Molecular Approaches to Neurobiology (eBook)

Ian R. Brown (Herausgeber)

eBook Download: PDF | EPUB

2014 | 1. Auflage
434 Seiten
Elsevier Science (Verlag)
978-1-4832-8229-9 (ISBN)

Infos

Molecular Approaches to Neurobiology deals with molecular approaches to the analysis of the nervous system. This book surveys the current state of knowledge in a number of areas of molecular neurobiology that includes the subcellular level of chromatin structure, complexity of RNA synthesis, role of hormones in cell differentiation, and molecular correlates between neuropeptides and behavior. A comprehensive review of procedures for the isolation of specific brain cells and their experimental use is also provided at the end of this text. This publication is useful to those working in the field of neurochemistry and those engaged in morphological and physiological approaches to the analysis of the nervous system, as well as molecular biologists and biochemists working with non-neural tissues.

Textauszug

1

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....

Produkt-Details

• Erscheint lt. Verlag: 28.6.2014
• Sprache: englisch
• Themenwelt: Sachbuch/Ratgeber ► Natur / Technik ► Naturführer
• Themenwelt: Naturwissenschaften ► Biologie ► Zoologie
• Themenwelt: Technik
• ISBN-10: 1-4832-8229-5 / 1483282295
• ISBN-13: 978-1-4832-8229-9 / 9781483282299