Physiological Genetics

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Preface

Physiological genetics is the study of gene action at all levels and encompasses the genic control of both metabolism and development. No sharp distinction can be drawn between these two broad aspects of experimental biology. However, although both concern ontogeny, they deal with it at different levels. Studies of metabolic systems involving different genotypes usually involve specific metabolic pathways, genetically dissecting individual biochemical reactions, and determining how these are affected by specific enzymes or sets of enzymes encoded in identifiable genes. Developmental studies often involve the identification of specific developmental differences, both temporal and spatial, and subsequently attempting to identify, usually by working backwards, the genetic differences that lead to such developmental diversification. Thus, physiological genetics is that sub-branch of the science of genetics dealing with the study of developmental events or processes through which genetic differences are eventually expressed as phenotypic differences. It is essential, therefore, that a thorough understanding of phenotypic differences involve an understanding of how such differences are generated both spatially and temporally, and how structural genes may respond to both external and internal signals to be so precisely expressed in time and place.

Until relatively recently the primary concern of genetics was to define the gene, its structure, function, mutation, and replication. This was achieved by the progressive molecularization of genetics. It was correctly assumed by most geneticists that it would have been virtually fruitless then to attempt to understand the intervention and sequential action of genes in the realization of the phenotype, especially when considering the complexity of higher organisms. Nevertheless, a number of early studies, with both plants and animals, combining genetics, experimental embryology, and physiology (e.g., pigment production in some animals and plants, and metabolic pathways in microbes) proved most constructive in efforts to understand gene action at least at the phenomenological level.

The basic problem of how genes function in the complex higher eukaryotes has only just recently come to the forefront of modern experimental biology. This is largely due to new and highly sophisticated knowledge and technology in the broad area of molecular biology which allows us to experimentally probe some old questions so that new ones can be generated and, hopefully, answered. Information generated as to how genes operate in lower organisms has suggested new and exciting approaches to problems in the higher and more complex forms. Differential gene function, responsible for generating a variety of cell phenotypes from a single genotype, has become the central problem of contemporary experimental biology.

In a rapidly developing field of science the ability to keep informed of progress becomes a major problem. Consequently, there is an occasional need to summarize and highlight some of the more interesting and, it is hoped, significant advances as a useful reference source for our interested colleagues. Additionally, the material should also prove useful for teaching and training at the graduate and advanced undergraduate levels. Only time will render a verdict on the success of these goals.

The chapters were not intended as comprehensive reviews, but represent personal accounts of developments best known to each writer who, in each case, was involved in the research that has led to significant advances in the respective areas. Each contributor provides a current and critical account of recent advances and a number of exceptional model systems for studies in physiological genetics. The book covers this area from several different points of view. Following an introductory chapter describing genetic factors in developmental gene regulation, there are discussions on enzyme differentiation, hormonal control of gene expression, biochemical genetics of morphogenesis, cytoplasmic male sterility in maize, plant somatic cell genetics, and the population dynamics of genetic polymorphism. We hope we have covered some material of lasting value in view of the rapid developments in this field.

I am indebted to all contributors for pausing in their research long enough to make their contributions on schedule. Special credit is due to all our colleagues who contributed by supplying figures and data or made material in press available to the various authors. A number of colleagues aided me in the review process and I am most sincerely thankful to them. Last but not least, I thank Penny, Artemis, Lisa, and Nikki for their love, patience, and understanding; the time I spent on this and other simultaneous projects has detracted from my precious time with them.

John G. Scandalios

1

Genetic Factors in Developmental Regulation

K. PAIGEN

Publisher Summary

The studies of temporal gene regulation of enzyme development in several organisms indicate that genetically determined nuclear programming is an important mechanism regulating macromolecular differentiation of cells. There is an implication that developmental control is a deterministic process, in which programming by a nuclear genetic machinery plays an important role. The developmental controls for each enzyme are relatively independent and appear to rely on a rather small number of discrete elements. The experiments on β-glucuronidase suggest the existence of a specific genetic regulatory process that functions to determine the glucuronidase developmental program. The proximate temporal gene site and the enzyme structural locus appear to be functionally distinct and physically separated.

I. Introduction

II. Temporal Gene Mutants

A. Proximate Loci

B. Distant Loci

C. Systems with Both Proximate and Distant Loci

D. Miscellaneous Proteins

III. Properties of Temporal Genes

A. Stage and Tissue Specificity

B. Proximate and Distant Loci

C. Enzyme Specificity

D. Molecular Level of Control

E. Additive Inheritance

F. The Nature of Temporal Regulation

G. Questions of Generality

H. Controlling Elements and Temporal Genes

I. The Coding Problem

IV. Homeotic Mutants

A. Background

B. Characteristics of Homeotic Mutants

C. Models and Questions

D. Conclusions

V. Intercellular Signals

VI. Conclusions

References

It gradually dawned on us that what we eventually came to identify as a differentiated cell of given type, shape, structure, colour, and behaviour, was really just the last scene of a long play of interactions, the earlier stages of which had been hidden from us. We came to realize that, in dynamic terms, the so–called stages of the development of a cell, an embryo, or a disease, represented simply cross-sections through a continuous stream of processes, recorded by whatever methods of assay and portrayal–whether in pictures or in graphs–we had at hand. The picture of an overt character thus emerged as merely the residual index of prior covert dynamics; form, as the registered outcome of formative processes; … fibres, as the result of the recruitment, polymerization, alignment, and bundling of masses of anisodiametric macromolecules; and so forth.

Paul Weiss (1973)

I INTRODUCTION

To describe the role of the genome in guiding development is in effect to ask what changes in developmental processes attend individual mutations. Two quite distinct frames of reference have been used for this purpose. One is to express the outcome in terms of nuclear differentiation—the changing pattern of gene activities that varies both temporally, as development proceeds, and spatially, from one cell type to another. The result of nuclear differentiation is that each cell type comes to produce its own characteristic array of RNA and protein molecules. The other frame of reference has been morphogenesis, or pattern formation, including both the development of visibly specialized cells and the morphological structures containing them. The two frames of reference are related by the obvious fact that morphogenesis is brought about by the characteristic complement of macromolecules each cell produces. As Paul Weiss has so evocatively stated it, form is the registered outcome of formative processes.

A major experimental dilemma has been the difficulty of deciding whether an observed developmental mutation affects the process of nuclear differentiation itself or the subsequent steps of morphogenesis. This difficulty has been especially acute for the group of mutants in which a normal act of differentiation fails, such as in the stunted wings of the Drosophila mutant vestigial(Lindsley and Grell, 1967) or the death of the visual cells in the mouse mutant retinal degeneration (Noell, 1958). For the great majority of these mutants it has not been possible to determine whether they are impaired in the structure of a protein required for morphogenesis or whether they are impaired in the genetic mechanism that arranges for adequate amounts of that protein to be present in the right cell at the right time. The development of a contractile muscle will fail whether an amino acid substitution in one of the myosin chains destroys its functional capacity or whether myosin is produced in inadequate amounts, or in the wrong cell, or at the wrong stage of development.

The reasons for this dilemma are not hard to find. The products of many structural genes are required to bring about even one morphogenetic event; each of these structural genes has a genetic regulatory apparatus associated with it, and the various proteins coded by these structural genes, as well as the metabolic products of the reactions they catalyze, react with each other in processes whose identities are unknown, much less their concentration dependence, stoichiometries, and cofactor requirements. This has made it quite difficult to identify the primary gene product affected in a given morphological mutant. Similarly, if several genes are identified as affecting the same morphogenetic step, there is no way to test whether they even involve the same or different gene products. Among the thousands of morphological mutants known, the affected protein has been identified for only a few. These, such as rudimentary in Drosophila (Rawls and Fristrom, 1975), have turned out to be simple enzyme deficiencies and relatively uninformative regarding the fundamental mechanisms of either nuclear differentiation or morphogenesis. Thus, the importance of morphological mutants has primarily been in defining morphogenetic interactions.

Two other groups of mutants have, however, provided some information regarding the role of the genome in guiding development. One group includes mutants altered in the developmental programming of individual proteins. Mutants of this kind have been described for maize, Drosophila, and mice, and the concept has developed that a set of genetic units exists, called temporal genes, whose function is the coding and expression of these programs (Paigen, 1964, 1971, 1977; Paigen and Ganschow, 1965). Temporal gene mutants appear to be specific in affecting only single proteins, and those known do not have grossly observable morphological consequences. These mutants are discussed in Sections II and III.

The second group includes mutants with a substitution of one morphogenetic pattern for another. These are the homeotic mutants of Drosophila, such as antennapedia, where a leg appears in place of an antenna (for reviews, see Oberlander, 1972; Postlethwait and Schneiderman, 1973; Gehring and Nóthiger, 1973; Ouweneel, 1976). Such mutants are largely confined to insects, and their existence reflects the unique metamorphosis of these organisms in which some adult body parts develop from a small number of discrete larval structures, the imaginal discs. Each disc normally gives rise to a fixed complement of adult structures, and the homeotic mutants are explicable as a partial substitution of one complement for another. These mutants are discussed in Section IV.

Before turning to the mutants, it is appropriate to consider the extent to which the sequence of nuclear differentiation is the result of an intrinsic nuclear programming machinery and the extent to which it is a sequential response to metabolic changes in the cell, each change inducing the next metabolic transition. It will be some time before a definite experimental decision can be made as to which is the predominant mechanism, or whether they participate relatively equally. But even now there are reasons to suspect that intrinsic nuclear programming is at least an appreciable part of the whole. This is encouraging, for it is apparent that if nuclear differentiation is largely or entirely directed by a complex sequence of metabolic interactions, the experimental effort required to reach an appreciable level of understanding will be immense. This is in contrast to the possibility that a much more discrete set of elements and rules defines the operation of a nuclear programming system.

Although the nature of any intrinsic programming mechanism is virtually unknown, especially at the molecular level, there is considerable independent evidence that such mechanisms do exist. It appears that encoded DNA is a machinery that can determine a later sequence of highly specific nuclear events and that this machinery operates relatively independently of metabolic processes. The pioneering studies of McClintock (1951, 1965, 1967), now corroborated and extended by others (for reviews, see Fincham, 1973; Fincham and Sastry, 1974), demonstrate that in both plants and animals a class of genetic controlling elements exists that is capable of programming future genetic events in nuclei.

The expression of controlling element action is generally bipartite, requiring both a distant locus initiating the genetic action and the presence of a suitably sensitive allele of the structural gene that will respond. That is, the DNA in or near the structural gene must be of such a nature that it can respond to the action of the distant element. Two types of control are seen. One is the continuing ability of the distant elements to modulate the activity of sensitive alleles of the structural gene. In this sense the distant element acts as a genetic regulator that functions in all cells. The other, more dramatic, capability is that of programming the later occurrence of somatic mutations in sensitive alleles of the structural gene, but not in other genes. These mutations occur only during certain stages of development and in some tissues. That the somatic mutations represent true changes in DNA structure is deduced from the observation that when they occur in tissues that eventually give rise to germ cells the new phenotypes are transmitted through meiosis to the next generation of offspring.

What is immediately relevant is that the existence and properties of controlling elements demand the presence of genomic components capable of determining, for specific genes, the subsequent timing and tissue location of somatic mutations. Once this larger fact was established, it became easier to consider the smaller notion that a system, similar or identical in composition to the controlling elements described by McClintock, might program the changes in levels of gene function that characterize differentiation.

The examination of mutants with altered developmental programs for specific gene products provides a preliminary comparison of the relative roles that intrinsic genetic programming and sequential metabolic responses may play. To the extent that sequential metabolic changes direct the course of nuclear differentiation, we anticipate that mutations at many sites will affect the ultimate programming of even a single enzyme. In addition to the metabolic changes in the cell lineage itself, each dependent on earlier metabolic events, there will be the changes induced by metabolic or hormonal signals reaching the cell from outside. These will involve multiple genetic functions. Conversely, we would expect a mutation altering an earlier metabolic process to have multiple consequences, and thus have pleiotropic effects on the programming of many enzymes. In general, we would also anticipate that mutations affecting enzyme programming through metabolic perturbations would be recessive. Experience with many mammalian mutants, both in humans and experimental animals, shows that while mutant alleles generally show codominant expression at the level of the primary gene product, physiological consequences are usually recessive, since 50% changes in protein activity are easily compensated for (Childs and Young, 1963; Harris, 1964; Paigen and Ganschow, 1965). For example, heterozygotes of human enzyme deficiencies characteristically exhibit a normal phenotype despite half normal enzyme levels, even though the homozygous deficiency may produce severe symptoms or be lethal.

Thus, to the extent that sequential metabolic processes are an important mechanism in determining the process of differentiation, we would anticipate that the programming of one enzyme will be affected by mutation at many sites in the genome; that mutation at any one site is likely to affect the programming of multiple enzymes; and that mutant alleles will show recessive–dominant inheritance patterns rather than codominant–additive inheritance. Although the list is still relatively short, the enzyme developmental mutants analyzed so far do not have properties matching these predictions. As detailed in Section II a very high proportion of mutants are located in close proximity to the structural gene of the enzyme being programmed. There is a great deal of specificity, so that even enzymes located in the same cellular organelle and whose activity changes are coordinate in development are under independent genetic control, and all of the systems tested show additive inheritance. To the extent that it is possible to generalize from the examples now available, it appears that a great deal of intrinsic programming occurs.

The simplest type of programming that can be considered is that of individual structural genes. The temporal gene mutants provide examples of this. The question then arises as to whether a higher level of programming also exists, one that chooses between ectoderm and endoderm, between becoming an erythroid stem cell or a myeloid stem cell. The alternative is to consider that these larger steps represent the summation of many individual decisions. The origins of this question are really historical. Classic descriptions and definitions of the problems of embryology and development were framed in morphological terms. One heritage of that is our tendency to consider development as a series of discontinuous steps leading to the formation of new cell types. In superimposing the past on the present, we are implicitly assuming that morphogenesis involves a simultaneous change in the functional state of many genes, a kind of regulation en bloc. Such a discontinuous step requires the existence of master controllers of some kind, agents capable of initiating changes at many genes simultaneously. Serious theoretical consideration has been given to models and mechanisms that might provide this ability (Britten and Davidson, 1969; Gierer, 1973; Brawerman, 1976), and the concept remains an important part of our contemporary thinking.

There is, however, no experimental requirement that master switches exist. It now appears that morphogenesis is not necessarily synchronous with, or even an accurate reflection of, nuclear differentiation. The appearance of morphologically distinct cells is not always correlated with the magnitude or extent of the changes in protein complement that differentiated cells undergo. The limited utility of morphological criteria as an estimate of nuclear differentiation is well illustrated by the behavior of early mammalian embryos. During the preimplantation development of the mouse there are two major changes in the complement of proteins being synthesized (Van Blerkom and Manes, 1977). However, both of these precede the first morphological differentiation that occurs, the separation of the morula cells into an outer trophectoderm and an inner cell mass. The trophectoderm will eventually give rise to the tissues that interface the embryo and the maternal uterus, and the inner cell mass will eventually give rise to the embryo proper. The differentiation into inner cell mass and trophectoderm is not accompanied by any major change in the pattern of protein synthesis. The two differentiated cell types each synthesize virtually the same complement of proteins as the other and the same complement of proteins that was synthesized by the progenitor cells from which they arose. Nevertheless, the concept of master switches remains an attractive one, and, if they do exist, the outstanding candidates for membership are the homeotic genes of Drosophila.

II TEMPORAL GENE MUTANTS

Mutants altered in the developmental expression of defined proteins have been described in several organisms including maize, Drosophila, and mice. They lack morphological consequences. The experimental strategy they offer is the possibility of distinguishing mutations affecting the production of specific proteins from mutations affecting the functional properties of these proteins. With this comes the ability to focus on the genetic apparatus involved in developmental programming of gene activity. For each structural gene a limited set of genetic sites, called temporal genes (Paigen, 1964, 1971, 1977; Paigen and Ganschow, 1965), has been found to participate in developmental programming. The basic pattern appears to include interaction between a proximate element located adjacent to the structural gene and one or several distant elements located elsewhere.

A Proximate Loci

1 Glucuronidase

a Background

The first temporal gene mutant described, and the one most extensively studied, controls the development of β–glucuronidase in various tissues of mice. Animals homozygous for this mutation undergo programmed changes in β–glucuronidase activity occurring at a different age in each tissue. Regulation is expressed as a control of enzyme synthesis; whether it involves a modulation of mRNA activity is unknown. The changes are enzyme specific in the sense that other enzyme activities, and especially other lysosomal enzymes, are not affected.

The variant phenotype was originally discovered in the C3H strain of mice, and present evidence suggests that this strain is altered in two regions of DNA that influence enzyme activity. One change is the production of enzyme molecules with altered structure and increased thermolability; the other change is a new age–dependent program of enzyme synthesis. Each change is determined by a single genetic site, and both sites map within the same small region of chromosome 5. The thermolability mutation, together with other structural variants exhibiting altered electrophoretic mobility, define the structural gene Gus for this enzyme. The failure to observe recombination between the structural locus and the genetic determinant for temporal regulation indicates that age–dependent synthesis is programmed by a chromosomal site Gut that is in close proximity to the structural gene. The limitations of fine structure mapping in the mouse do not allow a distinction as to whether the Gut site is adjacent to or integral with the structural gene. Immediately, the crucial point is that some DNA sequence is capable of programming age–dependent enzyme synthesis. The problems involved in deciding where this DNA sequence is located are presented in Section III.

Gut was historically important in providing the first evidence that a specific locus can program the level of a single enzyme during development; its close proximity to the structural gene and the fact that it acts by regulating enzyme synthesis imply that the mechanism is not trivial.

b Biochemistry and Genetics

Considerable information is now available on the structure and regulation of β–glucuronidase in mice (for reviews, see Paigen et al., 1975; Lusis and Paigen, 1977; Swank et al., 1978). The enzyme is a tetramer of molecular weight about 280, 000 and has been purified to homogeneity (Tomino et al., 1975). The structural gene for the enzyme is located near the distal end of chromosome 5 and directs the synthesis of a polypeptide chain of molecular weight from 70,000 to 75,000. From this primary polypeptide chain the cell produces two physically distinguishable tetrameric forms of the enzyme, known as L and X (Ganschow and Bunker, 1970; Swank and Paigen, 1973). L is the classic lysosomal form of the enzyme that is familiar to most investigators. Its subunit molecular weight is 71, 000. X is an alternate tetrameric form that is anchored onto the membranes of endoplasmic reticulum through complexing with a second protein, egasyn (Swank and Paigen, 1973; Tomino and Paigen, 1975). X differs from L in being 2000–3000 daltons heavier and in having a higher isoelectric point. The presence of egasyn requires the function of the Eg gene on chromosome 8 (Ganschow and Paigen, 1967; Tomino and Paigen, 1975; Lusis et al., 1977). The complexes with egasyn serve to anchor the enzyme to membranes. A variety of evidence confirms that these multiple intracellular forms of glucuronidase are, in fact, derived from a single structural gene. Other than the differences related to their intracellular location, the various enzyme forms are identical physically, chemically, catalytically, and immunologically. Most convincingly, genetic experiments show that the properties of all intracellular forms of the enzyme are determined by a single genetic site (Paigen, 1961b; Lalley and Shows, 1974). Substitution of one structural allele for another at the chromosome 5 site simultaneously alters the properties of the enzyme at both intracellular locations. Three alleles of the glucuronidase structural gene have been reported: Gusb, the standard form of the enzyme; Gush, present in C3H mice and determining the thermolabile enzyme mentioned above (Paigen, 1961b); and Gusa, an electrophoretically fast form (Lalley and Shows, 1974).

The same structural gene is expressed throughout development; mutation at the Gus locus affects the properties of the enzyme at all developmental stages, from the preimplantation embryo (Wudl and Chapman, 1976) through the neonatal and adult stages (Szoka, quoted in Paigen,