1st Edition

Functional Analysis of the Human Genome

    256 Pages
    by Garland Science

    An excellent review of the relationship between structure and function in the human genome, and a detailed description of some of the important methodologies for unravelling the function of genes and genomic structures.

    Contributors -- Abbreviations -- Preface -- Foreword /V.A. McKusick -- 1 Structure and function in the human genome /D.N. Cooper -- Introduction -- Chromatin structure and transcription -- Chromatin structure -- Nucleosome positioning -- Transcriptional domains -- Chiasmata, recombination and recombination hotspots -- Scaffold attachment regions -- Centromeres -- Telomeres -- Origins of DNA replication -- Genes -- Gene structure and organization -- seudogenes -- Functional organization of human genes -- Repetitive sequence elements -- Tandem repeats -- Alu sequences -- LINE elements -- Endogenous retroviral sequences -- Transcriptional regulation -- Promoter elements -- Enhancers -- Negative regulatory elements -- Locus control regions -- Boundary elements -- Trans-acting protein factors -- Sequences involved in transcriptional termination -- mRNA splicing and processing -- Sequences involved in determining mRNA stability -- Role of sequences in 5' untranslated regions -- DNA methylation -- Distribution of 5-methylcytosine -- Replication of the methylation pattern and de novo methylation -- Role of DNA methylation in the regulation of transcription -- Role of DNA methylation in X-inactivation -- Changes in DNA methylation during embryogenesis -- DNA methylation and imprinting -- References -- 2 Mapping the human genome /D.N. Cooper -- Introduction -- Markers -- Gene sequences -- DNA polymorphisms -- D-segments -- Sequence-tagged sites -- Inter-Alu PCR probes -- Allele-specific oligonucleotides -- Cytogenetic mapping -- Somatic cell hybrid analysis -- Radiation hybrid mapping -- Fluorescence in situ hybridization -- In situ PCR -- High-resolution physical mapping -- Yeast artificial chromosome cloning -- Contig assembly -- Pulsed-field gel electrophoresis and CpG island mapping -- Chromosome jumping /linking libraries -- DNA sequencing -- Progress in physical mapping -- Genetic mapping -- Transcription map of the human genome -- Comparative gene mapping -- References -- 3 Cloning the transcribed portion of the genome /P. Towner -- Introduction -- Gene detection -- Preparation of target material -- Isolation of total RNA -- Isolation of mRNA -- Preparation of cDNA -- Selection of specific genes -- Library-based cDNA cloning strategies -- Construction of a cDNA library -- Screening cDNA libraries -- Manipulation of identified cDNA sequences -- PCR-based isolation of genes from cDNA -- Primer design -- Mixed-pool or redundant oligonucleotide primers -- Primary PCR reaction -- Isolation of the 3' end of a cDNA -- Isolation of the 5' end of a cDNA -- Gene identification by differential display -- Expression systems -- Expression using E. coli -- Eukaryotic expression systems -- References -- 4 Retroviral insertional mutagenesis. F. Farzaneh, J. Gaken -- andS.-U. Gan -- Introduction -- The retroviral life cycle -- Host range -- Replication-defective retroviral vectors -- Packaging cell lines -- Conditions required for efficient mutagenesis -- Mechanisms involved in retroviral insertional mutagenesis -- Mutation frequency -- Multiplicity of infection -- Mutant selection procedures -- Cloning of the sites of pro virus integration -- Construction of genomic libraries -- PCR-mediated amplification -- Identification of the gene of interest -- Identification of common sites of provirus integration -- Library screening by nuclear run-on probes -- References -- 5 Gene entrapment. H. von Melchner and H.E. Ruley -- Introduction -- Gene trap vectors -- Cloning and analysis of flanking sequences -- Isolation and use of promoter-tagged sites -- Insertional mutagenesis in cultured cells -- Insertional mutagenesis in mice -- Identification of regulated genes -- References -- 6 Gene transfer studies. D. Darling and M. Kuiper -- Introduction -- What is transfection? -- What form should the DNA be in? -- Generalized requirements for eukaryotic gene transcription -- Eukaryotic gene transcription -- SV-based plasmids -- Specialized eukaryotic host cells -- Specialized plasmids -- Double insert plasmids -- Inducible expression -- Epstein-Barr virus-based plasmids -- Shuttle vectors -- Multifunctional plasmids -- Transfection procedures -- Calcium phosphate co-precipitation -- DEAE-dextran -- Electroporation -- Liposomes and lipid-based transfection -- Adenovirus and poly-L-lysine-conjugated complexes -- Alternative transfection procedures -- Assays for new protein synthesis -- Dominant selectable marker genes -- Reporter genes -- Analysis of cloned genes -- Identification of ligands for novel receptors -- Identification of transcription factors -- References -- 7 Foreign DNA integration and DNA methylation patterns /W. Doerfler -- Introduction -- The adenovirus system as a model -- Site selection in the integration of adenovirus DNA -- Modes of adenovirus DNA integration - a synopsis of data -- On the mechanism of integrative recombination -- Insertion of foreign DNA by a versatile mechanism -- Studies on the mechanism of integrative recombination in a cell-free system -- De novo DNA methylation of integrated foreign DNA -- De novo methylation of integrated foreign DNA: a cellular defence mechanism? -- Initiation of de novo methylation in mammalian cells is not predominantly dependent upon the nucleotide sequence of foreign DNA -- Methylation of triplet repeat amplifications in the human genome: manifestation of the cellular defence mechanism -- Alterations in patterns of cellular DNA methylation and gene expression as consequences of foreign DNA insertions into mammalian genomes -- DNA methylation and gene activity -- A fully 5'-CG-3' but not a 5'-CCGG-3' methylated late FV3 promoter retains activity -- Topology of the promoter of RNA polymerase II- and Ill-transcribed genes is modified by the methylation of 5'-CG-3' dinucleotides -- Impact of 5'-CG-3' methylation on the activity of different eukaryotic promoters: a comparison -- Uptake of foreign DNA through the gastrointestinal tract -- A concept of oncogenesis - implications for gene therapy and research on transgenic organisms -- References -- 8 Transgenic animals in human gene analysis /F. Theuring -- Introduction -- Methodology -- Transgenes to study gene regulation -- Transgenes to study gene function -- Functional analysis: gain-of-function -- Functional analysis: loss-of-function -- Conclusions -- References -- 9 Homologous recombination /A. Mansouri -- Introduction -- Embryonic stem cells -- Principles of homologous recombination in mammalian cells -- Targeting vectors -- Promoterless constructs -- Positive-negative selection procedure -- Hit-and-run and in-out targeting strategies -- Potential of homologous recombination in embryonic stem cells -- Developmental biology -- Animal models of human disease -- Homologous recombination and gene therapy -- Future perspectives: Cre-LoxP mediated gene targeting -- References -- 10 Complementation analysis /A. Patel -- Introduction -- Principles of somatic cell hybridization -- De novo and salvage pathways of nucleotide synthesis -- Purine nucleotide synthesis -- The HPRT gene -- HPRT variants -- Pyrimidine nucleotide synthesis -- Metabolic cooperation -- The HAT selection system -- Selection procedures for the isolation of hybrid cells -- Identification of complementation groups and topological relationships -- Extinction and activation -- Assignment of complementation groups in clinical diseases -- Assignment of complementation groups in senescence -- Assignment of complementation groups in biochemical pathways -- Assignment of complementation groups in cytokine activity -- Identification of the dominant /recessive nature of genetic lesions -- Chromosome segregation -- Dominant and recessive genetic changes involved in senescence -- Dominant and recessive nature of viral genes -- Dominant and recessive events in tumour progression -- Dominant nature of multi-drug resistance genes -- Dominant and recessive events involved in the immunological process -- Dominant and recessive developmentally regulated genes -- Microcell fusion: principles and application to the chromosomal localization of genes -- Introduction to microcell fusion -- General principles for microcell-mediated transfer -- Pinpointing chromosomes involved in specific disease processes -- Identification of tumour suppressor genes -- Identification of genes involved in cellular senescence -- References -- 11 Antisense oligonucleotides: a survey of recent literature, possible mechanisms of action and therapeutic progress /D. Pollock andj. Gaken -- Introduction -- Some examples of antisense action in different systems -- Targeting and design -- Uptake of antisense oligonucleotides -- Toxicity of antisense oligonucleotides -- Modifications to the structure of antisense oligonucleotides -- Possible mechanisms of action -- Steric inhibition -- RNase H-like cleavage of target RNA -- Triplex DNA formation -- Double-stranded oligonucleotides -- Circular oligonucleotides Ribozymes -- Non-specific cleavage of host RNA -- Therapeutic applications -- References -- Index.

    Biography

    Department of Molecular Medicine, King’s College School of Medicine and Dentistry, London, UK. Charter Molecular Genetics Laboratory, Thrombosis Research Institute, London, UK.

    "a very worthwhile attempt to draw together the various technologies involved in analysing the function of the human genome." and "it is informative and the gathering of material from several disciplines is helpful. The explosion of data about genes and