bluuebunny/crossref_metadata_2025_split huggingface dataset:
Domains and boundaries
Authors: Schedl Paul , Grosveld Frank 
Published: November 2023

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

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

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

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

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

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

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

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

Abstract
Chromosomal DNA replication must be coordinated with other events of the cell division cycle. This means that:
DNA replication should only occur when the cell is legitimately committed to undergoing cell division;
the entire genome must be fully replicated in each S phase, as any section of DNA left unreplicated at mitosis would lead to chromosome breakage;
no DNA should be replicated more than once in a single cell cycle, as this is likely to lead to an irreversible amplification of a section of the genome;
S phase and M phase must occur in strict alternation to maintain the ploidy of the cell.
Regulation of S phase in the fission yeast Schizosaccharomyces pombe [CT]
Authors: Forsburg Susan L 
Published: November 2023

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

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

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

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

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

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

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

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

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

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

Abstract
Nucleic acid hybridization is an important component of many molecular biology techniques. Filter hybridization methods exploit the specificity of molecular hybridization for the detection of rare sequences in a complex mixture. The applications of filter hybridization are widespread and are a fundamental part of any analysis of gene structure or gene expression.
In filter hybridization, denatured nucleic acids are immobilized on an inert support so that reannealing of sequences within the sample is prevented but the nucleic acids are still available for hybridization. A suitably-labelled probe is added and the filter is incubated under conditions that promote hybridization between the immobilized target and the nucleic acid probe. The filter is washed until only specifically hybridized probe remains and the label is detected. The intensity of the hybridization signal is an indication of the abundance of target sequences within the sample and, in several types of analysis, the position of the signal on the filter gives further qualitative information.
The first part of this chapter contains methods for the immobilization of nucleic acids on nylon and nitrocellulose filters. Most of the remainder of the chapter deals with the hybridization analysis of these filters. Parameters that affect both the hybrid stability and rates of hybridization are briefly described. Specific protocols for the basic techniques are given together with indications of how these can be adapted for related applications.
Transcript mapping
Authors: Smith Colin P 
Published: November 2023

Abstract
A detailed study of the structure and expression of any gene will normally include the determination of the end-points of the mature transcript derived from it. This type of analysis is generally conducted in order to identify the positions of DNA sequences potentially involved in the initiation or termination of transcription, or to identify the position of processing sites within the transcript. A knowledge of the ‘transcript map ‘ can also be very useful for designing subsequent genetic manipulations, including, for example, the construction of a transcriptional fusion of the test gene ‘s promoter to a reporter gene, or the cloning of the gene of interest into an expression vector.
Several methods are available for directly quantifying the amount of a specific transcript. These include northern hybridization analysis, slot-blotting and reverse transcription coupled with PCR (RT-PCR). However, these methods do not allow the mapping of transcript end-points (the subject of this chapter).
Mutagenesis
Authors: Grigliatti T A 
Published: November 2023

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

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