List of ContributorsPreface1 Current Genetic Approaches to the Mammalian Nervous System I. Introduction II. Inherited Variations in Genes and Gene Products III. Generating Genetic Variants IV. Assessing the Role of Heredity in Behavior and Disease V. Conclusion References2 Chromosomal Components in Brain Cells I. Introduction II. Organization of Chromatin III. Brain Histones IV. Nonhistone Chromosomal Proteins (NHCP) of the Brain V. DNA Polymerase VI. RNA Polymerase VII. Concluding Remarks References3 The Sequence Complexity of Brain Ribonucleic Acids I. Introduction II. RNA-DNA Hybridization Analysis III. Complexity of Gene Transcription in the Brain IV. RNA Sequence Complexity at the Level of Translation V. Regional and Cellular Distribution of Gene Transcripts VI. Gene Activity during Development and Aging VII. Concluding Remarks References4 Molecular Characterization of Synapses of the Central Nervous System I. Introduction II. Isolation and Characterization of Synaptic Junctional Complexes and Postsynaptic Densities III. Protein Composition of Isolated Synaptic Junctions and Postsynaptic Densities IV. Functional Properties of Synaptic Junctions and Postsynaptic Densities V. Development of the Synapse VI. Conclusions References5 Axonal Transport of Macromolecules I. Introduction II. Slow Axonal Transport III. Rapid Axonal Transport IV. Conclusions References6 Mechanistic Studies on the Cellular Effects of Nerve Growth Factor I. NGF and Its Target Cells II. Role of Transcription in NGF Mechanism of Action on Neurite Outgrowth III. Priming Model of Neurite Outgrowth IV. Transcriptional Regulation of NGF Responses Other Than Neurite Outgrowth V. NGF Receptors and Transcription VI. Nontranscriptional, Anabolic, and Rapid Effects of NGF VII. Genetic Approaches to NGF Molecular Mechanism VIII. NGF and Cell Proliferation IX. Concluding Remarks References7 Cell Interactions in Embryonic Neural Retina: Role in Hormonal Induction of Glutamine Synthetase I. Introduction II. Neural Retina of Chick Embryo III. Glutamine Synthetase (GS) in Avian Neural Retina IV. Induction of GS V. Cell Interactions and GS Induction VI. Cortisol Receptors VII. Comment References8 Sexual Differentiation of the Brain: Gonadal Hormone Action and Current Concepts of Neuronal Differentiation I. Introduction II. General Plan of Sexual Differentiation for Mammals III. Sexual Differentiation in Rats IV. Morphological Sex Differences in the Brain V. Sexual Differentiation in Relation to Current Concepts of Neuronal Development and Differentiation VI. Conclusions References9 Analysis of Protein Synthesis in the Mammalian Brain Using LSD And Hyperthermia as Experimental Probes I. Introduction II. Perturbation of Protein Synthesis in the Brain III. Effect of LSD on the Translational Apparatus of the Brain IV. Effect of Hyperthermia on the Translational Apparatus of the Brain V. Effect of LSD and Hyperthermia on Brain Protein Synthesis In Vivo VI. Effect of LSD and Hyperthermia on Subsequent Cell-Free Protein Synthesis in the Brain VII. Concluding Remarks References10 Neuropeptides as Putative Neurotransmitters: Endorphins, Substance P, Cholecystokinin, and Vasoactive Intestinal Polypeptide I. Introduction II. Endogenous Opioid Peptides III. Substance P IV. Cholecystokinin and Vasoactive Intestinal Polypeptide V. Conclusion References11 Molecular Correlates between Pituary Hormones and Behavior I. Introduction II. Behavioral Effects of Pituitary Hormones III. Molecular Correlates of Pituitary Peptide Hormones IV. Concluding Remarks References12 Macromolecules and Behavior I. Introduction II.
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CURRENT GENETIC APPROACHES TO THE MAMMALIAN NERVOUS SYSTEM
Xandra O. Breakefield, John E. Pintar and Michael B. Rosenberg
Publisher Summary
This chapter describes genetics as a set of concepts that provide unique insights into complex natural phenomena. It presents genetic concepts that are combined with new techniques in molecular biology, cell culture, biochemistry, and developmental biology to expand an understanding of the nervous system. Knowledge of the number, nature, and position of genes controlling neural properties explains the molecular basis of the expression, structure, function, and interaction of these properties. It is also possible to selectively alter the genotype of cells and to use these cells to create animals with known genetic lesions. Further, naturally occurring variations in DNA and protein structure can be assessed and the effects of these variations on neural function evaluated.
I Introduction
II Inherited Variations in Genes and Gene Products
A Identifying Genes and Their Products
B Locating Genes
III Generating Genetic Variants
A In Cell Culture
B In Animals
IV Assessing the Role of Heredity in Behavior and Disease
A Populations and Individuals
B Cells and Molecules
V Conclusion
References
I INTRODUCTION
Genetics, as other scientific disciplines, is a set of inherently consistent concepts which can provide unique insights into complex, natural phenomena. This chapter will examine how genetic concepts can be combined with new techniques in molecular biology, cell culture, biochemistry, and developmental biology to expand our understanding of the nervous system. Knowledge of the number, nature, and position of genes controlling neural properties can elucidate the molecular basis of the expression, structure, function, and interaction of these properties. Further, by perturbing neural function at the level of the gene, the relationships between specific molecules and behavior can be established. Genetic studies should contribute not only to an integrated, multidimensional view of the nervous system, but also to an understanding of the molecular etiology of inherited neurologic and psychiatric diseases. Here we will summarize genetic approaches currently available and their potential use in the study of proteins critical to neural function.
II INHERITED VARIATIONS IN GENES AND GENE PRODUCTS
A Identifying Genes and Their Products
1 Number and Nature of Genes Coding for Proteins
In analyzing a particular protein and its genetic determinants, it is first helpful to establish its structure. If a protein can be purified in sufficient quantity, this can be determined biochemically. If, however, the protein is difficult to purify or exists in more than one form, a genetic analysis becomes useful. A discrete set of genes is necessary for the expression of any protein. These include structural genes coding for the primary amino acid sequence of the subunit(s) and other enzymes involved in processing of mRNA, post-translational modification of the protein, and metabolism of associated molecules. In addition, regulatory genes may affect the expression of structural genes. Examples will be discussed in which families of genes, arising from gene duplication and evolutionary divergence, can give rise to a set of functionally related or functionally distinct proteins, and in which one gene can give rise to a number of different polypeptides.
These types of potential genetic diversity in neural proteins will be considered for the following proteins: tubulin, the acetylcholine receptor, monoamine oxidase (MAO), the insulin family of polypeptides [which includes nerve growth factor (NGF)], adrenocorticotropin (ACTH) β-lipotropin precursor polypeptide, somatostatin, and myelin basic protein. These studies serve to illustrate the extent to which variation in protein structure can result from differences in structural genes coding for these proteins, as well as in processing of mRNA precursors and post-translational modification of polypeptides.
a Related Genes Code for Functionally Related Proteins.
Multiple forms of tubulin, the major component of microtubules, have been identified by biochemical criteria. Two related forms of this protein, α- and β-tubulin have been identified (for review see Raff, 1979). As isolated from chick brain these forms do not differ in molecular weight, but can be distinguished on the basis of peptide maps and isoelectric points (Nelles and Bamburg, 1979). The structural similarity of these two tubulin proteins suggests that genes coding for them arose from a common precursor gene (Cleveland et al., 1980). DNA sequences containing genes coding for α- and β-tubulins have been identified using cDNA probes for them prepared from mRNA of chick brain, where tubulin represents % of the total cellular protein. Digestion of chick DNA with several restriction endonucleases reveals the presence of four unique fragments, which hybridize with both the 3′ and 5′ ends of each of the probes. This provides strong evidence that at least four separate genes code for each form of tubulin. Comparable analysis of human and rodent DNA reveals about ten genes coding for each form. Thus, what appeared to be two related proteins by biochemical criteria has been resolved into a family of eight to twenty related proteins by genetic criteria.
The nicotinic acetylcholine receptor, which mediates membrane events in synaptic transmission, represents another protein critical to nerve function for which two forms, junctional and extrajunctional, have been identified. Although in rat muscle these types of receptors can be distinguished on the basis of their affinities for d-tubocurarine, isoelectric points, and immunologic properties, they have the same subunit composition, and the subunits derived from them yield indistinguishable peptide maps (Nathanson and Hall, 1979). It is still not clear whether subunits of these two receptors originate from separate gene loci or represent different post-translational modifications of the same protein. Messenger RNA isolated from the electric organ of Torpedo can be used to direct the in vitro synthesis of polypeptides that cross-react with antibodies prepared against the acetylcholine receptor. Some of these newly synthesized polypeptides have apparent molecular weights different from the subunits of the receptor (Mendez et al., 1980). These data are consistent with the known post-translational modifications of these subunits (Raftery et al., 1980). Amino acid sequencing of the five subunits of the Torpedo receptor shows that two are identical and all are structurally related, suggesting that they arose from a common precursor gene. Analysis of the number and structure of genes coding for the mammalian acetylcholine receptor may provide insight into the relationship between junctional and extrajunctional receptors.
Monoamine oxidase, which degradatively deaminates biogenic amines throughout the body, also has two distinct functional types. The A type of MAO activity has a higher affinity for serotonin and norepinephrine and is inhibited by lower concentrations of clorgyline; while the B type has a higher affinity for phenylethylamine and benzylamine and is selectively sensitive to low concentrations of deprenyl (Murphy, 1978; Houslay et al., 1976). The flavin containing polypeptides of MAO-A and -B, isolated from rat and human sources, are structurally different on the basis of apparent molecular weight and peptide maps (Callingham and Parkinson, 1979; Cawthon et al., 1981; Brown et al., 1980; Cawthon and Breakefield, 1979). It is still not clear whether these structural differences result from variations in the primary amino acid sequence of different polypeptides or from post-translational modifications of the same polypeptide. For enzymes such as MAO, which are difficult to purify and for which there is no readily available source of mRNA, techniques of somatic cell genetics provide a means to resolve this issue (see Section II,B,1).
b Related Genes Code for Functionally Distinct Proteins.
A number of genes, which presumably have originated from gene duplications and evolutionary divergence in DNA sequence, can give rise to polypeptides that have substantial homology, but differ dramatically in function. Many “families” of related peptide hormones which serve both neural and endocrine functions have been described (Dockray, 1979; Stewart and Channabasavaiah, 1979). One family, the insulin-related polypeptides, includes insulin, relaxin, the insulin-like growth factors (Blundell and Humbel, 1980), and NGF (Bradshaw et al., 1974). Preliminary studies (L....
