bluuebunny/crossref_metadata_2025 huggingface dataset:
How the Protein Folds in the Cell
Authors: Hlodan Roman , Hartl F Ulrich
Published: November 2023

In order to fold in the cell, the newly synthesized polypeptide chain has to negotiate an environment far more complex than that faced by the unfolded chain in vitro. First, the cellular concentration of nascent chains can be quite high. In E. coli for example, the concentration of ribosomes is 50 micromolar (1) and local concentrations of nascent chains may well be even higher. Most proteins aggregate during refolding in vitro at much lower concentrations than those likely to be found in vivo. Secondly, unlike the test-tube which provides a single compartment, living cells are subdivided into several subcompartments. For example, many proteins synthesized in the eukaryotic cell have to be transported across the membranes of organelles such as mitochondria or the endoplasmic reticulum, and are only allowed to fold once they have reached their final destination. As the protein folds in cellulo, it may also be subject to various processing events, important both for its function and in determining its extra- or intracellular location.
| Topological selectivity in site-specific recombination
Authors: Marshall Stark W , Boocock Martin R
Published: November 2023

In conservative site-specific recombination, DNA molecules are broken in both strands at two separate predetermined points, and the ends are rejoined to new partners, without any synthesis or degradation of the DNA (Fig. la). The reactions are catalysed by specialized recombinase proteins and may involve other protein accessory factors. Many site-specific recombination systems have been identified in prokaryotes and eukaryotes, and several systems have been reconstituted in vitro. In studying these systems, one of the main aims has been to attempt to understand the mechanisms by which selectivity for recombination between sites in particular relationships is achieved. It has become apparent that DNA topology plays a crucial role in this selectivity. In vivo, site-specific recombination has a variety of functions, including switching of gene expression, control of plasmid copy number, resolution of plasmid multimers to monomers, bacteriophage integration and excision, and transposon co-integrate resolution.
Applications of chromosomal in situ hybridization
Authors: Levy Nicolas , Mattei Marie-GeneviÈve
Published: November 2023

In situ hybridization (ISH) is a technique which allows one to detect the sequences complementary to a given nucleic acid molecule in metaphase or interphase chromosomes, and in tissue specimens. This chapter will describe the applications of ISH to metaphase and interphase chromosomes. The application of ISH to tissues, to monitor the expression of specific genes, is covered in Chapter 8. The principle of ISH is simple, and is based on the complementarity of the two strands of the DNA molecule. The probe, a DNA or RNA sequence, is labelled with a radioisotope or chemical reporter group, and denatured. Target DNA, i.e. metaphase or interphase chromosomal DNA, is also denatured so as to be accessible to the probe. Complementary sequences in the probe and target are then allowed to hybridize. Depending on the type of probe used, the hybridized sites are then detected either by autoradiography or by using an immunofluorescence procedure.
Analysis of gene expression by PCR: a quantitative approach
Authors: Striker Liliane J , Peten Emmanuel P , Striker Gary E
Published: November 2023

The introduction of PCR has added a new degree of sensitivity and specificity in the study of gene expression in tissues or cells (1). It is now possible to obtain accurate and sensitive measures of gene expression in small samples of tissue, or in a few cells in vitro, using a variation of PCR which allows quantification in the attomolar range. This represents a considerable increase in sensitivity over other analytical methods such as Northern blot analysis or nuclease protection assays. In addition, it is possible to assay gene expression by PCR in tissue samples, without resorting to extraction of the mRNA.
Optimizing PCR
Authors: Kjijo K K , Ruano G
Published: November 2023

Although this entire volume is intended to provide explicit, practical guidelines and protocols for PCR in its myriad manifestations, we wish to start with a series of perspectives on various aspects of the several processes that are collectively called the polymerase chain reaction. No individual protocol will be optimal for all PCR reactions, nor will any single, simple set of variables to be optimized necessarily produce a functioning protocol for a specific case. Therefore, in order to optimize PCR, the researcher must have a mental picture of the underlying processes and be able to grasp how the various aspects of a protocol affect the chemical reactions. The relevance of most of the underlying physico-chemical principles can be understood mechanistically to allow a researcher to optimize a PCR reaction for a specific application. In this chapter, we present some of these principles and our perspective on the process without resorting to precise physico-chemical characterizations and quantification. If such a detailed treatment is required, it can be found in Ruano et al. (1).
Fingerprinting of DNA and RNA using arbitrarily primed PCR
Authors: Welsh John , Perucho Manuel , Peinado Miguel , Ralph David , Mcclelland Michael
Published: November 2023

Arbitrarily primed PCR is a modification of PCR that provides an informationrich fingerprint of genomic DNA. This method is based on the selective amplification of genomic sequences that, by chance, are flanked by adequate matches to an arbitrarily chosen primer. If two template genomic DNA sequences are different, their arbitrarily primed PCR products display different banding patterns. Such differences can be exploited in ways largely analogous to the uses of restriction fragment length polymorphisms, including genetic mapping, taxonomy, phylogenetics, and the detection of mutations.
Measurement of neurotransmitters and their associa.ted second messengers in vivo
Authors: Cadogan A K , Marsden C A
Published: November 2023

Measurement of neurotransmitters in vivo Neurotransmitter release has been measured in vivo over the last decades using a variety of techniques. Among the many innovations that have appeared are the cortical cup which was introduced in 1953 (1) and the push-pull cannula introduced in 1961 (2). One of the main drawbacks with the above methods was that they involved an open system in which the brain came into direct contact with the perfusion fluid. One method which overcame this problem was the introduction of brain microdialysis (also known as intracerebral dialysis, intracranial dialysis, and transstriatal/cortical dialysis). An alternative approach is the use of in vivo voltammetric electrodes which can also be successfully applied to brain slices forming an alternative to con ventional superfusion methods. The preparation and application of voltam metric electrodes have recently been reviewed in another book in this series (3). Microdialysis is in principle the only technique which can collect virtually any substance from remote brain regions with a limited amount of tissue trauma. Described as early as 1966 by Bito and colleagues (4), this method has been refined and undergone several technical improvements (5-7) to rapidly become a popular bioanalytical sampling tool in brain research. The major advantage of the in vivo microdialysis method is that it can be used in the freely-moving animal allowing one to make correlations between changes in extracellular neurotransmitter levels and behaviour.
Virulence plasmids
Authors: Tolmasky Marcelo E , Actis Luis A , Crosa Jorge H
Published: November 2023

The term plasmid was originally used by Lederberg (1) to describe all extra chromosomal hereditary determinants. Currently, the term is restricted to autonomously replicating extrachromosomal DNA. Their sizes range from 1 kb to more than 200 kb (2), and even larger plasmids were detected in Rhizobium (3). Although they replicate autonomously, plasmids rely on host encoded factors for their replication. Plasmids are not essential for the survival of bacteria, but they may nevertheless encode a wide variety of genetic determinants which permit their bacterial hosts to survive better in an adverse environment or to compete better with other micro-organisms occupying the same ecological niche. The medical importance of plasmids that encode for antibiotic resistance, as well as specific virulence traits has been well documented. In this chapter we describe techniques used in our laboratory for studying plasmid-encoded virulence factors such as the iron-uptake systems encoded by pJM1 in Vibrio anguillarum (4), and by pColV-K30 in Escherichia coli (5), as well as antibiotic resistance coded for by plasmids isolated from pathogenic Klebsiella pneumoniae strains (6).
Plasmids in Agrobacterium tumefaciens and Rhodococcus fascians
Authors: Desomer Jan , Marc Rolf Deblaere , Montagu Van
Published: November 2023

In this chapter, we would like to provide an overview of the main techniques used in our laboratory to identify, isolate, and analyse plasmids (and the functions they encode) in plant-tumourigenic bacteria from the Gram negative genus Agrobacteriumand from the Gram-positive genus Rhodococcus. Although these bacteria are evolutionary far apart, in both cases phytopatho genicity is determined by large conjugative plasmids, and similar genes have been detected on plasmids from both groups of bacteria (1). The plasmid based recombinant DNA technology developed for Agrobacterium can also be applied to other bacteria that interact with plants, such as the nitrogen fixing rhizobacteria (2).
Structure and replication of chloroplast DNA
Authors: Sugiura Masahiro
Published: November 2023

The demonstration of a unique DNA species in chloroplasts over 30 years ago (e.g. Ref. 1) has led to studies of the structure of chloroplast DNA (ctDNA). These studies were accelerated by development of techniques such as gene cloning and DNA sequencing in the mid-1970s. The first physical map of ctDNA was constructed for maize in 1976 (2). Within 10 years the entire sequences of ctDNAs were determined for tobacco, liverwort, and then rice (3-5). Sequences for defined regions of many other ctDNAs were also completed. These sequencing efforts have revealed an enormous amount of information about the chloroplast genome (6, 7). The genes and their organization in ctDNA were thought to be well conserved through land plant evolution (8). Each ctDNA was shown to contain nearly 100 different genes which could be classified into two main classes: genes involved in transcription and translation and those related to photosynthesis (9). Recent structural analysis of ctDNAs from a variety of other land plants and algae has modified previous understanding. Chloroplast genomes among the plant kingdom are much more diversified in gene content and gene organization than we thought. A non-photosynthetic parasitic flowering plant has been shown to contain a grossly reduced plastid genome that has lost both photosynthetic and chlororespiratory genes (10). Black pine, a gymnosperm, lacks all chlororespiratory genes (T. Wakasugi et al., in preparation).
Transcriptional and post-transcriptional gene regulation in plastids
Authors: Abrahamson Susan , Gruissem Wilhelm
Published: November 2023

Plastids perform several essential functions in plant cells, from photosynthetic carbon fixation to amino acid biosynthesis and fatty acid production. Since the discovery almost 40 years ago that plastids contain DNA, much of the molecular research in chloroplasts has focused on the structure and organization of the plastid genome structure and the expression of its approximately 150 genes (Chapter 3). This chapter will discuss our current understanding of the mechanisms that control gene expression in this unique organelle, and many aspects will focus on the photosynthetic chloroplast as an example. We will begin with a brief introduction to the biology of plastids and a historical overview of the study of plastid transcription. We will then discuss the components of the transcription machinery, the role transcription has in plastid gene expression, and the different types of contrgls that operate at the post-transcriptional level in the regulation of plastid gene expression during plant development.
Characterisation of industrial high methoxyl pectins
Authors: Kravtchenk T P , Voragen A G J , Pilnik W
Published: November 2023

Pectic substances are extracted industrially and are widely used in the food industry, mainly as a gelling agent. Food technologists are therefore primarily interested in their physical behaviour. Together with rheological measurements, the fine structure of extracted pectins has been intensively investigated during the last decades. These studies clearly showed that the strength of pectin gels is affected by numerous structural features such as molecular size and degree of methoxylation. However, the complexity of industrial pectin samples has prevented the attainment of the ultimate stage that would be the prediction of the physical behaviour from the chemical analysis.
Short chain fatty acids and metabolic effects in humans
Authors: Bornet F R J , Alamowitch C , Slama G
Published: November 2023

Short chain fatty acids are the major end products of dietary fiber degradation by anaerobic bacteria in the large bowel. The principal short chain fatty acids are acetate, propionate and butyrate. They are rapidly absorbed, stimulate salt and water absorption and provide an energy source for the colonic epithelium. Butyrate is mostly utilized as an energetic substrate by the colonocytes. It has been suggested that short chain fatty acids could contribute to the lipidemic and glycemic effects of fiber. In isolated hepatocytes, acetate inhibits glycolysis and stimulates gluconeogenesis while propionate has the opposite effects. Propionate inhibits cholesterol synthesis in vitro. They are very few studies in healthy humans. The acute intake of acetate does not seem to affect metabolism in man. No mention is made of its influence in case of chronic intake. The studies reveal the propionate tendency to reduce glycemia in fasting subjects and to enhance their sensitivity to insulin. The real part played by the short chain fatty acids in the glucose metabolism causes controversy and further investigations into their chronic intake in man are required.
Physical influences on the growth of food-poisoning bacteria in emulsion systems
Authors: Brocklehurst Tim F , Coleman Heather P , Gunning Paul A , Parker Maryl. , Robins Margaret M
Published: November 2023

In order to emulate the growth of food-poisoning bacteria in foods of a given chemical composition, microbiologists have traditionally used homogeneous culture media modified to have a similar chemical environment to the food in question. Hitherto, much less attention has been given to the influence of the physical structure of a food on the growth of bacteria. Experiments have shown that the microstructure of such colloidal systems can inhibit the growth of bacteria. Growth rates and microscopic observations of the growth of food-borne pathogenic bacteria in oil-in-water emulsions are reported and discussed with reference to physical characteristics of the emulsions.
European Brewery Convention
Authors: Narzillß L
Published: November 2023

Europe's illustrious Brewery Convention--held every two years--draws together brewers from around the world to exchange ideas on all aspects of the science and technology related to their craft. The papers presented here reflect this wealth of research and knowledge. They cover all stages of the brewing process, form raw materials and fermentation techniques to quality control and new innovations like low-alcohol beer. Each paper is presented in the original language with accompanying summaries in English, French, and German. The volume provides a valuable, state-of-the-art reference for all those involved in the brewing industry and fermentation biotechnology.
The economics and utilisation of brewing materials in the 1990’s
Authors: Brookes Peter A
Published: November 2023

Considerable improvement in the quality and efficient use of brewing materials has occurred over the past 40 years. Development work has been driven by the desire to more fully understand the mechanisms involved in the interactions taking place in malting, mashing, wort boiling and fermentation. The market place in the early 1990’s, however, is affected by recession, environmental concerns and a changing pattern of demand for alcoholic drinks. An analysis will be made of the most effective way of using cereals, sugar and hop resins to produce beer of the highest quality. The brewer who uses these materials with greatest efficiency will prosper.
The regulation of starch granule size in endosperm of developing barley grains
Authors: Tillett Ian J L , Bryce James H
Published: November 2023

A significant proportion of small “B-type” starch granules from barley is not degraded during malting and mashing. Our aim is to understand starch granule synthesis so that carbohydrate can be redirected to the large degradable “A-type” granules. Following digestion to remove cell walls and proteins from individual endosperms, the size and number of starch granules, and the wave pattern of their growth during grain development were determined using Image Analysis. SDS-PAGE and Western blotting with antibodies to starch synthases showed that enzymes of starch synthesis were specifically associated with both types of starch granules.
A simple and rapid test for gushing tendency in brewing materials
Authors: Vaag Pia , Riis Preben , Knudsen Anne-Dorte , Pedersen Stig , Meiling Ella
Published: November 2023

Gushing in beer may be caused by microbial components present in the brewing materials. Tests for gushing tendency are indispensable, but often laborious and time-consuming. A simple and rapid test has been developed, based on extraction of gushing factors from brewing materials, addition of the extract to bottles of non-gushing finished beer, followed by the gushing test. The test can be completed within four days and is more sensitive and more reproducible than a twelve-day mini-brew test used at present. Moreover, the extraction test can be used for determination of gushing tendency in adjuncts as well as in malt.
Lactic starter cultures in malting-a novel solution to gushing problems
Authors: Haikara A , Uljas H , Suurnäkki A
Published: November 2023

Lactic acid bacteria were tested as starter cultures during malting as natural preservatives for restricting the growth of Fusarium moulds in order to prevent gushing. Addition of Lactobacillus plantarum and Pediococcus pentosaceus cultures during malting diminished Fusarium and bacterial contamination of barley and malt due to the antimicrobial substances produced by these lactobacilli.
Yeast flocculation: biochemical characterization of yeast cell wall components
Authors: Garsoux G , Haubursin H , Bilbault S , Dufour J P
Published: November 2023

Comparison of flocculating and non-flocculating cells was performed with one brewery yeast strain. Cells cultivated at 10°C exhibited floes during the late exponential phase. However, cells grown at 28°C did never flocculate. The growth temperature did not inhibit cell-cell interactions but probably induced or repressed the synthesis of a cell wall component involved in flocculation. Surface analysis (hydrophobicity, electrostatic charge, chemical composition) and biochemical characterization of cell wall mannoproteins were carried out to identify the origin of this differential behaviour
HIV infection of macrophages
Authors: Collin Matthew , Montaner Luis J , Georges Herbin , Gordon Saimon
Published: November 2023

The susceptibility of mononuclear phagocytes to infection by HIV has long been recognized. The principal aims of recent research have been first, to describe the frequency and distribution of infected macrophages (m<|>) in the body and of the viruses that infect them and secondly, to understand the replicative cycle of HIV in m, and document the effects of HIV on m<|> function. The first area relies heavily upon histological studies and primary isolation of viruses, whereas the second, which is the main focus of this chapter, depends largely on infecting cells in vitro with established isolates of HIV. We describe the isolation of monocytes and their maturation in vitro, followed by methods to prepare and titrate the infectivity of m<|)-tropic HIV. The final section deals with general considerations for HIV experiments with m.
Detection of viral DNA by the polymerase chain reaction (PCR)
Authors: Krogstad Paul , Zack Jerome A
Published: November 2023

The polymerase chain reaction (PCR) has revolutionized the study of human retroviruses. Numerous methods have been employed to detect and analyse products resulting from amplification of retroviral DNA. These have included ethidium bromide staining of electrophoretically separated products, and hybridization of radiolabelled or chemiluminescent probes to dot blot DNA samples or Southern blot transfers (1). We and our collaborators have developed and employed a PCR method in which one of the oligonucleotide PCR primers is radiolabelled, allowing direct detection of electrophoretically separated products. We have used this method to quantitatively analyse DNA samples from experiments in which cells are infected in vitro, and for analysis of brain tissue, peripheral blood cells, and other tissues infected in vivo by human immunodeficiency virus type 1 (HIV-1) and human T cell leukaemia virus types I (HTLV-I) and II (HTLV-II) (2–5). In the sections that follow, we describe preparation of DNA samples and their analysis, using this PCR method.
Identification, isolation, and characterization of platelet glycoproteins mediating platelet adhesion
Authors: Mcgregor John L , Gayet Odile , Mercier Nathalie , Mcgregor Lilian , Chignier Elza
Published: November 2023

Platelets play a vital role in haemostasis by the formation of a plug or thrombus at a site of vessel wall injury. The function of these anucleated cells is largely dependent on the presence of a number of glycoproteins. Moreover, adhesion and aggregation of platelets to disrupted atheroma plaque, leading in some cases to vessel occlusion, is also mediated by glycoproteins. The importance of glycoproteins in platelet functions has led to extensive work, using various techniques, to identify structural/functional sites on these glycoproteins implicated in interacting with various ligands. The aim of this chapter is to describe a number of techniques used in studying glycoproteins involved in platelet adhesion to extracellular matrix proteins or to leucocytes.
Methods for measuring agonist-induced phospholipid metabolism in intact human platelets
Authors: Holub Bruce J , Watson Steve P
Published: November 2023

Stimulation of platelets by agonists causes marked alterations in phospholipid metabolism (1,2). The activation of phospholipase C results in the degradation of phosphoinositides, noteably phosphatidylinositol 4,5-bisphosphate (PI 4,5-P2), resulting in the production of the second messengers diacylglycerol (DG) and inositol 1,4,5-trisphosphate (Ins l,4,5-P3) (3,4). DG is further metabolized to phosphatidic acid (PA) via DG kinase. In addition to PI 4,5-P2 hydrolysis via phospholipase C with agonist stimulation, phosphatidylinositol (PI) is sequentially phosphorylated upon receptor occupancy to phosphatidylinositol 4-phosphate (PI 4-P) and PI 4,5-P2 via PI and PI 4-P kinase activities, respectively (5). Recently, novel phosphorylated phosphoinositide compounds, namely phosphatidylinositol 3,4-bisphosphate (PI 3,4-P2) and phosphatidylinositol 3,4,5-trisphosphate (PI 3,4,5-P3), have been identified in stimulated platelets (5). The activation of phospholipase A2 following agonist stimulation of platelets cleaves platelet membrane phospholipids, including PI, phosphatidylcholine (PC), phosphatidylethanolamine (PE), and phosphatidylserine (PS), at the sn-2 position producing the corresponding 1-acyl lysophospholipids and releasing predominately arachidonic acid (AA, 20 : 4n-6) (1,6). AA is further metabolized via cyclo-oxygenase to thromboxane A2 (TxA2), a potent potentiator of platelet activation (6).
lmmunocytochemical and electron microscopic studies of platelets
Authors: Israels Sara J , Gerrard Jon M
Published: November 2023

Although platelets were one of the first types of cells to be studied using the electron microscope, a clear understanding of normal platelet architecture using electron microscopy had to await improved techniques for the isolation, preservation, and fixation of these cells. Similarly, an understanding of the changes in blood platelet fine structure during clotting required improvements in preparative techniques. The preservation and fixation of certain platelet subcellular structures, notably dense granules and contractile filaments, have presented particular challenges. In this chapter, we describe basic fixation procedures for platelet transmission electron microscopy, and for immunofluorescent and immunoelectron microscopic studies, while providing a brief overview of some other aspects of platelet electron microscopic studies.
Glutathione
Authors: Anderson Marye.
Published: November 2023

Glutathione (GSH; v-γ-glutamyl-L-cysteinylglycine) is present in most mam malian cells. It is usually the most abundant cellular thiol (up to 10 mM); cysteine is usually present in lower levels (about 10% of GSH). GSH is an antioxidant, and it participates in many biological phenomena. It is involved in the transport of certain amino acids, it is a coenzyme for various enzymes, and it protects against oxygen radicals and toxic compounds (1–6).
Targeting precursor proteins to mitochondria
Authors: Lithgow Trevor , Schatz Gottfried
Published: November 2023

In order to grow and divide, eukaryotic cells need mitochondria. This statement is underscored most dramatically by the observation that deletion of any of the several genes essential for mitochondrial biogcnesis kills yeast cells (1) and that failure to direct at least one mitochondrion into the daughter cell after mitosis prevents the final steps of yeast cell division (2). Mitochondria cannot be made de novo (3). To assemble a new mitochondrion, a eukaryotic cell directs specific precursor proteins, synthesized in the cytosol, to the surface of its existing mitochondria, from where the precursors are directed into the organelle and sorted to one of the four submitochondrial compartments (Fig. 1). Assembly of new precursors into existing mitochondria then leads to the birth of new organelles through a poorly understood fission process. A long-standing question has been how eukaryotic cells target mitochondrial precursors specifically to that organelle.
HPLC of oligonucleotides and polynucleotides
Authors: Andrus Alex , Bloch Will
Published: November 2023

This chapter reviews applications of HPLC to two distinct classes of macromolecular analyte: single-stranded oligonucleotides and duplex polynucleotides. These two targets differ in several important respects apart from size and conformation, namely: origin; end-uses; position in a laboratory hygiene scheme; requirements for separation speed, throughput, and cost; goals of the separation; and maturity of the separation technology. Whereas optimized protocols are widely applied to oligonucleotide HPLC, polynucleotide HPLC is not yet popular enough to have evolved beyond the experimental stage of development. This contrast will be evident in what follows, which emphasizes refined procedures for oligonucleotide separations and tactical issues for polynucleotide analysis.

Modern molecular biology depends on nucleic acid separations at both ends of many procedures. Synthetic oligonucleotides should be purified and evaluated for quantity and purity before use as primers, probes, adapters, mutagenesis templates, anti-sense reagents, or ribozymes. In addition, qualitative or quantitative analysis of polynucleotide reaction products usually requires product resolution from excess reagents and side-products, either before or during the analysis. A multi-stage process, such as the sequencing or cloning of DNA amplified by the polymerase chain reaction (PCR), may require an intermediate purification so that preparative (e.g. amplification) reagents and side products do not interfere with downstream (e.g. sequencing) chemistry.
Glycosylphosphatidylinositols and the surface architecture of parasitic protozoa
Authors: Mcconville Malcolm J
Published: November 2023

A functionally diverse family of eukaryotic cell surface proteins are anchored to the plasma membrane via glycosyl-phosphatidylinositol (GPI) glycolipids (for recent reviews see Refs 1 and 2). These glycolipids constitute an alternative anchoring mechanism to the transmembrane polypeptide domain of type I membrane proteins. GPI-anchored proteins make up a variable proportion of the protein repertoire on the cell surface of complex metazoan organisms, but are usually less abundant than proteins with a transmembrane polypeptide domain. In contrast, GPI anchors are found on the most abundant surface proteins of many parasitic protozoa, including the African trypanosomes, Trypanosoma cruzi, Leishmania spp., Plasmodium spp. and Toxoplasma gondii (2) and are thus essential in maintaining the surface architecture of these organisms.
Standardization of immunochemical procedures and reagents
Authors: Thorpe Robin , Wadhwa, Meenu , Mire-Sluis Tony
Published: November 2023

A prerequisite for producing valid, correctly calibrated immunochemical results is that the procedures and reagents are appropriately standardized. This is also necessary if results obtained on different occasions and particularly in different laboratories are to be compared.
Expressing cloned genes in Escherichia coli
Authors: Bass Steve , Yang Maria
Published: November 2023

The expression of cloned genes in Escherichia coli for the production of recombinant proteins has provided a valuable system for studying protein structure and function. Production of recombinant proteins in L. coli is the basis or the multibillion dollar biotechnology industry. Proteins from E. coli or smaller eukaryotes like yeast arc usually easily over-produced in E coli. However. mammalian proteins are often more difficult. A combination of factors can conspire against the researcher to prevent high-level production or recombinant proteins.
Evolutionary theories of genomic imprinting
Authors: Hurst Laurence D
Published: November 2023

As deleterious recessive somatic mutations are hidden in diploids but not in haploids, and as most mutations are probably deleterious recessives, a multicellular organism is usually better off with two sets of chromosomes rather than one (for references and discussion see ref. 1). Why then do some genes (i.e. imprinted genes) behave as though they are haploid, with the same copy inactive in many tissues? A variety of hypotheses have been proposed to account for the evolution of imprinting. It is the function of this chapter to critically review and evaluate these. This is prefaced, however, with a brief word on how this chapter is different from the others in the book.
The instability of differentiation in hepatomas
Authors: Goss S J
Published: November 2023

In 1965, Henry Harris showed that viable heterokaryons could be made between HeLa cells and normal differentiated cells as distinctive as rat lymphocytes, hen erythrocytes, and rabbit macrophages; and in these heterokaryons he detected reactivation of RNA and DNA synthesis in the previously quiescent nuclei. Cell fusion was rapidly established as a powerful tool in the investigation of differentiation, as is clear from the intense activity in this field in the immediately following years (Ringertz and Savage 1976). In work with growing hybrid cells, it is liver differentiation that has been most extensively studied. Well-differentiated hepatomas are available that maintain a wide range of liver-specific traits even when they are grown rapidly in standard tissue culture media. Their phenotype is generally stable through repeated cloning (Deschatrette and Weiss 1974), and the usefulness of the system has been increased by the development of media and conditions that select either for or against liver-specific traits (Leffert and Paul 1972; Haggerty et al. 1975; Choo and Cotton 1977). When a hepatoma is fused to a non-liver cell, the simplest outcome is the cross-activation of liver-specific genes derived from the non-liver parent (Peterson and Weiss 1972; Darlington et al. 1974; Malawista and Weiss 1974; Brown and Weiss 1975).
Loss-of-function mutations in human cancer
Authors: Cavenee Webster K
Published: November 2023

The breadth of biological questions that have been addressed through the technique of somatic cell fusion is indeed impressive. Perhaps the greatest challenges for the approach are situations in which the question is directed at problems of tissue organization or where intertypic cellular activities are required.
Cancer suppression by the retinoblastoma gene
Authors: Lee Wen-Hwa , Goodrich, David , Lee Eva
Published: November 2023

A concentrated effort toward the molecular cloning of cancer suppressor genes had an important recent success: a candidate retinoblastoma (RB) gene was cloned, identified, and extensively characterized by us and others (Friend et al. 1986; Fung et al. 1987; Lee et al. 1987a). Our demonstration of the tumour suppression activity of this single gene when transferred into tumour cells containing inactivated endogenous RB genes constituted a critical milestone for the theory of cancer suppression (Huang et al. 1988). Since several other cancers are known to be familial, are associated with chromosomal deletions, or exhibit loss of heterozygosity, an extension of this concept to other genes and to other cancers is widely anticipated (Malkin et al. 1990).
Roles of the myc gene in cell proliferation and differentiation, as deduced from its role in tumorigenesis
Authors: Klein George
Published: November 2023

Myc, myb, fos, rel, and ski are oncogenes that code for DNA binding nuclear phosphoproteins that can trans-activate other genes and can stimulate DNA replication, directly or indirectly. They may contribute to tumour development and/or progression, after having been activated by structural and/or regulatory changes (Eisenmann 1989). The best known gene of this category, c-myc (Penn et al. 1990), is also most widely implicated in spontaneous tumour development in vivo. It is the cellular counterpart of v-myc, which has been isolated from four different acute avian leukaemia viruses. c-myc and v-myc can induce a broad spectrum of tumours, such as myelocytomatosis, endotheliomas, liver and kidney carcinomas, and erythroblastosis. One strain can also transform fibroblasts.
Human Linkage Mapping
Authors: Curran Julie L
Published: November 2023

The basic principles of linkage mapping have been used by scientists for many years, to establish whether a particular disease is associated with a specific chromosomal region. Linkage amongst simple Mendelian traits is often observable at the phcnotypic level. and the chromosomal rearrangements associated with some disorders could he located cytogenetically. But even in combination, these two approaches gave an extremely sparse map by today’s standards. One of the first loci to be mapped was that for zonal, pulverulent cataracts (CAEI) on lq21-q25, linked to the Duffy blood group (FY) at the same chromosomal location in 1969 (1).
Contig assembly by fingerprinting
Authors: Gregory Simon G , Soderlund, Carol A , Coulson Alan
Published: November 2023

A previous chapter in this series (1) described, primarily, the physical mapping of the 100 Mb Caenorhabditis elegans genome by fingerprinting of cosmid clones, and the linking of the contigs thus derived by YAC hybridization. At that time, the primary function of the map was to enhance the molecular genetics of the organism by facilitating the cloning of known genes, and to serve as an archive for genomic information. However, a clonal physical map—even with good alignment to the genetic map—carries only a tiny proportion of the information present in the genome. Consequently, the current objective of the C. elegans genome project (2) is to establish of the entire genomic sequence. The bacterial clone map, although incomplete by virtue of the uncloneability of regions of the genome in cosmid vectors (a factor which we shall discuss later in this chapter), has proved a sound basis for the systematic sequence analysis. The sevenfold cosmid coverage has a resolution sufficient to enable the selection of a subset of cosmids for sequencing such that, on average, each clone contributes 30 kb of unique sequence to the whole.
Separation by charge, size, and hydrophobicity
Authors: Millner Paul A
Published: November 2023

The material in this chapter, which covers perhaps the most generic forms of chromatography, is written very much from a pragmatic standpoint and results from experience of chromatography of every sort. Where appropriate, links have been drawn with later chapters, that cover more specific chromatographic approaches, although many of the procedures and precautions within this chapter are generic to all chromatographic procedures. General maxims for chromatographic work are to do the fewest chromatography runs possible and as quickly as possible. These aims stem not only from a desire for speed, but often from constraints imposed by the viability of the sample or target protein. The protocols presented are basic chromatographic procedures which cover the most common situations encountered.
Synthetic peptides as affinity ligands
Authors: Reynolds Gary J , Millner Paul A
Published: November 2023

There are many reasons for wanting to construct an immobilized peptide matrix. Purification of a specific antibody is one typical application. In this approach the antibody to be purified would either be raised against peptide conjugated to a carrier of some sort and the same peptide used to construct the matrix for purification. Alternatively, the antibody may have been raised against a whole protein and the peptide-matrix used to select antibodies binding to a specific epitope. The latter is the basis for many of today ‘s highly specific and powerful protein purification procedures using peptide affinity chromatography.
Cleavage using RNase to detect mutations
Authors: Nagase H , Nakamura Y
Published: November 2023

Recent advances in the human genome project will allow us to obtain information on the whole human genomic sequence and to map the positions of genes responsible for human hereditary diseases including both simple and complex genetic trait diseases. This information will provide a large number of candidate genes which will be used to identify the real responsible gene and assess its functions. For this purpose detection of mutations in genes is one of the most crucial techniques required. In order to screen for mutations, three methods have been frequently employed; detection of conformational changes between mutant and normal sequence, cleavage of mismatched heteroduplex, and direct sequencing. Conformational change detection, such as single-strand conformation polymorphism (SSCP) (1), denaturing gradient gel electrophoresis (DGGE) (2), and heteroduplex analysis (HA) (3), are most commonly utilized because of relatively easy handling and high sensitivity. However, there are some disadvantages in these methods. One main problem is a restriction of the fragment size for analysis; for SSCP this is less than 200 bp (4), for HA it is 200-300 bp, and for DGGE it is less than 600 bp (5). In SSCP and HA, sensitivity for special mismatches is condition-dependent, and DGGE require about 60 bp oligonucleotides with a G,C clamp to obtain high sensitivity (6). Chemical cleavage methods can analyse relatively long fragments, more than 1 kb. Among chemical cleavage methods, very early studies used SI and Mug bean nucleases.
Detection of mRNA in tissue sections with radiolabelled riboprobes
Authors: Simeone Antonio
Published: November 2023

Gene expression studies have been greatly advanced by techniques developed to visualize gene expression during embryonic development and tissue differentiation. Among these, in situ hybridization of sections with radiolabelled probes has a historical significance because the in situ expression of mRNA in invertebrate and vertebrate development was first described using this pro¬ cedure. Although alternative techniques are now available to identify gene transcripts in tissues and embryos (1, 2), in situ hybridization on sections with radiolabelled RNA probes is frequently found to be the more sensitive and reliable procedure. This chapter describes protocols for in situ hybridization with radiolabelled RNA probes on embryo and brain sections. In addition, a list is provided of diagnostic probes useful at specific stages of development for defining the expression domains of a given gene.
The amplified expression, identification, purification, assay, and properties of hexahistidine-tagged bacterial membrane transport proteins
Authors: Ward* Alison , Sanderson* Neil M , O’Reilly* John , Rutherford* Nicholas G , Poolmanꝉ Bert , Henderson* Peter J F
Published: November 2023

In bacteria, 3-15% of genes are predicted to encode proteins involved in membrane transport (1), a process which is vital for both the capture of nutrients and the excretion of waste products, toxins, and antibiotics. The energy required for these processes is derived from the electrochemical gradients of ions across the cell membrane (in bacteria the majority operate by proton-driven symport or antiport mechanisms but some utilize a sodium gradient) or from the hydrolysis of ATP (2). Whilst the 3-D structures of thousands of soluble proteins have been determined, to date only 21 membrane protein structures have been solved to atomic resolution (3, 4). The elucidation of the structures of membrane proteins using physical techniques is, therefore, a key area of research.
Spectroscopic and kinetic approaches for probing the mechanisms of solute transporters
Authors: Walmsley Adrian R
Published: November 2023

This chapter deals with the use of spectroscopic methods to monitor conformational changes in membrane transporters and kinetic approaches to time-resolve these changes. It outlines the use of fluorescence spectroscopy to monitor ligand-membrane protein interactions and the use of stopped-flow fluorescence spectroscopy to determine mechanisms of ligand binding and translocation. It also outlines the use of rapid-quench techniques for measuring the rate constants governing the translocation of the substrate across the membrane. To a large extent, these studies are illustrated by reference to the kinetics of substrate and inhibitor binding and translocation by various transporters studied in the author’s laboratory.
Detection and analysis of glucose transporters using photolabelling techniques
Authors: Gillingham Alison K , Koumanov Françoise , Hashimoto Makoto , Holman Geoffrey D
Published: November 2023

The use of photolabels to study biological systems has emerged as a powerful technique for tagging proteins, and the range of approaches and types of ligand that can be employed have been extensively reviewed (1). Although various affinity labelling approaches have been tried (for example using the highly reactive isothiocyanate group substituted into maltose (2) ), these have generally been unsuccessful in labelling glucose transporters. This difficulty can be largely attributed to the low affinity of glucose transporters for their substrate and the poor tolerance for groups substituted around the glucopyranose ring. Specificity studies (3) indicate that there is a close approach of the binding site around the sugar as it enters from the external solution. These properties of the glucose-transporter binding site have limited the range of approaches that can be used in photoaffinity labelling studies. Often photoreactive substituents are rather bulky and hydrophobic, and based on benzoic acid substituted with azides or diazirine substituents.
Mouse care and husbandry
Authors: Hetherington Colin M , Doe Brendan , Hay Donald
Published: November 2023

This chapter illustrates methods for the care and husbandry of mice. It out-lines various types of animal accommodation and methods of identification, record keeping, breeding systems, timed mating, superovulation and re-derivation. Mice may be housed under of a variety of conditions. The type of accommodation and husbandry will depend on the nature of the animal experimentation and financial considerations. The more sophisticated the methods used, the greater the cost of building, equipping and running the facility. A balance also must be struck between ease of access and working and the level of pathogen barrier used.
Radiolabelling of monoclonal antibodies
Authors: Mather Stephen J
Published: November 2023

Antibodies have been tagged with radionuclides since the 1940s when David Pressman, perhaps the first to recognize the potential of radiolabelled anti bodies in vivo, radiolabelled polyclonal antibody preparations with iodine-131, a radionuclide first produced by artificial means only a decade or so previously (1). Today the researcher may wish to radiolabel an antibody for a wide variety of different applications, many of which are described in other chapters in this book.
lmmunoassays
Authors: Peppard Jane V
Published: November 2023

This chapter describes methods by which monoclonal and also polyclonal antibodies may be used in order to quantitate antigens. Since the choice of format is extensive, the information is divided so that options for each stage of the assay, e.g. choices of label or solid support, which can be ‘mixed and matched’ according to requirements, are discussed individually. Finally, the most frequently used methods of immunoassay and their set-ups are described.
Immunofluorescent labelling of sections
Authors: North Alison J , Small J Victor
Published: November 2023

Over the past two decades, immunofluorescent labelling of cells and tissues has become one of the most powerful tools in cell biology. It is commonly performed in two ways: labelling of whole cells and labelling of sections. The problem with the first approach lies in rendering the intracellular targets accessible to the detecting antibodies. This necessitates some form of membrane permeabilization, generally using detergents or alcohols, or a direct physical approach such as micro-injection. Unfortunately such manipulations can lead to artefacts including ultrastructural changes and the loss or re distribution of the target antigen. Moreover, not all parts of the structure may be equally accessible to the antibody, even after extensive permeabilization. We have found this to be particularly pertinent in densely packed structures such as muscle (1). For this reason, the use of sectioned tissue, in which all cellular sub-structures are rendered unequivocally accessible at the section surface, can often prove to be the optimal procedure for consistent and reliable immunolocalization studies.
Green fluorescent protein in plants
Authors: Hawes Chris , Boevink Petra , Moore Ian
Published: November 2023

The introduction of the jellyfish green fluorescent protein (GFP) and its wavelength-shifted derivatives as reporter proteins for both gene expression and protein location has provided cell biologists with a powerful new tool with which to study cell structure and function in vivo. The protein has a number of intrinsic properties that make it attractive as a marker for cell biological studies. For instance, it is highly fluorescent and requires no co-factors or substrates other than oxygen to form the fluorochrome. It is relatively stable and pH insensitive, has a low level of cytotoxicity and in many instances does not interfere with the functioning of native proteins when expressed attached to them as chimeric constructs. Moreover, it has been genetically modified to alter its spectral and other properties such as folding rate, to make a family of tailor-made reporter proteins (1).