Abstract: Random X-chromosome inactivation makes the female of placental mammals a natural mosaic for clones of cells having either the maternally derived X (Xm) or paternally derived one (XP) genetically inactivated (Lyon 1961). In marsupials, on the other hand, preferential inactivation of the paternally inherited X chromosome seems to be a rule in most tissues (Sharman 1971; Cooper, Johnston, Murtagh, Sharman, VandeBerg, and Poole 1975). There are, however, instances in which inactivation is obviously not random in placental mammals (Lyon 1974). Nonrandomness was assessed from studies made on differentiated cells remote from early embryonic cells in which inactivation occurred. Thus it is not clear whether nonrandom inactivation, or a secondary event(s) after random inactivation, was responsible for the ultimate nonrandom expression.

Abstract: Ribulose bisphosphate (RuBP) carboxylase is truly a multifunctional protein. Not only does it exhibit the well-known carboxylase and oxygenase activities, but also its high concentration and turnover characteristics in the leaf fit the classification of a storage protein. RuBP carboxylase varies in concentration by species but can be up to 65% of the total soluble protein of grass or alfalfa leaves. It is assembled and sequestered in a discrete organelle, the chloroplast, wherein it is protected from the proteolytic enzymes in the cytoplasm. In fact, it appears that carboxylase degradation is a cytoplasmically driven process. After its synthesis and assembly, little turnover is detected until plants require its remobilization. RuBP carboxylase can then be mobilized during senescence or when the plant requires its reserves because of deficits of either nitrogen or carbohydrates. As such, the RuBP carboxylase concentration in the leaf is very responsive to environmental stresses.

Abstract: It is well established that the chloroplast synthesizes only a limited number of its own proteins. Many stromal enzymes and thyla-koid membrane polypeptides are made in the cytosol and must be transferred subsequently from their sites of synthesis to their final locations inside the chloroplast (1). The mechanism of intracellular transport of proteins across the two chloroplast envelope membranes and the events associated with it are unknown.

Abstract: Nonrandom X-chromosome expression in XX female mammals has been reported in several different situations. For example, when one X chromosome carries a deletion, only the normal X chromosome is expressed (Grumbach, Morishima, and Taylor 1963), but when one X chromosome is involved in a certain reciprocal X-autosome translocation* — Searle’s translocation in mice (reviewed by Eicher 1970) — only the abnormal X chromosome is expressed. Similar exclusive expression of one X chromosome has been reported in the blood of certain human X-inactivation mosaics (Nance 1964; Gandini, Gartler, Angioni, Argiolas, and Dell’Acqua 1968), and this observation is most consistently made when the absent blood-cell population would express a deleterious X-linked gene, such as an HPRT deficiency (Nyhan, Bakay, Connor, Marks, and Keel 1970). Less extreme cases of nonrandom X-chromosome expression have been reported in some tissues of humans (Nance 1964; Ropers, Wienker, Grimm, Schroetter, and Bender 1977) and mules (Hook and Brustman 1971), and for various stocks of mice apparently heterozygous for an X-linked controlling-element locus (Cattanach and Isaacson 1965; Grahn, Lea, and Hulesch 1970; Krzanowska and Wabik 1971; Falconer and Isaacson 1972; Ohno, Christian, Attardi, and Kan 1973).

Abstract: As a consequence of X-chromosome inactivation, one X chromosome becomes the sole determinant of the X-specified characteristics of the cell, leading to potential cellular mosaicism in females. If there were no differences between the maternal and paternal alleles at any X-linked locus, there would be no cellular mosaicism. However, unlike the relatively homogeneous laboratory mouse populations, the human population is very heterogeneous, largely attributable to heterozygosity at many loci. Based on estimates by Harris and Hopkinson (1972), it seems certain that most women are heterozygous at one and probably more than ten X-linked loci. Therefore, with respect to her X-linked genes, the phenotype of the female is determined by the nature of her individual heterozygosity, the effect of random inactivation on the proportions of cells of the two types in each tissue, and the result of selection following inactivation.

Abstract: During early development of female (XX) eutherian mammals, one or the other of the X chromosomes is rendered inactive in all, or most, of the cells of the embryo. This differentiation of the X chromosomes is irreversible, and the adult female is a mosaic with respect to clones of cells with either the maternally-derived or paternally-derived X chromosome inactive. The timing of X-chromosome differentiation has been a subject of considerable interest. Cytogenetic evidence suggests that it occurs around the time of implantation, or at the late blastocyst stage (e.g., see Takagi 1974; Mukherjee 1976). However, other genetic evidence (Gardner and Lyon 1971) suggests that both X chromosomes are active at this stage, at least in the inner cell mass cells of the blastocyst. The subject of X-chromosome inactivation has been extensively reviewed (e.g., see Lyon 1968, 1972, 1974; Eicher 1970; Gartler and Andina 1976; and Monk 1978).

Abstract: Rearrangements between mammalian X chromosomes and autosomes are valuable tools in the study of X inactivation, since they provide a karyotype in which the physical continuity of the X chromosome is interrupted. In reciprocal X-autosome translocations, T(X;A)’s, there are three chromosomes within each cell of a heterozygote — instead of the usual two — that contain X-chromosome material. This circumstance may be put to use in addressing questions of X-differentiation mechanisms and randomness.

Abstract: Mosaics are arrays of two or more alternatives which may be in one, two, or three dimensions, and for each of which there are many biological examples. Before we can discuss the biological material intelligently, it is essential to determine some of the parameters of purely random mosaics. The most obvious attributes of mosaics are patchiness, and the variation in patchiness. I shall use the terms run, patch, and cluster to describe aggregates of like cells in one-, two-, and three-dimensional mosaics, respectively.

Abstract: A long-standing and continuing problem concerning ribulose bisphosphate carboxylase/oxygenase is the discrepancy between its activity in vivo and in vitro (1, 2). The apparently low affinity of the enzyme for CO2 was one of the better reasons Warburg had for dismissing the C3 photosynthetic carbon reduction cycle (3). When assayed in vitro with the naturally occurring concentrations of CO2 (10 µ,M) and O2 (250 µM), the purified enzyme is incapable of fixing CO2 for sustained periods (>90 sec) at rates equal to or greater than those of photosynthesis. Similarly, sustained synthesis of phosphoglycolate under natural conditions for >2 to 3 min at rates equal to or greater than the in vivo rates has not yet been observed. Nevertheless, beginning with the observations of Bahr and Jensen (4, 5), some progress towards resolution of these discrepancies has been made, and, provided that both reactions are initiated with fully activated enzyme, adequate rates of carboxylation or oxygenation under natural conditions can be achieved or even exceeded, if only for a minute or two (6–12). Thereafter, progressive inactivation of the enzyme becomes apparent. At best this represent only a partial solution to the problem, for clearly there are no such restrictions upon the enzyme in vivo.

Abstract: When whole tissues or isolated chloroplasts are brightly illuminated they do not immediately commence to assimilate carbon at maximal rates. Instead there is an initial lag or induction period which may persist for several minutes (1). This is perhaps the best known and most readily observed example of photosynthetic regulation. Clearly the chloroplasts are potentially capable of rapid photosynthesis because they soon begin to evolve O2 and fix CO2 at high rates. Equally clearly this potential ability is slowed or regulated during the first few minutes of illumination. What is the nature and function of this regulation? Osterhout and Haas (2), who first observed induction at Woods Hole in 1918, suggested two possible causes. Either the lag represented a period during which substrates were built up to the level required for full activity or else the catalysts concerned might be activated in the light. Sixty years later there is little that can be added to these statements in principle, except of course the inevitable notion that it might be both. This article deals with these possibilities in regard to ribulose bisphosphate (RuBP) carboxylase and in particular to the role of ortho-phosphate in metabolic regulation.

Abstract: There are compelling reasons to believe that the initial atmosphere of the earth after its formation about 4.7 × 109 years ago was a reducing one consisting chiefly of methane, ammonia, water, and hydrogen (1). In the last two decades considerable research has been described in which numerous organic precursors of biopolymers have been synthesized under conditions simulating the primitive earth and its atmosphere (for a review see ref. 2). Formaldehyde and hydrogen cyanide are known to be formed so readily under a variety of these conditions that they are considered not only as products of ancient chemical evolution but as likely precursors of a variety of important components of the prebiotic soup, including sugars, amino acids, and purine bases (2).

Abstract: The spontaneous appearance of an individual with somatic and gonadal characters typical of both sexes is rather rare, but not unknown, among men and mice. Such individuals have been labeled intersexes, hermaphrodites, or gynandromorphs, depending upon the investigator’s emphasis. In the following discussion of BALB/cWt strain mice, we have adopted the term hermaphrodite because of its general usage vis-a-vis phenotypic morphology. However, we do not wish to imply that connotations associated with intersex or gynandromorph are unacceptable.

Abstract: Photorespiration is the light-dependent, oxygen-sensitive CO2 evolution from green leaves that originates from the metabolism of compounds in the glycolate pathway. It has been termed “an inevitable consequence of the existence of atmospheric oxygen” (1) and has been attributed to the ability of ribulose bisphosphate carboxylase to act also as an oxygenase catalyzing the reaction of oxygen with ribulose bisphosphate (RuBP) to yield phosphoglycolate (2), the precursor of the substrate for the glycolate pathway. In this reaction, oxygen not only produces the substrate for photorespiration but also competitively prevents the fixation of CO2 (3, 4). It has been known for some time that the inhibition of photosynthesis by oxygen is comprised of two components, inhibition of true photosynthesis and stimulation of photorespiration (5, 6). With the discovery of the oxygenase activity of RuBP carboxylase, both these components were attributed to the effect of oxygen on the enzyme (4, 7), and the joint action of oxygen and CO2 on the enzyme was proposed to be responsible for the regulation of soybean net photosynthesis (8). The oxygen concentration around a leaf—and in it, as the oxygen concentration is similar, (9)—can be quickly changed; and, if oxygen acts only on the oxygenase, one might expect a change in oxygen concentration to be accompanied by a corresponding rapid change in CO2 fixation to a new steady rate.

Abstract: When the X-linked enzyme, hypoxanthine guanine phosphoribosyl-transferase (HGPRT), was found to increase significantly in activity during the morula stage of preimplantation mouse embryonic development (Epstein 1970), it was obvious that this enzyme would be a useful marker for studying the control of X-chromosome expression. This conviction was strengthened by the finding that, while still present at the 2-cell stage, the gene dosage effect related to the number of X chromosomes present during oogenesis is no longer present after blastulation, indicating that the increase in HGPRT activity is determined by the embryonic genome (Epstein 1972).

Abstract: In recent years, studies on aggregation and injection chimaeras and on the genetic mosaics that result from X-chromosomal inactivation have done a great deal to elucidate what goes on in the early stages of mammalian development. Other kinds of genetic mosaic, namely those arising through forward and reverse mutation at particular loci, can also provide useful information. This is particularly so because the initial mutational event in these mosaics can occur at different stages in development, from the first cleavage division on, and can therefore provide us with a sort of running commentary on developmental processes; whereas the initiating events with respect to X-chromosome inactivation and the formation of chimeras are limited to very early stages in embryogenesis. Another point which may be of importance is that there would seem to be much less chance of preferential proliferation of one of the clonal components of such a mosaic than in most chimeras. In these mosaics, the genetic difference between the clones normally involves two viable alleles at a single locus controlling some aspect of coat colour. In chimeras, however, there are usually many genetic differences between the components.

Abstract: The version of the X-inactivation hypothesis presented by Lyon (1961, 1972) proposed that inactivation was a random event with respect to the parental origin of the X chromosome. Although there have been some suggestions that inactivation may not always occur randomly, the evidence has not been compelling. On the basis of enzyme phenotype in small numbers of blood samples from interspecies hybrids, it has been suggested that the paternally derived X chromosome of marsupials is inactivated preferentially; on the other hand, studies of other tissues have revealed that paternal alleles are expressed (Cooper 1971; Cooper, Johnston, Murtagh, Sharman, VandeBerg, and Poole 1974; Cooper, Johnston, Murtagh and VandeBerg 1975). Observations compatible with preferential expression of the maternal allele in the mule, the interspecies hybrid between female horse and male donkey (Hamerton, Richardson, Gee, Allen, and Short 1971), proved to be the result of a relative selective advantage for cells with an active horse X chromosome (Hook and Brustman 1971). Similarly, the nonrandom pattern of X-inactivation observed for structurally abnormal X chromosomes is now known to result from selection rather than X inactivation (Leisti, Kaback, and Rimoin 1975; Russell and Cacheiro 1978).

Abstract: The role of ribulose bisphosphate (RuBP) carboxylase in photosynthesis and photorespiration, its structure, and the genetics of subunit transmission have been well studied (1). Other aspects of the biology of RuBP carboxylase have received less attention; in particular, the timing and the genetic regulation of its synthesis and degradation require further study. Knowledge of the timing and the effects on it of environmental and developmental cues will contribute to an understanding of whether the amount of RuBP carboxylase protein is a limiting factor in photosynthesis. Examination of the large and small subunits separately should clarify whether the small subunit is necessary to induce synthesis of the large one. The orderly progression of plastid development (2) and of leaf senescence (3) indicates that these are well regulated processes. Nonetheless the genetic regulation of RuBP carboxylase synthesis and degradation must be characterized and described before these developmental processes can be fully understood.

Abstract: Depending on the point of view, the tobacco plant is either extolled for the solace it brings to those who smoke and/or for the secure economic rewards from its cultivation and manufacture, or branded a weed of unmitigated evil for its effect on health. The latter view now seems to be the more popular. However, there is another possibility. Tobacco plants can be used as a source of high-grade protein for human consumption. The exploitation of this possibility could turn tobacco into an agricultural commodity of undeniable value. Since the idea of using leaf protein is not new, the purpose of this paper is to present reasons for thinking that tobacco plants could be a superior source of supplemental protein in the human diet compared with leaf proteins from other plants.

Abstract: The agronomically important aspects of the comparative biochemistry of RuBP carboxylase are locating a natural enzyme, creating a mutant enzyme, or identifying compounds which differentially alter the enzyme so as to allow CO2 to be fixed more efficiently or O2 to be fixed less efficiently. Two interrelated approaches can be pursued in trying to increase the agronomic efficiency of the enzyme at atmospheric CO2 concentration. (i) The specific activity of the enzyme could be increased by increasing the maximal velocity of the enzyme’s action with respect to both substrates. This will cause a proportional increase in both photosynthesis and photorespiration, but, because the rate of CO2 uptake by photosynthesis exceeds the rate of CO2 loss by photorespiration under natural conditions, net photosynthesis will increase, (ii) The ratio of oxygenase activity to carboxylase activity could be decreased, thereby increasing net photosynthesis by decreasing photorespiratory CO2 evolution.

Abstract: The variegated patterns seen in tissues of chimaeras and mosaics should tell us something about the developmental histories of these tissues. A number of ingenious approaches have been made to try to analyse these patterns, but there is still considerable controversy about the interpretation and relevance of this type of analysis (Mintz 1974; McLaren 1976; West 1978). Comparative analysis between different groups of animals or tissues, or between individuals of different ages, offers one of the more reliable approaches since this type of analysis is not dependent on precise numerical estimates. Even this approach, however, requires some caution and, in the past, a number of inappropriate comparisons have been made. It is essential to understand the contribution of the different elements to the total variegated pattern before a meaningful comparison can be made. The purpose of this article is to consider the organisation of variegated patterns seen in chimaeras and mosaics, and how the degrees of cell mixing and clonal growth in different groups of animals may be compared.

Abstract: Dosage compensation for X-linked genes occurs by the process of X-chromosome inactivation (XCI) in mammalian somatic cells (Lyon 1972). The inactivation of one X chromosome in females takes place early in development, although the exact time is unknown. One method of ascertaining the time of XCI is to determine the activity of the X chromosome at different stages of development as measured by the activity of an X-coded enzyme. Prior to XCI, and in the absence of other dosage compensating mechanisms, the activity for an embryonically expressed X-coded enzyme should be twice as high in female embryos with two X chromosomes, as in male embryos with one X chromosome. The distribution of enzyme activities for single embryos from a litter would have two equal-sized peaks that are separated by a factor of two. The convergence of the two peaks into one would indicate that XCI had occurred.

Abstract: It is widely accepted that only one of two X chromosomes present in the somatic cells of female mammals is active (Lyon 1961). This results in a similar dosage relationship between functional X chromosomes and autosomes in females and males. This compensation occurs early in development between the 2-cell stage (Hoppe and Whitten 1972) and the time of implantation of the embryo into the uterus (Gardner and Lyon 1971). An important issue concerning X-chromosome differentiation is the functional state of X chromosomes during early embryogenesis, before irreversible dosage compensation occurs. That is, does X-chromosome differentiation involve a process of X-chromosome inactivation or X-chromosome activation? Choosing between these alternatives is important for understanding a number of features of X-chromosome differentiation, including: (1) the derivation of molecular and genetic models of this process, and (2) the determination of the timing of this differentiation.

Abstract: Although its results are subject to uncertainties in interpretation, chemical modification is a proven method for defining structure-function relationships in enzymes. Innumerable times, tentative conclusions based on chemical modification studies regarding identities of active-site residues, or even their precise catalytic function, have been substantiated by x-ray crystallography.

Abstract: It is hardly surprising that both the synthesis and the activity of the enzyme ribulose 1,5-bisphosphate (RuBP) carboxylase are highly regulated. This most abundant enzyme on earth catalyzes the entry of CO2 into the reductive pentose phosphate pathway (Calvin cycle) (1), the pathway leading to the reduction of CO2 to sugar phosphates in all green plants (2), including those with a prelimin ary C4 cycle (3) for CO2 accumulation. Such first reactions are often the sites of important metabolic regulation. The carboxylation reaction is one of four steps in the Calvin cycle unique to that cycle and not found in the oxidative pentose phosphate cycle (the other such reactions are the ones converting fructose and sedo heptulose bisphosphates to their respective monophosphates and the reaction converting ribulose 5-phosphate to RuBP) (Figure 1). All four of these reactions are inactivated or are less active in the dark, when the oxidative pentose phosphate cycle and the glycolytic pathway operate. The inactivation in the dark of these four reactions unique to the reductive cycle is required to prevent the oper ation of futile cycles.

Abstract: As a means of obtaining insights into the mechanisms for maintaining X-chromosome inactivation, we have carried out a series of experiments in an attempt to reverse the process. To identify cells in which the silent X has been derepressed, we have developed a model based on the one described by Comings (1966) using human fibroblasts heterozygous for the common A electrophoretic variant of glucose-6-phosphate dehydrogenase (G6PDA). In our model, however, the cells are also heterozygous for the Lesch-Nyhan mutation specifying deficiency of hypoxanthine guanine phosphoribosyl transferase (HGPRT-), so that we can select for rare cells in which reactivation has occurred (Migeon 1972). Clonal populations of skin fibroblasts from females heterozygous for both G6PDA and HGPRT-, but expressing only the alleles on the active X, are subjected to a variety of treatments, and the phenotype with regard to both loci is ascertained following treatment. The resultant phenotype is interpreted according to Table 1. The G6PD heteropolymer, because it is never found in mixtures of the two cells under conditions used for these studies, is a sensitive indicator of two functional X chromosomes within the same cell, while the presence of variants at two X-linked loci helps distinguish reactivation from other events such as reversion, somatic crossing over, or contamination with cells of other phenotype.

Abstract: The limiting factor in the use of mouse chimeras for the revelation of detailed fate maps during embryogenesis is the lack of suitable cell marker systems. This limitation is evident when the list of available chimeric or mosaic markers is examined and compared to the criteria for an ideal marker system. An ideal cell marker should (1) be expressed in all descendants of a cell; (2) be present in all cells of a tissue or organism; (3) be cell autonomous; (4) be cell localized; (5) exist in variant forms which are selectively neutral; (6) have variant forms which can be detected in histologic sections; and (7) have variants which are expressed early in embryogenesis.

Abstract: The control scheme for the expression of X chromosomes in female mammals can be considered to consist of two parts (Lyon 1972, 1974). The first of these involves the initiation of inac-tivation of one X chromosome (Kratzer and Gartler 1978; Monk 1978; Chapman, West, and Adler 1978). This initial choice may be either random, as it is in eutherian embryonic somatic tissues, or preferential, as occurs in extraembryonic components of some species (Takagi 1978; West, Papaioannou, Frels, and Chapman 1978) and in somatic tissues of marsupial mammals (Cooper, Johnston, Murtagh, Sharman, VandeBerg, and Poole 1975). This report will focus on the second part of the problem of X-chromosome differentiation: how genetic repression of the inactive X is maintained and propagated. It will consider specifically some of the insights into the regulatory mechanisms involved in the maintenance of repression that may be gained from experiments designed to derepress genes on the inactive X.

Abstract: Increased population and the dietary changes accompanying increased affluence are creating a need for a suggested doubling of world cereal grain production (a 3% per year compounding rate) and quadrupling of grain legume production (a 6% per year compounding rate) during this quarter century (1). CO2 enrichment of field-grown crops has demonstrated the possibility of enhancing RuBP carboxylase activity to achieve improved crop production; it increases the production of grain legumes by 50 to 100% and that of cereal grains, for which the studies are less complete, by perhaps 10 to 50%. Results of O2 alteration of growth-room legumes and cereal grains are consistent with the results of CO2 enrichment except for a second role of O2 in assimilate partitioning. It may be necessary to include other components of the system, e.g., additional soil fertility, especially for non-N2-fixing plants, to enable an improved RuBP carboxylase to increase production. No practical method—chemical, genetic, or physical—of improving RuBP carboxylase activity has been reported.

Abstract: The properties, distribution, biogenesis, function, and regulation of ribulose bisphosphate carboxylase/oxygenase, as described elsewhere in this Symposium, are mainly integrated around the mechanism of action of the carboxylase and oxygenase reactions. Recently, interest has been stimulated by the discovery of the oxygenase reaction (1, 2) and its role in the glycolate pathway of photorespiration (3), and by the desirability of differential regulation of the carboxylase and oxygenase activities, if possible, in favor of the former in order to increase photosynthetic productivity. A prerequisite for such regulation is a thorough knowledge of the relationships between structure and function in this protein.