bluuebunny/crossref_metadata_2025_split huggingface dataset:
Molecular methods
Authors: Savage David , Baxter-Lowe Lee Ann , Gorski, Jack , Middleton Derek 
Published: November 2023

Abstract
The application of molecular techniques to tissue typing has led to a fundamental change in methodology for HLA class II typing. The first part of this chapter describes the use of Southern blot (1) techniques which permit the identification of restriction fragment length polymorphisms (RFLPs) which correlate with known serological (HLA-DR/DQ) and cellular (HLA-Dw) defined specificities (2). The latter part of the chapter describes the enzymatic amplification of the second exon of HLA-DR/DQ class II genes using the polymerase chain reaction (PCR) (3) and analysis of the product with alleleand sequence-specific oligonucleotides (4). Compared to serotyping (see Chapter 3), DNA typing techniques provide more information on genetic variation. Other advantages include: • cell type, cell viability, or cell surface expression are generally unimportant • homozygosity can be confirmed
HLA class I gene and protein sequence polymorphisms
Authors: Little Ann-Margaret , Parham Peter 
Published: November 2023

Abstract
The unique feature of the HLA-A, -B, -C class I proteins is their polymorphism. Differences between class I proteins can be detected by alloantisera in the classical serological tissue typing assay, and it is this method which is most widely used to define and distinguish class I polymorphism (see Chapter 3). Comparison of class I sequences, together with the 3D structure of HLAA2 (1) has shown the majority of polymorphic residues are located within and around the peptide binding cleft (2). It is likely that certain residues found deep within this cleft are not directly accessible to allo-recognition mechanisms and go undetected by alloantisera. Additional techniques have been applied to define class I polymorphisms undetected by serology, these include analysis of variants recognized by cytotoxic T lymphocytes (CTL), electrophoretic heterogeneity of immuno precipitated class I molecules, and molecular structure determination via nucleotide sequencing. Collectively these techniques show that many more class I alleles exist than are detected at present by serology.
In vivo assays for multipotential and marrow repopulating cells
Authors: Lord B I 
Published: November 2023

Abstract
Until comparatively recently, the functional status of the bone marrow has been assessed purely on qualitative criteria; primarily the absolute and differential cellularities. The existence of a stem cell population, responsible for the generation of all the recognizable haemopoietic cells, had been assumed but only about 0.5% of cells in the marrow are multipotent. Morphological assessment was clearly out-functional assays were necessary (see Appendix 1). The advent of radiation for military, peaceful and clinical purposes made objective assessment of haemopoietic tissue essential since haemopoiesis appeared to be the process most critically affected by it. Thus was spawned a major offensive in radiation biology. Jacobson and his colleagues (1, 2) reported a circulating factor that could prevent haemopoietic death and accelerate haemopoietic recovery. This was explained (3) on the basis of a repopulating cell. It was found that haemopoietic recovery (at least in mice) could be anticipated by the appearance of splenic nodules (4) the significance of which was recognized by Till and McCulloch (5).
Enrichment of progenitor cell populations from murine and human haemopoietic tissue
Authors: Lord B I , Marsh J C W 
Published: November 2023

Abstract
The progenitor cells exist in very small numbers, the pluripotent cells representing about 0.4% of the total bone marrow cells and the more restricted committed progenitors being perhaps 3% of the population (see Appendix 1). To study direct effects of growth factors on progenitor cells, to assess their performance, or to carry out biochemical studies, therefore, it is desirable to enrich the concentrations of the appropriate cells. Historically cells are separated on physical parameters--density and size-and these properties are incorporated into the modern techniques which employ electronic sorting of cells tagged with fluorescent molecules, or a process of continuous flow centrifugal elutriation. It is these two processes that will be outlined. Numbers of animals and/or cells used will be stated merely as a guide to convenient and practical handling of a cell sort. They may of necessity vary with specific practical requirements. For all procedures involving the use of mice, local and/or national legislation governing their use must be observed.
In vitro clonal assays for murine multipotential and lineage restricted myeloid progenitor cells
Authors: Heyworth C M , Spooncer E 
Published: November 2023

Abstract
The first experiments describing the clonal growth of mouse haemopoietic progenitor cells immobilized in a soft-gel matrix in vitro were reported in the mid-sixties (1, 2). The significance of these observations was readily apparent to research workers in haematology and their pioneering studies allowed quantification of primitive progenitor cells that possess an ability to proliferate, differentiate, and develop into phenotypically and functionally mature myeloid cells. Indeed, this early work, along with the spleen colonyforming assays developed by Till and McCulloch in 1961 ((3) and Chapter 1), forms the basis of modern haematology and has culminated in the clinical use of the colony-stimulating factors.
Development of haemopoietic cells in liquid culture
Authors: Whetton O Kan A D , Heyworth C M 
Published: November 2023

Abstract
The development of haemopoietic stem and progenitor cells to form mature cells can be regulated by soluble peptide growth factors, or alternatively via specific interactions with other cell types (such as bone marrow stromal cells). The original work which defined the target cells for the various haemopoietic growth factors used soft-gel assays which permitted the clonal development of haemopoietic cells from several distinct lineages to be assessed (see Chapter 3, Appendix I). This approach demonstrated that the progenitor cells require haemopoietic growth factors for survival as well as proliferation with accompanying development. Whilst these soft-gel assays have permitted the identification and characterization of novel growth factors and specific progenitor cell populations, they do not readily lend themselves to a study of the molecular mechanisms activated by haemopoietic growth factors to stimulate proliferation and development.
Analysis of growth factor binding to haemopoietic cells
Authors: Walker F , Heyworth C M 
Published: November 2023

Abstract
Haemopoietic cells must receive and act upon information from other parts of the body or from the local environment. Both mature haemopoetic cells and also their precursor cells in the bone marrow receive some of this information in the form of messenger molecules known as cytokines. The type and concentration of these cytokines presented to the cells governs the response observed. The cytokines which govern haemopoietic cell proliferation (haemopoietic growth factors) are believed to elicit their effects through cell surface receptors which display specific high affinity, reversible binding of these growth factors (1). The expression of the appropriate receptors on the cell surface is a prerequisite generally required for the cell to be capable of responding to the growth factor in question. Similarly cells which are incapable of responding to a specific growth factor, e.g. FDCP-1 cells (see Chapter 6), which cannot proliferate in response to M-CSF, can be transfected with the M-CSF receptor and acquire the ability to proliferate in response to M-CSF (2).
Retroviral gene transfer into haemopoietic cells
Authors: Fairbairn L J , Spooncer E 
Published: November 2023

Abstract
The transfer and expression of foreign genes in haemopoietic cells is emerging as a powerful and important tool for the study of the effects of oncogene products and other biologically active molecules. In the past five to ten years, a plethora of gene transfer methods have become available, including calcium phosphate precipitation, scrape loading, and lipofection. However, although these methods have been used successfully with adherent cell lines, (e.g. 3T3 mouse fibroblasts), haemopoietic cells appear less susceptible to gene transfer using these techniques. Electroporation has been successfully used to transfer DNA to haemopoietic cells by some groups, but often involves timeconsuming optimization of conditions coupled with extensive cell death and results in a low frequency of DNA transfer. In addition, techniques like electroporation generally lead to incorporation of multiple copies of the transferred gene in the recipient genome, which can cause problems of stability of transfer and expression.
Assays of the haemopoietic microenvironment
Authors: Friedenstein A J , E. Luria, , Molineux G , 
Published: November 2023

Abstract
Prolonged blood cell production both in vivo and in vitro depends upon a complex of haemopoietic and non-haemopoietic stromal cell types and extracellular matrix molecules which are collectively called the haemopoietic microenvironment (HM, (1)). They provide both the physical framework within which haemopoiesis occurs, and probably also play some role in directing the processes by synthesizing, sequestering, or presenting haemopoietic growth factors. The precise role of each of the cell types composing the HM has yet to be defined and quantitative assays of their individual components are only available for the fibroblastic cells. In the absence of any specific functional assays for the various components of the HM several approaches have been devised to measure the functional capacity of the entire complex.
Visualization of haemopoietic and stromal cell types and their interactions in bone marrow culture
Authors: Allen T D , Wynter E A De 
Published: November 2023

Abstract
The growth of both haemopoietic and stromal cells in tissue culture conditions is a well established practice (see Chapters 4, 5, and 11). The methods outlined in this chapter will be restricted to liquid culture systems, as agar and methylcellulose-based milieu do not readily lend themselves to in situ morphological examination at the ultrastructural level.
Molecular analysis of clonality and transplantation in haemopoiesis
Authors: Marsh J C W , Hampson I N , Cowling G J 
Published: November 2023

Abstract
In this chapter Southern analysis of genomic DNA (1) is used to define
the clonality of abnormal cells found in some haemopoietic diseases
the fate of transplanted haemopoietic cells
In the first of these applications the phenomenon of X-chromosome inactivation, coupled with the analysis of DNA polymorphisms at X-linked loci, is used to determine whether haemopoietic cells in females are of monoclonal or polyclonal origin. Such methods were first described by Vogelstein et al. (2), who analysed the clonality of tumours in various diseases including acute and chronic non-lymphocytic leukaemias, uterine leiomyomas, and certain monoclonal solid tumours. In the second application, Southern analysis of Y-chromosome linked loci is used to distinguish between male donor and female recipient cells following allogeneic bone marrow transplantation in animal models (3).
Structural characterization of mucin-type O-linked oligosaccharides
Authors: Piller Friedrich , Piller Veronique 
Published: November 2023

Abstract
O-glycosylation is one of the most common post-translational modifications of proteins. O-glycans are found on a variety of proteins with widely different functions. O-linked saccharides are classified according to the sugar involved in the O-glycosidic linkage with a hydroxyamino acid: GalNAcαl→SerfThr: found in secreted and membrane proteins (1) Xylβ1→Ser: encountered in proteoglycans and in some membrane glyco-proteins (2) Galβ1→Hyl/Hyp: present in collagen and in complement Clq (3) GlcNAcβ1→SerfThr: discovered in nuclear and cytoplasmic proteins (4) Fuc/Glcβ1→Serffhr: found in some serum proteins (5, 6)
Characterization of nuclear and cytoplasmic glycoproteins
Authors: Berlin William K , Hanover John A 
Published: November 2023

Abstract
Traditionally, the O-linked and N-linked glycoproteins were thought to be synthesized within the lumen of the endoplasmic reticulum and Golgi apparatus and ultimately delivered to the cell surface via the secretory pathway. Work carried out in the past few years has revealed that glycoproteins also exist in the cytoplasm and the nucleoplasm (that part of the cytoplasm surrounded by the nuclear envelope), and are not restricted to the membrane bound organelles as biosynthetic intermediates. Nuclear and cytoplasmic proteins modified by O-linked N-acetylglucosamine are now known to comprise a major class of glycoproteins, with strong evidence that other classes of glycoproteins and proteoglycans are present in the nuclear and cytoplasmic compartments of the eukaryotic cell (1–3).
T cell antigen receptor genes
Authors: Davis Mark M , Chien Yueh-Hsiu 
Published: November 2023

Abstract
The characteristics of T cell recognition and the nature of their antigen receptors has long been a difficult and controversial area for immunologists. However, the development of cloned T cell lines and hybrids (as reviewed in ref. 1), the genera tion of antisera (2) and monoclonals (3-7) enabling the identification of polymor phic T cell receptor (TCR)-heterodimer-CD3 complexes (8-11) and the isolation of four different TCR genes (o:, 13, -y, 8) (12-18) has generated a large amount of information in the past decade. It has also shifted the focus of this area from the question of 'What molecules mediate T cell recognition?' to 'How do they work?' and 'How are they selected in the thymus?' Although antibodies and their genes (see Chapter 1) are a natural touchstone for understanding some aspects of TCRs, the emphasis in this chapter will be on the unique features of the different T cell antigen receptors and their possible biological significance.
T lymphocyte signal transduction
Authors: Terhorst Cox , Spits Hergen , Staal Frank , Exley Mark 
Published: November 2023

Abstract
That the immune system combines an extraordinary specificity of recognition with extremely complex control mechanisms has been amply demonstrated by the studies of antigen recognition by T lymphocytes. In response to antigen recogni tion, resting T lymphocytes are required to undergo a complex series of events collectively known as T cell activation. Although much has been learned about T cell activation in the last years, an orderly array of the molecular events involved in the whole process remains to be established. There is heterogeneity in the effector cells within the T cell compartment comprising T helper (TH) and cytotoxic T lymphocyte (CTL) subsets.
Structure and function of MHC class I and class II antigens
Authors: Jewell David A , Wilson Ian A 
Published: November 2023

Abstract
The major histocompatibility complex (MHC) is a contiguous set of genes that code for polymorphic cell surface receptors involved in antigenic peptide presentation (reviewed in ref. 1). Although originally observed in transplant rejection (2), it is now clear that the biological role of MHC proteins is to initiate cell-mediated immune responses. The co-ordinated activities of MHC proteins act to rid the body of xenobiotic infections (3). In order to combat a wide spectrum of pathogens, the class I and class II antigens have evolved to act as indicators of foreign intrusion at the surface of the host cell. The MHC antigens bind to peptide fragments, derived from the pathogen's proteins, while within the cell, and thereby direct peptides to the cell surface for presentation to immune cells. Antigenic peptide fragments (4, 5) tend to bind with high-affinity to the MHC molecules by virtue of their 'bound' shape and chemical complementary to the MHCs peptide binding groove. The lifetime of these MHC-peptide complexes on the cell surface is an important parameter of MHC restriction. Stable or long-lived peptide-MHC com plexes help to increase the probability that the immune system will detect the presence of foreign antigens.
Flow cytometric selection of responsive subclones and fluorimetric analysis of intracellular Ca2+ mobilization
Authors: Ransom John T , Dunne John F , Sharif, N A 
Published: November 2023

Abstract
With commonly utilized cell lines of clonal origin, we have observed that a substantial percentage of the population often fails to respond to a particular stimulus while a smaller percentage responds with a clearly detectable response. This suggests that, with multiple culture passages over time, genetic variants have developed that exist in the population and which exhibit heterogeneous response phenotypes that may not be morphologically apparent.
Confortnational studies on oligosaccharides
Authors: Homans Steven W 
Published: November 2023

Abstract
Since the initial demonstration by Burnet that the influenza virus requires a specific carbohydrate signal on the erythrocyte for attachment and infectivity (1), oligosaccharides have been implicated as recognition signals in a variety of biological processes. Indeed, in the field of cell adhesion, we now have knowledge of the oligosaccharide ligands and a detailed knowledge of their protein receptors has given rise to the term 'selectins' for this class of membrane proteins (reviewed in references 2, 3, see also Chapter 4). As techniques for the purification and sequence analysis of oligosaccharides improve in efficiency and sensitivity, we can anticipate the characterization of the precise oligosaccharide ligand and its receptor in other important biological processes.
Strategies for using gene probes
Authors: Lamb Richard F , Birnie George D 
Published: November 2023

Abstract
One of the most common questions, asked both by newcomers and experienced researchers alike, in designing a strategy for nucleic acid hybridization, is ‘which is the “best” nucleic acid probe for a particular experimental application?’ Since both the number of possible applications of nucleic acid hybridization and the choice of possible labelling and detection systems available have increased dramatically over the last ten years, it is useful before embarking upon a particular strategy to ask a few basic questions of the detection system to be employed in order to maximize the chance of success. The most critical parameters which have to be considered in designing a strategy for using a particular gene probe successfully are those outlined in Table 1. The first and most important question is, how sensitive does the detection system need to be? The answer to this question is often unknown, but can usually be guessed at intelligently. Moreover, the answer very often dictates both the choice of probe and the method of labelling used. For example, in situations requiring maximum sensitivity, such as the detection of single copy genes in mammalian DNA where sequences equivalent to about 10 fg per µ,g of DNA must be revealed, long DNA or RNA probes randomly labelled throughout their length with 32P are likely to be successful in most filter hybridization experiments.
| The discovery and significance of mobile genetic elements
Authors: Shapiro James A 
Published: November 2023

Abstract
One of the most intriguing facts about the history of mobile genetic elements is that no-one set out to discover them. In every case, they were initially found by geneticists studying other problems: chromosome breakage and rejoining in maize, transfer of genetic information between bacterial cells, lysogeny, regulation of gene expression, or antibiotic resistance. Is there a message in this repetitive serendipity? I think so. The message appears to concern the power that unspoken assumptions hold over our thinking (in this case, the idea that the genetic material is basically stable) and the need to be alert for those unexpected findings which overturn those assumptions and suddenly open a whole new universe for scientific exploration. Such major reorientations in thinking are inevitable in science Genetics has travelled a long conceptual road from the Constant Genome paradigm of the 1940s, when McClintock first described transposable controlling elements in maize, to the Fluid Genome paradigm that reigns today. There is an even longer and more fascinating voyage ahead as we come to terms with the implications of mobile genetic elements and molecular discoveries about genome organization and reorganization. This historical chapter affords a rare opportunity to trace the outlines of the intellectual journey already completed as well as to attempt some projections of where we might be going. The emphasis here will be on new ways of looking at genetic problems, on key observations, and on the unexpected convergence of discoveries in separate experimental systems. Accounts of the history of particular systems with a more mechanistic focus can be found in the first Mobile Genetic Elements book (2).
| Transposable elements as tools for molecular analyses in bacteria
Authors: Berg Claire M , Berg Douglas E 
Published: November 2023

Abstract
The transposable elements of bacteria vary in size and structure, and in the ways they move. Some half-dozen elements and their engineered derivatives have been exploited as research tools since the late 1970s. Their use in conjunction with recombinant DNA methods has revolutionized the practice of microbial genetics. The value of transposons as molecular genetic tools stems from their ability to move as discrete DNA segments to new locations where they disrupt the target DNA. Natural and engineered transposons can serve as mobile sources of a variety of phenotypic or physical markers, including selectable (or sometimes counter-selectable) genes, predetermined restriction sites, reporter genes, promoters, and replication origins.
Analysis of protein expression by immunocytochemistry
Authors: Brown Dennis 
Published: November 2023

Abstract
Immunocytochemistry is a major technique in unravelling the roles of proteins expressed by cells. This is true whether the protein is endogenous, or whether it is expressed as a result of transfection of a cell with a specific cDNA. Remarkable insights into the complex physiology and cell biology of cells, tissues, and organs have been obtained by combining immunocytochemical observations with the powerful tools of molecular biology, and with measurements of physiological and cellular function. In view of the increasing use of such detection techniques in multidisciplinary studies, it becomes important to understand the strengths and weaknesses of the many methods that are available, in order to choose the correct procedure for each particular antigen.
Cosmid cloning of yeast DNA
Authors: Stucka Rolf , Feldmann Horst 
Published: November 2023

Abstract
After in vitro packaging into premature phage heads, recombinant cosmids can easily be introduced into Eschericha coli cells and propagated with high efficiency like plasmids. Depending on the vector, genomic DNA fragments in the size range 35–45 kb can be accommodated: 78–105% of the length of wild-type 11. DNA (38–55 kb) can be packaged efficiently. The in vitro packaging systems give an approximately 10-fold higher yield of transformants per µg of DNA than the standard CaC12 transformation procedure.
The construction and use of cDNA libraries for genetic selections
Authors: Mulligan John T , Elledge stephen J 
Published: November 2023

Abstract
The popularity of the yeast Saccharomyces cerevisiae as a research system is due in large part to its facility as a genetic organism and its usefulness as a model eukaryote. It combines the straightforward collection and analysis of mutations with the ability to isolate the corresponding genes by transformation and complementation. All of the genetic elements necessary for promoting chromosomal replication and segregation reside on small fragments which have been incorporated, along with selectable markers, into small cloning vectors capable of replicating in Escherichia coli or yeast.