Molecular Neurobiology: Recombinant DNA Approaches

ISBN-13: 9781461574903 / Angielski / Miękka / 2012 / 297 str.

Steve Heinemann; James Patrick

Opis

This book is a collection of papers describing some of the first attempts to apply the techniques of recombinant DNA and molecular biology to studies of the nervous system. We believe this is an important new direction for brain research that will eventually lead to insights not pos- sible with more traditional approaches. At first glance, the marriage of molecular biology to brain research seems an unlikely one because of the tremendous disparity in the histories of these two disciplines and the problems they face. Molecular biology is by nature a reductionist approach to biology. Molecular biologists have always tried to attack central questions in the most direct approach possible, usually in the most simple system available: a bacterium or a bacterial virus. Important experiments can usually be repeated quickly and cheaply, in many cases by the latest group of graduate students entering the field. The success of molecular biology has been so profound because the result of each important experiment has made the next critical question obvious, and usually answerable, in short order. Studies of the nervous system have a very different history. First, the human brain is what really interests us and it is the most complex structure that we know in biology. The central question is clear: How do we carry out higher functions such as learning and thinking? How- ever, at present there is no widely accepted and testable theory of learn- ing and no clear path to such a theory.

Spis treści

1. The Molecular Biology of the Na,K-ATPase and Other Genes Involved in the Ouabain-Resistant Phenotype.- 1. Introduction.- 2. Molecular Cloning of the Na,K-ATPase Catalytic Subunit.- 3. Organization and Expression of the Rat Sodium Pump a-Subunit Gene.- 4. Organization and Expression of the a-Subunit Gene in Ouabain-Resistant Cell Lines.- 5. Isolation and Characterization of a Ouabain-Resistance Gene.- 6. Transfer of the Sodium Pump a-Subunit Gene Confers Ouabain Resistance to Ouabain-Sensitive Cells.- 7. Isolation of Genes Related to the Sodium Pump and Their Expression in a Ouabain-Resistant Cell Line.- 8. Conclusions and Future Prospects.- References.- 2. Molecular Biology of the Genes Encoding the Major Myelin Proteins.- 1. Introduction.- 2. Formation and Structure of the Myelin Sheath.- 3. Myelin-Specific Proteins.- 3.1. Myelin Basic Protein.- 3.2. Po.- 3.3. Proteolipid Protein.- 3.4. Other Myelin Proteins.- 4. Conclusion.- References.- 3. Molecular Biology of the Neural and Muscle Nicotinic Acetylcholine Receptors.- 1. Introduction.- 2. Isolation of cDNA Clones Coding for the Acetylcholine Receptor Expressed in the Torpedo Electric Organ.- 2.1. Torpedo ?-Subunit.- 2.2. Torpedo ?-Subunit.- 2.3. Torpedo Clone 3C1.- 3. Isolation of cDNA Clones Coding for Mouse Skeletal Muscle Acetylcholine Receptor.- 3.1. Mouse ?-Subunit.- 3.2. Mouse ?-Subunit.- 3.3. Mouse ?-Subunit.- 3.4. Mouse ?-Subunit.- 3.5. Expression of Mouse Receptor in Oocytes.- 4. Structure of the Acetylcholine Receptor.- 4.1. Primary Structure.- 4.2. Folding through the Membrane.- 4.3. Acetylcholine-Binding Site.- 5. Expression of the Acetylcholine Receptor Genes in Skeletal Muscle.- 5.1. Skeletal Muscle Denervation.- 5.2. Synaptic versus Extrasynaptic Acetylcholine Receptor.- 6. Brain Receptors.- 6.1. Neuronal a-Subunit Clone.- 6.2. Structure of the Neuronal ?-Subunit.- 6.3. Expression of the Neural ?-Subunit RNA.- 6.4. Evidence for a Gene Family.- 7. Conclusion.- References.- 4. Molecular Biology of Muscle Development: The Myosin Gene Family of Caenorhabditis elegans.- 1. Introduction.- 2. Genetics of the unc-54 Locus.- 2.1. Selection of unc-54 Mutations.- 2.2. Deletions and Duplications of the unc-54 Locus.- 2.3. Orientation of the unc-54 Gene.- 2.4. In Situ Hybridization to Chromosomes.- 2.5. Interactions with Other Genes.- 3. Immunological Identification and Localization of Myosin Isoforms.- 3.1. Production of Four Sarcomeric Myosin Heavy-Chain Isoforms by Caenorhabditis elegans.- 3.2. Monoclonal Antibodies Specific for Different Myosin Isoforms.- 3.3. Two Myosins Required to Make the Body Wall Thick Filament.- 4. Molecular Cloning of the unc-54 and myo-1,2,3 MHC Genes.- 4.1. Cell-Free Translation of MHC mDNA.- 4.2. unc-54 cDNA Clones.- 4.3. Cloning the unc-54 Gene.- 4.4. Identification of the myo-1,2,3 MHC Genes by Homology to unc-54.- 5. Immunological Identification of the Products of the myo-1,2,3 MHC Genes.- 6. Structural Organization of the Nematode MHC Genes.- 6.1. Highly Conserved Head Sequences and Variable Rod Sequences.- 6.2. Selective Loss of Introns from the Nematode MHC Genes.- 6.3. Unusual Features at Intron Junctions.- 6.4. Terminal Sequences.- 7. Molecular Anatomy of the Myosin Molecule.- 7.1. Topography of the Head.- 7.2. Rod Sequences.- 8. Sequences and Molecular Interpretation of unc-54 Mutations.- 8.1. Rapid Cloning and Sequencing of Mutations.- 8.2. Correlation of Physical and Genetic Fine-Structure Map.- 8.3. Deletions in the Rod.- 8.4. Nonsense Mutations and Suppressors.- 8.5. Mutations Affecting the Synthesis of MHC.- 8.6. A Dominant Assembly-Defective Missense Mutation.- 8.7. Mutations in the Head.- 8.8. Mechanisms of Mutagenesis in the Nematode..- 9. Myosin Protein Expression and Mutagenesis in E. coli.- References.- 5. Small Cardioactive Peptides in A and B: Chemical Messengers in the Aplysia Nervous System.- 1. Introduction.- 2. The Distribution of SCP-Immunoreactive Neurons in the CNS.- 3. The SCP Gene and Precursor Protein.- 3.1. Cloning and Characterization of the SCP Gene.- 3.2. The SCP Precursor Protein.- 4. SCP Gene Expression.- 5. Subcellular Localization.- 6. Coexistence of Multiple Transmitters.- 6.1. SCPA and SCPB.- 6.2. Acetylcholine in Neuron B2.- 7. Physiological Activities.- 8. Conclusions.- References.- 6. Molecular Biology Approach to the Expression and Properties of Mammalian Cholinesterases.- 1. Introduction: Expression of Cholinesterases as a Research Subject—Scientific Significance, Advantages, and Difficulties.- 1.1. Detection of Enzymatic Activity.- 1.2. Polymorphism of Cholinesterases.- 1.3. Tissue and Cell-Type Specificity.- 1.4. Putative Biological Role(s).- 1.5. Genetic Evidence for Allelic Polymorphism.- 1.6. Molecular Approach to Cholinesterases.- 1.7. General Research Strategy: Simultaneous Experiments Approaching Various Levels of Gene Expression.- 2. Expression of Cholinesterase mRNAs in Microinjected Xenopus Oocytes.- 3. Identification of Drosophila DNA Fragment that Hybridizes with Cholinesterase mRNA.- 3.1. Assignment of Drosophila Transcripts that Hybridize with DroS.- 3.2. Hybrid Selection of Cholinesterase-Inducing mRNA by DroSR.- 3.3. RNA Blot Hybridization Reveals Homology between DroSR and mRNA from Human Cholinesterase-Expressing Tissues.- 4. Isolation and Partial Characterization of Human DNA Fragments Homologous to DroSR.- 4.1 Isolation and Characterization of a Human Genomic Fragment: Huachel.- 4.2. Hybridization of HuachelR DNA with Poly(A)+ RNA Species from Fetal Human Brain.- 4.3. Hybrid Selection of Acetylcholinesterase-Inducing mRNA with HuachelR DNA.- 4.4. Immunoprecipitation of Acetylcholinesterase Polypeptides from Oocytes Injected with Hybrid-Selected mRNA.- 4.5. Preliminary Characterization of HuachelR DNA.- 4.6. Isolation of HuachelR-Homologous Genomic DNA Fragments.- 4.7. Preparation of Huache1R-Homologous Fetal cDNA Clones.- 4.8. Conclusions.- 5. Preparation of Synthetic Oligonucleotide Probes According to the Consensus Sequence at the Organophosphate-Binding Site.- 5.1. Strategy for Preparation and Selection of the Correct Mixture of Synthetic Oligonucleotides.- 5.2. Probing of Selected DNA Fragments with the Synthetic Oligonucleotide.- 5.3. Screening for OPSYN-Containing cDNA Sequences from cDNA Libraries in Xgt Vectors.- 6. Preliminary Characterization of Neuroche cDNA Clones.- 6.1. Blot Hybridizations with Restricted Genomic DNA (Human and Mouse) and with mRNA.- 6.2. Identification of Acetylcholinesterase-Immunoreactive Fusion Protein by Crossed Immunoelectrophoresis and Immunoprecipitation.- 7. Summary and Conclusions.- References.- 7. Genes and Gene Families Related to Immunoglobulin Genes.- 1. Introduction.- 2. Immunoglobulin Genes and the Immunoglobulin Domain.- 3. The T-Lymphocyte Cell-Surface Receptor for Antigen.- 4. Class I MHC Genes.- 4.1. H-2 K, D, and L.- 4.2. Qa/TLa Genes.- 4.3. ?2-Microglobulin.- 5. Class II MHC Genes.- 6. Cell-Surface Receptors for Transepithelial Transport of Immunoglobulin.- 7. The T-Cell Accessory Molecules T4 and T8.- 8. Related Members of the Immunoglobulin Supergene Family Expressed in the Nervous System.- 8.1. The Thy-1 Glycoprotein.- 8.2. The OX-2 Antigen.- 9. Conclusion: The Evolution of the Immunoglobulin Super family..- References.- 8. Specificity of Prohormone Processing: The Promise of Molecular Biology.- 1. Introduction.- 1.1. Tissue-Specific Processing of Opioid Peptides.- 1.2. Limitations of Classic Approaches to the Study of Prohormone Processing.- 2. Gene-Transfer Systems.- 2.1. Transfer of Proenkephalin into Mouse Pituitary Cells.- 2.2. Other Gene-Transfer Studies.- 2.3. Reduction of Enzyme Activity with Antisense RNA.- 3. Enzymatic Studies.- 3.1. Trypsinlike Processing Enzymes.- 3.2. Carboxypeptidase E (Enkephalin Convertase).- 4. The Internal Environment of Secretory Granules.- 5. Perspectives.- References.