bluuebunny/crossref_metadata_2025 huggingface dataset:
A model of glandular epithelium for studying secretion
Authors: Adessi Gérard L , Beck Laurent , Mahfoudi Abderrahim
Published: November 2023

The endometrium is a complex tissue consisting of the endometrial epithelium supported by a mesenchymal tissue called the endometrial stroma. The endometrial epithelium comprises the luminal epithelium, which faces the uterine lumen, and the glandular epithelium which invaginates into the endometrial stroma. The endometrium is a classic target tissue for ovarian steroids. It becomes a proliferative tissue under the action of oestrogens and, after ovulation, is changed by progesterone into a secretory tissue which accommodates ovum implantation. The cyclic morphological and functional differentiation of the whole endometrium under the effect of hormones represents the response of individual cell types within a complex signalling network.
Elements of chromatin structure: histones, nucleosomes, and fibres
Authors: Holde Kensal Van , Zlatanova Jordanka , Arents, Gina , Moudrianakis Evangelos
Published: November 2023

The subject of this book is chromatin, its structure, and how this structure functions in regulation of the eukaryotic genome. The biochemists’ operational definition of chromatin is probably the best we can do at present: chromatin is the DNA-protein complex extracted from lysed interphase nuclei. Just which of the multitudinous substances present in a nucleus will constitute a part of the extracted material will depend in part on the techniques each researcher uses. Furthermore, the composition and properties of chromatin vary from one cell type to another, during development of a specific cell type, and at different stages in the cell cycle. Nevertheless, we can provide at least a rough description of the features common to chromatin of most eukaryotic cells (1-3).
Metabolic Flux Analysis
Authors: Holms William H
Published: November 2023

Flux analysis is a method of organizing data which shows the quantitative relationships among all the metabolic events going on in a fermentation. It is a simple description of what is actually happening in the fermentation and does not, in itself, consider the theory of how fermentations are limited or controlled. However, once the analysis is complete, it defines the ballpark within which the game is actually being played and the interpretation of this can often suggest what might be the factors limiting the process engineer’s aspirations for his/hers defined goals. By definition, the biomass always does the best it can with what is available, unless human intervention has deliberately deranged its genetic machinery or put it into a deliberately misleading environment.
2-5A-Antisense Chimeras for Targeted Degradation of RNA
Authors: Silverman R H , Maran A , Maitra R K , Waller C F , Lesiak K , Khamnei S , Li G , Xiao W , Torrence P F
Published: November 2023

The 2-SA-antisense approach under development in our laboratories seeks to substantially enhance the efficiency of antisense oligodeoxyribonucleotides (oligos) in vivo by harnessing the activity of a ubiquitous intracellular endoribonuclease called 2-SA-dependent RNase (RNase L) (l-3). In this strategy, an activator of RNase L, the 2’,5’-linked tetraadenylate pA(2’p5’ Ah, is conjugated through linkers to antisense oligos. As a result, the cell’s ability to control RNA decay is reprogrammed to degrade RNA molecules of choice. We have used this strategy to demonstrate selective and catalytic destruction of target mRNA molecules in assays containing homogeneous RNase L, in crude cell-free systems and in intact human cells. Here we present our protocols to synthesize and analyse the activity of2-5A-antisense species for the ablation of target RNA molecules. Current work, to be described elsewhere, aims to further enhance the stability, activity. and mode of synthesis of2-5A-antisense.
Antisense Applications in Dictyostelium: A Lower Eukaryotic Model System
Authors: Hildebrandt Martin
Published: November 2023

The cellular slime mould Dictyostelium discoideum is a single-cell haploid amoeba, which, upon starvation, enters a well characterized developmental cycle. During this process, multicellular aggregates, also called pseudo plasmodia are generated, which later form a fruiting body and differentiate into a tube of non-viable stalk cells and a sorocarp of encysted spore cells. Since the many Dictyostelium proteins involved, for example, in signal transduction or cell movement are very similar to their mammalian homologucs, antisense RNA experiments in Dictyustelium are an easy and inexpensive model system to generate and study mutants lacking a protein of general interest.
Antisense Rescue
Authors: Holt Jeffrey T
Published: November 2023

Genetic manipulation of cells has provided a powerful approach to study gene function and the regulation of gene expression. Although genetic manipulation includes overexpression and ‘undcrexpression’ strategies, methods which inhibit gene function arc particularly important because they probe the usual function of a gene product within an appropriately expressing cell. Antibody injection (1), antisense methods (2-6). and gene targeting (7) can be used for gene inhibition; and each approach has its strengths and weaknesses.
Cell cycle control of DNA replication in Saccharomyces cerevisiae
Authors: Schwob Etienne , Nasmyth Kim
Published: November 2023

Living organisms grow and divide to produce genetically identical progeny. To divide continuously, a eukaryotic cell must duplicate all of its constituents including DNA, proteins, ribosomes, organelles, etc., and segregate them more or less equally to daughter cells. To perpetuate its genetic information, a cell must, how-ever, duplicate and segregate its chromosomes with high fidelity 10-6 mutation/bp and 10-5 chromosome loss/ cell division in yeast). An important question in cell biology is therefore how cells manage to replicate and segregate faithfully their chromo-somes? Furthermore, how is cell division regulated by cell growth and external conditions? This formidable task is achieved following the instructions of a programme, the cell cycle, which integrates the many biochemical events needed for chromosome duplication, transmission, and cell separation over time and space. For example, this programme ensures that DNA is replicated before sister chromatids begin to separate, or that sisters move to opposite poles.
Production of single-chain Fv monomers and multimers
Authors: FILPULA DA YID , Mcguire Jeffrey , Whitlow Marc
Published: November 2023

The Fv fragment (25 kDa) is a two-chain heterodimer of the antibody variable domains (VL and V8) and is regarded as the minimal structural component of an antibody required for antigen-binding activity. Fvs have been found to dis sociate into VL and V8 domains at low protein concentrations and under physiological conditions (1). This limited stability may be overcome by incor poration of a designed linker peptide to bridge isolated VL and V8 domains into a single polypeptide (2). Basic research on recombinant single-chain Fv (scFv) proteins produced from Escherichia coli has recently provided some answers to at least two basic questions about this new antibody technology.
Mapping chromatin structure in Drosophila
Authors: Swede Marci J , Wallrath Lori L , Elgin Sarah C R
Published: November 2023

Most of the DNA making up a eukaryotic genome is packaged into nucleosomes. Discontinuities in the nucleosome array are typically due to the binding of non-histone chromosomal proteins. These nucleosome-free regions (about 50–200 bp in length) were first identified as sites hypersensitive to digestion with DNase [ and other nucleases (reviewed in refs 1. 2). DNase I hypersensitive sites have been found 5’, 3’, and within introns of genes in organisms ranging from plants to humans (3–6). DNase I hypersensitive sites frequently map to locations of gene regulatory elements. Nucleosomes have been shown to block gene transcription when positioned over cis-acting regulatory elements in the promoter region of a variety of genes (reviewed in refs 7–10); this suggests that the formation of DNase I hypersensitive sites encompassing cis-acting elements might be a critical regulatory event providing accessibility for trans-acting regulatory factors. Consistent with this idea, the appearance of DNase I hypersensitive sites can be tissue specific and/or developmentally regulated (11–13).
Transcriptional and structural analysis of chromatin assembled in vitro
Authors: Pazin Michael J , Kadonaga James T
Published: November 2023

The biochemical analysis of the transcriptional properties of DNA packaged into chromatin, as it is in vivo, promises to increase our understanding of gene regulation (1–4). This chapter focuses on the assembly and transcription of chromatin by using two different extracts derived from Drosophila melanogaster embryos. We have also included techniques for the analysis of chromatin structure to verify the proper assembly of chromatin as well as to examine mechanisms of gene regulation. Hence, this chapter describes methods for the assembly of chromatin (Protocols 1-3), in vitro transcription of the reconstituted chromatin templates (Protocol 4), and analysis of chromatin structure and the binding of transcription factors to the nucleosomal templates (Protocols 5–7).
Getting started in molecular biology
Authors: Brown T A
Published: November 2023

In several respects molecular biology is not a user-friendly discipline. The new comer is faced with a bewildering array of techniques, some unhelpful jargon, and the impression that only the complicated experiments are worth doing. In fact, most of the apparent problems are illuso1y and a series of relatively straight forward experiments will allow any competent research worker to obtain in formation on the structure and mode of expression of a gene. This is true not only for scientists in established molecular biology laboratories but also for researchers from other areas wishing to apply DNA analysis techniques in their own projects.
Generation and identification of recombinant clones
Authors: Brown T A
Published: November 2023

The cells are plated on to a selective medium (or a series of media) that enables recombinant colonies (i.e. those that contain recombinant DNA molecules) to be identified. There are several different selection strategies; the appropri ate one depends on the type of vector that is being used {see also Chapter 9).

This chapter begins with methods for introducing recombinant DNA molecules into bacteria and then provides details for recombinant selection with the most important vectors. The protocols deal solely with the use of Escherichia coli as the host organism, E. coli being the host that is used for all basic gene cloning experiments.
The polymerase chain reaction
Authors: Brown T A
Published: November 2023

The polymerase chain reaction is a deceptively simple but remarkably versatile procedure that has applications in virtually all areas of molecular biology research. All that a PCR does is to make multiple copies of a target DNA sequence, the amplification process enabling microgram quantities of DNA to be obtained from a single starting molecule. Any segment of any DNA or RNA molecule can be amplified, provided that the sequences on either side of the targeted region are known. If genomic DNA is used as the starting material the PCR can be used in an analogous fashion to cloning, to provide a pure sample of a single gene or other DNA sequence, with the advantage that the gene is obtained after a few hours work, rather than the weeks needed to prepare and screen a genomic c. If RNA is the starting material then PCR can achieve the same objective as cDNA cloning, again much more rapidly. Only the limitation that some sequence information is needed in order to design appropriate primers for a PCR, plus the difficulty in obtaining PCR products longer than 5-10 kb long, has prevented PCR from completely replacing cloning as a means of gene isolation.
Functional analysis of DNA replication accessory proteins
Authors: Hübscher Ulrich , Mossi Romina , Ferrari Elena , Stucki Manuel , Jónsson Zophonías O
Published: November 2023

This chapter summarizes practical experiments that can be performed with DNA replication accessory proteins. The DNA, in preparation for DNA synthesis, has to become single-stranded to serve as a template for the replicative DNA polymerases (pols). For this a set of proteins have been identified that support the replicative and repair pols in performing processive, accurate, and rapid DNA synthesis. Furthermore these proteins prevent damage to the transient single-stranded DNA. The three best known are the proliferating cell nuclear antigen (PCNA), replication factor C (RF-C), and replication protein A (RP-A). DNA replication accessory proteins provide particular functions that are mandatory for pols. Such functions include the recruitment of particular pols when needed, the facilitation of pol binding to the primer terminus, the increase in pol processivity, the prevention of non-productive binding of the pol to single-stranded DNA, the release of the pol after DNA synthesis, and the bridging of pol interactions to other replication proteins. Thus it is not surprising that proteins carrying out such functions have been universally found in nature (reviewed in ref. 1). The eukaryotic auxiliary proteins RF-C and PCNA appear to be functional homologues of the bacteriophage T4 proteins gp44/62 and gp45, and subassemblies of the E. coli pol III holoenzyme -y-complex and β-subunit, respectively (reviewed in ref. 2).
Differenzierung von Sommer- und Wintergersten und Gerstensortenbestimmung mit der Polymerase Chain Reaction (PCR)
Authors: Schuhbeck T , Vogeser G , Geiger E
Published: November 2023

Many barley varieties, especially winter barleys, are of only limited suitability for brewing. This causes production problems with mixtures of varieties. Therefore batches of pure varieties, free of winter barley, are required. Distinguishing DNA by the Polymerase Chain Reaction (PCR) was investigated for the monitoring of varietal purity.
Nachweis von Fusarium graminearum und Fusarium culmorum unter Verwendung einer sektionsspezifischen Polymerase-kettenreaktion
Authors: Leiβner Christian E W , Niessen Martin L , Vogel Rudi F
Published: November 2023

Fusarium graminearum, F. culmorum, F. avenaceum, and Microdochium nivale are the most serious funga.l contaminants found in cereal grain. F. graminearum and F. culmorum may produce mycotoxins and have been found to be closely linked to the induction of gushing in beer. A pair of PCR primers was designed to amplify a 288 bp fragment from the DNA isolated from fusaria of the Discolor section. This PCR selectively detects F. graminearum and F. culmorum in sample material without being sensitive to other organisms present. The method described can be used in quality control in breweries and malt production.
Thermotolerant fungal glucanase in malting barley
Authors: Mannonen L , Ritala A , Nuutila A M , Kurten U , Aspegren K , Teeri T H , Aikasalo R , Kauppinen J Tammisola & V
Published: November 2023

Molecular breeding is becoming a useful breeding tool in improvement of plant raw material e.g. for more easy processing. The gene for fungal thermotolerant ß-glucanase was introduced into the malting barley variety Kymppi. The gene is regulated by barley e-amylase promoter and is therefore functional only during germination. First results indicate that the enzyme in transgenic seeds is active and thermotolerant. Its structure is different from the original protein but resembles the previously reported enzyme made by cultured barley cells. Further studies are currently underway. Risk assessment for cultivation of barley transformed with marker-gene was performed 1996 to give information of dispersal of transgenic pollen in field.
Evaluation de la qualité sanitaire de l’orge par le dosage de l’ergostérol
Authors: Clamagirand V , Boivin P
Published: November 2023

Les moisissures sont des micro-organismes infectant I’orge et pouvant entrainer des alterations qualitatives et sanitaires de celle-ci, particulterement par la syntitese de mycotoxines. Les rrtethodes couramment utilises pour estimer la contamination fongique sont le plus souvent non quantitatives et longues. L’ergosterol est un constituant spScifique des parois cellulaires des moisissures et son dosage sur orge a 6te effectu6 par HPLC-UV. Une relation entre la quantity d’ergosterol (de 3 ci 50 mg/kg) et celle de deoxynivalenol a 6te faite. Cette ntethode, tepStable et facile mettre en oeuvre, permet dtevaluer rapidement la qualite sanitaire de I’orge.
Image analysis as practical tool in barley-malt-beer chains
Authors: Laarhoven H P M van , Douma A C , Angelino S A G F
Published: November 2023

An automated image analysis system was evaluated for its potential in the rapid.assessment of purity and quality parameters of barley batches. The system performed purity analysis and thousand kernel weight determination successfully. For kernel size grading it was necessary to process data for each variety separately to obtain satisfactory results. Batch identification was also feasible for varietal indentity and region of growth. Elevator companies and maltsters may implement the system as a chain management tool for fast monitoring and improved control of their raw materials quality.
Hop-derived phytoestrogens in beer?
Authors: Keukeleire Denis De , Milligan Stuart R , Cooman Luc De , Heyerick Ame
Published: November 2023

Hops proved to be a rich source of phyto-oestrogens as established via a very sensitive and selective in vitro bioassay. Since hop-derived phyto-oestrogens may occur in beer, this could have significant health-related effects on consumers (e.g. protection against hormone-dependent cancers). The phyto-oestrogenic fraction of different hop varieties was obtained by selective solvent extraction, analysed by reversed phase gradient HPLC and identified via diode array UV and mass spectroscopic techniques. A procedure based on solid phase extraction of beer and subsequent HPLC analysis was developed to fingerprint beer phyto-oestrogens.
Expression systems
Authors: Grisshammer Reinhard , Kambach Christian , Tate Christopher G
Published: November 2023

The over-expression of DNA-binding proteins or their domains is essential for their purification, characterization and three-dimensional structure determination. Unfortunately, isolation of functional protein in quantities sufficient for crystallization trials or for NMR experiments is still not routine. In this chapter, we discuss two expression systems that have been used successfully to produce large amounts of soluble protein, namely Escherichia coli and the baculovirus expression system. Many proteins can be expressed in E. coli, although it is often necessary to optimize expression parameters to achieve successful production of a desired protein. Methionine-auxotroph strains allow the expression of seler10methioninyl protein which can be used for multi-wavelength anomalous diffraction techniques during structure determination. However, some proteins, such as the euka1yotic telomere binding protein, TRFI, cannot be produced in E coli for unknown reasons (T. de Lange and L. Fairall, personal communication). This protein was successfully expressed in insect cells (1), therefore, we will describe the baculovirus system in this chapter. Owing to space limitations, we will not consider yeast systems: the reader is referred to two excellent reviews containing detailed protocols (2, 3).
Gel electrophoresis and bending in cisplatin-modified linear DNA
Authors: Malinge Jean-Marc , Schwartz Annie , Leng Marc
Published: November 2023

Recent studies have shown that some natural DNA fragments are bent and that the bend can be deduced from the DNA electrophoretic mobility in polyacrylamide gels (1-4). Structural distortions are induced in DNA by the covalent binding of compounds such as mutagens, carcinogens or anti tumour drugs (5). The purpose of this chapter is to show the relations between electrophoretic mobility and some aspects of the structural distortions induced in linear DNA by the binding of the antitumour drug cisplatin (cis-diamminedichloroplatinum(ll)). This chapter comprises of four parts which deal, respectively, with: (i) gel electrophoresis in polyacrylamidegels; (ii) platination of oligonucleotides; (iii) ligation of the platinated oligonucleotides into multimers and their separation by gel electrophoresis;
Mapping histone positions in chromatin by protein-directed DNA crosslinking and cleavage
Authors: Travers Andrew
Published: November 2023

1 Introduction

The precise mapping of protein-binding sites on DNA can be facilitated by the covalent conjugation of an activatable reagent to a specific amino acid residue on the protein of interest. ln this procedure, the conjugated protein is first bound lo DNA and then the reagent is activated. Two general methods are currently used. The activated reagent can crosslink to DNA in van der Waals’ contact <1nd thus sensitize the backbone to alkaline hydrolysis in the immediate vicinity of the crosslink.
Quantitative DNase I kinetics footprinting
Authors: Mollah A K M M , Brenowitz Michael
Published: November 2023

Nuclease protection or ‘footprinting assays’ have been extensively applied to the study of nucleoic acid-ligand interactions and nucleic acid structure. The principal virtue of foot printing is that it can precisely localize individual sites of ligand binding or tertiary contact on nucleic acid molecules. Nucleic acid molecules hundreds or thousands of nucleotides long can be analysed. By choosing any of a number of chemical or enzymatic nucleases, changes in different aspects of the nucleic acid structure Gill be visualized. Quantitative footprinting protocols can also provide individual site-specific characterization of the thermodynamics and kinetics of reactions involving nucleic acids. These protocols have> been recently reviewed (1).
Molecular processing of DNA folding anoinalies in Escherichia coli
Authors: Leach David R F
Published: November 2023

For evolution to proceed, there must be a balance between genetic stability and rearrangement. Chromosomal arrangements that prove beneficial to an organism are expected to be maintained while retaining the potential to generate radically new organizations over an evolutionary time-scale. In order to maintain this balance, cells have evolved mechanisms to reduce the frequency of chromosomal rearrangements. An important threat to DNA stability comes from sequences that have the potential for unusual folding. Unusually folded DNAs also pose problems for DNA replication, which may be particularly critical for organisms where there is an evolutionary advantage associated with rapid cell duplication. In principle, these problems can be reduced by lowering the genomic content of foldable DNA, reducing its frequency of folding, unfolding it when it does fold, and destroying folded structures if all else fails. It should be remembered, in this context, that ‘random’ single-stranded DNA sequences will fold upon themselves, so the structures studied within non-random sequences may best be considered as extreme examples of what is possible. Studies of the effects on DNA instability of sequences with the potential to form unusual secondary structures in the bacterium Escherichia coli have revealed a number of interesting rearrangements, which may have general relevance to living cells. E. coli has also provided a simple experimental system where questions concerning the formation and processing of unusual secondary structures can be addressed in vivo. Finally, the detailed knowledge of E.coli genetics is being used to identify genes and proteins that process unusually folded DNA.
The ATM gene and stress response
Authors: Lavin Martin F , Khanna Kum Kum
Published: November 2023

Reactive oxygen intermediates (ROI) are generated as by-products of normal oxidative metabolism in the mitochondria and peroxisomes, and are associated with specific cell functions such as cell killing by cytotoxic T cells and natural killer (NK) cells (1, 2). While ROI can, in some cases, be utilized as part of a destructive mechanism (cell killing) there is increasing evidence that they also function as second messengers in signal transduction pathways (3, 4). Under normal conditions the potential damaging effects of the free radicals generated are neutralized by enzymes such as catalase, peroxidases, and superoxide dismutase (1). Perhaps more critical to cell survival are the damaging effects of ROI produced by exogenous agents such as ionizing radiation (5), hypoxia (6), and a variety of other compounds. ROI so produced can interact with cellular macromolecules in abstraction or addition reactions to generate additional radicals. These can either have direct effects, as in damage to DNA, or indirect effects, by initiating a cascade of reactions giving rise to membrane changes or damage (7).
Protein–protein interactions in papillomavirus replication
Authors: Mccance Dennis J
Published: November 2023

Certain members of the human papillomavirus genus are the etiological agents of lower genital tract cancers, in particular cervical cancer, and so an understanding of the protein-protein interactions involved in viral replication would be important if antiviral therapy were to become a reality. Viral DNA replication occurs in the upper half of stratified epithelium and this poses a problem for the virus. The viruses infect the basal epithelial cells and when these cells divide, one daughter cell moves up through the epithelium and undergoes terminal differentiation (Fig. 1A). This situation, if allowed to develop, will inhibit the ability of the virus to replicate, since differentiating cells will not contain the replicative machinery for DNA replication. Therefore papillomaviruses have acquired a strategy which involves stimulating cells into S-phase of the cell cycle where abundant replicative enzymes will be present. However, there is an important caveat to this scenario, that is, for viral DNA amplification to proceed, a certain level of keratinocyte differentiation is necessary for the switch from the early promoter to the late promoter and capsid protein production. Therefore the virus has the task of uncoupling S-phase progression from partial cell differentiation. In this chapter I will concentrate on what is known of the protein – protein interactions involved in the replication of HPV-16, but will refer to other HPV types in comparative terms or where little or nothing is known for this particular HPV type.
Molecular interactions in herpes simplex virus DNA replication
Authors: Stow Nigel D
Published: November 2023

The family Herpesviridae comprises a diverse and interesting group of viruses characterized by their distinctive virion morphology and the possession of large linear double-stranded DNA genomes which range in size from approximately 120-250 kb (for review see refs 1, 2). Many vertebrate species are natural hosts to herpesviruses including fish, amphibians, reptiles, birds, marsupials, and mammals, and eight viruses that infect man have been identified. The family has historically been sub-divided into the Alpha-, Beta-, and Gammaherpesvirinae subfamilies on the basis of bio-logical properties; a classification largely (but not completely) supported by our rapidly advancing understanding of the genetic relationships amongst these viruses based upon DNA sequencing studies. The prototype and most extensively studied herpesvirus is herpes simplex virus type 1 (HSV-1), a human pathogen belonging to the Alphaherpesvirinae which is most frequently responsible for cold-sore lesions around the lips and mouth. In common with most other herpesviruses, HSV-1 is able to enter a latent state in its natural host. The latent viral genomes reside in a quiescent form within neuronal cells of the sensory ganglia but a variety of stimuli can trigger sporadic reactivation of viral replication giving rise to the recurrent symptoms of disease.
Analysis of DNA virus proteins involved in neoplastic transformation
Authors: Hall Kersten T , Zajdel Maria E Blair , Blair G Eric
Published: November 2023

The small DNA tumours viruses, simian virus 40 (SV40), polyoma viruses, human adenoviruses (e.g. Ad2, Ad5, and Ad12), and human papillomaviruses (e.g. HPV16 and HPV18) encode at least one oncogene product responsible for transformation of cells in culture and formation of tumours in animals. These oncogene products modulate signal transduction and other cellular pathways to abrogate normal cell cycle control mechanisms, and this results in rapid cell proliferation. To achieve this, virus oncogene products frequently interact with cellular proteins such as pRb, p53, or c-Src. Here we give a broad overview of the properties of the major transforming proteins encoded by the small DNA tumour viruses, and their cellular targets. A more detailed treatment of these topics is given in articles in Fanning (1). In addition, a number of reviews give thorough background information on the topics of transforming proteins of human adenoviruses (2-5), SV40 (6), polyoma viruses (7), and human papillomaviruses (8, 9). A good general source book for information on oncogenes, including those of DNA viruses, is Hesketh (10).
Reverse genetics
Authors: Barstead Robert J
Published: November 2023

The nematode Caenorhabditis elegans has about 19000 genes. Approximately 2000 of these have been identified in forward genetic screens for animals with mutant phenotypes. The generation and analysis of such mutants has been the key to the success of the C. elegans model because the in vivo function of a gene can be inferred from its mutant phenotype. In principle, it is possible to identify mutations in the remaining 17000 genes using various forward genetic screens. In practice, however, it is unlikely that forward genetics will lead to such an end in the foreseeable future. Further, now that nearly all the sequence of C. elegans is known, many genetics projects run in reverse: where a gene is identified first through its sequence and then mutations are recovered that inactivate or alter its function. Several imperatives have driven the effort to develop strategies for the targeted inactivation of genes in C. elegans. One might expect that such strategies would include recombination mediated gene replacement, where a wild type copy of a gene is replaced with a mutant copy. Regrettably, recombination mediated gene replacement, though effective in many other systems, has not worked with C. elegans. However, this chapter will describe four strategies that have worked. First, I will describe a method for the epigenetic inactivation of genes using double stranded RNA from the target locus. Following this I will describe three strategies to generate germline mutations at specific target loci. Each of the latter involves a mutagenesis step followed by the selective identification of animals carrying mutations at the target locus.
One-bead one-compound combinatorial library method
Authors: Liu Gang , Lam Kit S
Published: November 2023

The ‘one-bead one-compound’ combinatorial library was prepared by a ‘split synthesis’ method (1-3, see also Chapter 1) such that each solid phase bead displays only one chemical entity although there are approximately 1013 copies of the same compound on a 100 μm bead. This bead-supported library is then assayed for a specific biological activity using on-bead binding (1), or functional assays (4). Alternatively, the compound on each bead is released from the solid support via a cleavable linker for subsequent solution phase assays (5). In either solid phase or solution phase assays, the positive beads are then identified, physically isolated, and subjected to structural determination. If the compound on the bead is a peptide, each individual positive bead is inserted into a cartridge of an automatic protein sequencer for sequence analysis. Alternatively, the peptide or non-peptide compound on each bead is released chemically or photochemically and the releasate is analysed by mass spectrometry. If the structure of the compound cannot be easily determined by mass spectrometry, one can use chemical encoding (6-8) or radiofrequency tagging (9, Chapter 4) strategies.
Combinatorial library synthesis using polymer-supported catalysts
Authors: Kobayashi Shu
Published: November 2023

Combinatorial synthesis (Chapters 1-7), a synthetic strategy which leads to large numbers of structurally distinct molecules, in association with high-throughput screening techniques is making a significant impact not only on the field of drug discovery (1, 2) but also on the development materials sciences and catalysis (Chapters 14, 16).Owing to the availability of a myriad of high-yielding and highly selective reactions, complex and structurally diverse natural and synthetic compounds can be prepared by traditional organic synthesis strategies. However, these reactions do not necessarily provide useful methods for the synthesis of large chemical libraries where each member must be obtained in high yield and selectivity. Thus, we are facing the challenge of developing new reliable synthetic methodologies to prepare these libraries, just as new methodologies for natural product synthesis were required forty years ago (3).
Combinatorial approaches to chiral catalyst discovery
Authors: Snapper Marc L , Hoveyda Amir H
Published: November 2023

In most combinatorial approaches to drug discovery, lead compounds are generally identified from large, structurally diverse libraries (1). Preliminary findings are then often optimized through the subsequent design and examination of more limited libraries that focus and expand on the initial results. In a similar manner, this layered approach to drug discovery and optimization can be adapted to catalyst development (Scheme 1, see also Chapters 14 and 15). In the first phase, a wide range of catalyst candidates can be screened to select specific complexes that effect a reaction of interest. Once a catalyst has been identified that demonstrates the desired reactivity, efforts can then tum toward optimizing the conditions and system to yield the desired selectivity. This two-tiered development strategy can offer distinct advantages in the discovery and identification of catalysts for asymmetric reactions. If the catalyst discovery and optimization protocols are sufficiently rapid and reliable, there is no prerequisite for finding general solutions to catalytic reactions; each reaction can enjoy a catalyst that is designed specifically for the substrate and transformation of interest.
The use of RT-PCR differential display in single-celled organisms and plant tissues
Authors: Nuccio Michael L , Hsieh, Tzung-Fu , Thomas Terry L
Published: November 2023

Differential display of eukaryotic mRNA is a fast and powerful PCR-based technique developed to compare relative variation in gene expression between RNA populations (1, 2). This technique provides a way to monitor differentially expressed genes across various tissues, developmental stages, or physiological states by systematically amplifying cDNA derived from mRNA populations and displaying them on a polyacrylamide gel. The bands that are identified as potential targets of interest can subsequently be isolated, reamplified, and cloned for further verification. Differential display is flexible in two ways. First, it is unnecessary to begin experiments with a poly(A) enriched RNA fraction; they can be performed with as little as 1 µ,g of total RNA per sample. Secondly, many comparisons between samples can be made using various primer combinations in the assay.
Use of suppression subtractive hybridization strategy to identify differential gene expression in brain ischaemic tolerance
Authors: Wang Xinkang , Feuerstein Giora Z
Published: November 2023

Differential gene expression has important functional implications in normal cell growth, differentiation, and in pathophysiological conditions. Ischaemic pre-conditioning (PC) is a phenomenon produced by a short duration of ischae mia that results in a subsequent resistance to severe ischaemic tissue injury (i.e. ischaemic tolerance). This phenomenon has been described in heart and brain (1-3). To identify genes that are specifically regulated in PC, we applied a tech nique using a PCR-based cDNA subtractive strategy termedsuppression subtractive hybridization(SSH) (4).
Construction and applications of gene microarrays on nylon membranes
Authors: Mooney Jeffrey L , Kayne Paul S , O‘brien Shawn P , Debouck Christine M
Published: November 2023

The rapid advancement of expressed sequence tag (EST) and genomic sequencing has been paralleled by the need to develop methodologies for the functional characterization of newly identified genes. Since the expression pattern of a gene provides direct or indirect information about its function, a number of new approaches have been developed to determine the normal tissue distribution of large numbers of genes and/or to identify genes that are differentially expressed between two or more given tissues or cell types. Some of these methods are designed for thede novaisolation of genes expressed at different levels in compared tissues or cell types, e.g. differential display (DD) (1), and related methods such as RNA fingerprinting by arbitrary primer PCR (RAP-PCR) (2), also representation difference analysis (RDA) (3), and suppressive subtractive hybridization (SSH) (4). The other methods exploit high-throughput sequencing and sequence databases either to estimate transcript abundance, e.g. EST numerical analysis (5) and serial analysis of gene expression (SAGE) (6), or to construct DNA microarrays of thousands of gene sequences derived from the databases, e.g. oligonucleotide arrays (7) or gene fragment arrays (8). Individual laboratories need to balance the pros and cons of each technique, including labourintensiveness and the need for expensive equipment, and decide which of these methodologies is best suited to study their particular biological problem.
Enzymes involved in the synthesis of PtdIns(4,5)P2 and their regulation: PtdIns kinases and PtdInsP kinases
Authors: Tolias Kimberley F , Carpenter Christopher L
Published: November 2023

Phosphatidylinositol 4,5-bisphosphate (PtdIns(4,5)P2) is a glycerol-phospholipid found predominantly in the inner leaflet of eukaryotic plasma membranes (Fig. 1). Although it constitutes less than 0.05% of total cellular phospholipids, PtdIns(4,5)P2 plays a critical role in intracellular signalling. PtdIns(4,5)P2 is best known for its ability to serve as a precursor for the second messengers inositol 1,4,5-trisphosphate (Ins(1,4,5)P3) and diacylglycerol (DAG). Ins(1,4,5)P3 and DAG are generated from the hydrolysis of PtdIns(4,5)P2 by phospholipases C (PLCs) following agonist stimulation (1, 2). Production of Ins(1,4,5)P3 triggers a transient rise in intracellular calcium (Ca2+), while the generation of DAG activates protein kinase C (PKC) family members.
Regulation of chloride channel conductance by Ins(3,4,5,6)P4; a phosphoinositide-initiated signalling pathway that acts downstream of Ins(1,4,5)P3
Authors: Ho Melisa W Y , Carew Mark A , Yang Xiaonian , Shears Stephen B
Published: November 2023

The Ins(1,4,5)P3-mediated mobilization of cellular Ca2+stores is a well-known consequence of stimulus-dependent PtdIns(4,5)P2turnover (1). Thus, reviews on the actions and metabolism of Ins(1,4,5)P3, together with reviews on the Ca2+channels that are gated by Ins(1,4,5)P3, often feature prominently in multichapter publications that are devoted to ‘phosphoinositide’ biology. However, much less attention has typically been given to a receptor-dependent metabolic event that occurs downstream of Ins(1,4,5)P3, namely, the Ins(1,3,4)P3-regulated Ins(3,4,5,6)P41-kinase/ Ins(1,3,4,5,6)P51-phosphatase metabolic cycle (Fig. 1). The interconversion of Ins(1,3,4,5,6)P5with Ins(3,4,5,6)P4is significant because Ins(3,4,5,6)P4is a cellular signal that inhibits the ionic conductance through Ca2+-activated Cl–channels in the plasma membrane (2). These ion channels contribute to the regulation of salt and fluid secretion, and may also participate in osmoregulation, pH balance, and control of smooth muscle excitability (3–6). Thus, Ins(3,4,5,6)P4may regulate several important physiological processes. The inclusion of a review of this topic in a volume devoted to phosphoinositides provides a timely opportunity to draw more attention to this signalling process.
Gene expression analysis via cDNA microarrays of laser capture microdissected cells from fixed tissue
Authors: Salunga Ranelle c , Guo Hongqing
Published: November 2023

The enormous number of DNA and mRNA sequences available to researchers, coupled with cDNA microarray technology, now allows for gene expression to be studied on a massive scale. Using microarrays, thousands of genes from organisms such as S. cerevisiae (1, 2) and A. thaliana (3), as well as those from mammalian species (4), have been analysed. However, most methods that have been described to date have relied on acquiring microgram quantities of messenger RNA or poly(A) RNA from pure populations of cells, in order to synthesize cDNA probes (1-6).
Expression data and the bioinformatics challenges
Authors: Heller Michael J , Tu Eugene , Holmsen Anita
Published: November 2023

First, and foremost, although every gene is present on its respective chromosome in every cell of an organism, it is not necessarily being transcribed into an active gene product. In fact, the current assumption is that many of the genes are silent or expressed at very low levels in certain cell types. In other words, the number of instances of many genes’ mRNA is, presumably zero. So, although we might know exactly what function a gene might code for-its capability or use value-in practice, it might have no utility at all in a particular cell under certain conditions.
Threading methods for protein structure prediction
Authors: Jones David , Hadley Caroline
Published: November 2023

As the attempts to sequence entire genomes increases the number of protein sequences by a factor of two each year, the gap between sequence and structural information stored in public databases is growing rapidly. In stark contrast to sequencing techniques, experimental methods for structure determination are time-consuming, and limited in their application, and therefore will not be able to keep pace with the flood of newly characterized gene products. The develop ment of practical methods for predicting protein structure from sequence is therefore of considerable importance in the field of biology.
Protein family-based methods for homology detection and analysis
Authors: Henikoff Steven , Henikoff Jorja
Published: November 2023

Most methods for homology detection have traditionally relied upon pairwise comparisons of protein sequences, and in recent years, several improvements in pairwise methods have been introduced (see Chapter 8). But, with sequence data becoming available at an accelerating rate, there is an increasing opportunity to use multiple related sequences for improved homology detection. Even when functional information is lacking for known members of a protein family, these members can be aligned and the alignments used in searches. Protein multiple alignments have been shown to improve performance of secondary structure prediction methods by identifying constraints on positions (1, 2), and so it seems reasonable to expect that improvements will be likewise obtained by using multiple alignments for homology detection and analysis. In this chapter were view some of the numerous methods that are aimed at achievement of this goal.
Epithelial stem cell identification, isolation and culture
Authors: Hudson David
Published: November 2023

This chapter will discuss the ways in which the identification and characterization of stem cells from various epithelial tissues can be approached. Specific protocols are given for techniques that have been shown to be effective in studies involving one of the best-characterized epithelial stem cell models, epidermal keratinocytes. First, methods involved in the separation of an epithelial cell type from other cells will be examined, followed by ways in which the proliferative capacity of such a cell type can be assessed. Secondly, methods used for the maintenance of primary stem cells in culture and ways of characterizing stern cells using immunocytochernistry will be described. Details of Basic Cell Culture have been described in another edition of The Practical Approach series (1).
High performance liquid chromatographic assays
Authors: Syed Shabih E H
Published: November 2023

Liquid chromatography is a separation process in which a mixture is separated into its individual components followed by their detection with a suitable monitor. Optimization of the resolution of the separated components, as well as speed of separation, has been a major interest of many researchers for a very long time. It is well known that the above parameters are directly affected by the size and nature of stationary-phase particles. In conventional liquid chromatography, where gravity was usually used to pull the solvent or mobile phase through a column packed with a stationary phase, a lower limit on the size of particles was eventually reached beyond which flow under gravity completely diminished.
Electrochemical assays: the pH-stat
Authors: Brocklehurst Keith
Published: November 2023

pH-stat assays are used to monitor the progress of chemical reactions in which protons are liberated or taken up. This is achieved by measuring the quantity of base or acid that needs to be added at various times to keep the pH essentially constant. The technique was developed in 1923 by Knaffle-Lenz (1) in connection with his work on esterases. It has since been applied widely in many biochemical systems (see ref. 2 for early examples and refs. 3 – 5 for more recent examples). In a more general sense, ‘stat’ techniques are used to monitor an even greater variety of reactions in which a chemical species that is either generated or consumed can be detected by using electrodes. Reactions involving metal ions can be monitored by using ion-selective electrodes and reactions involving redox changes by using platinum electrodes.
Plant cell biology
Authors: Ceriotti Aldo , Vitale Alessandro , Paris Nadine , Frigerio Lorenzo , Neuhaus Jean-Marc , Hillmer Stefan , Robinson David G
Published: November 2023

The ability to express heterologous proteins in plant cells, and to study the subcellular localization of heterologous and endogenous polypeptides is central to many studies in the field of plant cell biology. Heterologous expression and metabolic labelling are, together, two powerful tools to study intracellular transport and protein-protein interactions. However, it is often the combination of biochemical and immunohistochemical approaches that gives the more comprehensive view of what is really happening within the cell. Accordingly, the first part of this chapter will present a protocol for transient expression in tobacco protoplasts, while two other sections will be devoted to immuno labelling for confocal microscopy observation and immunogold electron micro scopy, respectively.
Protein purification
Authors: Slade Susan E , Dalton Howard
Published: November 2023

This chapter outlines the main features and requirements for the successful purification of a target protein from either its native or recombinant host. For each of the commonly used separation techniques an experimental protocol is described. The aim of purification is to obtain pure target protein in a structurally or functionally active form from a crude lysate. In many cases, pure protein is required only so its gene can be isolated using amino acid sequence information, in which case only a small quantity of protein may be required. Therefore, it is important to establish the purity and quantity of protein required from the procedure before embarking on any purification method. Often this decision is determined by the availability of raw material and the concentration of target protein within the host cell or culture supernatant. The increasing use of mass spectrometry to determine molecular mass, subunit composition, amino acid sequence, and establish the nature of protein-ligand interactions, requires a high level of target protein purity