bluuebunny/crossref_metadata_2025_split huggingface dataset:
Cell Transplantation in Xenopus
Authors: Altaba Ariel Ruiz I 
Published: November 2023

Abstract
Transplantation of cells in Xenopus embryos can be easily achieved. Early embryonic cells, blastomeres, can be separated, dissociated, and cultured with ease due to their large size and the fact that all cells contain a storage of yolk. This chapter describes simple procedures for transplanting single labelled blastomeres and for grafting small groups of cells into host embryos.
RNAase protection assays
Authors: Altaba Ariel Ruiz I 
Published: November 2023

Abstract
The development of a sensitive assay with which reliably to measure and map messenger RNAs (1, 2) has proved very useful in the study of the molecular mechanisms underlying inductive and differentiation events. The transcription of specific genes is often taken as a reliable marker of the commitment of the expressing cell towards one fate or another. For example, embryonic cells expressing a-actin are thought of as cells differentiating along a muscle pathway and those expressing N-CAM along a neural pathway. Early regulatory genes, the expression of which may be crucial in the commitment of a cell to a given fate, are also used as markers, although the expression of these genes should always be interpreted cautiously. Here I describe a method, based on that of Krieg and Melton (3), to measure the levels of mRNAs in embryonic cells by RNAase digestion of RNA:RNA hybrids. For general molecular techniques the reader is advised to consult ref. 4.
Construction of representative genomic DNA libraries using phage lambda replacement vectors
Authors: Kaiser Kim , Murray Noreen E , Whittaker Paul A 
Published: November 2023

Abstract
In the years since this article first appeared, the technology it describes has become much more routine. This is in large part due to the contribution that the biotechnology industry has made to the provision of reliable reagents, ready-prepared vector DNA (cleaved and dephosphorylated if necessary), packaging extracts, host strains, and even kits to take one through the entire procedure from beginning to end. Failing all else one can send away a tissue sample and receive, if at some cost, a library in the post.
Procedures for cDNA cloning
Authors: Watson Christine J , Demmer Jerome 
Published: November 2023

Abstract
The first mammalian cDNA was cloned almost two decades ago (1) and the construction and screening of cDNA libraries has since become one of the fundamental procedures of molecular biology. A considerable number of advances have been made during the past 20 years, including the availability of highly purified, cloned, and modified enzymes which has improved the efficiency of cDNA synthesis and cloning. The development of high efficiency cloning systems has allowed the construction of a representative cDNA library to become a straightforward procedure which can be successfully carried out in any molecular biology laboratory. More recently, the polymerase chain reaction (PCR) has revolutionized cDNA cloning and in some circumstances it will not be necessary to construct a library for the isolation of a particular cDNA clone.
Production of monoclonal antibodies against proteins expressed in E. coli
Authors: Picksley Steven M , Lane David P 
Published: November 2023

Abstract
The production of monoclonal antibodies against the product of a genetically identified open reading frame (ORF) is often a major starting point in its characterization. The procedure is more complex than that described in Chapter 2 for the production of polyclonal antibodies and should be seen as a complementary technique. Ideally, both procedures should be employed to exploit the full potential of immunochemical analysis. For while the major benefits of monoclonal antibodies, namely their unlimited supply and defined specificity, come into their own during the advanced stages of characterization, polyclonal antibodies do allow simple detection and immunodepletion studies. The preparation of monoclonal antibodies is, however, preferred for the quantitative assay of the ORF product and for rapid purification of the product in a biologically active form. Moreover, the fine mapping of the antibody binding sites (epitopes) on the ORF product, when correlated with their effect on biological activity, provides a valuable insight into possible structure-function relationships.
Production of transgenic rodents by microinjection of cloned DNA into fertilized one-cell eggs
Authors: Waller Sarah Jane , Ho Mei-Yin , Murphy David 
Published: November 2023

Abstract
Since the pioneering demonstration in the early 1980s that the introduction of recombinant growth hormone DNA into the pronuclei of single cell mouse embryos could result in inheritable alterations in the growth of these mice (1), the techniques of mammalian transgenesis have led to significant ad vances in our understanding of the regulation of numerous complex biological processes. The power and appeal of transgenesis lies in its ability to allow the study of almost any protein within a truly physiological environment, that of the whole organism. Thi& permits detailed studies of the developmental and physiological regulation of the protein as well as its function in context with the normal processes occurring in the animal.
Databases, computer networks, and molecular biology
Authors: Fuchs R , Cameron g. N 
Published: November 2023

Abstract
Since M. Dayhoff established the first public macromolecular sequence data collection in the mid-1960s databases have become indispensable research tools in molecular biology (1). The number of databases of interest to biologists has grown steadily since then, and in 1993 the ‘database of databases’ LiMB (Listing of Molecular Biological Databases) presented more than a 100 databases in this area (2). Regular consultation of databases for nucleotide or protein sequence information, mapping data, or the description of the functional role of a particular protein in the living cell is now essential to the work of any molecular biologist. All biological databases today are available, often exclusively, in electronic form. Thus a certain amount of computer literacy is crucial for a modern biologist, even though computing has so far not been a key element of biological training and education. A biologist who mastered the principles of using a computer to support him in his paperwork may still face apparently insurmountable obstacles when he tries to find a small piece of relevant information in the mass of information provided by current databases.
Complement genetics
Authors: Schneider Peter M , Rittner Christian 
Published: November 2023

Abstract
With the advent of recombinant DNA methodology about 20 years ago, tremendous progress has been made in the definition of the genetic basis of the complement system. Most genes coding for complement components and related proteins have been cloned, partially or completely sequenced, their chromosomal location determined, and a variety of mutations leading to partial or complete genetic deficiency described. Nevertheless, genetic typing of complement polymorphisms is still being carried out routinely for almost all components at the protein level. Protein typing technology using electrophoretic as well as serological methods has been proven to be a reliable tool for the definition of polymorphic variants. The analysis of complement polymorphisms has been applied to a wide range of problems, such as association with diseases, structure–function relationship, phylogenetic as well as ethnological studies, and paternity testing. The analysis of complement polymorphisms at the DNA level has provided an even more complex picture by revealing further variants, which subdivide alleles defined at the protein level. DNA sequence analysis of protein allotypes of a number of complement components has demonstrated that most sequence variations are not confined to short stretches of genomic DNA, but are rather scattered throughout the entire gene. Therefore it appears to be technically difficult to replace protein typing methods completely by direct DNA typing using PCR methodology. In contrast, PCR typing has been established successfully for the direct genetic typing of HLA class II genes. A combination of typing methods both at the protein and DNA level has to be applied to obtain complete genetic typing results for complex polymorphic complement components as well as for the detection of complement deficiency.
Map-based cloning in Arabidopsis
Authors: Putterill Joanna , Coupland George 
Published: November 2023

Abstract
Novel plant genes are often first identified as mutations that disrupt or modify plant processes. However, in most species it is still extremely difficult to isolate the affected genes if nothing is known of their sequence or the biochemical properties of the proteins they encode. Insertional mutagenesis using transposons or T-DNA has proved a successful method of isolating such genes in maize, Antirrhinum, Arabidopsis, and Petunia (1-5), as long as appropriate alleles caused by insertion of one of these elements are available or can be identified (see also Chapter 7). However, the isolation of such alleles is always laborious and is often the rate-limiting step in this approach. More recently map-based cloning has proved to be a powerful alternative to insertional mutagenesis in tomato (6) and Arabidopsis (see below). This involves isolating the gene after first accurately locating a mutation on the chromosomes relative to previously isolated DNA markers. The approach therefore overcomes the major difficulty of T-DNA or transposon tagging, in that a rare allele caused by insertion of one of these elements is not required.
Comparative polymerase chain reaction
Authors: Kossodo Sylvie De 
Published: November 2023

Abstract
Comparative PCR may be used to compare the expression of cytokines in a series of diverse settings: for example, in different tissues or cell lines; in the same tissues or cell lines; before and after specific treatments; during kinetic studies; in uninfected versus infected organs etc. In each case the small amount of tissue, low cell-numbers or low copy-number would limit the utilization of Northern blots or the RNAse-protection assays described in Chapter 3. In contrast to the techniques described in Chapter 4, the method detailed here does not necessitate the construction of a multispecific control fragment. Comparison is achieved by subjecting samples to amplification, in sub-saturating conditions, of the constitutively expressed glyceraldehyde phosphate dehydrogenase (GAPDH) gene and the specific cytokine, either in parallel, or preferably, in the same tube, in the presence of a radiolabelled deoxynucleotide. The relative product amounts are quantitated by measuring the incorporated radioactivity of the specific bands from dried gels. This can be done by phosphor-imager analysis 01, after exposure to film, by conventional densitometry. The values obtained by measuring the incorporated radioactivity in the cytokine amplification products are normalized to the values obtained in the GAPDH amplification products.
Measurement of cytokine induction of PC-specific phospholipase C and sphingomyelinases
Authors: Schötze Stefan , Kronke Martin 
Published: November 2023

Abstract
Cytokines play a vital role as mediators of intercellular communication within the immune system. One of the hallmarks of cytokine function is the marked redundancy of biological activities. The overlapping biological functions of TNF/IL-1 and GM-CSF/IL-3 are just two examples. The redundancy of cytokine activities may be based on the utilization of shared key components of intracellular signalling pathways. The principal architecture of major signalling pathways used by cytokines consists of series of proteins including G-proteins, second messenger generating enzymes, protein kinases, and phosphatases. Receptor-mediated hydrolysis of cellular phospholipids by phospholipases of distinct specificities (PLA2, PLC, PLD, SMase) is a ubiquitous event of central importance in cellular signalling. Activation of these enzymes results in generation of early second messenger molecules which further transduce receptor signals to cytoplasmic target proteins. Since multiple intracellular targets exist for a given second messenger, the cytokine signal may branch off to more complex and diversified signals.
Assays for chemotaxis
Authors: Damme Jovan , Gs Rene Con In 
Published: November 2023

Abstract
In view of the recent developments of cytokine research, interest in chemotaxis assays has received a real revival. Indeed, within a short time a number of novel chemotactic cytokines have been identified. These low molecular weight proteins are different from other cytokines such as interleukin-1 (IL-1) and tumour necrosis factor (TNF), previously reported to be chemotactic. They belong to a novel supergene family and include proteins exerting their chemotactic activity specifically on granulocytes or monocytes (1-4). Some members of this family of inflammatory proteins were already biochemically characterized, but their chemotactic effect has only recently been demonstrated. The lack of sensitive and specific bioassays for measuring chemotaxis, as well as the complexity of these tests, can in part explain the late discovery of this family of chemotactic cytokines. However, the assays that were used to detect their activity during purification were basically identical to those already used for a long time, such as the Boyden chamber assay (5) and the agarose method (6) described in this chapter. Since many substances can exert chemotactic activity, their identification rather had to wait for techniques to produce sufficient amounts and to purify them to homogeneity.
Assessment of DNA damage in mammalian cells by DNA filter elution methodology
Authors: Bertrand Richard , Pommier Yves 
Published: November 2023

Abstract
A variety of DNA lesions can be measured in mammalian cells by the DNA filter elution methodology. All DNA elution methods were developed in Dr Kurt W. Kohn’s laboratory (Laboratory of Molecular Pharmacology, NCI, NIH, Bethesda, MD). DNA elution is commonly used to study the effects and mechanisms of action of chemotherapeutic drugs and carcinogens (1-3). The basic DNA elution filter methods were originally designed to assay DNA damage in intact cells or tissues from living animals (1). More recently, the DNA elution filter assays were adapted to study drug mechanisms in isolated nuclei (4-8) and in a reconstituted cell-free system (9-12). Altogether, the various elution methods are currently applied to study the DNA effects of a variety of anticancer agents (topoisomerase inhibitors, alkylating and crosslinking drugs) in cells in culture and to analyse the DNA fragmentation associated with programmed cell death (apoptosis) (9-12).
Immunomethods: magnetic, column, and panning techniques
Authors: Dyer P A , Brown P , Edward R 
Published: November 2023

Abstract
Cell separation techniques mediated by antibody-antigen interactions are now widely used in both research and clinical laboratories. This has largely come about with the advent of monoclonal antibodies and their exquisite specificity for defined antigen cpitopes. Cell surface molecules to which mono clonal antibodies have been developed arc identified with the internationally recognized nomenclature of cluster of differentiation (CD) antigens. Each CD antigen is assigned a unique number following Leucocyte Typing Workshops (1). Some commonly occurring CD antigens, such as CD4 and CD8 present on T lymphocytes, have been recognized for many years while others, largely those with higher CD numbers, have been defined only recently.
Analysis of human antibody sequences
Authors: Walter Gerald , Tomlinson Ian M 
Published: November 2023

Abstract
Diverse repertoires of antibody genes are generated during B-cell develop ment by the combinatorial rearrangement of relatively small numbers of basic building blocks: VH (variable), D (diversity), and JH (joining) segm nts for the heavy (H) chain variable domain and VL and h segments for the light( L) chain variable domain (for review see ref. 1). The antigen-binding site comprises three loops from the heavy chain variable domain (Hl, H2, and H3) and three from the light chain variable domain (Ll, L2, and L3), which are supported by a conserved f3-sheet scaffold (2).
Converting rodent into human antibodies by guided selection
Authors: HOOGENBOOM HENNIE R , ALLEN DEBORAH J , ROBERTS ANDREW J 
Published: November 2023

Abstract
Hybridoma technology, developed 18 years ago, has given rise to many rodent antibodies with exquisite specificity. Unfortunately, the therapeutic potential of rodent antibodies in humans has not yet been fully realized owing, to a great extent, to the immune responses they elicit, which can hamper or abolish their activity. Obtaining human antibodies directly by hybridoma technology has been relatively difficult, particularly in the case of those antibodies with specificity for ‘self’ antigens. Efforts to convert rodent antibodies into less immunogenic, more human structures have led to the construction of humanized antibodies (1), where the regions of the murine antibody responsible for antigen binding are transferred on to a human back bone (or framework) (Chapter 7). As a consequence, a substantial portion of the antibody (the six complementarity-determining regions or CDRs) remains non-human, which, in some cases can still lead to an anti-idiotype response, compromising the therapeutic benefit of the antibody treatment.
Extraction and separation of core histones and non-histone chromosomal proteins
Authors: Thorne Alan W , Cary Peter D , Crane-Robinson Colyn 
Published: November 2023

Abstract
The participation of the nucleosome in DNA processing events such as transcriptional activation is now well established (see Editor’s Introduction). It has been shown. for example, that certain transcription factors can access their recognition sites within a nucleosome alone or with the help of the SWI/SNF complex. The involvement of core histone modifications.
In vivo footprint and chromatin analysis by LMPCR
Authors: Riggs Arthur D , Singer-Sam Judith , Pfeifer Gero P 
Published: November 2023

Abstract
In vitro footprinting, as introduced by Galas and Schmitz (1 ). has become a common procedure which uses cloned DNA and purified DNA binding proteins or nuclear extracts. In vivo footprinting, performed by genomic sequencing after treatment of nuclei or intact cells with dimethyl sulfate (DMS). was introduced by Church et al. (2, 3) and improved by Becker et al. (4) and Saluz and Jost (5). However, for mammalian single-copy genes, the technique required the use of high levels of radioactivity and remained difficult until the advent of ligation-mediated PCR (LMPCR) (6, 7). LMPCR takes advantage of the sensitivity of PCR and the specificity of nested PCR. This enables a high-resolution, nucleotide-level analysis of chromatin, and only a microgram or less of total genomic DNA is needed. LMPCR was introduced by Mueller and Wold (6) for OMS footprinting and extended by Pfeifer and his colleagues for use in DNA sequencing (7), methylation analyses (7, 8). DNase I footprinting (9, 10), nucleosome analyses (9), and UV photofoot-printing (11, 12). In essence, LMPCR is a procedure for detecting the presence of rare breaks in the phosphodiester backbone of DNA. Generally, the breaks or nicks are the result of enzyme or chemical agents that directly, or indirectly after additional treatment, provide a 5’ phosphate.
Recovery of DNA from electrophoresis gels
Authors: Towner Paul 
Published: November 2023

Abstract
During molecular biology projects it is frequently necessary to isolate DNA fragments for further manipulations. The DNA could be from many sources but would typically be from PCRs or restriction enzyme digestion of recombinant plasmids. The simplest way to separate DNA is by agarose gel electrophoresis in the presence of ethidium bromide, the required DNA bands being identified using a UV transilluminator. The quality of agarose is now so good that concentrations as high as 8% (w/v) can be cast and used to separate DNA fragments down to 30 bp with very good resolution, allowing virtually all double-stranded DNA fragments to be isolated.
CTL epitopes
Authors: Elliott Tim , Haurum John S 
Published: November 2023

Abstract
Cytotoxic T lymphocytes (CTL) are primarily responsible for the clearance of acute virus infections. Thus, for example in mice, recovery from influenza A infection correlates with the induction of CTL before a neutralizing antibody response emerges (1). In humans, a rise in the frequency of influenza-specific CTL correlates with a reduction in virus titres in nasal washes (2, 3), and similar observations have been made for RSV (4), HSV (5), measles virus (6), and hepatitis A virus (7). CTL may also be important in controlling chronic viral infections such as EBV (8), CMV (9), and HIV (10). Some intracellular bacterial infections such as Listeria monocytogenes (11) as well as parasitic infections such as Theileria parva (12) and Plasmodium yoelii (13, 14) also appear to be susceptible to attack by CTL. The protective effect of CTL has been amply demonstrated in several cases by adoptive transfer experiments (3–5, 12, 15–19). In addition to providing protection against infectious agents, CTL are thought to provide some degree of protection against spontaneous tumours, by virtue of their ability to detect subtle quantitative and qualitative antigenic differences in transformed cells (reviewed in ref. 20).
Poles apart? Spindle pole bodies and centrosomes differ in ultrastructure yet their function and their function and regulation are conserved
Authors: Hagan Iain M , Gull Keith , Glover David M 
Published: November 2023

Abstract
One of the most overpowering images in biology has to be the simplicity symmetry of the apparatus that forms in order to segregate the eukaryotic genome before cell division, the spindle. Nothing illustrates more graphically the complexity of structure that can be generated by regulating the function of biological polymers. The essential bipolarity of the spindle requires the function of specialized microtubule organizing centres (MTOCs) at its poles, termed centrosomes in animal cells and spindle pole bodies (SPBs) in fungi. These specialized organelles differ markedly in structure from one class of organisms to another.
Eco-controlling - a tool to implement value-based environmental management
Authors: Storm Andreas , Wittke Anja 
Published: November 2023

Abstract
Managerial eco-controlling is based on the basic process of financial controlling.[1] Eco-Controlling envisages a strategic approach to environmental issues and proposes a systematic management procedure with various steps from target and strategy formulation to data management, decision support, control, implementation and communication [Schaltegger & Sturm 1995]. The concept is specifically developed to link environmental strategy with the financial and strategic targets of top management. The system therefore focuses on the improvement of eco-efficiency.