bluuebunny/crossref_metadata_2025 huggingface dataset:
Site-Specific Attachment Of Labels To The DNA Backbone
Authors: Conway Nancy E , Fidanza Jacqueline A , O’donnell Maryanne J , Narekian Nicole D , Ozaki Hiroaki , Mclaughlin Larry W
Published: November 2023

Sequence-specific attachment of reporter groups, drug derivatives, or chemically reactive species to DNA sequences has the potential to provide new materials for detailed spectroscopic and biophysical studies as well as a host of new DNA therapeutics and diagnostics. The covalent binding of a variety of such agents at specific locations within the nucleic acid sequence can be achieved by a number of procedures depending on whether the nucleobase, carbohydrate, or phosphate residue is employed as the site of attachment. These procedures often exploit the availability of specific functional groups (such as terminal phosphomonoesters, see Chapter 8) or the reactivity of selected sites on the purine or pyrimidine building blocks (such as the CS position of pyrimidines, see Chapter 11) in order to attach an appropriate linker or the active agent directly. While the manner in which the nucleic acid is labelled may be dictated by the specific study involved, in general the principles of simplicity and versatility arc best employed to guide the choice of labelling procedure.
Oligodeoxynucleotides For Affinity Chromatography
Authors: Blanks Robert , Mclaughlin Larry W
Published: November 2023

A number of proteins such as polymerases, repressor proteins, transcription factors, and restriction and modification enzymes, bind to their recognition or initiation sites with relatively low dissociation constants (10-10 M) and exhibit a high degree of nucleic acid sequence specificity. Specific binding between a protein and a small molecule is commonly used for the affinity purification of proteins. In these procedures, the specific ligand or substrate analogue is covalently attached to an insoluble chromatographic matrix in such a manner as to facilitate interaction of the ligand with the protein. After introduction of the protein mixture to the matrix, only those proteins which bind effectively to the attached ligand arc retained, and thus separated from the mixture introduced to the matrix.
Oligonucleotides Attached To Intercalators, Photoreactive And Cleavage Agents
Authors: Thuong N T , Asseline U
Published: November 2023

During the past few years there has been a growing interest in the use of modified oligonucleotides as research tools in molecular biology and as specific inhibitors of gene expression. The potential therapeutic application of oligonucleotides as artificial gene control agents depends on the specificity of their binding to the target nucleic sequence under physiological conditions, their resistance to nuclease, and their uptake by intact cells. The calculation based on the statistical distribution of the nucleic bases showed that short oligonucleotides (11-15 nucleotides in length) could be used to recognize specifically a single target in the mRNA population of the human cell (1).
Analysis of barley and malt parameters by partial least squares loadings plots
Authors: Lebouille J L M , Drost W C
Published: November 2023

To predict the quality of brewing barley, many parameters are determined. Most of the parameters are empirical, and a molecular basis is often lacking. By using the combination of near-infrared (NIR) analysis and the spectral loadings of partial least squares regression, the “spectrum” of a parameter can be visualized. By comparing these loadings spectra with those of pure and mixed constituents, one can predict the relative involvement of each constituent. This opens the possibility to use the data of NIR calibrations of complex brewing parameters for analytical purposes.
Transformation of brewing yeast: use of a vector conferring resistance to cycloheximide
Authors: Delgado M , Pavón M C , Esteban M
Published: November 2023

A new cloning vector YEpCR20 carrying a dominant resistance to cycloheximide has been constructed. Because brewing yeasts are very sensitive to cycloheximide selection of transformants is unambiguous. YEpCR20 has been transferred from a karl laboratory strain to an industrial brewing yeast. Transformants show the same fermentation behaviour and electrokaryotype than the brewing parental.
Authors: Goossens E , Debourg A , Villanueba K D , Masschelein C A
Published: November 2023

In order to circumvent this problem, an integrative plasmid was constructed and used to transform haploid and brewing yeast. Brewing performance in 2L EBC-fermentors, flavour profile, effect on vicinal diketone production and isomeroreductase activity of these transformants were determined. ILV5 m-RNA levels from control yeast and transformants obtained with multi-copy and integrative plasmids were also compared.
Application of the gas-lift principle to the improvement of deep cooling efficiency in one tank operation
Authors: Lemer C , Taeymans D , Masschelein C A
Published: November 2023

Pilot trials were carried out in two-hectoliter cylindro-conical vessels. Mixing and cooling efficiency being closely related, we investigated the effect of the power generated by CO2 bubbles on the recirculation distribution-time of a tracer. Results obtained showed total mixing as well as a significant reduction of the time required for deep cooling.
The development of brewing processes: The impact of European biotechnology regulations
Authors: Hammond J R M
Published: November 2023

In recent years, it has been demonstrated at the research level that new biotechnological techniques could be usefully applied within the brewery industry. The ability to transfer genes fro other organisms into brewing yeasts using genetic modification methods has provided a significant advance over the use of conventional techniques. Similar approaches for the development of brewing raw materials are under investigation. However, the implementation of these advances may be jeopardised by the stringency and inappropriateness of E.C. legislation for regulation of genetic modification and by the different approaches being adopted by product-based and environment-based regulatory authorities. The implications for the brewing industry are discussed.
Effect of dextrins composition on flavour characteristics
Authors: Louant G , Dufour J -P
Published: November 2023

The natural dextrins content of beer or their extra addition (as adjunct) appears as an interesting approach to improve the flavour profile of low or non-alcoholic beer. Data are presented which indicated that the effect of dextrins on higher alcohols and esters depend on the polymerisation degree of the dextrins and the characteristics of the aroma compound. In this respect, the quantitative and physico-chemical analysis of the dextrins appears as a perequisite for a rational use of these interactions. Two chromatographic techniques using the pulsed amperometric detector or viscometric/light scattering/refractive index detectors have been tested. The two techniques give complementary informations.
Immunological detection of Fusarium in barley and malt
Authors: Vaag P
Published: November 2023

High levels of fungal infection of barley are incompatible with high quality malt and beer. In particular Fusarium infection is known to be involved in eliciting gushing in beer, although the actual causative component remains unknown. Antibodies raised to Fusarium have been used to establish immunological procedures (ELISA’s) for determination of Fusarium levels in either single seeds or larger samples.
Gellan gum: effect of composition on gel properties
Authors: K.Baird J , A.Talashek T , Chang H
Published: November 2023

Gellan gum is the extracellular hetero polysaccharide produced by Pseudomonas elodea. The primary structure comprises a tetra saccharide repeat with glyceryl and acetyl substituents. X-ray diffraction analysis indicates that the molecule forms a parallel halfstaggered double helix in which each polysaccharide chain is a left handed 3-fold helix. The native product forms elastic gels whereas the completely deacylated product forms brittle gels. The alkali treatment conditions used during recovery determine both the composition and rheological properties of gellan gum. The controlling influence of the glycerate substituent on gel properties is consistent with the proposed secondary structure. The potential to vary the gel properties, in a systematic and controlled manner, should lead to enhanced functionality and wider application in food and industrial applications.
Methods in solid tumour cytogenetics
Authors: Mandahl N
Published: November 2023

It is now firmly established that the acquired chromosome abnormalities of neoplastic cells are non-randomly distributed throughout the genome and that certain aberrations are associated with specific histologic tumour types (1, 2). In Human Gene Mapping 10.5 (3) no less than 179 non-random structural chromosome changes, identified in 51 different types of neoplastic disorders, have been listed. The number of neoplasms with cytogenetic aberrations investigated by chromosome banding techniques amounted, by September 1990, to 14141 cases (4). The majority of these, 68%, were leukaemias, 11% were lymphomas, and 21% were solid tumours. The reasons why so relatively few solid tumours have been investigated are the technical problems encountered. Good methods for cytogenetic analysis of solid tumours have only become available since the mid-1980s. As a consequence, from 1983 to 1990 the number of reported solid tumours has increased from about 500 to 3000 (4).
Purification and assay of mammalian protein (serine/ threonine) kinases
Authors: Wang Yuhuan , Roach peter J
Published: November 2023

Phosphorylation of proteins at serine and threonine residues is one of the most frequent forms of post-translational modification leading to changes in biological function. The first protein (serine/threonine) kinase, phosphorylase kinase, was discovered in the study of the regulation of glycogen metabolism in the 1950s. Since then, well over a hundred protein (serine/threonine) kinases have been identified and many have been purified and characterized—it is likely that the number of protein (serine/threonine) kinases will escalate with time. Protein kinases and protein phosphorylation essentially pervade every area of cellular regulation so that the biochemical characterization of new protein kinases will continue to be an important aspect of research on signal transduction.
Characterization of protein (tyrosine) phosphatases
Authors: Tonks Nicholas K
Published: November 2023

It is now well established that the phosphorylation of tyrosyl residues in proteins is an essential feature of many cellular processes, including the control of both normal and neoplastic cell growth (1). Initial studies of protein (tyrosine) phosphorylation highlighted the fact that this is a reversible process. Thus, in cells infected by temperature-sensitive mutants of Rous sarcoma virus, transformation occurs at the permissive temperature and is accompanied by elevated levels of phosphotyrosine (2). However, if the transformed cells are shifted to the non-permissive temperature, a rapid decline in the level of phosphotyrosine is detected, resulting from the action of protein (tyrosine) phosphatases (PTPases). While a plethora of protein (tyrosine) kinases (PTKs) have been identified, it is striking that phosphotyrosine accounts for less than 0.1% of the total protein bound phosphate in the cell (1). A component of this strict negative control of the PTKs is the family of PTPases (3). One should not lose sight of the fact that the net level of phosphotyrosine observed in a target substrate in vivoreflects the balance between the competing action of PTKs and PTPases. Thus a complete understanding of the physiological role of tyrosine phosphorylation and its potential as a mechanism for the reversible modulation of enzyme activity must necessarily encompass the characterization of the PTPases.
Retrograde tracers for light and electron microscopy
Authors: Llewellyn-Smith Ida J , Pilowsky Paul , Minson Jane B
Published: November 2023

Retrograde neuronal tract tracing is used to establish where neurones send their axons, and is based on the ability of axons to take up substances from the extracellular fluid and transport them back to cell bodies. Retrograde tracing was introduced to the field of neuroanatomy just over twenty years ago (1, 2). Since then, retrograde tracers have become invaluable tools for the study of nerve pathways in the central and peripheral nervous systems. Initially, horseradish peroxidase (HRP) was the only retrograde tracer available to neuroanatomists. However, in the late 1970s and in the 1980s, a number of new tracers were developed, including fluorescent molecules and fluorescent latex microspheres, HRP conjugated to lectins or toxins, gold labelled tracers, cholera toxin B subunit (which is detected immunocyto chemically), and most recently, viruses. These developments have lead to an explosion in the number of retrograde tracing studies, since there is now a retrograde tracer suitable for virtually any kind of study that the neuroanatomist can imagine. This chapter deals with non-viral retrograde tracers that can be used for both light and electron microscopic studies on neuronal connectivity, i.e. HRP-based tracers, colloidal gold-labelled tracers, and un conjugated cholera toxin B subunit.
Preparation and Analysis of Intercellular Fluid
Authors: Hammond-Kosack Kime E
Published: November 2023

The apoplast represents a key domain within the plant. It is a continuum with both the external environment and the plant cell, and, therefore, its contents can potentially be modified by both internal and external stimuli. The molecular events occurring peripheral to the plasma membrane are of immense importance to studies of plant-pathogen interactions, cell-to-cell interactions and to cell wall metabolism. This section describes a rapid, simple, and effective method to selectively analyse the apoplastic domain. The technique involves the recovery of intercellular fluids (IF). Such fluids, which are retrievable from all plant tissues, contains both free and ionically-bound polysaccharides, glycoconjugates, proteins, and enzymes from the cell wall, the surface of the plasma membrane and the external aqueous layers.
Biochemical Analysis of Chitinases and β -1,3-Glucanases
Authors: Boller Thomas
Published: November 2023

Plant hydrolytic enzymes play an important role in host-pathogen interactions (1). Chitinase and 13-1,3-glucanase are of particular interest for three reasons: The activities of both enzymes increase markedly in many plant tissues in response to pathogen invasion, to treatment with fungal cell wall components (so-called elicitors), or to the presence of the plant stress hormone, ethylene indicating a possible role in the plant stress response to a given pathogen (1, 2). Both enzymes strongly inhibit the growth of many fungi in culture, indicating that they may have a direct antibiotic function in plant-fungus interactions (3-5).
Isoflavonoid Phytoalexins and their Biosynthetic Enzymes
Authors: Edwards Robert , Kessmann Helmut
Published: November 2023

Phytoalexins are low molecular weight antimicrobial compounds which accumulate in plant tissues after exposure to micro-organisms. In the case of isoflavonoid phytoalexins , the rates of synthesis and accumulation of these compounds in leguminous plants can be correlated with the resistance of the host to the pathogen (for recent reviews see references 1-3). The biosynthesis of isoflavonoid phytoalexins (for example medicarpin) proceeds via a multistep pathway which begins with phenylalanine (Figure J). The pathway may also be extended to produce more complex isoflavonoid phytoalexins, such as the prenylated pterocarpans phaseollin and the glyceollins. In this review we will use the biosynthetic route to medicarpin to illustrate the methods used to analyse isoflavonoids and to measure the activities of the enzymes involved in their synthesis. The exact methods of treatment for induction of phytoalexins will depend on the biological system used. Whole plant tissues may be elicited with suspensions of fungi (spores or mycelia) or bacteria either by surface application or by infiltration. Alternatively, abiotic elicitors such as heavy metal salts, may be used at a final concentration of 10-2- 10-3 M. However, biotic elicitors derived from micro-organisms or plants (see Chapter 8) are frequently the inducing agents of choice. When treating suspension-cultured plant cells the sterilized elicitor is added in aqueous solution with sterile water serving as a control treatment. In the case of crude elicitor preparations from fungal cell walls elicitation may be optimized in the range 10-100 mg glucose equivalents per litre of cell culture medium. Elicitor active fractions may be most conveniently obtained from yeast (4).
Retroviral vectors
Authors: Stoker Andrew W
Published: November 2023

Retroviruses can transfer genetic material efficiently into vertebrate cells, and incorporate it stably into the cellular genome. Many differentiated and undifferentiated cell types of rodents, birds, and primates can be infected, and high viral expression levels can be achieved. Furthermore, retroviral gene transfer is effective in vivo from early embryonic stages onwards, as well as in cultured cells. Given this versatility, retroviruses are excellent tools for gene transfer in experimental animal systems, and are potential vehicles for gene therapy in humans. Engineered derivatives of naturally-occurring retroviruses, the retroviral vectors, are being used increasingly, and with great success, for the transfer and expression of foreign genes (1-3). This chapter describes the structures of retroviral vectors, their varied applications, and methods for their construction and use.
Expression of genes by vaccinia virus vectors
Authors: Smith Geoffrey L
Published: November 2023

Jenner introduced the use of cowpox virus as a vaccine against smallpox in 1798, and, as he predicted, this practice led to the eradication of smallpox (in 1977), although in the modern era it was vaccinia, not cowpox, virus which was used as the smallpox vaccine. Today, variola (smallpox) virus is confined to two high-security laboratories in Moscow and Atlanta, and the World Health Organization plans to destroy these last stocks in 1993 after the genomes of representative strains have been sequenced. Cowpox and vaccinia viruses have clearly played important roles in the history of human medicine, since they represent the first human vaccine and the only vaccine whose use has resulted in the eradication of a human disease. Surprisingly, interest in poxviruses in general, and vaccinia virus in particular, increased substantially after the disappearance of smallpox. This interest stemmed, in a large part, from the development of vaccinia virus as an expression vector and from the potential use of vaccinia virus recombinants as live vaccines against pathogens other than variola virus. In this chapter, concepts and protocols central to the use of vaccinia virus as an expression vector are described. Readers should refer to another chapter in this series (1), which provides an earlier account of this topic and contains several protocols which remain essentially unaltered, and to another recent review on the construction and applications of vaccinia virus recombinants (2).
Cellular methods
Authors: Reinsmoen Nancy L
Published: November 2023

The mixed lymphocyte culture (MLC) is perhaps the most widely used of cellular assays, and represents a functional assay of cellular response to stimulatory determinants associated predominantly with HLA class II molecules including HLA-DR, and -DQ, and to a lesser extent HLA-DP molecules. The first descriptions of this assay as a measurement of cellular immunity (1, 2), together with the development of a one-way method of stimulation, allowed the correlation of proliferative responses between siblings and the conclusion that a single genetic locus or region, now known as HLA, controlled the MLC reactivity. The recognition of disparate HLA class II molecules and the resulting T cell activation as measured in MLC are thought to represent an in vitro model of the afferent arm of the in vivo allograft reaction.
Use of antibodies to visualize neurotransmitter receptor subtypes
Authors: Levey Allan I
Published: November 2023

Methods for visualization and quantification of receptors have provided information that is fundamental for understanding the roles of neurotransmission and specific receptors in development, synaptic plasticity, learning, memory, control of movement, and a variety of other neural processes. Receptor autoradiography is one of the most widely used methods for this purpose because it is a relatively easy and sensitive technique. However, this technique is limited by the specificity of available radioligands and by its relatively poor spatial resolution. These factors have become more problematic in the past several years as a result of the molecular cloning of large gene families of neurotransmitter receptors.
Molecular cloning of glycosyltransferase genes
Authors: Schachter Harry
Published: November 2023

Oligosaccharides differ from proteins and nucleic acids, the other two classes of large biological polymers, in two important characteristics-they are usually highly branched molecules and their monomeric units are connected to one another by many different linkage types. Proteins and nucleic acids are linear and there is only a single type of linkage between the monomers of these molecules (amide bonds for proteins and 3'-5'-phosphodiester bonds for nucleic acids). Although all three classes of macromolecule share an almost infinite capacity for information storage, branching and multiple linkage types bestow on oligosaccharides an ability to store more information per unit of molecular size-more information 'bang' for the molecular weight 'buck'. Perhaps it is not surprising that a major site for information exchange mediated by oligosaccharides is at membrane surfaces, for example interactions between cells and their fluid or cellular environments. The cell surface and its environment are busy places and the spatial efficiency of oligosaccharides may make them uniquely suitable for these interactions.
The Protein Folding Probletn
Authors: Creighton Thomas E
Published: November 2023

To be biologically active, proteins must adopt specific folded three-dimensional, tertiary structures. Yet the genetic information for the protein specifies only the primary structure, the linear sequence of amino acids in the polypeptide backbone. Many purified proteins can spontaneously refold in vitro after being completely unfolded, so the three-dimensional structure must be determined by the primary structure (1). How this occurs has come to be known as 'the protein folding problem'. It had been primarily of academic interest, but the advent of protein engineering and the ability to produce any protein, often in an insoluble, unfolded, inactive, and useless form, has made it also of great practical importance (Chapter 9).
Assembly of Multi-Subunit Structures
Authors: Price Nicholas C
Published: November 2023

Many proteins associate specifically to generate a range of novel structures and functions. These include enzymes and proteins involved in key cellular control mechanisms. The process of subunit association in these relatively simple systems also represents a model for understanding the assembly of more complex biological structures such as large multi-enzyme complexes, ribosomes, and viruses. X-ray crystallography has provided a great deal of detailed information concerning the nature of the interactions between the subunits in the native state of multi-subunit proteins, but can give only limited insight into the mechanisms of association. Several fundamental questions are posed by such assembly processes.
Use of speciality phosphoramidites in PCR
Authors: Mclean Michael J
Published: November 2023

In the decade or so since the introduction of automated oligonucleotide synthesizers, the ability to use these machines for the incorporation of unnatural residues into nucleic acids has led to the chemical synthesis of a wide variety of phosphoramidite derivatives. When included in an oligomer synthesis, these entities provide a source of site-specific, stable modifications of the backbone, thus enabling a rigorous determination of their biochemical effects.
Chemical methods for 5’ non- isotopic labelling of PCR probes and primers
Authors: Andrus Alex
Published: November 2023

Synthetic oligonucleotides are now accessible to most laboratories conducting molecular biology experiments. The decreases in preparation time, costs, and the expertise required have enabled the annual production and use of several million oligonucleotides (1). PCR undoubtedly consumes more of these sequences than any other application. Many PCR experiments require the covalent attachment (labelling, tagging, conjugation) of small molecules to perform ancillary functions, in addition to base-specific annealing to complementary nucleic acid and 3’ chain extension by polymerase. The labels (reporter groups) conduct or facilitate detection, quantification, isolation, sizing, and location of the amplified sequence. Covalent attachment of molecules, such as fluorescent dyes, biotin, and proteins, can be made at virtually any site on the oligonucleotide, including the 5’- and 3’-termini, through the sugars and bases, and by internucleotide phosphate modification (2). The sequence- and base-specific hybridization properties must be preserved, as well as the chain extension capacity for PCR primers. The methods described in this chapter will focus only on examples of specific, chemical 5’-labelling of oligonucleotides, for use as PCR primers and probes of PCR products (3). Post-synthesis protocols such as cleavage/deprotection, analysis, purification, and 5’-labelling operations are described. Enzymatic labelling methods conducted before, during, or after PCR have been reviewed elsewhere (4).
Solid phase PCR
Authors: Stamm Stefan , Brosius Jurgen
Published: November 2023

The method of solid-anchored PCR uses specific oligonucleotides coupled to a solid phase as primers for cDNA synthesis (1) and results in cDNA that is covalently linked to the solid phase. Typical solid phase matrices are agarose (1), acrylamide (1), magnetic (2-4), or latex beads (5). A solid phase with cDNA attached, generated using oligo(dT) as a primer, contains sequence information similar to a cDNA library, that is, it represents a ‘solid phase library’ (1, 3, 4). The cDNA that is attached to the solid phase can be used directly as a template for PCR reactions or can be modified enzymatically prior to the PCR reaction. Oligonucleotides that are attached to a solid phase can also serve for affinity purification of RNA (2). RNA isolated in this way can be directly reverse transcribed, using the primer that is coupled to the solid phase. Subsequent PCR reactions can employ this primer with or without additional internal primers. Since the cDNA is coupled to a solid phase, changing buffer conditions or primer composition is conveniently achieved by washing the solid phase and resuspending it in a different PCR reaction mixture. The general principle of solid anchored PCR is shown in Figure 1.
Solid phase sequencing of PCR products
Authors: Wahlberg Johan , Hultman Thomas , Uhlén Mathias
Published: November 2023

Solid phase methods have proven to be extremely useful for the separation and synthesis of biomolecules. The advantages of solid phase approaches are a combination of good yields and reproducible results. Furthermore, the facilitated separation of the solid phase from the reaction solution allows routine methods suitable for automation to be designed. This chapter describes protocols for the use of magnetic beads in combination with the biotinstreptavidin system for solid phase DNA sequencing of PCR products. The use of a solid support for the preparation of sequencing templates enables efficient production of single-stranded DNA with simultaneous removal of PCR buffers, dNTPs, and PCR primers. The immobilization of the PCR product also makes it possible to avoid competition of the sequencing primer with the complementary strand of the template obtained when using the double-stranded DNA fragments directly.
cDNA cloning by RT-PCR
Authors: Edwards Jean Baptiste Dumas Milne , Ravassard Philippe , Icard-Liepkalns Christine , Mallet Jacques
Published: November 2023

The methods available for the characterization of mRNAs have progressed enormously since the mid 1970s. This includes the isolation of RNAs, cloning and bacterial amplification of specific mRNAs, Northern blotting studies, in situ hybridization, and nuclease protection assays that allow the description of mRNA metabolism in cells.
Measurement of mRNA stability
Authors: Brown Alistair J P
Published: November 2023

The level of an mRNA in the cell is determined both by its rate of synthesis and by its rate of decay. Since mRNA half-lives range from about 1 min to over 1 h in the yeast, Saccharomyces cerevisiae (1–4), differential rates of mRNA decay have a fundamental influence upon the overall pattern of protein synthesis in this model eukaryote. However, the structural features which determine the stability of an mRNA in yeast and the mechanisms by which they mediate their effects remain largely obscure. Multiple parameters influence the stability of an mRNA. They include the 3′ poly(A) tail, the 5′-cap, mRNA translation, and various structural determinants that lie within an mRNA (5–10). Also, many of these parameters are inter-related. For example, the inactivation of the poly(A)-binding protein leads to a decrease in mRNA translation (11), and some mRNA destabilizing elements are known to depend upon translation for their function (6). An increasing number of groups are demonstrating how significant contributions can be made to the field by combining molecular, genetic, and biochemical approaches with careful RNA analysis (1–11).
Virus-like particles: Ty retrotransposons
Authors: Kingsman Alan J , Adams Sally E , Burns Nigel R , Martin-Rendon Encarna , Marfany Gemma , Hurd Douglas W , Kingsman Andsusan M
Published: November 2023

The Saccharomyces cerevisiae genome contains a number of movable genetic elements known as retrotransposons. In moving from one genomic location to another they use an RNA intermediate and a reverse transcriptase reaction that is reminiscent of stages in the mammalian and avian retrovirus life cycles (1, 2). Like retroviruses, the yeast retrotransposons package their RNA intermediate into particles. These are known as virus-like particles (VLPs). The particles of one of the yeast elements, Tyl, is the subject of this chapter. They have been used to produce fusion proteins, authentic proteins, and novel vaccines. In addition, they are used to study fundamental aspects of retrotransposon biology.
The Kl killer toxin: molecular and genetic applications to secretion and cell surface assembly
Authors: Brown Jeffrey L , Roemer Terry , Lussier Marc , Sdicu, Anne-Marie , Bussey Howard
Published: November 2023

The killer phenomenon is widespread among yeasts, having been demonstrated in at least eight genera (1). The best characterized is the Kl killer system of Saccharomyces cerevisiae (2-5). Kl killer strains are immune to the toxin they produce, and secrete a small pore-forming toxin that kills sensitive strains. Kl killer strains maintain a double-stranded RNA (dsRNA) viral genome of two distinct species: M1, a medium-sized dsRNA (1.8 kb) encoding the killer toxin and immunity precursors, and L-A, a larger dsRNA (4.6 kb) required for the replication and maintenance of M1. These two dsRNAs are packaged separately with a common capsid protein (encoded by L-A) to form virus-like particles that are transmitted cytoplasmically during vegetative growth and conjugation (6).
Introduction and basic set-up for protein transfer
Authors: Dunbar Bonnie S
Published: November 2023

Since the first studies on the electrophoretic transfer of proteins from acrylamide or other gels to paper or membrane ‘matrices’, there have been numerous modifications to the types of equipment used for these purposes. Detailed studies have also been carried out which include the development of methods for sample preparation and the electrophoretic separation of complex protein samples and for the transfer techniques and renaturation of molecules. The modifications of methods for specific techniques illustrate the need for an in-depth understanding of the basic principles of protein transfer techniques.
Detection and characterization of glycoprotein carbohydrate chains after electrophoretic separation
Authors: Irimura Tatsuro , Kawashima Hiroto
Published: November 2023

Lectins and carbohydrate-specific antibodies are often used for the detection of carbohydrate chains after electrophoretic separation of glycoproteins. Lectin blotting and antibody blotting can be applied before or after electrophoretic transfer of the glycoproteins to nitrocellulose membranes (1). The structural information from these blotting experiments is based on the carbohydrate binding specificity of the immunochemical agents applied. The information can be substantiated by partial modification of the glycoprotein carbohydrate chains prior to the reaction with immunochemical agents (2). To further elucidate the carbohydrate structures of electrophoretically separated glycoproteins, a simple method for the release of oligosaccharides from glycoproteins has also been developed (3). In this method, glycoproteins are electroblotted onto Immobilon transfer membranes. A small piece of the membrane with a glycoprotein band is cut out, dried in a desiccator and subjected to gas-phase hydrazinolysis. The released oligosaccharides, after re-N-acetylation, can be characterized by lectin affinity chromatography, gel permiation chromatography, and other means of oligosaccharide analysis.
Plasmid replication and maintenance
Authors: Nordstrom Kurt
Published: November 2023

All the essential genetic information in bacteria is located in one double stranded, circular DNA molecule, the chromosome. In addition, it is the rule, rather than the exception, that bacteria contain other DNA molecules, plasmids, that live in harmony with their hosts. These are separate entities, replicate autonomously, and control their own replication. They are present in defined copy numbers (average number of molecules per cell) during exponential growth of their hosts. The copy number is determined by genes present on the plasmids but is also affected by the host and by growth conditions (1). In this chapter, replication of plasmids as well as the control of replication will be discussed, with particular emphasis on plasmids that replicate in Escherichia coli.
Nuclear genes involved in chloroplast gene expression
Authors: Rochaix Jean-David
Published: November 2023

Chloroplast biogenesis depends on the concerted action of two distinct genetic systems located in the nucleus and in the plastids. While the informational content of chloroplasts is well known from the complete sequences of several plastid genomes (1,2,3,4; Chapter 3), the role of nuclear genes involved in this process is less understood and appears to be rather complex. A large number, most likely over 1000 nuclear genes, are involved in chloroplast biogenesis. Many of these genes encode components of the photosynthetic apparatus and of the chloroplast genetic and protein synthesizing systems. Others encode polypeptides from the stroma and from the plastid envelope, and several participate in various chloroplast metabolic processes.
Transgenic plants with special emphasis on photosynthetic carbon metabolism
Authors: Riesmeier JÖrg W , Rossmann Jens , Müller-röber Bernd , Willmitzer Lothar
Published: November 2023

Carbon metabolism in photosynthetically active cells of higher plants is of central importance for the allocation of carbohydrates formed to tissues that are dependent on the supply of carbon and energy. Carbohydrates are needed either to fuel housekeeping functions or for the formation of storage pools. Many control mechanisms influence carbohydrate formation and partitioning into different pools. Photosynthetic carbon metabolism will therefore be discussed with respect to the main pathways occurring in photosynthetically active tissues and the regulation of these pathways.
Non-isotopic detection of RNA in situ
Authors: Howard Pringle James
Published: November 2023

The synthesis of RNA plays a crucial role in the molecular biology of the cell and is, therefore, an important process to study. Although RNA can provide a source of genetic information in the genome of RNA viruses, its main role in the cell is to aid the transfer of genetic information from genomic DNA to functional proteins during gene expression. The process is regulated at each step in the pathway from DNA to RNA to protein (1). This includes the control of transcription of heterogeneous nuclear RNA (hnRNA), hnRNA processing to produce mature mRNA via the spliceosomes, mRNA transportation to the cytoplasm, and mRNA translation into proteins. The majority of cellular RNAs including tRNAs, rRNAs, and small nuclear RNAs (snRNA) have catalytic functions in this process (2, 3).
Combination of non-radioactivein situ hybridization andimmunocytochemistry
Authors: Mullink H , Vos W , Jiwa N M , Horstman A , Rieger E , Meijer C J L M
Published: November 2023

i>In situ hybridization is a powerful tool for the detection and localization of nucleic acid sequences in cells and tissue sections. In this way, the presence of specific DNA fragments as well as (messenger) RNA products can be demonstrated with preservation of morphology (1-3). As morphology may be impaired during in situ hybridization, additional information regarding the cells involved may be required, depending on the scientific or diagnostic problem being studied. Since a particular cellular phenotype is characterized by the presence of specific proteins or antigens, immunocytochemical staining is required to reveal it. This phenotype can be studied by careful comparison of adjacent tissue sections stained by in situ hybridization and immunocytochemistry respectively. However, a more accurate and easier method is to analyse both simultaneously in one single, double-stained section. Moreover, only this approach allows the study of individual cells.
PCR analysis of RNA
Authors: Yokozaki Hiroshi , Tahara Eiichi
Published: November 2023

The polymerase chain reaction (PCR) has become an invaluable technique for amplifying small amounts of DNA for a wide range of molecular biological analyses. First strand cDNA, which is the product of reverse transcription of messenger RNA, can also serve as a template for PCR, such that gene expression can be analysed from only trace amounts of mRNA. It can be applied not only to the molecular cloning of genes, but also to RNA blot analysis (1), nuclease protection assay (2), and mRNA phenotyping (3) for the study of low copy number transcripts. The enzyme reverse transcriptase (RT) synthesizes first strand cDNA using mRNA as template. By combining this reaction with subsequent polymerase chain reaction (PCR) amplification, low abundance mRNA species can be analysed in cells or tissue specimens. In this section, conventional protocols for the extraction of total RNA, the RT reaction, and for PCR after the RT reaction are given.
Protein-stabilized water-in-oil-in-water emulsions
Authors: Dickinson Eric , Evison Jane , Owusu Richard K , Williams Andrea
Published: November 2023

Multiple water-in-oil-in-water (W/0/WJ emulsions are inherently less stable than ordinary oil-in-water (0/W) emulsions. In addition to the usual processes of creaming, flocculation and coalescence of the outer (oil) droplets, there is-with a multiple emulsion-the possibility of destabilization due to swelling and coalescence of the inner droplets, leading to rupture of the duplex film between inner and outer aqueous phases. These tendencies are exacerbated by the presence of both hydrophilic and hydrophobic emulsifiers which may migrate between inner and outer oil-water interfaces with consequent detrimental effects for emulsion stability. The use of a two-stage process to make a W/0/W emulsion that is stable to creaming requires extremely vigorous emulsification conditions during the second stage in order to produce droplets no bigger than a few micrometres in diameter. These vigorous conditions have the undesirable potential for disrupting the primary water-in-oil (W/0) emulsion droplets, thereby reducing the yield of multiple emulsion.
Xanthan polytetramer: conformational stability as a barrier to synergistic interaction
Authors: Foster T J , Morris E R
Published: November 2023

The genetically-engineered polytetramer variant of xanthan, in which the terminal mannose residues of each sidechain are absent, shows a thermoreversible conformational transition in solution (as monitored by DSC, CD and optical rotation). As found for xanthan, there is no detectable thermal hysteresis on cooling, and lffm decreases linearly with In I (where Tm is the transition midpoint temperature and I is ionic strength), but the Tm values for polytetramer are substantially higher. Native polytetramer shows normal solution rheology, but develops some xanthan-like ‘weak-gel’ properties after heating and cooling through the temperature-range of the conformational transition. We suggest that the polymer, as biosynthesised, is fully ordered along each chain, but renatures with shorter ordered sequences that promote network formation. Polytetramer shows no evidence of synergistic association with locust bean gum or konjac glucomannan, in contrast to xanthan and (particularly) de-acetylated xanthan, which gave massive viscous interactions even in very dilute solution. The difference is attributed to the enthalpic stability of the polytetramer helix (Afl “‘ 8.5 J/g, in comparison with -4.0 J/g for xanthan) preventing conformational rearrangement into heterotypic junctions. Non-acetylated polytetramer (also geneticallyengineered) gave a similar high value of AfI and again showed no evidence of synergistic interaction. As found for xanthan, the absence of acetate groups lowers Tm, indicating that they contribute to the stability of the ordered structure.
Barley germination and mass spectrometry
Authors: Angelino S A G F , Tas A C , Vos G F La , Mocking H C M
Published: November 2023

During barley germination in malting, various changes take place which affect the composition and internal structural features of the kernel. These changes can be monitored using a sophisticated mass spectrometric technique coupled with data analysis for interpretation of the extensive mass data sets obtained.
Comparaison de divers supports d’immobilisation cellulaire
Authors: Ruhaut L , Barbeau J Y , Shingleton M , Marc I
Published: November 2023

To build a continuous fermentation process, we have studied, in three steps, a large selection of potential yeast carriers. This study contained: a cell immobilization capacity evaluation in agitated process a mercury porosimetric characterisation a comparative wort fermentation on various material beds.
Beer flavor stability improvement and correlations to carbonyl profile
Authors: Bright David R , Patino Hugo , Schroedl David T , Nyarady Stefan A
Published: November 2023

Improvement of the flavour stability of packaged beer is a goal of many brewers. As part of this objective, knowledge of changes in the carbonyl profile of packaged beer during warm storage was targeted as an important step toward understanding the mechanisms of flavour staling. Using a new GC analytical method developed specifically to reduce artifact formation and shorten analysis time, 92 aldehydes, ketones, esters, alcohols and unknown compounds of several test and control beers were monitored during warm storage. Significant differences in carbonyl concentrations were observed between different beers and between different storage temperatures.
The specific role and interaction of yeast enzymatic systems in the removal of flavour-potent wort carbonyls during fermentation
Authors: Debourg A , Laurent M , Dupire S , Masschelein C A
Published: November 2023

In this work, we have studied the in vivo removal of wort carbonyl compounds by brewing yeast strains transformed with different genes encoding enzymes involved in aldehyde degradation. Moreover, using mutant strains and yeast cell-free extracts in the presence of specific substrates and inhibitors, we have investigated the role of different enzymatic activities on the reduction of wort carbonyl compounds. Results indicated an important heterogeneity in the enzymatic carbonyl compounds reduction systems. Nevertheless, it is the first time that these activities as well as the cofactor requirements are well characterized in yeast.
Application of machine vision methods to in-line measurement of yeast (and other particulate matter) in beer
Authors: Forrest I S , Grant I J
Published: November 2023

A knowledge of the concentration and size of particulate material in wort and beer is important at various stages in brewing. In particular, since yeast concentration is critical for beer quality and flavour during fermentation, filtration and cask conditioning, a measure of its concentration is essential. This paper describes the application of machine vision methods in the development of a general purpose, non-contacting, and non-intrusive instrument. The system determines the concentration and size distribution of yeast and other particulate matter from 2 to 100 microns. Examples are presented of the application of the instrument to in-line measurements during the brewing process.
The use of lux gene technology for rapid detection of spoilage bacteria
Authors: Davies Anne M , Hammond John R M , Lindhardt Charlotte
Published: November 2023

Most bacteria do not produce light, but can be induced to do so by infection with a bacteriophage containing the Lux genes coding for bacterial bioluminescence. The modified bacteriophage used in these studies is able to infect Obesumbacterium proteus. While initial light emission requires a certain threshold cell density, cells infected during logarithmic growth emit light in proportion to cell number. Sensitivity of the test is influenced by strain, age and metabolic condition of target cells,5 assay medium and presence of yeast cells. The test is able to detect 4 x 10 cells per ml of original sample, within a working day.
Monitoring and information system applied to the bottling area
Authors: Cavalli Roberto
Published: November 2023

The bottleshop has become a key part of the brewery where competitiveness is gained under the technical, economical and quality point of view. Good information is the base for a correct decision making process. The complexity and wideness of the problems has brought to the development of a computer-based system for the optimization of production lines. Two plants 7 lines (5 bottles, 1 keg and 1 can) have been linked to the system for several years. A substantial performance improvement has been reached with the help of a wide variety of analysis functions and with tools for processing data which are then turned into useful information.
Chemical speciation - a new factor in product and process management
Authors: Williams David R
Published: November 2023

Knowledge of why living systems and the products of living systems respond differently to the same chemical inputs can lead to greater efficiency, enhanced safety and reduced environmental impact. The secret lies in the chemical speciation of the elements - the different chemical forms (or species) in which the elements can be present. The chemical species present in a system frequently differ from the chemical forms added; in addition, knowledge of the chemical species that are present is also vastly more useful than a simple analysis of total elemental content.