human peripheral blood lymphocytes or cultured clone cells, in a nontoxic, biologically compatible buffer system. MultiPin Technology type peptides developed at Chiron Mimotopes Pty Ltd. (see also Chapter 2) are used in the procedures described in this chapter.
Phage display libraries
Authors: Williams Samantha , Logt, Paul van der , Germaschewski Volker 
Published: November 2023

Abstract
Two independent developments were brought together in the late 1980s to create a technique which since then has been shown to prove extremely powerful for the determination of amino acid sequences recognized by antibodies. First, the construction of synthetic peptide libraries on solid matrices, for example plastic pins (1) offered the possibility to screen for peptides that were bound by an antibody of interest (see also Chapters 2 and 3). Although peptides were isolated on the basis of binding the antibody used for screening the library, in many cases the amino acid sequences of the peptides bore no resemblance to any part of the native sequence of the antigen against which the antibody was originally raised. Rather, these sequences mimicked native epitopes and Geysen consequently called them ‘mimotope libraries’.
Chromosome mechanics; the genetic manipulation of aneuploid stocks
Authors: Gubb David 
Published: November 2023

Abstract
The use of chromosomal aberrations for cytogenetic mapping has been critical in the development of Drosophila as a model genetic organism. In 1932 Muller (1) designed a series of genetic tests, using chromosomal deletions and duplications, which define five classes of mutation; broadly corresponding to lack of function (amorphic and hypomorphic) and gain of function (hypermorphic, neomorphic, and antimorphic) alleles. Although Muller’s terminology is ignored frequently, his basic insight, that duplications and deletions could be used to characterize the nature of mutant alleles, remains true. In recent years, a large number of genes have been mapped between the breakpoints of chromosomal deletions or associated with the breakpoints of translocations, transpositions, or inversions. As a result there is an increasingly fine-scale correlation between the genetic map and cytological positions in polytene larval salivary glands (2). New mutations can be mapped by recombination and then rapidly assigned to a cytogenetic region using the available chromosome aberrations. Deletions are particularly useful for this purpose as failure to complement a mutant phenotype localizes the mutation within the cytological boundaries of the deletion. Translocation and inversion breakpoints can give a precise localization, if one end-point is mutant for the target locus, but breakpoints more frequently fall between transcription units. In this situation, it is often possible to construct a synthetic deletion from the aberration breakpoint. Similarly, the cytogenetic map position of cloned sequences can be identified by in situ hybridization to wild-type polytene chromosomes and refined by probing chromosomal aberrations. An up to date catalogue of available aberrations is maintained by FlyBase (Chapter 1).