bluuebunny/crossref_metadata_2025_split huggingface dataset:
Selective amplification of specific alleles
Authors: Syvänen A -C 
Published: November 2023

Abstract
A variety of methods have been developed to detect known mutations and polymorphisms. Many of these methods are based on the polymerase chain reaction (PCR) because a segment of DNA can be amplified more than one millionfold for study or analysis. The amplified DNA may be analysed by chemical cleavage, restriction enzyme digestion, single-strand conformation polymorphism, gel electrophoresis, or hybridization to specific oligonucleotides.These methods, however, all require additional steps following PCR. Therefore, one of the simplest and most rapid techniques is to modify the PCR for amplification of specific alleles by using specially designed oligonucleotides. This approach we have termed PCR amplification of specific alleles (PASA) (1, 2). Other laboratories also have developed this method to distinguish between specific alleles (3-6). Synonymous terms for PASA include allelespecific amplification (ASA), allele-specific PCR (ASP), and the amplification refractory mutation system (ARMS). The method is generally applicable for the detection of known point mutations, small deletions and insertions, polymorphisms, and other sequence variations.
The protein truncation test (PTT)
Authors: Luijt Rob B Van Der , Fodde, Riccardo , Dunnen Johan T Den 
Published: November 2023

Abstract
The detection and characterization of point mutations in genes responsible for inherited disorders has become one of the most common practices in human molecular genetics. Several protocols are nowadays available: single strand conformation polymorphism (SSCP) (1), RNase protection (2), hydroxylamine and osmium tetroxide (HOT) chemical cleavage (3), and denaturing gradient gel electrophoresis (DGGE) (4, 5). These techniques are generally aimed at the identification of single base substitutions, insertions, or deletions in relatively small DNA fragments (50-500 bp). However, when dealing with large genes, it is desirable to analyse larger fragments of the coding region to speed up the analysis and pin-point the location of the alteration prior to the nucleotide sequence determination. To this aim, mRNA-based mutation detection protocols are often employed. Moreover, if the spectrum and the type (i.e. missense, nonsense, or frame-shift) of the prevalent mutations at a given diseasecausing gene has already been established, one would prefer to employ a protocol ‘targeted’ for the detection of the most frequent type of mutations.
In situ hybridization of mRNA with hapten labelled probes
Authors: Xu Oiling , Wilkinson David G 
Published: November 2023

Abstract
In situ hybridization with hapten labelled probes offers several advantages compared with S-labelled radioactive probes, including a single cell resolution of the signal, the ability to directly visualize spatial patterns by hybridizing whole tissues, and the ability to visualize different transcripts simultaneously by use of multiple labelling and detection methods. Several haptenized nucleotides are available, including digoxigenin- (DIG), fluorescein-, and biotin-DTP that can be used to make labelled RNA probes. DIG and fluorescein can be detected with antibodies, and biotin with streptavidin, to which enzymes such as alkaline phosphatase (AP), J3-galactosidase, or horse-radish peroxidase have been conjugated. For each of these enzymes, a variety of chromogenic substrates are available that yield different coloured products. Currently, the reagents that have found widespread favour since they give a high sensitivity and low background are DIG labelled RNA probes detected with an AP-conjugated anti-DIG antibody and the chro¬ mogenic substrate mix of 5-bromo-4-chloro-3-indolyl-phosphate (BCIP) plus 4-nitroblue tetrazolium chloride (NBT). Fluorescein labelled probes detected with an AP-conjugated anti-fluorescein antibody and BCIP/NBT give similar results. In this chapter, we describe methods for non-radioactive in situ hybridization of whole mount tissues and of tissue sections.
Biological image processing and enhancement
Authors: Horgan G W , Reid C A , Glasbey C A 
Published: November 2023

Abstract
This chapter considers simple ways to enhance digital images, both to make visual interpretation easier and to make the images more amenable to subsequent automatic analysis. It is typically the stage in image analysis that follows image capture. Methods include adjustment of brightness or contrast, edge detection and edge sharpening, removal of small irrelevant features (termed noise), and adjustment for background intensity variation. Image enhancement can only improve and clarify information that is already present in an image: it cannot recover what was never there in the first place! Therefore, at the stage of image capture it is essential to seek the best image quality by suitable sample preparation, adjustment of instrumentation, choice of lighting, etc. We state this as a principle:
Computer prediction of transporter topology and structure
Authors: Hong Rong-I , Sansom Mark S P 
Published: November 2023

Abstract
One of the resources which may be exploited in studying membrane transport proteins is the enormous (and increasing) body of genome sequence data. At the time of writing, complete genomic sequences are known for 21 prokaryotes (1), a yeast (2), and for Caenorhabditis elegans (3). Current estimates suggest that integral membrane proteins comprise about 20–30% of most genomes (4, 5). This provides a strong motivation to exploit this information in terms of trying to understand the relationship between membrane protein sequence and structure (and eventually function). Prediction of protein structure is fraught with difficulties and despite intense efforts, ab initio methods for structure prediction remain elusive (6). However, for membrane proteins (or at least those whose membrane domains are composed largely of (l-helices), the prospects of prediction are somewhat brighter as the lipid bilayer restricts the number of degrees of freedom of the system. Prediction of transmembrane (TM) Cl-helices is widespread and a number of different methods and hydrophobicity scales have been devised to aid such predictions (7). Other methods allow the prediction of preferred orientations of such TM helices, thus placing restrictions on how they may be packed together in the intact protein.
Mapping phenotypic trait loci
Authors: Taylor Benjamin A 
Published: November 2023

Abstract
When a genetic locus is known only by a phenotype, mapping generally re quires analysis of segregating crosses (i.e. meiotic mapping). Phenotypic trait loci vary from major mutations affecting simple qualitative trait differences such as coat colour, to quantitative trait loci (QTLs) that exert small statistical effects on complex traits such as body weight. If the phenotypic trait variant is a new mutation, a new cross must be generated to detect linkage. On the other hand, if the new locus represents a variant that distinguishes inbred strains, special genetic systems, such as recombinant inbred strains, may provide an attractive alternative to a traditional linkage cross.
Characterization of cellular antigens using monoclonal antibodies
Authors: Hynes Gillian 
Published: November 2023

Abstract
Monoclonal antibodies are powerful biological tools due to their ability to distinguish an antigen molecule, typically a protein, from a complex mixture of macromolecules. A number of techniques have been developed to detect cellular antigens using monoclonal antibodies as specific probes. Consequently, a characterization study can be advanced considerably after the generation of a monoclonal antibody reagent to a target protein, and a possible strategy is outlined below:
Analysis of signal transduction pathways using protein kinase inhibitors and activators
Authors: Naito Yasuhito , Kobayashi Ryoji , Hidaka Hiroyoshi 
Published: November 2023

Abstract
A complete understanding of the organization and functioning of the protein phosphorylation system requires the pooling of expertise in several different fields, such as molecular pharmacology, genetic manipulation, biochemistry, and cell biology. The advent of a new class of effective pharmacological agents is always an event of considerable interest, in particular for new types of antagonists, i.e. potentially important groups of compounds that act by specifically blocking one or more of the steps in intracellular signalling systems (1). There is now convincing evidence that intracellular protein phosphorylation systems are of fundamental importance in biological regulation. A large number of cellular mechanisms involving protein phosphorylation have been revealed over the past decade. However, interrelationships amongst signal cascades are complex and uncertainties concerning the cellular responses remain. In order to obtain improved understanding, sophisticated methods for estimating changes in the activities of cellular response elements after extracellular stimulation have to be developed. While progress has been made in the biochemistry and molecular biology of protein kinases, it has proved very difficult to elucidate their function in intact cells. For this reason researchers studying second messenger systems and protein phosphorylation have long sought specific and effective inhibitors that would permit definitive determination of physiological roles of protein kinases (2).
Functional characterization of co-activators using mammalian cell microinjection
Authors: Rose D W , Mullek T M , Rosenfeld. M G , Glass C k. 
Published: November 2023

Abstract
The past few year; have led to an appreciation that the transcriptional activity of the nuclear receptor family is regulated by binding of the receptor. in a ligand-dependent manner. to a multicomponent complex of co-activator proteins. many of which possess histone acetyltransferase activity. The activity of these co-activators is; also further regulated by a variety of signal transduction pathways. Many of these co-activator molecules have now been isolated, often on the basis of their interaction with the nuclear receptors. The functional characterization of these protein complexes has been facilitated by the use or a series of powerful single cell microinjection assays that employ the use of specific anti-co-activator antibodies used in conjunction with reporter constructs specific for each of the nuclear receptors and expression constructs for wild-type and mutant forms of the co-activators.
G proteins II: Gq, G12, and Gz
Authors: Glick Jennifer L , Meigs Thomas E , Casey Patrick J 
Published: November 2023

Abstract
The ability of the cell to sense and communicate with its environment is critical for its survival, and therefore for the survival of the organism. Simpler life forms such as Dictyostelium sense toxic or unfavourable environments and rely on the process of chemotaxis to steer away from these areas and toward areas where conditions are more suitable. During the development of multicellular organisms, progenitor cells must migrate appropriately and coordinate their differentiation with one another in order to produce a viable embryo. Adult organisms rely on sensory cells such as photoreceptors, the olfactory epithelium, and taste buds, as well as a central nervous system, to guide them out of danger and track down food or potential mating partners. Further still, in multicellular organisms, cells must be able to respond to complex signals that originate from many regions of the body. A classic example of this is the well-known ‘fight or flight’ response, wherein an animal senses imminent danger and the body immediately prepares itself for the necessary action (for instance, with a faster heartbeat to increase blood flow to its musculature). Many of the processes involved in this type of response (vision, memory, smell, taste, reflex) are in large part regulated by heterotrimeric G proteins (G proteins).
Mammalian MAP kinase pathways
Authors: Kyriakis John M 
Published: November 2023

Abstract
Mitogen-activated protein kinase (MAPK) signal transduction pathways are among the most widespread mechanisms of cellular regulation. All eukaryotic cells possess multiple MAPK pathways, each of which is preferentially recruited by distinct sets of stimuli, thereby allowing the cell to respond in parallel to multiple divergent inputs. Mammalian MAPK pathways can be recruited by a wide variety of different stimuli ranging from hormones, such as insulin and growth hormone, to mitogens (i.e. epidermal growth factor, EGF; platelet-derived growth factor, PDGF; fibroblast growth factor, FGF), vasoactive peptides (angiotensin-II, endothelin), inflammatory cytokines of the tumour necrosis factor (TNF) family, and environmental stresses such as osmotic shock, ionizing radiation, and ischaemic injury.
ATM and DNA-PK: the sensing, signalling, and repair of DNA damage
Authors: Bristow Robert , Bluyssen Hans , De Klein Annelies , Van Gent Dik 
Published: November 2023

Abstract
A significant and ongoing problem for every living cell is the occurrence of DNA damage caused by exogenous and endogenous agents. For example, DNA base damage and DNA strand breaks are induced as consequences of the normal cellular processes of DNA replication and oxidative stress, or after exposure to exogenous DNA-damaging agents including UV-light or ionizing irradiation. As a result, cells have developed a sophisticated approach to the integration of DNA damage-sensing and subsequent DNA repair as a means of preserving genomic stability. This chapter will mainly focus on the cellular activity and biochemistry of two members of the PI3K-related protein kinase family named ATM (for ‘mutated in ataxia telangiect-asia’), and DNA-PK (DNA-dependent protein kinase), which are thought to play important roles in the sensing, signalling, and repair of DNA strand breaks.
Detection and quantification of heterogeneous viral targets: HIV as a model
Authors: Kwok Shirley 
Published: November 2023

Abstract
The diversity of the human immunodeficiency virus (HIV) genome presents it as one of the most difficult targets for the development of nucleic acid-based assays. The extensive genetic variability of HIV is primarily due to the high error rates of the viral reverse transcriptase (3.4 x 10-5 per base incorporated) and the absence of proofreading mechanisms (1). HIV-1 and HIV-2 comprise the two groups of viruses that cause AIDS. The HIV-ls are divided into three groups: M (for Major), 0 (for Outlier), and N (for non-M, non-0). The HIV-1 group M and HIV-2 viruses have been further divided into subtypes A-K and A-G, respectively, based on phylogenetic analysis of proviral sequences (ref. 2; see Chapter 8). The group O viruses are as heterogeneous as the group M viruses and comprise a number of distinct genetic subtypes that remain to be identified and characterized (3). The recently identified, HIV-1 group N viruses are distinct from group Mand O (4). Within individuals, HIV-1 undergoes continuous genetic changes; patients usually harbour a swarm of highly related but individually distinguishable viral variants, which are referred to as quasispecies. Genomic recombination between two different HIV-1 populations frequently occurs in vivo, which further contributes to HIV genetic variability.
PCR-based methods of HLA typing
Authors: Erlich Henry A , Trachtenberg Elizabeth A 
Published: November 2023

Abstract
Over the last two decades, molecular genetic techniques have been used to isolate the genes encoding the HLA class I and class II molecules and to characterize their genomic organization as well as their sequence diversity. The allelic sequence polymorphism at the HLA class I and II loci, revealed by extensive sequencing studies on a variety of human populations, is far greater than the antigenic variation detected by conventional serological typing (1). For example, there are about 280 DRB1 alleles defined by the second exon sequence but only about 15 different DR serological specificities or serotypes. Similarly, for the HLA-B locus, more than 450 alleles have been reported, compared to about 48 serological specificities. For the DPA1 and DPB1 loci, which encode the DP molecule, no serological typing system has been available and, consequently, most functional investigations of DP polymorphism, such as disease association studies or the analysis of DP mismatching in transplantation, have been possible only since the development of DNA-based HLA typing methods (2). The HLA class I and class II loci are the most polymorphic coding sequences in the human genome. The study of their allelic sequence diversity as well as the development of simple and rapid DNA-based typing methods has been greatly facilitated by the development of PCR amplification in the mid 1980s (3–6).
Fluorescence and fluorochromes
Authors: Ormerod Michael G 
Published: November 2023

Abstract
Fluorescence occurs when a molecule excited by light of one wavelength returns to the unexcited (ground) state by emitting light of a longer wavelength. The exciting and emitted light, being of different wavelengths, can be separated from one another using optical filters. Fluorescence has been used to visualize certain molecules (and hence structures) by light microscopy for many years. It is a sensitive technique because a positive signal is detected against a negative background. It has found widespread application in flow cytometry and, together with light scatter, is the major type of measurement made in most machines. The ability to detect fluorescence simultaneously from two, three, or four com pounds fluorescing at different wavelengths enables several parameters to be measured and opens up the field of multi-parametric analysis.
Signal transduction in flamentous fungi: regulation of protein kinases
Authors: Ye Xiang S , Wu Liping , Osmani Stephen A 
Published: November 2023

Abstract
In eukaryotes, including fungi, protein kinases are key components of signal transduction pathways. Normally a cascade of reversible protein phosphorylation/ dephosphorylation is the major mechanism of signal transduction, which provides both signalling specificity and diversity. Although second messengers, such as Ca2_, IP3, cAMP, and G proteins have pivotal roles in many signalling events, their physiological functions are normally mediated through regulation of protein kinases.
Biochemical methods for filamentous fungi
Authors: Döhren Hans von 
Published: November 2023

Abstract
Although biochemical approaches in the study of filamentous fungi may be considered more complex than bacterial or yeast studies, this is not obvious from current protocols. Fairly little attention has so far been given to morphological specialities (except for mycelium and spore structures) and ultrastructural compartmentation. This will hopefully improve in the future with rising interest in cell biological questions. Biochemistry of fungi includes the description of constituents, and here focus is given to proteins and metabolites. At this time, the genomes of filamentous fungi have not been fully sequenced, and/or no genome sequence is as yet available in the public domain. Still, proteomics is entering the field, and will be involved in problem solution in the near future.
Lymphocyte culture
Authors: Clouston H J 
Published: November 2023

Abstract
Cytogenetic analysis of blood samples is the largest concern of most constitutional cytogenetics laboratories. There are two main reasons why peripheral blood remains the tissue of choice for postnatal studies. First, blood samples are comparatively easy to obtain. Second, simple, relatively inexpensive culture techniques can be used to rapidly generate abundant metaphases of optimum length and quality. These preparations are ideal for high-resolution G-banding and FISH studies.
This chapter describes the techniques and rationale of lymphocyte culture and chromosome preparation, and includes protocols with which the author has achieved considerable success.
The application of cytogenetic investigations to clinical practice
Authors: Wolstenholme J , Burn J 
Published: November 2023

Abstract
Cytogenetic investigations are expensive. It is inappropriate to perform cytogenetic screening on unselected populations or to apply all the available culture and staining techniques to every sample. This is particularly so for the explosion of FISH-based approaches which are currently moving from the research environment to the diagnostic laboratory. At present, many are prohibitively expensive, extremely time-consuming and require specialized equipment for analysis. Their place in mainstream diagnostic laboratories is thus restricted to certain referral categories, but based on previous experience, this may change rapidly as these drawbacks are overcome.
Fluorescent probes for living plant cells
Authors: Fricker Mark , Parsons, Andrew , Tlalka Monika 
Published: November 2023

Abstract
There are several reviews that cover the biological questions that have been addressed with fluorescent techniques in plants (1-3) and there is extensive on line documentation on the properties of the fluorescent probes themselves (e.g. Molecular Probes, . This chapter focuses on optimization of the systems used for live cell imaging, the methods used to mount plant specimens and load them with dyes, a compendium of dyes that have proved useful in plant tissues, and a series of protocols to convert qualitative observations to quantitative measurements. The hazards associated with these dyes are, in the main, not known, so all should be treated as potentially harmful.
Flow cytometry
Authors: Pena Teodoro Coba de la , Brown Spencer 
Published: November 2023

Abstract
Single cell biochemistry, single cell screening, and assessment of heterogeneity in cell populations have become feasible in both fundamental and industrial situations. These strategies have benefited from the following happy marriage. On one hand we have development of thousands of fluorescent markers for probing cellular functions, physiological states, or gene expression (Chapters 2, 4, 5), to which one must add the diagnostic tools of in situ hybridization (Chapter 12) and immunolabelling (Chapters 10, 11). On the other hand we have the numerical power, simplicity, and sensitivity of cytometry. This term conveys cell-by-cell quantification of structure or, typically, of fluorescent signals. This may be achieved by quantitative microscopy-or by flow cytometry, in which case no image is obtained but fluorescent intensities are measured with remarkable ease and precision. There is not space to handle cell physiological studies here, although vacuolar properties (1), pH (2), relative membrane potential (3), and membrane viscosity (4) can be assessed by cytometry.
Chaperones in secretion pathl’Vays of E. coli
Authors: Harms Nellie , Luirink Joen , Oudega Bauke 
Published: November 2023

Abstract
Cytoplasmic proteins once synthesized at the ribosome fold into their native conformation, whereas proteins with destinations outside the cytoplasm are targeted to the cytoplasmic membrane and have to be prevented from premature folding and from degradation. For the majority of these latter proteins, folding occurs outside the cytoplasm. The precision and speed of these processes is ensured by different chaperones, targeting factors and folding catalysts. Most of these helper proteins, present in the cytoplasm, the periplasm, and in the membranes have a general role; some, however, are dedicated to special processes. This review focuses on the targeting and folding processes that function in a number of secretion pathways of E. coli. Since the field of protein secretion is very broad, not all pathways and their concomitant chaperones will be described.