Molecular Approaches to Neurobiology

Netherlands.

PREFACE

The neurosciences have experienced marked growth over the past few years as evidenced by the rapid expansion of journals and societies devoted to brain research. This field is of importance not only to those who traditionally have studied the nervous system but to a broad spectrum of biomedical scientists since it represents a frontier research area in which new discoveries are frequently made. The disciplines of anatomy, physiology, and neurochemistry have contributed greatly to the development of the neurosciences. More recent is the contribution of cellular and molecular biology which has opened avenues of investigation into mechanisms regulating differentiated function in cells of the nervous system.

This book deals with current molecular approaches to the analysis of the nervous system. The scope of topics ranges from the subcellular level of chromatin structure and complexity of RNA synthesis to the role of hormones in cell differentiation and molecular correlates between neuropeptides and behavior. The author of each chapter presents an overview of his or her area of speciality, highlighting recent advances and, where appropriate, pointing out current problems which might be impeding progress. Each contribution includes current results from the author’s laboratory and concludes with a viewpoint on future directions. The last chapter includes a comprehensive review of procedures for the isolation of specific brain cells and their experimental use.

This book should be useful both to those working in the field of neurochemistry and to those engaged in morphological and physiological approaches to the analysis of the nervous system. Molecular biologists and biochemists working with non-neural tissues may also be interested in this book which surveys the current state of knowledge in a number of areas of molecular neurobiology.

Ian R. Brown

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. Villa-Komaroff, personal communication) using a DNA probe for the human insulin gene show that human brain contains at least six distinct mRNA species with substantial homology to this probe; some of these messages may code for as yet uncharacterized neurotropic peptides. Insulin will be considered here because it has been studied in the greatest detail and it serves as a prototype for neuronally active peptides. Insulin is synthesized as a larger molecular weight precursor polypeptide, pre-proinsulin. The initial cleavage in processing involves loss of an amino acid signal sequence. The resulting molecule, proinsulin, then folds with the formation of two disulfide bonds. Two subsequent site-specific cleavages yield two peptide chains joined by the disulfide bonds (insulin) and a free peptide which is subsequently lost. Using cloned cDNA fragments that correspond to mRNA coding for pre-proinsulin, it has been established that most mammalian species contain only one gene for insulin. However, some rodents have two separate gene loci for this protein which differ only slightly in structure (Cordell et al., 1979; Lomedico et al., 1979). Both forms of insulin are synthesized in equal amounts and appear to be functionally equivalent.

The biologically active subunit of NGF, β-NGF, is also derived from a larger molecular weight precursor pro-β-NGF (Berger and Shooter, 1977), which in turn may be derived from an even larger MW precursor (Wiche, 1979). Genes for β-NGF and insulin are believed to have arisen from a common precursor gene (Bradshaw et al., 1974). However, proinsulin and β-NGF polypeptides have only about 30% sequence homology indicating that a substantial degree of diversification has occurred since the putative gene duplication event. These differences in amino acid sequence suggest that these two polypeptides may differ in conformation, and, in fact β-NGF is not able to compete with insulin for its receptor (Blundell and Humbel, 1980).

c One Gene Codes for Many Proteins.

An interesting example of one gene transcript giving rise to a number of functionally distinct polypeptides is provided by the ACTH-β-lipotropin precursor molecule synthesized in the pituitary (Roberts et al., 1978; Eipper and Mains, 1980; Herbert, 1981) and possibly in the brain (Krieger and Liotta, 1979). Different subsets of biologically active peptides are generated from similar and possibly identical precursor molecules by differential post-translational modifications mediated by site-specific proteolysis and glycosylation (Fig. 1; Roberts et al., 1978). In the anterior lobe of the pituitary, ACTH and β-lipotropin are the prominent end-product peptides, whereas in the intermediate lobe, α-melanotropin, corticotropin-like intermediate lobe peptide, and β-endorphin are the major end-products. These differences in processing are apparently lobe specific throughout development (J. Pintar and R. Allen, personal communication). Although there appears to be only one bovine gene coding for the ACTH β-lipotropin precursor (Nakanishi et al., 1980), there appear to be two rodent genes (J. Roberts, personal communication), and thus differential processing in this latter species may result from differential gene expression.

Fig. 1 The ACTH-β-lipotropin precursor polypeptide and different modes of processing. in the anterior lobe of the pituitary proteolytic cleavages and glycosylation events yield primarily ACTH and β-lipotropin (β-LPH) whereas in the intermediate lobe of the pituitary these events yield α-melanotropin, corticotropin-like intermediate lobe peptide (CLIP), γ-lipotropin (γ-LPH), and β-endorphin. (Reprinted from Herbert, 1981, with permission.)

Another example where one gene produces two structurally different, but functionally equivalent, peptides is provided by human somatostatin, a growth hormone produced by the pituitary. Two forms differing in molecular weight can be identified; the smaller is identical to the larger except that it lacks an internal sequence of amino acids (Lewis et al., 1980). Nucleotide sequencing of the gene for somatostatin reveals that the smaller form is generated when an error occurs during excision of an intron region of the precursor mRNA (Fiddes et al., 1979). Such processing errors can be explained by regions near the splicing signals which are similar in sequence to them. The occurrence of several common modes of processing of the same precursor mRNA may also explain the four forms of myelin basic protein normally present in mice which are identical in sequence except for the presence or absence of two internal sequences (Barbarese et al., 1977).

These findings show that the relationship between gene structure and protein structure is not always apparent, and that even functionally identical proteins are not necessarily the product of a single gene locus. Even in the case of monomeric proteins, structural diversity may result from differences in the sequence of separate gene loci or different alleles at the same locus, as well as in post-transcriptional processing of precursor mRNA and post-translational modification of polypeptides. A number of techniques are available to elucidate structural differences in proteins. The most complete involves amino acid sequencing, but not all proteins can be obtained in sufficient quantities for this procedure. Other techniques include electrophoretic separation of proteins on the basis of molecular weight, charge, and conformation; electrophoretic and chromatographic separation of peptides generated from proteins by site-specific proteases; and identification of antigenic differences between molecules using antisera or monoclonal antibodies. Although these latter techniques are not sensitive enough to resolve all possible differences in protein structure that could affect the functional capacity of the molecule, they have allowed resolution of some inherited variations in the structure of neural proteins (Sections II,A,2 and IV,B).

2 Mutant Proteins

Although all changes in DNA sequence can be classified as mutations, only a few of these mutations result in functionally defective gene products. A few examples will be discussed here in which disruption of function has resulted from mutations leading to production of structurally altered forms of tubulin, insulin, the neurophysins, myelin basic protein, and two components of the hormone-sensitive adenylate cyclase response.

Structurally altered forms of β-tubulin which interfere with microtubular assembly and confer resistance to antimitotic drugs have been described in Drosophila and in cultured Chinese hamster cells. In Drosophila, a unique form of β-tubulin specific to the testis has been demonstrated by two-dimensional gel electrophoresis (Kemphues et al., 1979). A phenotypically dominant mutation that produces sterility in males is characterized by the production of equal amounts of a normal and an altered form of this testis-specific β-tubulin, which differs from the normal in apparent molecular weight and charge. The ultrastructure of the testes of heterozygous flies strongly suggests that coassembly of the mutant and wild-type tubulins leads to formation of structurally defective microtubules; meiotic events and formation of the sperm tail axoneme are disrupted.

A structurally altered form of β-tubulin has also been found in cultured Chinese hamster ovary cells following mutagenesis and selection for resistance to the antimitotic drugs colcemid and griseofulvin (Cabrai et al., 1980). The variant form of tubulin has a different isoelectric point from the normal. Mutant cells express both normal and variant forms of tubulin. In vitro translation of poly(A) mRNA isolated from these cells has revealed the presence of messages coding for both forms. Thus, the variant tubulin does not result from an alteration in post-translational modification, but must be due to a difference in gene structure or post-transcriptional processing of mRNA. Studies using cloned cDNA probes for β-tubulin have indicated that there may be up to ten copies of this gene in mammals (see Section II,A,1). It appears, then, that even a single mutation in one of the twenty allelic copies of this gene can alter the structure of microtubules sufficiently to change their sensitivity to antimitotic drugs. This phenomenon presumably results from the random insertion of altered tubulin subunits into microtubules composed predominantly of normal tubulin. The structural disorientation of a polymeric molecule by a minor component illustrates how phenotypically dominant mutations can occur even in multigene families.

Two interesting structurally altered forms of human insulin have been described which also show dominant phenotypic expression. A patient with diabetes mellitus produces insulin molecules which contain a single amino acid substitution (Tager et al., 1979). This substitution does not affect the charge of the molecule, but results in a biologically inactive form of insulin that antagonizes the action of normal insulin. Another altered form of insulin has been described in which a single amino acid substitution in one of the two sites of proteolytic cleavage of the proinsulin molecule leads to formation of a larger, circulating proinsulin-like molecule (Gabay et al., 1979). This altered form of the molecule is present in greater quantities than its normal counterpart, possibly due to a slower rate of degradation. However, this proinsulin-like molecule has some biologic activity and the condition asymptomatic.

The peptide hormones vasopressin and oxytocin are produced by separate neuronal populations in the posterior pituitary and are stored in granules along with their respective carrier proteins, the neurophysins (Pickering and Jones, 1978). These neuropeptides and their carrier proteins appear to be synthesized from a common precursor molecule. Brattleboro rats (an inbred strain) are deficient in production of the antidiuretic hormone vasopressin, and have diabetes insipidus (Valtin et al., 1974). These rats lack vasopressin, its associated neurophysin, and the normal precursor molecule for both peptides; while they retain oxytocin, its neurophysin, and their precursor (Brownstein et al., 1980). Since inbred strains are homozygous at most alleles, this deficit presumably results from a mutation in both copies of a gene coding for this precursor molecule and/or for an enzyme involved in its processing.

Preliminary studies (J. H. Carson, personal communication) on the shiverer mutant in mice suggest that this recessive mutation leading to loss of myelin basic protein is responsible for the lack of myelin in the central nervous system of these animals. Although myelin basic protein is also absent in the peripheral nervous system, myelin is still present there and appears to be structurally normal. These mutants lack poly(A) mRNA coding for myelin basic protein, indicating either a change in the gene for myelin basic protein such that it is not transcribed, or an altered form of the message which is not processed normally and/or is degraded rapidly.

A series of mutant cell lines deficient in several aspects of hormone-responsive adenylate cyclase have been generated using lymphosarcoma cells that normally die in the presence of high levels of dibutyryl cyclic AMP (Coffino et al., 1978) (also see Section III,A). In three of the mutants, structurally altered or missing proteins appear to be responsible for these deficits. The hormone-responsive adenylate cyclase system involves at least three membrane components: a receptor protein (e.g., a β-adrenergic or prostaglandins receptor), a coupling protein(s) (termed G/F factor or GTP binding protein), and the enzyme adenylate cyclase. The G/F factor can be labeled with ³²P-NAD, through ADP ribosylation mediated by cholera toxin, and resolved into two bands on SDS Polyacrylamide gels of apparent MW 42,000 and 52,000 (Cassel and Pfeuffer, 1978). One class of mutants (AC−) able to grow in the presence of high levels of dibutyryl cyclic AMP has receptors and adenylate cyclase activity (with Mn²+-ATP, but not Mg²+-ATP as the substrate; Ross and Gilman, 1977), and lacks both protein components of the G/F factor (Johnson et al., 1978). Another class of mutants (UNC−), selected for growth in the presence of a stable agonist of the β-adrenergic receptor and a phosphodiesterase inhibitor, has receptors and normal adenylate cyclase activity, but has an altered form of the G/F factor with the same apparent molecular weight 42,000 and 52,000 as the wild type, but an altered charge (Ross et al., 1979; Schleifer et al., 1980). These studies have helped to elucidate the role of the G/F factor in the hormone coupled adenylate cyclase response.

Variations in cyclic AMP dependent protein kinase have also been obtained by selecting lymphosarcoma cells able to grow in the presence of dibutyryl cyclic AMP (Hochman et al., 1975; Steinberg et al., 1977). One class of mutant cells required about ten times as much cyclic AMP, as wild-type cells, to dissociate protein kinase into its subunits and thus activate its catalytic activity (Hochman et al., 1975; Nimmo and Cohen, 1977). Mixing experiments using subunits from both cell types revealed that the mutant regulatory subunit had an altered binding affinity for the catalytic subunit. By using affinity chromatography to enrich for the regulatory subunits and analyzing these polypeptides by two-dimensional gel electrophoresis, the mutant subunits were shown to have the same apparent molecular weight, but an altered net charge as compared to the wild type.

Although there are only a few current examples of inherited structural alterations in neural proteins that affect their function, still it is clear that by generating a set of mutations in different regions of the same peptide, one can elucidate the portions of the molecule critical to its binding to other subunits, substrates, and cellular components. Similiarly, by obtaining mutants altered in the same function, it is possible to identify the number of different genes and proteins which contribute to that function.

3 DNA and RNA Structure

Since most of this volume deals with molecular biology, we will consider these techniques only briefly in this chapter. To fully understand the genetic control of a protein it is necessary to understand not only its structure and processing, but also its origins in RNA and DNA.

In tissues in which a protein of interest is being synthesized in substantial quantities, precursor polypeptides have been studied by isolating total poly(A) mRNA, using it to direct in vitro translation, and then immunoprecipitating and analyzing the immediate translation products. This approach has been used, for example, in studies of β-NGF from mouse submaxillary gland (Wiche, 1979), somatostatin from mouse and rat hypothalami (Joseph-Bravo et al., 1980), and the acetylcholine receptor from the electric organ of Torpedo (Mendez et al., 1980). In all three cases precursor molecules differing in apparent molecular weight from the final polypeptides were identified by SDS polyacrylamide gel electrophoresis. Because of potential cross-reactivity between proteins (e.g., Julliard et al., 1980), it is important to establish the authenticity of the immunoprecipitated molecules. This can be done by comparing proteins precipitated with preimmune and immune sera, and by identifying protein structure by peptide mapping.

Total mRNA or partially purified mRNA (e.g., obtained by sucrose density gradient centrifugation or by immunoprecipitation of polyribosomes) can be used to generate cDNA probes. These probes can be used to study the structure of the mRNA. Sequencing of the probe can elucidate features of the untranslated portion of the mRNA molecule which may have a role in processing and turnover of mRNA, positioning of the mRNA and nascent polypeptides within the cell, and rates of protein translation. By hybridizing this probe with cellular RNA it is also possible to identify and characterize precursor forms of the mRNA.

DNA probes can also be used to identify specific gene sequences in the genome and to determine the number and structure of these genes, including intervening and flanking sequences. Probes can be generated not only from cDNA transcribed from mRNA, but also from synthetically synthesized nucleotide sequences which code for a unique amino acid sequence in the molecule, and from random fragments of nonrepetitive DNA sequences generated by restriction endonuclease digestion of total cellular DNA (Maniatis et al., 1978).

Probes for neural proteins are just beginning to be obtained. The cloning of the cDNA sequences for brain tubulin and actin has recently been reported by Cleveland et al. (1980) and Ginzburg et al. (1980). Examples of the kinds of information that such probes can provide are illustrated by studies with probes for other types of proteins. Studies of globin genes have helped elucidate the evolutionary relatedness of the different globins, the position of these genes in the genome, the number of copies of each type of gene, and individual variations in gene number and structure in coding, noncoding, and flanking sequences (for review see Forget, 1979). For insulin genes, it has been shown that whereas two nonallelic loci of the gene occur in some rodents, most species including humans have only one locus (Cordell et al., 1979; Lomedico et al., 1979). Further, sequencing of genes for insulin in humans has revealed the presence of two allelic forms of the gene, which differ only in noncoding regions (Ullrich et al., 1980). In addition to being used to analyze inherent variations in gene structure associated with normal and abnormal variations in protein structure and function, probes can also be specifically mutated in vitro and introduced into cells or organisms to create new variants (see Section III,A and B).

B Locating Genes

1 Mapping the Chromosomal Location of Genes

The assignment of specific structural genes to individual chromosomes and to regions within chromosomes has been carried out extensively for human and rodent cells. Major advances in this field were gained by staining techniques that allow identification of individual chromosomes and specific regions within them (e.g., Francke and Oliver, 1978) and by somatic cell genetic techniques that allow introduction of chromosomes from donor cells into recipient cells (Giles and Ruddle, 1973). Donor chromosomes can be transferred into the recipient cell by fusion with the donor cell, with the subsequent loss of some donor chromosomes (Ringertz and Savage, 1976), or by fusion with donor cell vesicles (microcells), which contain only one or a few intact chromosomes (e.g., Fournier and Ruddle, 1977). Clone panels have been established in which a series of hybrid clones contain a small complementary set of donor chromosomes (e.g., Mellman et al., 1979).

To map a gene it is necessary that the donor gene product is expressed in hybrids and can be distinguished from the recipient gene product, or that the presence of the donor gene itself can be established. Because it is difficult to construct appropriate hybrid cells that express differentiated neural properties, this latter approach may prove more useful in mapping neural genes. When the chromosome containing the gene of interest has been identified, one can use a series of hybrid lines which contain various portions of this chromosome to assign the gene to a particular region within the chromosome.

We have recently mapped the chromosomal location of a structural gene coding for human MAO using somatic cell hybridization techniques (Pintar et al., 1981). Hybrids were formed between mouse neuroblastoma cells and normal human skin fibroblasts (Fig. 2). The neuroblastoma parent lacked hypoxanthine phosphoribosyltransferase (HPRT) activity and MAO activity (Breakefield et al., 1976), both of which were present in the human parent. Following fusion, the cells were grown in medium containing hypoxanthine, aminopterin, and thymidine (HAT) (Littlefield, 1964), as well as ouabain (Mankovits et al., 1974), to select against growth of the mouse and human parent cells, respectively. HAT medium also selects for hybrid cells which retain the human × chromosome since it bears the gene for HPRT (Pearson et al., 1979). All hybrid lines selected in HAT medium expressed MAO activity (Table I); the A form, not the B form, of the enzyme predominated. Conversely, all hybrids except one cloned in medium containing 6-thioguanine (6TG), to select for cells which had lost HPRT activity and the human × chromosome, lost MAO activity. The MAO-A in HAT selected hybrids was shown to be of human origin by limited proteolysis and peptide mapping of [³H]pargyline-labeled MAO molecules (Fig. 3). [Pargyline binds irreversibly to the flavin containing subunit of MAO (Chuang et al., 1974)]. Thus the structural gene for the flavin-polypeptide of MAO-A is on the human × chromosome.

Table I

Analysis of Neuroblastoma × Fibroblast Hybridsa

aReprinted from Pintar et al., 1981, with permission. Copyright 1981 by Williams & Wilkins, Baltimore.

bHybrid lines were cloned and maintained in nonselective medium (NS), or in medium containing HAT or 6TG.

cMAO activity was measured using [³H]tryptamine as described (Costa et al., 1980). Values below the level of detection (<1.0 pmoles/min/mg protein) are designated as negative.

dHPRT activity was measured using [¹⁴C]hypoxanthine as described (Pintar et al., 1981). Values below the level of detection (<12 pmoles/min/mg protein) are designated as negative.

eThe presence of the human × chromosome was determined by scanning metaphase chromosome spreads stained by the Giemsa banding technique (Francke and Oliver, 1978).

fHuman forms of these enzymes were detected by electrophoresis on cellulose acetate thin layer sheets and staining for enzyme activity (Pintar et al., 1981).

Fig. 2 Hybridization scheme used in mapping human gene for MAO. Variants of mouse neuroblastoma line N1E-115 were selected by growth in 6-thioguanine (6TG). One of these variants N1E-115TG2 lost both MAO-A and HPRT activities. This variant was fused to normal human skin fibroblasts from a male which expressed MAO-A and -B, as well as HPRT activities. Following fusion, cells were plated in medium containing HAT and ouabain. Survivors were plated at cloning density with no selection; colony clones were isolated, tested, and recloned in medium containing either 6TG or HAT. (Reprinted from Pintar et al., 1981, with permission. Copyright 1981 by Williams & Wilkins, Baltimore.)

Fig. 3 Peptide maps of [³H]pargyline-labeled MAO-A from human, rodent, and hybrid cell lines. First, labeled proteins from crude mitochondrial preparations were resolved by SDS polyacrylamide gel electrophoresis, then the portion of the gel containing labeled MAO was cut out and placed in the well of another gel with Staphylococcus aureus V8 protease, and electrophoresis was carried out as described by Cawthon and Breakefield (1979). The distribution of peptides in the second gel was determined by slicing and counting. The migration relative to the bromphenol blue dye (Rf) is plotted on the lower abscissa; the migration of standard molecular weight markers on the upper abscissa. Radioactivity in each 2 mm slice is plotted on the ordinate. A representative map illustrating all peptides observed is shown for each line. The leftmost peak represents intact mao molecules. (I) Mouse neuroblastoma line N1E-115 from which the parent line N1E-115TG2 was derived and which expresses MAO-A activity. (II) Human neuroblastoma line BE(2)-C (kindly provided by Dr. J. Biedler) which illustrates the peptide map also found for human MAO A from fibroblasts and placental tissue (unpublished data). (III-V) Hybrid lines. (Reprinted from Pintar et al., 1981, with permission. Copyright 1981 by Williams & Wilkins, Baltimore.)

The one exceptional hybrid clone selected for loss of HPRT activity that continued to express human MAO-A has provided some information about the relative position of the MAO gene on the human × chromosome. These hybrid cells expressed the human form of phosphoglycerate kinase (PGK), but not the human form of glucose-6-phosphate dehydrogenase (G6PD) (Table I); genes for both of these enzymes are on the human × chromosome (Pearson et al., 1979). Further, these cells contained no recognizable portion of the human × chromosome, but did contain several fragments of human chromosomal material, both free and attached to mouse chromosomes (Pintar et al., 1981). This hybrid clone, then, appears to have retained the portion of the q arm of the human × chromosome that bears the genes for PGK and MAO.

It is also possible to map the chromosomal location of genes by digesting total cellular DNA from hybrids with specific restriction endonucleases and identifying fragment(s) containing the gene(s) of interest by hybridization to labeled DNA probes. Since the position of restriction endonuclease sites can vary among species, the same gene from different species may be identified by the length of the DNA fragment(s) in which it is found. The advantage of this approach is that the gene need not be expressed to be mapped. Thus the human insulin gene was mapped by digestion of DNA from hybrids lines in a clone panel constructed from fusions between Chinese hamster ovary cells and human fibroblasts (Owerbach et al., 1980). Only hybrids that contained human chromosome 11 had the human DNA fragment containing the insulin gene.

Radiolabeled DNA probes homologous to specific genes of interest can also be used to determine the chromosome location of these genes by using in situ hybridization techniques (Henderson et al., 1978). Metaphase chromosome spreads are incubated under conditions that denature double stranded helices, a single stranded ¹²⁵I-labeled DNA probe is hybridized to these denatured chromosomes, and autoradiography is carried out to identify the position of the probe. This procedure works best for genes present in a number of tandem copies (e.g., genes for histones and ribosomal RNA genes, or amplified genes) and is usually not sensitive enough for unique copy sequences.

As DNA probes become available for neural genes, mapping can proceed rapidly using these techniques of somatic cell and molecular genetics. Establishing the chromosomal location of genes coding for proteins critical to nerve function should provide insight into their evolutionary relationships and their regulated expression during development, as well as aid in the construction of detailed linkage maps.

2 Linkage Analysis of Gene Loci

Assigning a gene locus to a particular chromosomal region is a relatively gross method of determining its relationship to nearby genes, since one microscopically distinct band on a chromosome may contain up to 100 gene loci. Linkage analysis can provide more detailed information about the position of genes relative to each other. Several approaches will be discussed here, including classical techniques which use recombination frequencies and protein polymorphisms, as well as newer methods which use DNA restriction endonuclease site polymorphisms and gene transfer.

Classical methods of linkage analysis of mammalian genes involve animal breeding and examination of human pedigrees to determine the frequency of co-inheritance of specific traits. The more frequently two traits are inherited together, the less recombination has occurred between the genes coding for them during meiosis, and the closer the genes are to each other on a linkage map. Although information derived from linkage analysis can be applied to a physical map of the chromosomes, most chromosomes contain more than one linkage group [unlinked genes are transmitted randomly to offspring], and the distances on a linkage map are not proportional to physical distances on metaphase chromosomes. In the past, linkage analysis has depended on the demonstration of phenotypic diversity (polymorphisms) in specific gene products. Traits with common polymorphic forms in the population provided the basis for the construction of linkage maps, and rare traits were positioned relative to these common polymorphic markers. In mice, where extensive breeding analysis has been carried out, out of 69 genes known to affect neural function, 48 have been positioned on the genome (Searle, 1979; 1980; Sidman et al., 1965).

In humans classical linkage mapping has not yet provided any information about the chromosomal location of neuronally important genes, and has offered only limited insight into whether certain inherited neurologic and psychiatric diseases of unknown etiology are the result of variations in the structure of one or more gene loci. Limitations in these analyses result from the small size of family pedigrees and the relatively small number of common polymorphic traits in humans. (The 30 commonly used traits can provide linkage information for only about one-third of the genome.) Although some workers have reported that a gene responsible for a sex-linked form of bipolar (manic-depressive) illness is linked to genes on the × chromosome (Mendlewicz et al., 1979), other workers have disputed these claims (Leckman et al., 1979). Linkage analyses of schizophrenia (Elston et al., 1973), Huntington disease (Beckman et al., 1974; Hodge et al., 1980), and unipolar depressive illness (Tanna et al., 1976), to name a few, have not revealed the number or location of responsible gene loci.

Against this background of frustration in attempts to use classical linkage analysis to identify human genes affecting neural function, recent advances in molecular genetics have provided a single technique that can generate a large number of common polymorphic markers which can be used to establish linkage over the entire genome. In this approach polymorphisms in the sequence of the DNA itself are utilized as linkage markers (Botstein et al., 1980). Probes for unique copy sequences in the genome are generated by cloning either random sequences from the total genome (see Maniatis et al., 1978) or specific sequences homologous to mRNA’s or proteins of interest. Total DNA from several individuals is digested with site-specific restriction endonucleases and fragments are separated on the basis of molecular weight in agarose gels. If the position of at least one of the restriction endonuclease sites on either side or within the sequence in the genome homologous to the probe is different among chromosomes from the same or different individuals, the probe will hybridize with fragments of different molecular weight (Fig. 4). Two or more hybridizing bands will be identified either when an individual is heterozygous for a given restriction endonuclease site polymorphism and/or when the site lies within the region of the genome homologous to the probe. A number of laboratories are actively engaged in identifying probes that will reveal common polymorphism in DNA sequences in the human population. For example, a number of polymorphisms adjacent to the globin gene have been identified; one is tightly linked to a variant form of the β-globin gene responsible for sickle cell anemia (Kan and Dozy, 1978), another to the variant form of this gene causing β-thalassemia (Little et al., 1980). At least eight polymorphic alleles in the control population have been characterized in sequences surrounding a random sequence probe (Wyman and White, 1980). It will take approximately 150 probes that reveal common polymorphisms in DNA sequences and are spaced evenly along the DNA to establish a linkage map for the entire human genome (Botstein et al., 1980). By pedigree analysis the linkage relationship of other DNA probes and phenotypic traits can then be established.

Fig. 4 Use of restriction endonuclease fragment length polymorphisms in prenatal diagnosis. (A) Restriction endonuclease sites for a specific endonuclease in a portion of DNA which is homologous to the probe used. The polymorphism (*) is the presence or absence of one of these sites. (B) In this pedigree one of the children (112) is affected with an autosomal recessive disorder. Amniotic fluid cells are removed during a subsequent pregnancy (114). (C) DNA is isolated from family members of the pedigree and digested with the endonuclease. Fragments are separated on the basis of molecular weight in agarose gels and the position of the fragment of DNA containing the sequence homologous to the probe is determined. (The gene causing this disorder is contained within this same fragment of DNA.) In this family the mutant gene cosegregates with lack of the polymorphic restriction endonuclease site. Thus the fetus (114) has two copies of the mutant gene and will be affected. (Adapted from Fig. 1, Botstein, D., White, R. L., Skolnick, M., and Davis, D. W. Construction of a genetic linkage map in man using restriction fragment length polymorphisms, Am. J. Hum. Genet. 32, 314–331. Copyright 1980 by American Society of Human Genetics, Chicago, Illinois.)

This restriction endonuclease site linkage map can be used to establish the number and location of the gene(s) responsible for inherited neurologic and psychiatric diseases. Within a family, restriction endonuclease site polymorphisms which are tightly linked to the responsible gene can be used to detect the presence of the gene in heterozygous carriers, presymptomatic individuals, and affected fetuses. The advantages of this approach are apparent in a condition such as Huntington disease, which is inherited as an autosomal dominant mutation with late onset and where neither the biochemical lesion nor the chromosomal position of the defective gene has been established. Although it is not technically feasible at this time, eventually it should be possible to isolate the defective gene by walking closer and closer to it through adjacent restriction endonuclease site polymorphisms, and to identify the nature of the gene by in vitro transcription, translation, and characterization of the gene product. Antibodies could be raised against this protein and be used to determine its cellular location and to block its function in nervous tissue.

DNA mediated transfer of genes can also be used in linkage analysis (Ruddle, 1980; Pellicer et al., 1980). It is possible to introduce foreign DNA into cultured cells, eggs or early embryos by endocytosis or microinjection directly into them. DNA can be added in the naked state (Wigler et al., 1977) or in chromosomes (McBride and Ozer, 1973). Most of the DNA introduced into cells is degraded and lost, but a small portion is able to survive as self-replicating entities or through incorporation into the host genome (usually at random sites). These foreign genes may be expressed in the host cells and may be detected using homologous DNA probes and specific restriction endonucleases. In the case of cultured cells, it is convenient to have the foreign DNA contain a gene coding for a product that confers a selective advantage on the cells, e.g., drug resistance or nutritional auxotrophy. This allows the selection of the small number of cells in the population (usually about 1 in 10⁵–10⁶) to which genetic information has been successfully transferred. One can then establish whether other genes were co-transferred. The frequency of cotransfer of two genes is a measure of their proximity to each other in the genome. As selective systems for neural properties are developed, this approach should have more applications.

III GENERATING GENETIC VARIANTS

A In Cell Culture

Continuous cell lines, including neuroblastoma, glioma, and pheochromocytoma cells, are available which express a large number of neural properties in culture. In addition, other types of cell lines express some properties important in neural function, e.g., lymphosarcoma cells possess β-adrenergic receptors (Bourne et al., 1975), L-cells produce NGF (Pantazis et al., 1977), and hepatoma cells have MAO-A and -B activities (Hawkins and Breakefield, 1978). Such lines can be used to generate mutant or variant cells which are altered in these properties. Mutants can be used to understand the structure-function relationship of a particular protein by obtaining a series of different mutations in the gene locus or loci coding for this protein. Both mutants and variants can be used to dissect the functional components of a response by establishing the number of complementary lesions that can restore the