bluuebunny/crossref_metadata_2025_split huggingface dataset:
Nuclear protein sorting in heterokaryons and homokaryons
Authors: Ringertz Nils R 
Published: November 2023

Abstract
Our interest in nuclear protein sorting began 25 years ago and was triggered by Professor Harris’ report that it was possible to reactivate RNA and DNA synthesis in inactive chick erythrocyte (CE) nuclei by Sendai virus-induced fusion of chicken red cells with human HeLa cells (Harris 1965, 1967). This finding suggested that human tumour cells contain factors capable of activating chick genes and raised a number of interesting questions about the nature of cytoplasmic signals regulating transcription and replication. In collaboration with Professor Harris we carried out a quantitative cytochemical study of chromatin changes during nuclear reactivation in CE x HeLa heterokaryons. Individual nuclei in fixed heterokaryons were analysed using microspectrophotometry, microfluorimetry, and microinterferometry (Bolund et al. 1969). During early stages of reactivation the chick chromatin underwent drastic biophysical changes resulting in an increased ability to bind intercalating fluorochromes to DNA, altered thermal stability of the DNA, etc. These changes were followed by a marked dispersion of the condensed erythrocyte chromatin. Model experiments with isolated CE nuclei suggested that the biophysical changes, which we named ‘chromatin activation’ (Ringertz 1969), were triggered by ionic signals causing conformational changes in the deoxyribonucleoprotein complexes. The drastic increase in nuclear size noted by Harris (1967) and illustrated in Fig. 3.1, was paralleled by a dramatic increase in nuclear dry mass and protein content. An interesting aspect was that the nuclei grew in size and weight before there was any significant increase in chick RNA and chick proteins. Using species-specific antibodies to nuclear proteins (Ringertz et al. 1971) we subsequently found that the chick nuclei grow because they selectively take up and concentrate human nuclear proteins contributed by the HeLa fusion partner. This led us to examine which human proteins entered the chick nuclear compartment and which chick proteins, if any, were lost from the inactive CE chromatin. We also tested if different cytoplasmic environments, i.e. different fusion partners, resulted in activation of the same or of different genes.
Some insights into the replication of damaged DNA in mammalian cells
Authors: Johnson R T , Downes C S , Godfrey D B , Hatton D H , Ryan, A J , Squires S 
Published: November 2023

Abstract
In a volume devoted to the legacy of cell fusion, this article on cellular responses to DNA damage contains only a single example of the use of the technique, though the information gained thereby has transformed our view of the mutant cell whose phenotype is described at some length below. In the wider sense cell fusion continues to play an invaluable role in the analysis of DNA repair problems, most notably in complementation analysis using heterokaryons (Giannelli et al. 1982) or permanent hybrid cells (Johnson et al. 1989). In this way the number of genes involved in a repair pathway can be identified, by separating a set of repair-defective mutant cells into different complementation groups.
The role of the nucleolus in the transfer of information from nucleus to cytoplasm
Authors: Hatanaka Masakazu 
Published: November 2023

Abstract
When the nucleus of a hen erythrocyte is introduced into the cytoplasm of a human or mouse cell in culture, it resumes the synthesis of RNA (Harris 1965, 1967). The reactivated erythrocyte nucleus undergoes great enlargement, but it does not, for at least 2 or 3 days, develop nucleoli. During this period, the heterokaryon, although it may contain active erythrocyte nuclei, does not synthesize any hen-specific surface antigens. But when, later, the erythrocyte nuclei do develop nucleoli, hen-specific antigens reappear on the surface of the heterokaryon and progressively accumulate. These and other nucleolar inactivation experiments suggested that the nucleolus may play a decisive role in the transfer of information from the nucleus to the cytoplasm (Harris et al. 1969; Sidebottom and Harris 1969; Deak et al. 1972; Harris 1972).
Transcription by immobile RNA polymerases
Authors: Cook P R 
Published: November 2023

Abstract
An unstated assumption in current models for transcriptions is that a polymerase tracks along the template as it synthesizes RNA. However, experiments using ‘nucleoids’ derived by lysing cells in a non-ionic detergent and 2 M NaCl originally suggested that RNA was synthesized by a polymerase attached to an underlying nucleoskeleton. These experiments were subject to the criticism that the associations seen resulted from the artefactual aggregation of nascent RNA. Further experiments using more physiological conditions have confirmed the existence of an intermediate-filament-like nucleoskeleton and have shown that active polymerases resist electroelution from nuclei, presumably because they are attached to the skeleton. Whether immobilization affects polymerase activity has also been tested directly by attaching to plastic beads a pure enzyme that is widely used for transcription in vitro-the RNA polymerase of bacteriophage T7. Although initiation is inhibited, immobilization has no effect on elongation. It is suggested that genes become active by binding to an attached polymerase and then transcripts are generated as the template moves past the fixed enzyme.
Lateral mobility of membrane proteins-a journey from heterokaryons to laser tweezers
Authors: Edidin Michael 
Published: November 2023

Abstract
Soon after virus-induced heterokaryons were described (Harris and Watkins 1965) it was observed by several laboratories that species-specific surface markers of the parent cells were randomly distributed over the heterokaryon surface (Watkins and Grace 1967; Harris et al. 1969; Gordon and Cohn 1970). All of these observations were made hours after the fusion of the parent cells, and were made with fairly low spatial resolution. Larry Frye, then a graduate student in my laboratory, decided to refine the experiments by resolving the time required for randomization of surface markers from parent cells. He also improved the spatial resolution of the distribution by using fluorescent antibodies to the class I MHC (major histocompatibility complex) molecules of the mouse cell parent and antibodies to undefined, but membrane-integral, antigens of the human cell parent of the heterokaryons. With this system, Frye was able to show that randomization of surface marker antigens began within minutes of fusing mouse and human cells with virus (Frye and Edidin 1970). Controls, including fixation of newly formed heterokaryons, showed clearly that redistribution was due to diffusion and not to cell metabolism (Edidin and Wei 1977). Together with studies of lipid motion in the bilayer, these experiments set us on the road to viewing cell membranes, especially cell surface membranes, as ordered fluids whose components are free to move in the plane of the membrane. The experiments that showed diffusional motions of lipids and proteins are summarized in an early review on the subject (Edidin 1974); the model embodying them, together with data on the solubility of membrane proteins is the well-known ‘fluid mosaic’ model of Singer and Nicolson (1972).
Radiation hybrid mapping: an idea whose time has finally arrived
Authors: Cox David R 
Published: November 2023

Abstract
In 1975, Stephen Goss and Henry Harris published a short paper in the journal Nature that promised to revolutionize the field of human gene mapping. Entitled ‘New method for mapping genes in human chromosomes’, this paper described a systematic approach for determining the linear order of human genes and estimating the distances between them. Previous workers had used rodent-human somatic cell hybrids to map genes to specific human chromosomes or to specific chromosomal segments in those regions of the genome where human translocation chromosomes were available. However, such maps were very crude in comparison with the genetic maps available for many other organisms. By exposing diploid human cells to a lethal dose of ionizing radiation followed by fusion to rodent cells to obtain viable hybrid clones segregating human chromosomal fragments, Goss and Harris were able to measure the frequency with which pairs of human genes were cotransferred after irradiation, thereby creating maps of much higher resolution than was possible previously.
Genes in control of cell proliferation and tumorigenesis in Drosophila
Authors: Mechler Bernard M 
Published: November 2023

Abstract
Cancer is generally considered as a failure in the normal progression of differentiation. As a result, the cancer cells escape the mechanisms controlling normal growth and proliferate. When the overgrowth of neoplastic cells reaches a critical threshold, complex syndromes arise and, ultimately, lead to the death of the organism. A genetic basis for cancer is now well established and includes the discovery of protooncogenes, whose activation or altered expression is associated with the cancerous state and tumour suppressor genes which appear to be lost or functionally inactivated. About 25 years ago, pioneering studies in the field of Drosophila, mouse somatic cell, and human genetics revealed that neoplasia may result from recessive mutations in regulatory genes controlling cell growth and differentiation (Gateff and Schneiderman 1967, 1969; Harris et al. 1969; Knudson 1971).
Somatic cell genetics and the search for colon cancer genes
Authors: Thomas H J W , Frischauf A -M , Solomon E 
Published: November 2023

Abstract
As this volume is in honour of Professor Sir Henry Harris, it seems appropriate, under the general topic of genetics of colon cancer, to focus specifically on the role of somatic cell genetics in the isolation of genes responsible for this disease. Two rather different types of somatic cell genetics have been used in the identification of the genes mutated in colon cancer. The first type might be called tumour somatic cell genetics and has developed from the fact that loss of genetic material from tumour cells can be detected either microscopically, as whole or partial chromo­some loss or by loss of heterozygosity (LOH) (for reviews see Goddard and Solomon, (1993) and Lasko et al., (1991)). This genetic loss in tumours has come to be recognized as the hallmark of tumour suppressor genes.
The use of flow-sorted chromosomes in genome mapping
Authors: Ross Markt. , Langford cordelia f. 
Published: November 2023

Abstract
Flow-sorted chromosomes are an invaluable resource for genome mapping. Sorted material can be cloned efficiently in E. coli to produce chromosomespecific libraries with moderate insert sizes which arc suitable for contig building, gene identification, and genomic sequencing. Alternatively, smallinsert libraries, which provide a source of chromosome-specific landmarks. can be generated either directly or following amplification of the sorted DNA by the polymerase chain reaction. This chapter gives a brief introduction to now-sorting itself. but the principal emphasis is on the use of flow-sorted material for the construction of libraries and as a PCR template for the chromosomal assignment of markers.
Long-range restriction mapping of genomic DNA
Authors: Tümmler and Burkhard 
Published: November 2023

Abstract
Long-range restriction maps are important tools to determine the arrangement and distance of gene markers, and a prerequisite before committing oneself to costly and time-consuming cloning and sequencing projects in order to identify gene organization and rearrangements. For the construction of a low-resolution physical map of a genome or chromosomal region, the intact DNA is digested with a restriction endonuclease that cleaves infrequently and the fragments are separated by pulsed-field gel electrophoresis (PFGE) (1, 2). Restriction fragment patterns of small genomes, such as those of prokaryotes or lower eukaryotes, can be visualized directly by gel staining. With larger genomes, the complex mixture of fragments is blotted onto membranes after electrophoresis, and hybridized with suitable probes to detect fragments of interest. The order of probes and of fragments is established from the combinatorial analysis of partial and complete digests with one or more restriction enzymes. In the simplest case, two probes will hybridize to the same restriction fragment, thus revealing physical linkage between them. However, independent confirmation must always be sought to exclude hybridization to distinct but co-migrating bands. This may be accomplished by, for example, hybridization of the two probes in question to the fragments produced by partial digestion with the same enzyme as was used in the complete digest: both probes should detect the same bands (and with the same relative intensities) in this more complex mixture of fragments. Combined usage of several different enzymes should then allow construction of a macrorestriction map around both loci, which may be refined by double digestion analysis.
RFLP analysis using heterologous probes
Authors: Aquadro Charles F , Noon William A , Begun, David J , Danforth Bryan N 
Published: November 2023

Abstract
DNA sequence variation is ideally assayed by direct determination of nucleotide sequences representing independent copies of a gene region or other homologous segment of the genome sampled from a natural population. We will refer to such multiple copies of single regions as 'alleles'. An estimate of the level and distribution of sequence polymorphism segregating in a population requires multiple alleles to be sampled and sequenced. The sequences must also be determined with great accuracy, since a 1% error rate in deter mination of the sequence would generate an apparent level of nucleotide polymorphism higher than that actually seen for many species. The large sampling variances of most of the descriptive statistics of variation require the investigator to sample perhaps 10 or more alleles in order to obtain statistic ally reasonable estimates. The development of the polymerase chain reaction (PCR) and rapid sequencing methodologies has made such efforts practical for many genes and organisms.
Avidin-biotin immobilization systems
Authors: Wilchek Meir , Bayer Edward A 
Published: November 2023

Abstract
Over the past two decades, the avid in–biotin system has evolved to be an extremely useful tool in the fields of biology, biochemistry, and biotechnology. The extraordinary affinity (Kd ∼ 10−15 M) of avid in (or its bacterial relative, streptavidin) for the vitamin biotin forms the basis for using this system. This approach has found broad application, mainly for localization studies, medical diagnostics, and recombinant genetics. The principles and scope of avidin–biotin technology have been extensively described and catalogued, and the reader is referred to the series of reviews which are available in the literature (1–5) as well as the book which has recently appeared (6).
Preparative gel electrophoresis
Authors: Lee Kelvin H , Harrington Michael G 
Published: November 2023

Abstract
Protein analysis has long served an important role in the biological and biomedical sciences. The most commonly used methods for separating protein mixtures are chromatography and gel electrophoresis. The essence of these two methods are the same because, in both, a sample mixture is separated into components by various molecular interactions while passing in a ‘liquid phase’ though a ‘solid phase’ matrix. The difference lies in the force used to drive the liquid phase through the solid phase: pressure in chromatography and an electric field in electrophoresis. Among its many applications, analytical protein electrophoresis can be used to determine biochemical characteristics such as the isoelectric point and mass of a protein, to catalogue protein content in complex mixtures with two-dimensional electrophoresis, and to identify the relative quantity of a particular protein in a mixture.
Polymer solution mediated size separation of proteins by capillary SDS electrophoresis
Authors: Guttman András , Shieh, Paul , Karger Barry L 
Published: November 2023

Abstract
Researchers in the late 1960s found that the molecular weight of protein molecules can be easily derived from their electrophoretic mobility, measured by SDS-PAGE (1). The methodology was later significantly improved (2) and progressed as a standard molecular weight determination method for proteins in slab or rod gel electrophoresis methods (3, 4). The gel acts both as a molecular sieve, enabling separations based on the size (hydrodynamic volume) of the SDS: protein molecules, and as an anti-convective medium, minimizing the conductive transport and reducing diffusion of the analyse molecules during the electrophoretic separation. Classical slab and rod gel electrophoresis is still performed manually, requiring gel casting, manual sampling, and laborious staining/destaining procedures for detection of the separated proteins.
Conventional isoelectric focusing in gel slabs, in capillaries, and immobilized pH gradients
Authors: Righetti Pier Giorgio , Bossi, Alessandra , Gelfi Cecilia 
Published: November 2023

Abstract
This chapter both updates our chapter in the previous edition of this book (1) and covers the most recent aspects of the technique, including capillary isoelectric focusing (cIEF), the rising star in the field of IEF methodologies. We also draw the reader’s attention to two textbooks on conventional IEF (2) and on IPGs (3). For capillary IEF (cIEF), we also recommend two recent reviews (4, 5). Fractionations which rely on differential rates of migration of sample molecules, for example along the axis of a chromatographic column or along the electric field lines in electrophoresis, generally lead to concentration bands or zones which are essentially always out of equilibrium. The narrower the band or zone the steeper the concentration gradients and the greater the tendency of these gradients to dissipate spontaneously. This dissipative trans port is thermodynamically driven; it relates to the tendency of entropy to break down all gradients, to maximize dilution and, during this process, to thoroughly mix all components (6). Most frequently, entropy exerts its effects via diffusion which causes molecules to move down concentration gradients and so produces band broadening and component intermixing.
Two-dimensional gel electrophoresis
Authors: Hanash Samir M 
Published: November 2023

Abstract
Two-dimensional gel electrophoresis (2DE) has been and remains a unique and powerful approach to resolve proteins and polypeptides in complex protein mixtures. However, in the past this approach has caused as much frustration as satisfaction to investigators because of its complexity, the difficulty of achieving good resolution and reproducibility, and the perceived existence of alternative molecular approaches, primarily at the nucleic acid level, to uncover gene products or gene expression patterns of interest. Thus, 2DE as a methodology has had a limited appeal to investigators. This situation has gradually been reversed in the past few years, due to a number of important developments, some technological and others related to a renewed emphasis on ‘going back to the proteins’ for a greater understanding of important bio logical processes, such as signal transduction, growth and differentiation, and cellular responses to a variety of agents and conditions. The renaissance in 2DE can also be linked to the greater availability of ready-made products for this approach, to an enhanced resolution over a wider pH range from the most acidic to the most basic, to the increased feasibility of identifying proteins separated by 2DE, and to the burgeoning availability of relevant resources on the Internet. A description of approaches and resources for the two-dimensional separation of proteins is provided in this chapter. The procedures described are all in use in the author’s laboratory and have been developed or adapted from published monographs.
Sequence analysis of gel-resolved proteins
Authors: Simpson Richard J , Reid Gavin E 
Published: November 2023

Abstract
It is well recognized that polyacrylamide gel electrophoresis, particularly 2DE (1), is one of the most powerful techniques for rapidly analysing complex protein mixtures. Indeed, when the human genome is fully sequenced, predicted to occur by the turn of the century (2, 3), the attention of biologists will move away from de novo protein sequencing and towards establishing the function of mature proteins in terms of their post-transitional modifications, spatial and temporal characteristics. In this post-genome era, 2DE, undoubtedly, will play a key role in strategies aimed at analysing the full protein complement expressed by a cell or tissue type. Moreover, electrophoretic techniques will continue to play an important role in dissecting biological processes such as development, differentiation, and signal transduction which involve the co ordinated expression of proteins that interact with one another in a regulated fashion.
Engineered growth factors and receptors, and their applications
Authors: Mckay Ian A , Bustin Atephen A , Brown Kenneth D 
Published: November 2023

Abstract
This chapter serves as an introduction to the rest of the book. In it you will find described the various types of growth factors and receptors, and their potential applications. This is followed by a discussion of systems for getting expression of recombinant growth factors and receptors, with protocols describing specific examples of in vitro and mammalian cell expression. Other examples will be found in Chapters 3, 4, 5, 7, and 9 in this volume.
In the first edition of Growth factors: a practical approach (1), published in 1993, were listed approximately 130 different known growth factors. By the time of the first reprint in 1995 this number had grown to over 170. At the time of going to press about 200 are known (see Appendix 1). Therefore it is apparent that new growth factors are still being discovered.
Structure-activity relationships of chemokines
Authors: Clark-Lewis Ian , Anderson Jennifer , Owen Philip , Vo Luan , Gong Jiang-Hong 
Published: November 2023

Abstract
This chapter focuses on structure-activity relationships (SAR) of protein growth and activation factors citing examples from our work on chemokines (1, 2). The emphasis is on general principles and approaches to SAR of secreted and soluble proteins, the primary function of which is to bind specific integral membrane receptors leading to a distinct cellular response. The study of SAR requires changing the covalent structure of a pro tein and determining the effects of the changes in terms of function and three-dimensional (3D) structure. The structure could be changed by either chemical modification or by de novo synthesis. Compared with chemical modification, the synthesis approach has the advantages that a large repertoire of changes can be introduced into any position and that the covalent structure is defined. Thus, the de novo synthesis approach will be the subject of this chapter.
Generation and selection of RNA ligands that inhibit the interaction of vascular permeability factor/vascular endothelial growth factor (VPF/VEGF) with its receptors
Authors: Green Louis S , Janjic Nebojsa 
Published: November 2023

Abstract
Storage and transmission of genetic information by nucleic acids is critically dependent on their ability to interact with proteins. The formation of specific protein:nucleic acid complexes is a function of precisely defined three dimensional structures of both binding partners. Like proteins, nucleic acids are capable of folding into a wide variety of three-dimensional structures. The ability of nucleic acids to form secondary and tertiary structures such as Watson-Crick base pairing, Hoogstein, and other non-canonical base pairing, pseudoknots, and G-quartets is determined by their sequence. Sequence randomized nucleic acid libraries therefore contain a collection of binding specificities.