bluuebunny/crossref_metadata_2025 huggingface dataset:
Enzymology of the replication fork
Authors: Burgers Peter M J
Published: November 2023

The assembly of the replisome is an ordered process mediated by both protein DNA and protein-protein interactions. The initial steps in this process, origin recognition by the initiator proteins and localized melting of the origin into a replication bubble, will be discussed in Chapters 2 and 3 of this volume. This chapter focuses on the elongation process of DNA replication and the activities of the proteins at the fork. It combines our knowledge of individual replication proteins from mammalian systems and their application to SV40 DNA replication with genetic and biochemical studies of the analogous proteins in yeast. Comprehensive reviews detailing each of these fields have been published (1-10). With the exception of the putative DNA helicase that unwinds the DNA, all enzymes and factors that are predicted to function at a chromosomal replication fork from studies with model systems have been identified in these two eukaryotes, and most of their genes have been cloned. The eukaryotic replication fork contains both DNA polymerase a and DNA polymerase 8 and their accessory factors. Although not evident from viral replication studies, DNA polymerase £ may also participate at the chromosomal DNA replication fork. Finally, recent progress on the maturation of Okazaki fragments initiated by ribonuclease Hl and a 5’-3’ exonuclease will be discussed.
The initiation of DNA replication in the yeast Saccharomyces cerevisiae
Authors: Marahrens York , Stillman Bruce
Published: November 2023

The discovery that DNA consists of two complementary antiparallel strands associated by hydrogen bonds immediately led to the realization that the genetic material duplicates by using the preexisting strands as templates (1). DNA poly­merase was discovered shortly thereafter (2) and marked the onset of increasingly detailed enzymological studies of the template-driven reaction whose mechanism is now understood in great detail (Chapter 1). Insight into how DNA replication is initiated was gained much more slowly. Most progress has been made by studying the replication of the genomes of prokaryotes, bacteriophages and some eukaryote viruses, and the basic mechanism has been deduced for some of these organisms. How eukaryote cell chromosomes initiate DNA replication remains an unsolved problem.
Role of nuclear structure in DNA replication
Authors: Hozák Pavel , Jackson Dean A , Cook peter R
Published: November 2023

The idea that DNA polymerases track along the template as they duplicate the DNA pervades our thinking (Figs 1a-c) (1, 2; and this volume). We imagine that it must be the small polymerase that moves along the very much larger template and that each polymerase acts relatively independently of others. However, recent evidence suggests many active polymerases are concentrated together into large nuclear structures - ‘factories’ - and that replication occurs as a number of templates slide past the many enzymes fixed in the factory; the template moves whilst the enzyme remains stationary, rather than vice versa (Figs ld-f). Then, higher-order structure dictates how and when DNA is replicated. Discussion concentrates on eukaryotic enzymes, but as the catalytic domains of all polymerases are structurally related (3), prokaryotic enzymes probably work in the same way.
Affinity maturation of antibodies using phage display
Authors: Johnson Kevin s. , Hawkins robert e.
Published: November 2023

Protocols described in this section will deal with alteration of the properties of existing antibodies, formatted as scFv and displayed on bacteriophage. Although the title implies a focus on improving affinities, provisions are made for alteration in off-rate and specificity. Where the aim of the ‘matura tion’ process is a shift in specificity, this can be achieved by introducing bias into the selection process.
Human antibody repertoires in transgenic mice: manipulation and transfer of YACs
Authors: Davies Nicholas p. , Popov andrei v. , zou xiangang , bruggemann marianne
Published: November 2023

Therapeutic applications of an accessible human antibody repertoire are vir tually limitless. Considerable interest in the techniques for producing human antibodies are apparent from clinical results which highlight their benefits (1). The problems that occur with immunogenicity, when antibodies of a foreign species are being used for therapy, unambiguously point out that the affinity and authenticity of the antibody are critical (1-3 and references therein).
Measuring antibody affinity using biosensors
Authors: Hefta Laura J , Wu Anna M , Neumaier Michael , Shively John E
Published: November 2023

Antibodies are often engineered to improve their specificity and affinity for a given antigen. One of the first goals in evaluating newly engineered anti bodies is to measure their affinity constants. Recently, a new technology has become available that allows the measurement not only of antibody equilib rium constants, Ka (association) or Ka (dissociation), but also of their kinetic rate constants, k0n and koff, where Ka= k0fflk00 The biosensor-based technol ogy permits real-time mass measurements using either surface plasmon reso nance (SPR, BIAcore) or a resonant mirror (IAsys, Fisons). In general, a protein sample (ligand) is coupled to the sensor chip via a hydrogel (car boxymethyl dextran) and solutions containing different concentrations of a binding protein (ligate) are allowed to flow across the chip while monitoring binding with mass sensitive detection (association step). At the end of a bind ing experiment, it is possible to flow buffer across the chip and observe the dissociation step. This is usually done at saturating conditions, where rebind ing of the ligate is not appreciable. Appropriate analysis of the binding and dissociation steps allows the calculation of kinetic constants k00 and k0ff, and as mentioned above, Ka can be calculated from the ratio of the two kinetic constants.
Assembly of chromatin and nuclear structures in Xenopus egg extracts
Authors: Almouzni Genevieve
Published: November 2023

The structural organization of DNA within the eukaryotic nucleus is tightly defined to accommodate the functional properties of a given cell type. Nuclear functions such as replication and transcription operate in a highly regulated fashion using chromatin as a template. During every cell cycle, each nucleus has to replicate itself, requiring duplication not only of DNA but also of the individual chromosomes together with all their structural and regulatory features. Chromatin organization is known to influence gene expression ( 1 ) and its remodelling during replication and cell cycling can affect the transcription process. To study the relationship between the structural organization of DNA and macromolecular events such as replication and transcription, an adequate system is required, accurately reproducing both structural and functional features of the eukaryotic cell.
Purification of DNA
Authors: Towner Paul
Published: November 2023

TI1is chapter explains how to purify genomic DNA and the DNA of different types of vectors. Genomic DNA can be obtained from any microorganism, plant or animal at any time duringdevelopment and provides DNA which contains a copy of every gene from the organism. Vector DNA refers to plasmid and phage DNA.
Epitope mapping of carbohydrate binding proteins using synthetic carbohydrates
Authors: Nilsson U J , Magnusson G
Published: November 2023

Specific recognition of carbohydrates by proteins is involved in a number of important biological phenomena. For example, carbohydrates may act as cell surface antigens, e.g. in the ABO, Lewis, P, and I blood group systems or as tumourspecific antigens; they control growth and tissue development in an embryo, and are involved in fertilization; they regulate nerve growth and neural cell adhesion; and they direct the recruitment of leukocytes to damaged tissue (1, 2). Furthermore, carbohydrates often act as recognition sites for pathogenic bacteria and viruses (3). As a consequence, detailed knowledge on a molecular level concerning the recognition mechanisms and driving forces would open up possibilities to specifically interfere with, or promote, biological phenomena stemming from carbohydrate-protein interactions (i.e. development of therapeutics that target carbohydrate-binding proteins) (4).
Transposons—gene tagging and mutagenesis
Authors: Grigliatti T A
Published: November 2023

The discovery of mobile genetic elements and their association with ‘spontaneous’ mutations in nature has led to novel ways of cloning genes, of germline transformation, and has added methods for creating certain types of mutations. To date, the P element is the most exploited transposable element in Drosophila, but there are many others, several of which have been characterized sufficiently to allow similar applications with perhaps only a little more effort. For example, the hobo element has been used for both transformation and gene tagging, the IS elements have been used for mutagenesis and gene tagging, and mariner has been used for transformation. These other mobile elements may have properties which, in the future, make them better for certain applications, such as target site specificity, efficiency of movement, or homologous target integration (gene replacement). But, this chapter focuses on some of the common applications of the P transposable element technology for creating useful mutations. Specifically it focuses on:
Looking at embryos
Authors: Wieschaus Eric , Nusslein-Volhard Christiane
Published: November 2023

In Drosophila, the pattern observed in mutant embryos often provides the first indication for the role of the wild-type gene in normal development. The loci which affect embryonic pattern, represent only a small fraction of the total genome and their phenotypes are often very discrete. With practice, new mutations in a given locus can be recognized strictly on abnormalities observed among the unhatched embryos from a mutagenized line. The easy recognizability of embryonic phenotypes greatly simplifies mutagenesis experiments and has proved very useful in subsequent analysis of the mutations obtained.
Immunolabelling of Drosophila
Authors: White Robert A H
Published: November 2023

Specific antibodies provide the easiest way, in general, of studying the distribution and subcellular localization of proteins. Such knowledge can give important clues to their role in development and cell biology. Using an antibody to detect the protein product of a specific gene is a convenient way of following gene expression patterns in development and allows the analysis of both transcriptional and post-transcriptional gene regulation. Antibodies are also widely used as markers for individual tissue and cell types enabling these to be followed during morphogenesis (see ref. 1). Multiple labelling can be used to correlate the expression patterns of different proteins. The use of immunolabelling is now so established that many research papers merely say ‘Antibody labelling was by standard methods’. However, it is clear that appropriate fixation, labelling, and detection can make a huge difference to the sensitivity and clarity of the result, and indeed whether the labelling actually gives a tolerably faithful representation of the in vivo protein distribution.
Eukaryotic DNA polymerases
Authors: Wang Teresa S -F , Conger krista L , Copeland William C , Arroyo Martha P
Published: November 2023

DNA polymerases are the fundamental enzymes that synthesize cellular DNA to produce two identical daughter chromosomes. Cellular DNA polymerases in conjunction with DNA repair enzymes play critical roles for the transmission and maintenance of error-free genetic information from one generation to the next. In the last decade, genetic studies in yeast, biochemical studies of in vitro replication of the simain virus 40 (SV40) origin-containing DNA, and isolation of genes and cDNAs of DNA polymerases from budding yeast, fission yeasts, and mammalian cells led to the identification of five distinct DNA template-dependent DNA polymerases, named polymerase α, β, γ, δ, and ε(1,2).
Saccharomyces cerevisiae as a model system to study DNA replication
Authors: Foiani Marco , Liberi Giordano , Piatti Simonetta , Plevani Paolo
Published: November 2023

The budding yeast Saccharomyces cerevisiae represents a powerful tool to study DNA replication and the cell cycle regulatory pathways controlling entry into, and progression through S phase. In fact, the availability of the complete sequence of the S. cerevisiae genome, the identification of several DNA replication genes, together with the possibility of combining molecular genetic tools with classical techniques of genetics, biochemistry, and cell biology, make this system particularly useful to improve our knowledge of the DNA replication process at the molecular level. Moreover, by considering that the structure and function of most of the replication proteins and the regulatory circuits connecting DNA replication to other cell cycle events have been highly conserved during evolution, the information obtained using the S. cerevisiae or the S. pombe systems can be generally extended to other eukaryotic cells.
Cell cycle checkpoints: safe passage through mitosis
Authors: Yu Kristina R , Duronio Robert J , Sullivan William
Published: November 2023

Mitosis results in the production of two daughter cells containing identical genetic complements. Achieving this requires a carefully orchestrated series of nuclear and cytoplasmic events leading to the accurate replication and segregation of the chromosomes. A powerful combination of molecular genetics, cell biology, and biochemistry have led to the isolation of key structural, enzymatic, and regulatory components governing these events. One of the most gratifying aspects of this research has been the realization that many of these components are highly conserved throughout the phyla. Therefore many of the lessons learned from one system will probably apply to others.
Meiosis: chromosome behavior and spindle dynamics
Authors: Karpen Gary H , Endow Sharyn A
Published: November 2023

Meiosis is an alternative pathway of development that leads to the production of haploid cells, usually spores or gametes, for sexual reproduction, rather than the identical or near identical diploid cells produced by mitosis. The recent discovery of analogous proteins that are involved in the regulation of both meiosis and mitosis makes it increasingly apparent that meiosis is closely related to mitosis, and can be regarded as a highly specialized mitotic division. Although meiosis differs from mitosis in several respects, many of these differences are variations of events that occur during mitosis. Some, however, are not. Probably the most radical departure from mitosis occurs during meiosis I and concerns the behavior of the meiotic chromosomes: homologous chromosomes in most organisms undergo pairing (initial alignment of homologs in zygotene), synapsis (intimate association of homologs at pachytene) and recombination (physical exchange) during meiotic prophase, which are not observed in mitotically dividing cells. These events are followed by two successive divisions without an intervening replication, instead of the single division that occurs in mitotic cells, resulting in reduction of the chromosomes to the haploid number (Fig. 1).
Modern biotechnology and the consumer - Unilever’s perspective
Authors: Vet Bernard A De
Published: November 2023

The second reason, interested but not publicly so, is a dangerous course to choose. And the first one, not interested because of fear for controversy is one which calls for an assessment of the nature of that controversy and of the interest of individual consumers. It also requires a view on the further development of the discussion in the various countries.
Changes occuring in polyphenol substances content during wort production
Authors: Srogl Jiri , Kosar Karel , Mikyska Alexandr , Bousová Pavla
Published: November 2023

This article deals with the release of polyphenol substances to the solution during the wort production. The hop polyphenols are being released to the wort easily. Addition of neither unmalted barley nor of husks during mashing will increase the content of polyphenols in the wort, not even in the mixture with a significant surplus of well modified malt. In the course of mashing the polyphenol substances are not being released, not even from the malt. Decisive factor influencing the content of malt polyphenols in the wort is the malting process.
Rate analysis of dimethyl sulfide volatilization during wort boiling
Authors: Mitani Yutaka , Ishida Fumito , Akiyama Hiroshi , Tamaki Teruo
Published: November 2023

The decrease in dimethyl sulfide (DMS) in boiling wort indicates how much volatile compounds have evaporated during this operation. DMS volatilization behaviour could be analysed as a consecutive process.
High gravity brewing and its negative effect on head retention
Authors: Bryce James H , Cooper Dan , Stewart Graham G
Published: November 2023

The aim was to establish why beer brewed using high gravity techniques has poor head formation and retention. Work focused on measuring hydrophobic polypeptides, identified as important foam stabilising molecules, throughout the brewing of lagers at high (20°P) and low (10°P) gravities. The efficiency of hydrophobic polypeptide extraction during high gravity mashing was significantly less than during the mashing of normal gravity wort. Furthermore, high gravity fermentations had a greater loss of hydrophobic polypeptides. Thus, the low gravity lager had up to 40 % more hydrophobic polypeptides. Modifications to high gravity brewing can now be recommended to maximise foam potential.
New molecular probes for assessing stress conditions in brewing yeast: biotechnological implications and applications
Authors: Lopez Victoria , GiJU Rosario , Navarro José V Carbonell & Alfonso
Published: November 2023

Modem day brewing stimulates technological innovations allowing more rapid processing and greater consistency and contml over the fermentation and the physiological condition of pitching yeast. However, we are still far from being able to predict and control yeast performance. Trying to choose the right probe to allow us to detect and monitor stress situations in yeast, we have targeted our attention to 20S RNA and 23S RNA which are single stranded yeast replicons whose copy number is greatly induced under stress conditions. The molecular characterization of brewing yeast strains in relation to the presence or absence of these yeast RNA viruses together with their potential applications in process control are described.
Encapsulation of a-acetolactate decarboxylase to avoid diacetyl formation
Authors: Dulieu Claire , Boivin Patrick , Malanda M’Baka , Dautzenberg Horst , Poncelet Denis
Published: November 2023

Elimination of diacetyl at the end of fermentation can be achieved by the enzyme a acetolactate decarboxylase. This enzyme transforms a-acetolactate directly to acetoin, thus preventing the formation of diacetyl, a contributor to off-flavour. We have developed a technique of encapsulation of a-acetolactate decarboxylase. The twin objectives were re-use of the enzyme and absence of the enzyme from the final product. The enzyme was encapsulated in the active form: transformation of a acetolactate to acetoin was demonstrated in neutral buffer. Studies on stability of the encapsulated enzyme, and trials on beer, are currently in progress.
Novel method for determination of glucose concentration during fermentation by Saccharomyces cerevisiae
Authors: IerseJ Martijn van , Favier Jan Paul , Meersman Eric
Published: November 2023

An optothermal method for quantitative analysis was developed and applied to monitor sugar during fermentation. The measurements were compared to those obtained from HPLC data and a good correlation was found (r = 0.996; detection limit for glucose 0.14 g 1-1). The ability to measure glucose in a complex matrix with a good reproducibility (within 2 %) and a low relative error (typically

0.5 %) suggests the potential of the new method for use in beer brewing. Further enhancement of the detection limit and sensitivity are anticipated when using a differential scheme with automatic subtraction of the background signal.
Very high gravity fermentations with ale and lager yeast strains
Authors: Borthwick R , Stewart G G , Rees E M R , Jones H L , Hodgson J A
Published: November 2023

The successful fermentation of ever stronger worts is the objective of many breweries. This study investigated the fermentation performance of ale and lager yeast strains in 15 and 20°P worts (all malt and high adjunct). Four successive fermentations were conducted in EBC tall tubes, with yeast storage and acid washing periods between each fermentation. The lager strain tolerated the increased stresses associated with very concentrated worts. It out performed the ale strain in 20°P wort, regardless of wort type, with improved viability, intracellular trehalose and beer flavour matching. Currently, large scale production trials are being conducted, focused at increased wort strengths.
Beer filtration with regenerable filter aid
Authors: Harmegnies F , Bonacchelli B , Tiget R
Published: November 2023

The efficiency of kieselguhr filters for beer filtration is well recognized but they are more and more questioned by economical and ecological considerations. A concept of “ecological filtration” has been developed, based on the use of a regenerable filter aid of variable porosity for prefiltration or clarification of beer. PVPP stabilisation can be carried out simultaneously to clarification. According to the economical study of an industrial installation, the investment and operating costs are lower than in the case of a conventional system.
Schnellbestimmung zur Vorhersage der Geschmacksstabilitat von Bier
Authors: Klein H , Krammer R , Natter M
Published: November 2023

The consumer expects the brewery to guarantee comprehensive and consistent quality to the “best-before date” within the limits of the process. Therefore a knowledge of taste stability is a significant factor in control of quality. Since classical assessment by tasting is expensive and gives highly subjective results, we developed a single rapid but highly informative analytical method. Fresh beer is steam distilled and the distillate measured spectrophotometrically, so providing as an “absorption integral” a value that correlates well with tas!e panel assessment. Therefore this new procedure used alone can provide brewery laboratories with an objective method to monitor the taste stability of their beers.
Measurement and significance of oxidation-reduction levels in beer
Authors: Buckee G K , Mom M , Nye J W S , Hammond R V
Published: November 2023

Conflicting views have been expressed in the literature concerning the measurement and significance of oxidation-reduction (redox) potentials in the brewing process. Causes of hitherto unstable measurements and parameters influencing redox values have been identified. A measuring cell has been developed which consists of a pH electrode and two platinum electrodes connected to a millivoltmeter. The second platinum electrode facilitates electrolytic cleaning and electrode polarization. Redox values can be correlated with dissolved oxygen levels and physical stability of packaged beer. Other applications are prediction of flavour stability and in/on-line monitoring of the brewing process.
The measurement of beer foam using a new laser-based video device
Authors: Mulroney A R , Wenn R V , Oortwyn J , Williamson R R
Published: November 2023

A new laser-based video device which offers a non-invasive approach to the measurement of foam collapse in beers under a variety of dispense conditions has been tested. The device proved capable of accurately following beer foam collapse and has application within the industry to address issues of foam quality and quality control. Differences in foam stability were observed between beer foams derived from both carbonated and nitrogenated beers. Foam collapse profiles proved highly reproducible under consistent dispense conditions. For nitrogenated beer foams the device was sensitive to very small foam volume changes. Temperature was shown to influence the foam collapse parameter.
Quantitation of total sulphur dioxide in beer by automated headspace gas chromatography
Authors: Lowe Luis E , Dreyer Thomas
Published: November 2023

An automated method for the analysis of total SO2 was needed to take care of the increasing number of samples being analysed. The analysis relies on the formation of gaseous SO2 after acidification and heating of the sample. A volume of the SO2 gas generated is sampled by a headspace sampler and analysed by GC with a sulphur chemiluminescence detector. The accuracy and the precision of the method are equal to or better than those of the colorimetric ASBC method. Results from comparative analyses of beers using these two methods gave a correlation coefficient of r = 0.949 (149 samples).
Piezoelectric ceramics: a new concept for in-line density measurements
Authors: Gesnot M , Perrier L , Debourg A
Published: November 2023

In-line measurement of wort and beer gravity offers the opportunity of continuous process monitoring. The principle of the gravity sensor developed is based on a dynamic measurement of the resonance frequency between two piezoelectric membranes. The laboratory trials with this sensor have shown that yeast cells, suspended trub and CO2 bubbles do not interfere with specific gravity measurement. The accuracy and reliability of this system allow specific gravity measurement in a range from 1.0000 to 1.3000 with a variability of 0.002. The results were confirmed by trials in industrial fermentation vessels and in-line measurements of wort specific gravity.
The use of robots improves the performance in the bottling hall
Authors: Swinkels F L M
Published: November 2023

At the time Bavaria N.V. had its packaging lines equipped with the latest in conventional palletizing technology. Always in search of new technology or methods we ran into robots. We researched and tested this technology based on our future specifications for our end-of-line packaging requirements. This proved drastic reduction in floor space, reduced energy consumption, reduced scrap losses, minimal product change-over time, packaging material reduction, labor reduction. The proven results have made us decide to replace a!I conventional equipment by robots.
Keg tracking - a method to control a keg fleet; experiences and advantages
Authors: Till Volker
Published: November 2023

Keg tracking is a mostly unknown tool to breweries in order to increase profit and quality. It allows to register all external influence on the kegs and is used to give total control for the keg handling, keg washing and filling in the brewery as well. Systems in operation in Switzerland, France, Germany, Australia and South America proof the effectiveness. The paper summarizes the latest practical solutions and supplies guidelines how to introduce and install a system. It ends with an overview of cost and return on investment supplied from data of existing systerm; in operation.
microscopy and cryo-microscopy
Authors: Cam Eric Le , Delain Etienne
Published: November 2023

Characterization of the DNA curvature and flexibility, intrinsic or induced by protein, re-mains controversial and depends on the method used. Such an analysis is even more difficult with respect to the confom1ational changes in DNA induced by non-specific binding proteins such as histone-like proteins. Electron and recent near-field microscopes now offer different potentials to visualize and analyse individual molecules. These microscopes have the capacity to determine the average behaviour of a population of molecules observed individually and can thus provide a better appreciation of variability within the series of molecules than biochemical or other biophysical methods.
Mapping DNA target sites of chromatin-associated proteins by formaldehyde cross-linking in Drosophila embryos
Authors: Cavalli Giacomo , Orlando, Valerio , Paro Renato
Published: November 2023

The method described here allows the mapping of protein-DNA interactions through the ability of formaldehyde to cross-link proteins and nucleic acids in living cells. Formaldehyde is a very reactive dipolar compound which reacts with the amino groups of proteins and amino acids (1, 2). It shows no reactivity, however, towards free double-stranded DNA, and thus does not cause the extensive DNA damage seen after prolonged exposure to other crosslinking reagents such as UV. Each formaldehyde molecule has the capacity to interact with two amino groups. Therefore DNA-protein, protein-protein, and RNA-protein cross-links are rapidly formed after formaldehyde treatment, creating a stable structure which prevents the redistribution of cellular components. Furthermore, a simple heat treatment is sufficient to reverse the reaction equilibrium, and to allow the isolation of pure DNA for further analysis (3). Formaldehyde crosslinking combined with chromatin immuno precipitation (IP) is a way of mapping the in vivo distribution of chromatin• associated proteins. As such, this technique is of great value in the analysis of protein-DNA interactions, even more so when studying proteins which do not show specific DNA binding activities in vitro. This has recently been demonstrated for several chromatin associated proteins, such as Polycomb (PC) in Drosophila (4) and the Silent Information Regulators (SIR) proteins in budding yeast (5). Additionally, analysis of the crosslinking pattern can not only allow mapping of sites of protein-DNA interaction, but can also give an estimation of the relative binding affinity to different sequences across a large genomic region.
Chromosome fragmentation in vertebrate cell lines
Authors: Farr Christine J
Published: November 2023

Experimental approaches to understanding chromosome structure and function fall into two main categories: the first involves assembling candidate DNA elements in vitro to construct an artificial chromosome. This approach was used to create yeast artificial chromosomes in both Saccharomyces cerevisiae and Schizosaccharomyces pombe. More recently a related approach has lead to the generation of mammalian minichromosomes in the human somatic cell line HT1080, although the precise DNA organization and requirements of these mammalian minichromosomes has yet to be fully elucidated (1). The alternative approach involves manipulation of an existing chromosome to generate smaller minichromosome derivatives suitable for further study/fragmentation. This chapter will focus on the use of short stretches of the vertebrate terminal repeat DNA sequence (TTAGGG)n to 'seed' the formation of de novo telomeres when introduced into vertebrate cell lines, simultaneously truncating the chromosome at the integration site (2, 3). Combined with elaborate selection schemes and homologous recombination this approach has led to the generation of a series of minichromosomes derived from native human chromosomes (4, 5).
Enzyinology of huinan nucleotide excision repair
Authors: Naegeli Hanspeter
Published: November 2023

Damaged bases are eliminated from human genomes by two completely different excision mechanisms: repair by base excision and repair by nucleotide excision. Base excision involves the hydrolysis of N-glycosidic bonds and is restricted to a rather narrow range of defective or inappropriate DNA constituents (1, 2). Nucleotide excision repair (NER), on the other hand, proceeds through dual endonucleolytic cleavage of damaged strands and is capable of removing a nearly infinite variety of base lesions with little regard for the modification itself. The extraordinary substrate versatility of NER is necessary to cope with bulky (helix distorting) injuries to DNA that arise mainly from exposure to UV radiation and chemical carcinogens (3-6). NER is, in fact, the sole mechanism for the error-free removal of bulky DNA adducts in human cells and, as a consequence, provides a key line of defence against such genotoxic adversities. In addition, NER displays a backup role for the repair of apurinic-apyrimidinic (AP) sites, thymine glycol, 8-hydroxyguanine, O6-methylguanine, and similar non-bulky base lesions that are normally processed by more specific repair enzymes, primarily AP endonucleases, DNA glycosylases, or O6- methylguanine-DNA methyltransferase (7, 8). To exert these multiple DNA repair functions, human NER lacks selectivity for a particular type of damage, i.e. a limited number of gene products accommodates and processes a large repertoire of unrelated base modifications while ignoring the normal conformational fluctuations of undamaged DNA. This flexibility is achieved by means of a repair strategy that avoids the direct processing of modified deoxyribonucleotide residues, but, instead, operates exclusively on unmodified segments of native DNA around each lesion site.
Investigation of DNA virus genome structure
Authors: Skinner Michael A , Laidlaw Stephen M
Published: November 2023

The nucleotide sequence of the DNA genome of bacteriophage cpX174 (5386 bp) was determined in 1977 (1), and that of bacteriophage A (48 502 bp) followed in 1982 (2). In these early years of sequencing, virus genomes provided the targets to push the development of nucleotide sequencing technology forward. This situation continued until the completion of the sequences of the genomes of the large DNA viruses such as the herpesviruses and the poxviruses. Notable landmarks have been the first complete sequence of a herpesvirus, varicella zoster virus (124 884 bp) in 1986 (3), and of a poxvirus, vaccinia virus (191636 bp) in 1990 (4). The intervening years have seen the determination of the complete sequences of other members of these families of large viruses, representing examples of different genera and with different hosts. Examples of these for the herpesviruses are human and equine herpes viruses I (5, 6) and II (7, 8), cytomegalovirus (9), HSV 7 (10), channel catfish virus (11); and for the poxviruses, smallpox (12), and molluscum contagiosum virus (13). The sequences of other large DNA virus genomes, such as African swine fever (14), and a baculovirus (15), have also been determined. These projects represent various approaches to the problem; clearly there are alternative strategies that can be employed successfully. Moreover, it is no longer the viruses that provide the targets for the technological push, but the larger genomes of bacteria (16-19) and yeast (20), now superseded by the chromosomes and indeed entire genomes of eukaryotes. The tools currently available to the virus genome sequencer are therefore considerably more powerful than those used to elucidate the first sequences. We can therefore look forward to the routine determination of the sequences of a wide range of large DNA viruses with different host ranges and phenotypes, bringing new opportunities for the study of virus evolution and virus-host interactions.
Identification and analysis of trans-acting proteins involved in the regulation of DNA virus gene expression
Authors: Whitehouse Adrian , Meredith David M
Published: November 2023

We have been interested in identifying and characterising the genes that regulate viral gene expression in Herpesvirus saimiri (HVS). This is a lymphotrophic rhadinovirus (γ-2 herpesvirus) of squirrel monkeys (Saimiri sciureus), which persistently infects its natural host without causing any obvious disease. However, HVS infection of other species of New World primates can result in lymphoproliferative diseases (1). The genome of HVS (strain All) consists of a unique internal low G + C-content DNA segment (L-DNA) of approximately 110 kbp which is flanked by a variable number of 1444 G bp high + C-content tandem repetitions (H-DNA) (2). Analysis indicates that it shares significant homology with other ‘Y herpesviruses: Epstein-Barr virus, bovine herpesvirus 4, Kaposi’s sarcoma-associated herpesvirus (or human herpesvirus 8), and murine gammaherpesvirus 68.
Herpes simplex virus and adenovirus vectors
Authors: Scarpini Cinzia , Arthur Jane , Efstathiou Stacey , Mcgrath Yvonne , Wilkinson Gavin
Published: November 2023

Gene therapy has the potential to treat a wide variety of acquired and inherited disorders, including inborn errors of metabolism, infectious disease, and cancer. For each of these applications an appropriate vector system is required to deliver the therapeutic gene efficiently to the nucleus of the target cell, in order to mediate either transient or long-term expression of the required gene product. In this chapter we consider the biological properties and methods associated with the construction and propagation of two large DNA virus vector systems: herpes simplex virus and adenovirus.
The genome project and sequence homology to other species
Authors: Blaxter Mark
Published: November 2023

Caenorhabditis elegans has a small genome (1 3). At 97 megabases (Mb) it is one thirtieth the size of the human (and other mammalian) genomes. Since C. elegans is a metazoan, its genome might be expected to be significantly more complex than those of single celled eukaryotes such as yeasts and protozoa, yet it is only three times the size of the malaria genome and eight times that of fission yeast. Within this relatively small gene set lie all the instructions for the development and functioning of a fully differentiated animal. These include genes for developmental regulation and embryogenesis, the functioning of complex organ systems, and the integration of the nervous system to control behaviour (4 8). At the inception of the C. elegans genome project, the idea of determining the complete sequence (never mind the structure) of such a vast genome was nearly unthinkable. Thanks to the vision, drive, and sheer hard work of a core group of researchers (in particular John Sulston, Alan Coulson, and Bob Waterston), the dream of having a fully mapped, and fully sequenced genome is complete. The genome project has been central to the success of C. elegans as a model organism, as it allows researchers to home in on the molecular basis of observed genetic defects without having to spend years in the forest of conventional, mapless cloning projects (9). The genome sequence yields the ultimate in genotyping and is the basis on which the next generation of ‘post genomics ‘ research will be based. In addition, the finding of a potential homologue of a gene of interest in the C. elegans genome sequence dataset is often the first inkling a researcher will have that this small nematode might be of interest to them.
C.elegans and the Web
Authors: Thierry-Mieg Jean , Thierry-Mieg Danielle , Potdevin Michel , Stein Lincoln
Published: November 2023

With the completion of the C. elegans sequencing project, it is now a frequent event for a biologist working on another species to detect a homology to a nematode sequence during a routine BLAST search. Starting from such a link, or from a text search, we show, step by step, how a scientist, who does not have specialized knowledge of C. elegans biology, may use the Web to explore the C. elegans data. We show how to access the genomic DNA and cDNA sequences, look at the graphic displays of the sequence and the genetic map, and search for mutant phenotypes. Finally, we explain how to contact the relevant C. elegans laboratories to ask for further help, information, and possibly strains, clones, or other material from which may develop a new and fruitful collaboration.
Microscopy
Authors: Schnabel Ralf
Published: November 2023

This chapter is designed to help you to analyse the phenotype of your favourite worm or embryo by looking through a microscope. The worm is perfectly suited to analysis by light microscopy. The dimensions of the embryo (about 55 µm) or the adult (1 mm) are just in the right range, such that the whole embryo or significant parts of the body of larvae and adults can be viewed with the highest numerical aperture and therefore with the best achievable resolution. Thus, subcellular details can be seen without losing sight of the whole animal. Although a wealth of very helpful, molecular markers of cell fate are available (e.g. antibodies, transgenic lines with green fluorescent protein (GFP) or [3 galactosidase labelled proteins, or specific probes for RNA in situ), differentiated cells show very characteristic features when viewed directly through a light microscope fitted with Nomarski (DIC) optics, so that determining which tissue or organ a cell belongs to (e.g. pharynx, gut, hypodermis, nervous system, or body wall muscle) is quite easy. The use of 4D microscopy, which allows the development of whole embryos to be documented, adds a new level to the analysis of cells since the ‘behaviour ‘ of cells can also be followed. It is an interesting question whether the expression of a single molecular marker really defines the fate of a cell unambiguously. However, if a cell looks like, migrates, and intercalates like a hypodermal cell it is very likely a hypodermal cell. Many investigators have now become interested in C. elegans because the function of their favourite molecule can be readily analysed in this nematode. It is therefore the general aim of this chapter to introduce molecular biologists to the basics of microscopy. To determine the function of the gene, at least its biological function as opposed to its ‘chemical ‘ function, one has to look through a microscope. Although this is mainly a book about methods, concepts are also very important methods and therefore a short conceptual discussion about what is required to determine the function of the gene, that is characterization of the ‘phenotype ‘ of the organism lacking a gene function, is included in the section discussing 4D microscopy.
Neurobiology
Authors: Thomas James h. , Lockery Shawn
Published: November 2023

The nervous system of C. elegans is simple and uniquely well described. There are 302 neurons in the adult hermaphrodite that fall into 118 classes based on morphology and other characters (1). These neurons are interconnected by about 5000 chemical synapses and 600 gap junctions. The structure of each neuron is nearly invariant from animal to animal and the cell bodies are invariantly positioned so that each neuron can be identified in vivo (2). Despite its small size and simplicity, this nervous system regulates a wide variety of behaviours. These include several mechanosensory responses, many chemosensory responses, thermotaxis, complex responses to food, locomotion, feeding, egg laying, defecation, and male mating. Neurons that control most of these behaviours have been identified, and in many cases they use identified transmitters and act in defined circuits.
Molecular biology
Authors: Johnstone Iain L
Published: November 2023

This chapter deals with some of the common, molecular biological methods essential for general aspects of gene characterization. It includes the purification of DNA, RNA, and some simple, protein extraction methods useful for Western blot analyses. Because much of what is discussed here is not specific to the nematode and extensive coverage is available in other texts (1), I have emphasized the modifications to those more general methods relevant to working with the worm. The availability of the genome sequence makes an immense body of data available to any researcher. It is an extremely valuable resource which, for many of us in the field, has resulted in a great reduction in the amount of general molecular biology performed in the laboratory. However, it is important to avoid complacency and remember that most of the genes indicated by the genome project are predictions, and that the different aspects of such predictions, e.g. intron/exon organization and gene endpoints, must be con firmed experimentally. This is particularly important when considering the construction of reporter gene fusions and the interpretation of expression patterns so obtained (see Chapter 9). The predictions, around the start of genes in particular, are not always accurate. In this post genome sequence era, in addition to confirming predicted data, general molecular methods are still necessary for many purposes including the analysis of gene expression and gene function, as well as for investigating the nature of mutant strains.
Synthesis of combinatorial libraries using the portioning-mixing procedure
Authors: Furka Árpad , Hamaker Linda K , Petersonꝉ Mark L
Published: November 2023

Combinatorial chemistry is a young, yet intensely popular branch of science. Beginning in the 1980s, several innovative synthetic methods were published (1-7) which radically changed the theory and practice of designing and preparing new substances for pharmaceutical research and other areas of application. The field of combinatorial chemistry evolved as a result of these new approaches and has revolutionized the way scientists think about drug discovery. Combinatorial processes have since spread outside of pharmaceutical research and are gradually expanding to other areas of chemistry including catalysis and materials science. One of the first and most influential combinatorial synthetic methods was the portioning-mixing (split-mix) procedure (3, 8, 9) (see also Chapters 2 and 3).
Positionally addressable parallel synthesis on continuous membranes
Authors: Wenschuh Holger , Gausepohlꝉ Heinrich , Germeroth Lothar , Ulbricht╪ Mathias , Matuschewski§ Heike , Karmer* Achim , Volkmer-Engert* Rudolf , Heine* Niklas , Ast* Thomas , Scheme* Drik , Schneider-Mergener* Jens
Published: November 2023

Spatially addressable high-throughput solid phase synthesis of large arrays of compounds has generated intense interest over the past few years (1). Besides parallel synthesis on resin beads (see Chapters 1-3), polymeric pins and chips (see Chapter 4), SPOT synthesis using continuous membrane supports has been shown to be an efficient solid phase synthetic alternative. The development of this approach was fuelled by the need for a facile and economical complement to the classical solid phase synthesis procedures with increased flexibility and amenability to miniaturization and automation (2).
Regulation of cytoskeletal proteins by inositol lipids
Authors: Flanagan Lisa A , Janmey Paul A
Published: November 2023

Since the initial finding that phosphatidylinositol bisphosphate (PtdIns(4,5)P2) regulates the affinity of profilin for actin and promotes actin assembly from profilin–actin complexes in vitro (1), a variety of inositol lipids have emerged as potential regulators of different forms of cytoskeletal assembly in vivo. At the same time, the recognition and availability of different inositol lipid species and identification of PtdIns(4,5)P2-binding sequences in proteins have confirmed in many cases the biochemical specificity of these protein–lipid interactions. This chapter aims to synthesize recent studies of phosphoinositide effects on cytoskeletal proteins to suggest how these lipids may trigger cytoskeletal changes in response to cellular signals.
Physiological functions of phosphatidylinositol transfer proteins
Authors: Rivas Marcos P , Li Xinmin , Bankaitis Vytas A
Published: November 2023

Eukaryotic cells organize their interior by establishing and maintaining a diverse set of intracellular organelles. These organelles represent membrane-enclosed structures that function to compartmentalize physically the multitude of biochemical machines dedicated to the execution of specific cellular functions. This compartmentation function is reflected by the unique sets of resident proteins and by the lipid compositions that comprise each individual organelle. That cells would evolve elaborate mechanisms to maintain unique lipid compositions in the various organelle membranes is not a difficult concept to grasp. A more challenging problem is the elucidation of how the biochemical compartmentation of lipids is maintained in the face of robust membrane trafficking pathways that should act to randomize lipid distribution. As a result, the problem of how specific organelles maintain their signature lipid compositions is a complex one. It involves issues of where lipid synthesis and degradation reactions are localized, how these metabolic processes are coordinated, and how cells regulate and execute lipid sorting and transport reactions.