Genetic Dominance and the Heat-Shock Response

The Heat-shock Response and the Molecular Basis of Genetic Dominance

by D. R. Forsdyke The Journal of Theoretical Biology (1994), 167, 1-5
(With copyright permission from Academic Press)
(Received on 10 April 1992, Accepted in revised form on 3 June 1993)

1. Two Theories of Dominance
2. The Dose-Response Theory
3. Difference between Rate-limiting and Non-rate-limiting Steps
4. "Extreme Environmental Disturbances"
5. The Heat-shock Response
6. X-Chromosome Dosage Compensation

End Note


Abstract. Wild-type alleles are usually dominant over deleterious mutant alleles. For a particular pair of such alleles possible populations include a wild-type homozygote population, a heterozygote population, and a mutant homozygote population. 

    Fisher's theory that dominance would evolve by selection acting on the heterozygote subpopulation has lost ground in favour of the "dose-response" theory under which dominance is an incidental consequence of selection acting on the wild-type homozygote population. This postulates a "margin of safety" in the quantity of wild-type gene product so that heterozygotes with only one copy of a wild-type allele still have sufficient product for normal function. The selective force postulated to lead to the evolution of this margin of safety is some unspecified "extreme environment disturbance". 

    The author has proposed elsewhere that the heat-shock response evolved very early as part of an intracellular system for self/not-self discrimination. This paper proposes that the rapid decrease in quantity of most normal proteins occurring in the heat-shock response would have provided a sufficient selective force for the margin of safety to have evolved.

1. Two Theories of Dominance

Ronald A. Fisher (1890-1962)

Wild-type alleles are usually dominant over deleterious mutant alleles (Mendel, 1866). Possible mechanisms of dominance have been the subject of much debate, notably the classic dual between Fisher and Wright. Fisher (1931) proposed that heterozygotes begin with codominant expression of alleles, which are then subject to modification by the products of other genes (modifier genes); eventually, following evolutionary selection acting on the heterozygote subpopulation, only the wild-type phenotype is observed in heterozygotes.

    Growing evidence against the generality of Fisher's theory (Charlesworth, 1979; Orr, 1991), makes it appropriate that an alternative theory, the "dose-response" or "physiological" theory (Haldane, 1930; Muller, 1932; Wright, 1977), should receive closer scrutiny. A detailed enzyme kinetic analysis of the latter has been provided by Kacser & Burns (1980). A simpler enzyme kinetic model presented here provides a better basis for understanding the possible role of heat-shock proteins in the evolution of dominance.

2. The Dose-Response Theory

The dose-response theory states that the quantity of the product of a dominant wild-type gene in a homozygote is so much in excess of the needs of the organism that halving this quantity, as might occur in a heterozygotic containing a wild-type allele and a mutant allele (encoding a non-functional product), will not change the phenotype. Figure 1 shows a plot of some quantifiable phenotypic feature against the dose of the product of a gene concerned with that phenotype.


phenotype as function of dose of gene product

FIG. 1. Hypothetical in vivo dose-response curve showing some quantitative measure of phenotype (such as the rate of formation of enzyme product) as a function of the dose of a gene product which contributes to that phenotype (such as the quantity of an enzyme). 

    The vertical arrow indicates the normal concentration of a non-rate-limiting gene product in a diploid wild-type homozygous cell. Halving this dose (Y to Y') has a minimal effect on phenotype. X and X' indicate corresponding points for a rate-limiting gene product. 

    Added is a chart of the flow of information from gene to phenotype, as considered in this paper. It is assumed that in most circumstances gene-product concentration is directly proportional to gene dosage.


    At low doses of gene product (indicated by point X on the curve) the phenotype depends directly on gene-product quantity. Halving the dose (point X') halves the phenotypic parameter. At high doses of gene product a plateau is approached when some other factor becomes limiting (e.g. the availability of substrate A; see later). Under these conditions increasing the quantity of gene product has a minimal effect on the phenotype. 

    In the case of the wild-type homozygote it is postulated that the normal amount of gene product corresponds to a point well along the plateau of the curve (indicated by point Y). This creates a "factor of safety" (Haldane, 1930), or "margin of stability and security" (Muller, 1932), so that halving the amount of the product (to point Y') has no effect on phenotype.

3. Difference between Rate-limiting and Non-rate-limiting Steps

In many cases the phenotype will be the result of a series of enzyme catalyzed reactions such as shown in Fig. 2.


metabolic pathway with feed-back inhibition by the end product

FIG. 2. A hypothetical metabolic pathway catalyzed by enzymes E1, E2 and E3. A is the substrate of E1, which catalyzes the rate-limiting step in the pathway. A is also metabolized by another pathway. B is the product of E1 and the substrate of E2, which is normally not rate limiting. C is the product of E2 and the substrate of E3, which is also normally not rate limiting. D, the product of E3, is responsible for the phenotype and for pathway regulation by feedback inhibiting E1.


Substrate A is converted to end-product D through a series of intermediates (B, C), catalyzed by enzymes E1, E2, and E3. The first step of the pathway, the rate-limiting step, is subject to feedback inhibition by D. Using radioactively labelled A and short incubation periods, it has been shown experimentally that the system may be modeled as if in vivo the intermediate steps had no effect on the reaction rate (Forsdyke, 1971).


in vivo dose-response curves, showing
how the actual concentration of a metabolic intermediate relates to the curve

FIG. 3. Hypothetical in vivo dose-response curves for enzymes E1 , E2 , and E3 . Rates of formation of products (B, C, and D) are expressed as functions of the concentrations of substrates A, B, and C, respectively. The vertical arrows refer to the normal in vivo concentrations of these substrates as they interact with the enzymes.


    Figure 3 shows hypothetical in vivo substrate dose-response curves for the three steps in the pathway. The vertical arrows indicate the normal substrate concentration existing in vivo. In the case of the rate-limiting enzyme El the in vivo concentration of A must, by definition, correspond with the plateau of the dose-response curve, so that enzyme concentration, and not substrate concentration, is rate limiting. This would correspond to point X on the plot of reaction rate versus enzyme concentration (Fig. 1). 

    In the case of the non-rate-limiting enzymes E2 and E3, the normal substrate concentration corresponds to the ascending limbs of the corresponding substrate dose-response curves (Fig. 3). There is ample enzyme to accommodate fluctuations in availability of the substrates B and C (the products of El and E2, respectively). This quantity of enzyme would correspond to point Y on a plot of reaction rate versus enzyme concentration (Fig. 1). 

    Thus, after a molecule of A has squeezed through the "bottle-neck" El to become B, subsequent chemical modifications by E2 and E3 do not influence the rate of accumulation of the end-product D.

    It can be seen that the situation with the non-rate-limiting enzymes (E2, E3) corresponds to the "margin-of-safety" scenario. In the case of the rate-limiting enzyme E1, halving of gene-product concentration (X to X' in Fig. 1) would have consequences for the phenotype. 

    However, in general, rate-limiting enzymes are subject to complex controls by products of intermediary metabolism. The most common of these is end-product inhibition (Fig. 2). A decrease in D would decrease the inhibition. This would increase the activity per molecule of E1, so that the reaction rate would become the same as in the homozygote. Thus, in this case, feedback inhibition provides another "margin of safety" allowing a heterozygote to maintain the wild-type phenotype.

 4. "Extreme Environmental Disturbances"

A major problem with what we might now call the margin-of-safety theory, is in determining what selective forces would have created and sustained the margins of safety. Some have argued that this can be explained entirely in metabolic terms (Kacser & Burns, 1980). In the case of a rate-limiting enzyme (E1) the margin could indeed be a simple consequence of the evolution of metabolic controls. It is in the case of non-rate-limiting enzymes that a further explanation must be sought. What sustains the enzyme activity in a wild-type homozygote at point Y rather than at point Y' (Fig. 1)?

     Haldane interpreted dominance in metabolic terms, but adopted Fisher's view that weak selective evolutionary forces acting on heterozygotes would be sufficient to maintain the margin of safety. Thus Haldane (1930) wrote:

J.B.S. Haldane (1892-1964)

    

"If we imagine a race whose genes were only just doing the work required of them, then any inactivation of one of a pair of genes would lead to a loss of total activity. Thus if "A1 A1" can just oxidize all of a certain substrate as fast as it is formed, its inactivation will produce a zygote "A1 a" which can only oxidize about half. If now A1 mutates to A2, which can oxidize at twice or thrice the rate of A1, if necessary, no effect will be produced, i.e. "A1 A2" and "A2 A2" zygotes will be indistinguishable from "A1 A1". But "A2 a" will be normal. Hence "A2 a" zygotes will have a better chance of survival than "A1 a", and A2 will be selected."

Herman Joseph Muller (1890-1967)

Muller (1932), however, thought that the evolutionary selection would act at the homozygote level. He postulated:

"that the mutations favouring dominance .. . have been selected and are maintained, not so much for their specific protection against heterozygosis at the locus in question, but as to provide a margin of stability and security, to insure the organism against weakening or excessive variability of the character by other and more common influences -- environic and probably also genetic".

 

Along similar lines Wright (1977) stated that:

"because of extreme environmental disturbances, a considerable excess [of gene product] is advantageous. This is likely to be so great that the correlated response of the rare heterozygote is also brought fairly close to the asymptote, thus giving a high degree of dominance."

The possible nature of the "extreme environmental disturbances" was not specified.

 

Sewall Wright (1889-1988)

 5. The Heat-shock Response

The heat-shock response follows a sudden change in various physical or chemical features of the environment, and is particularly notable following an increase in temperature (Nover, 1989). The response is detected as a rapid increase in the intracellular concentrations of a set of evolutionarily conserved "heat-shock" proteins, which is accompanied by a decrease in the concentrations of most normal proteins. The notion that the response is predominantly concerned with protection against thermal and other types of "stress" has recently lost ground (Bader et al., 1992; Fisher et al., 1992).

    It is proposed elsewhere (Forsdyke, 1985, 1991, 1992, 1995), that the response has evolved as part of a mechanism for distinguishing the proteins of intracellular pathogens ("not-self") from normal intracellular proteins ("self"). The proteins of the crowded cytosol exert a collective pressure tending to make individual protein species aggregate when their concentrations exceed their individual solubility limits. These concentrations have been fine-tuned over evolutionary time so as not to exceed these limits. Not-self proteins more readily "trip" an intracellular surveillance system because their concentrations have not been so fine tuned.

    The aggregations, however, being primarily entropy driven (Lauffer, 1975), are strongly increased by an increase in temperature. The organism exploits this (e.g. fever) to promote the aggregation of the proteins of a foreign pathogen (Nguyen et al., 1989). In this process self proteins might also be aggregated. To avoid this, the concentrations of normal proteins decrease. However, in turn, this decreases the collective pressure exerted by the cytosolic proteins to make the proteins of the pathogen aggregate. To compensate for this, a special set of proteins, the heat-shock proteins, are produced (Forsdyke, 1995).

    The main point to be made about the heat-shock response in the present context is that it probably reflects a fundamental process which appeared early in evolution when sets of replicators encased in a membrane (prototypic cells) had to be protected against invasion by foreign replicators (prototypic viruses; Forsdyke, 1991). All subsequent evolutionary developments would potentially be influenced by this pre-existing system. The sudden general fall in the concentration of normal self proteins as part of the heat-shock response would severely compromise cell function if there were not a margin of safety regarding function. Thus the heat-shock response would constitute a powerful evolutionary force acting on wild-type homozygotes. 

    This would lead to the general evolution of proteins of a specific activity sufficient to sustain (or facilitate the recovery of), cell function, at a time when protein concentration had fallen. An incidental outcome of this would be that a heterozygote would normally have sufficient gene product so that it would be phenotypically indistinguishable from the wild type.

    However, having only one copy of the allele in question, a heterozygote would have sacrificed part of its margin of safety and this might have some impact on viability. Indeed, heterozygotes for lethal mutations do show some reduction in viability compared with the homozygous wild types and may be more temperature-sensitive (Plunkett, 1932; Simmons & Crow, 1977). 

    A viral infection might trigger a fever and an associated heat-shock response; the quantity of a non-rate-limiting protein might then fall from level Y' to X in Fig. 1. Thus a protein unable to respond to normal metabolic controls (e.g. show increased activity in response to a loss of end-product inhibition), would now become rate limiting. The consequence of this would depend on timing and the nature of the end-product involved. A viral infection at a key developmental stage might have disastrous consequences.

 6. X-Chromosome Dosage Compensation

The hypothesis offers a new way of looking at the problem of X-chromosome dosage compensation. This was first described as the process by which the function of the single X-chromosome in male fruit flies is made equivalent to the function of both X-chromosomes in females (Muller, 1948). Without dosage compensation, the situation would be formally equivalent to the points Y (in females) and Y' (in males) as shown in Fig. 1. 

    Muller postulated that these "exceedingly minute" phenotypic differences (differences between the point Y and point Y' phenotypes) would constitute a sufficient selection pressure for dosage compensation to have evolved. The heat-shock response, however, in shifting heterozygote (male) gene product concentrations from point Y' to point X (Fig. 1), would have created a much greater selection force for the evolution of a margin of safety in males. Although this might have been a factor in the evolution of dosage compensation, it is argued in the accompanying paper (Forsdyke, 1994) that the major factor is probably the need to fine-tune protein concentrations, rather than protein functions, to be equal in male and female cells.

This work was supported by the Medical Research Council of Canada and the Leukaemia Research Fund of Toronto.

REFERENCES

BADER, S. B., PRICE, B. D., MANNHEIM-RODMAN, L. & CALDERWOOD, S. K. (1992). Inhibition of heat-shock gene expression does not block the development of thermotolerance. J. Cell. Physiol. 151, 56-62.

CHARLESWORTH, B. (1979). Evidence against Fisher's theory of dominance. Nature, Lond. 278, 848-849.

FISHER, B., KRAFT, P., HAHN, G. M. & ANDERSON, R. L. (1992). Thermotolerance in the absence of induced heat-shock proteins in a murine lymphoma. Cancer Res. 52, 2854-2861.

FISHER, R. A. (1931). The evolution of dominance. Biol. Rev. 6, 345-368.

FORSDYKE, D. R. (1971). Application of the isotope-dilution principle to the analysis of factors affecting the incorporation of 3H-uridine and 3H-cytidine into cultured lymphocytes. Biochem. J. 125, 721-732.

FORSDYKE, D. R. (1985). Heat-shock proteins defend against intracellular pathogens: a non-immunological basis for self/not-self discrimination. J. theor. Biol. 115, 471-473.

FORSDYKE, D. R. (1991). Early evolution of MHC polymorphism. J. theor. Biol. 150, 451-456 (1991).

FORSDYKE, D. R. (1992).Two signal model of self/not-self discrimination: an update. J. theor. Biol. 154, 109-118.

FORSDYKE, D. R. (1994). Relationship of X chromosome dosage compensation to intracellular self/not-self discrimination: a resolution of Muller's paradox. J. theor. Biol. 167, 7-12.

FORSDYKE, D. R. (1995). Entropy-driven protein self aggregation as the basis for self/not-self discrimination in the crowded cytosol. J. Biol. Systems 3, 273-287.

HALDANE, J. B. S. (1930). A note on Fisher's theory of the origin of dominance and on a correlation between dominance and linkage. Am. Nat. 64, 87-90.

KACSER, H. & BURNS, J. A. (1980). The molecular basis of dominance. Genetics 97, 639 666.

LAUFFER, M. A. (1975). Entropy-driven Processes in Biology. New York: Springer-Verlag.

MENDEL, G. (1866). Versuche uber Pfanzenhybriden. Verhandl. Naturforsch. Ver. Brunn 4, 3-47 (1866).

MULLER, H. J. (1932). Further studies on the nature and causes of gene mutations. In: Proc. 6th Int. Cong. Genet. 2, (Jones, D. F. ed.) pp. 213-255. Menasha, WI: Banta.

MULLER, H. J, (1948). Evidence on the precision of genetic adaptation. Harvey Lect. 43, 165-229.

NGUYEN, V. T., MORANGE, M. & BENSAUDE, 0. (1989). Protein denaturation during heat shock and related stress. E. coli beta-galactosidase and Photinus puralis luciferase inactivation in mouse cells. J. Biol. Chem. 264, 10487-10492.

NOVER, L. (1989). 125 years of experimental heat-shock research. Genome 31, 668-670.

ORR, H. A. (I 99 1). A test of Fisher's theory of dominance. Proc. natn. Acad. Sci. U.S.A. 88, 11413-11415.

PLUNKETT, C. R. (1932). Temperature as a tool of research in phenogenetics. In: Proc. 6th Int. Cong. Genet. 2, (Jones, D. F. ed.), pp. 158-160. Menasha, WI: Banta.

SIMMONS, M. J. & CROW, J. F. (1977). Mutations affecting fitness in Drosophila populations. A. Rev. Genet. 11, 49-78.

WRIGHT, S. (1977). The evolution of dominance. In: Evolution and the Genetics of populations, Vol, 3, pp. 498-526. Chicago: The University of Chicago Press.

End Note

The basic idea in this paper was advanced as early as 1909 by G. H. Shull (The "presence and absence" hypothesis. American Naturalist 43, 410-419). 

"Having arrived at the conclusion that all the Mendelian characters are dependent upon chemical relations, we may return to the question of dominance, and the relation between the two kinds of homozygotes and the heterozygote, and see to what extent the known facts may be interpreted in terms of chemical experience.

    A fundamental principle in this connection is the law that the extent of a reaction between two chemicals is determined by the amount of the reagent which is present in less relative quantity, and not by the one which is present in excess. When the positive homozygote ... and the heterozygote ... are alike, i.e., when there is complete dominance of presence over absence, it may mean that already the presence of the one unit A of the heterozygote is sufficient to result in the maximum reaction, in which case the doubled factor AA of the positive homozygote can do no more. 

    When, on the other hand, one unit A is not sufficient to produce a maximum reaction with the other factors present, the AA of the homozygote produces the corresponding character in greater intensity, and the heterozygote will be intermediate between the two homozygous parents." 

 

Regarding his dispute with Wright, in correspondence (1934) Fisher wrote:

"It is quite obvious that in a chemical reaction one ingredient may be present in excess, in the sense that small variations in its amount have very little effect on the speed of the reaction, while a large diminution of it would slow the reaction down. That this is probably the case with the products of some genes is shown by Stern's 'bobbed' allelomorphs. 

    It is a relatively obvious way of producing dominance against mutations which partially inactivate the mutant genes. It might, as far as my theory is concerned, be the only mechanism by which dominance is produced, though I do not imagine that this is so. 

    But if this were so, the occurrence of dominance would be just as much in need of explanation as if dominance were produced by some other mechanism. For the fact that one component of a reaction is present in excess implies that its speed is regulated by other components, and that mutations affecting these, if they occurred, would not be recessive, whether the mutation reduced the activity or enhanced it. 

    On the theory of components in excess we should have to say that the organism had been so modified that the speeds of all biochemical processes were regulated only by the products of genes incapable of mutation."

Natural Selection, Heredity and Eugenics. Edited by J. H. Bennett. Oxford Univ. Press 1983. p. 235


Relationship of X-Chromosome Dosage Compensation to Intracellular Self/Not-self Discrimination: A Resolution of Muller's Paradox?

The Journal of Theoretical Biology (1994) 167, 7-12.

(With copyright permission from Academic Press)

(Received on 10 April 1992, accepted in revised form on 3 June 1993)

1. Introduction
2. Aneuploidy and the Evolution of Dosage Compensation
3. "Exceedingly Minute Differences"
4. Fine-tuning of Intracellular Protein Concentrations
5. Differential Aggregation of Foreign Proteins
6. Role of X Chromosome Inactivation
7. Two Active X Chromosomes in Oocytes and Embyros
8. Compensation in Z/W Chromosome Systems


Abstract. Muller's paradox is that X chromosome dosage compensation seems to have evolved in spite of there being only "exceedingly minute differences" between compensated and uncompensated phenotypes. The paradox can be resolved by considering, not the specific functions of individual proteins, but the collective functions of proteins per se. 

    One such function could be the collective pressure exerted by proteins in the crowded cytosol to drive individual protein species from simple solution (aggregation) when their concentrations exceed specific thresholds. It is proposed that over evolutionary time, individual genes, both on X-chromosomes and autosomes, would have fine-tuned factors such as transcription rates and protein stabilities to this collective pressure. 

    However, without X-chromosome dosage compensation the total concentration of cytosolic proteins, and hence the collective pressure, would have fluctuated between male and female generations. Fine-tuning, a process of vital importance for intracellular self/not-self discrimination, would have been severely compromised.

1. Introduction

    In many species the sex of an individual depends on which of two alternative sex chromosomes he/she inherits (Bull, 1983). In some species (e.g. fish, amphibia) the sex chromosomes are similar (homomorphic) and appear to differ only in the genes affecting sexual differentiation. The latter are kept from recombining with their alleles by inversions or by some other mechanism (Ohno, 1967). In other species (e.g. mammals, birds) the sex chromosomes differ in size (heteromorphic). One of the chromosomes appears to have degenerated losing most of its genes (Charlesworth, 1991). One sex, the homogametic sex, contains two copies of the normal, undegenerate, chromosome (X or Z). The other sex, the heterogametic sex, contains one normal chromosome and one degenerate chromosome (Y or W). These show sequence similarity only in a short (pseudoautosomal) segment.

    The essential haploidy (hemizygosity) of the sex chromosomes in the heterogametic sex has three important implications:

  • (i) Deleterious recessive mutations are expressed. Individuals containing these mutations will tend not to reproduce and so fewer deleterious mutations should be passed on to future generations.

  • (ii) It is not possible to repair DNA damage (or excise transposed elements; Steinemann, 1982) on the basis of information contained in an allelic chromosome. Without accurate repair the load of deleterious mutations passed on to future generations would increase (Bernstein & Bernstein, 1990). Eternally locked in heterogametic generations, the Y and W chromosomes would never have had the opportunity to repair damage on this basis and this may have contributed to their degeneration.

  • (iii) Since gene dosage is often a major determinant of gene product dosage, the dosage of X- and Z-encoded gene products (proteins) in the heterogametic sex should be half those in the homogametic sex. This discrepancy might either be accepted by the organism (dosage tolerance or dosage imbalance), or be adjusted (dosage compensation or dosage balance).

     Studies by Muller with the fruit fly Drosophila melanogaster in the late 1920s established the existence of a mechanism to adjust the concentrations of X-encoded gene products (excluding some concerned with sexual differentiation and a few others), so that these were equalized between the sexes (Muller, 1948). It is now known that this involves an increase in the expression of the single X-chromosome in the male fruit fly to equal the combined expression of the two X chromosomes in the female (Kuroda et al., 1991). In mammals equalization is achieved by inactivating one of the X chromosomes in the female (Lyon, 1992).

    A necessary condition for the evolution of dosage compensation was the degeneration of one of the sex chromosomes to create the Y-chromosome. However, with no clear justification, the assumption has grown that the degeneration of the Y-chromosome was alone sufficient to drive the evolution of dosage compensation. Thus Haldane (1933) wrote:

"The tendency to balance may be regarded as a secondary effect of the accumulation of recessive lethals on the Y-chromosome. Indeed, this accumulation can only proceed as the X develops internal balance" (my italics).

This coupling of Y degeneration and dosage compensation is implied by some modern authors. Charlesworth (1978) wrote that the gradual degeneration of the Drosophila Y-chromosome itself:

"Creates a selection pressure for differentially increasing the activity of the X-chromosome in heterogametic individuals" (my italics).

Of the mammalian type of dosage compensation, he wrote (Charlesworth, 1991) that this:

"Creates a selective advantage to reducing X activity in the homogametic sex" (my italics),

and added that degeneration of the Y-chromosome and dosage compensation are "

processes which must almost inevitably occur"

   This paper describes the attempts of Muller and others to understand the forces driving the evolution of dosage compensation. It is argued that dosage compensation can best be understood by taking into account the possibility that proteins influence phenotypes not only on the basis of their individual specific functions, but also on the basis of their collective functions as proteins per se. While still in need of experimental verification, the strength of this paper's hypothesis is that it brings together a variety of observations in fields which have not previously been seen as related.

2. Aneuploidy and the Evolution of Dosage Compensation

    The issue of what drove the evolution of dosage compensation was partially addressed by Ohno (1967) who noted that a heterogametic individual can be regarded as aneuploid for the X-chromosome.

"The hemizygous existence for all the genes on the X should be very perilous since monosomy for even the smallest autosome (only one fourth the size of the X) is apparently lethal in man".

Why aneuploid states are lethal was not discussed. In Drosophila Lindsley and coworkers (1972) found that:

"the deleterious effects of aneuploidy are, in the main, caused by the additive effects of genes that slightly reduce viability and not by the individual effects of a few aneuploid-lethal genes among a large array of dosage-insensitive loci" (my italics).

Orr (1990), when considering why polyploidy is rarer in animals than in plants, took the issue further. He noted first:

"The difficulty in establishing a tetraploid line in organisms with a genetically degenerate sex chromosome: although polyploid speciation does not necessarily disrupt sex determination in such species, it does invariably disrupt the balance of X chromosome relative to autosomal gene products normally maintained by dosage compensation" (Orr's italics).

He noted further that in mammalian females:

"The number of Xs that remain active increases with the number of autosomal sets"

He then concluded that:

"The doses of X-linked and autosomal genes have been fine-tuned by natural selection to ensure proper interactions between these loci" (my italics).

The nature of the "proper interactions" and why they had to be "fine-tuned" were not discussed.

    The importance of the ratio of sex chromosomes to autosomes both for dosage compensation and for sex-determination is well recognized in fruit fly and nematode systems (Hodgkin, 1990). Since in mammalian systems ratio-dependence seems to have been retained only for dosage compensation (Book & Santesson, 1961; Harnden, 1961; Jacobs & Midgeon, 1989), it is likely to be critical for this process. 

    It is difficult to imagine how sex chromosomes and autosomes could sense each other's dosage directly, and it seems more likely that the quantitation is carried out at the gene-product level. Furthermore, noting that polyploid cells are larger than euploid cells (thus presumably keeping the concentration of cytoplasmic components constant), it seems likely that the quantitation involves some relationship between the concentrations of the products of sex chromosomes and autosomes (rather than a relationship between their absolute quantities).

3. "Exceedingly Minute Differences"

    Muller (1948) approached the problem of the evolution of dosage compensation in fruit flies by first noting the relationship between the concentration of a protein and the activity of that protein as observed in a quantifiable phenotype. As a dose of a gene (and hence of the corresponding protein) is increased, there would be an approximately linear increase in the character assayed.

     However, this would only hold as long as the dose of the protein was limiting. Above a certain dose something else would become limiting, and the dose-response curve would tend to plateau (see Forsdyke, 1994 for further elaboration). Muller noted that the dose of most wild-type gene products corresponds to a point well along the plateau, so that a decrease in dose by half, due to hemizygosity, would still leave sufficient gene product to guarantee maximum activity (i.e. the phenotype would still correspond to a point on the plateau of the dose-response curve). In this circumstance there would have been no selection pressure, based on differential gene-product function, for dosage compensation to have evolved. He described this paradox as follows:

"The effects of individual genes, whether on the X or other chromosomes, are so near their saturation levels as to make direct discrimination between one and two doses impossible. Should not the very fact that most of these genes are so near their saturation level make dosage compensation unnecessary? Why should there be a perceptible advantage in going through the motions of equalizing them still further?"

    The paradox was not formally acknowledged by Muller because the following quasi-metaphysical answer, understandable at a time when little was known of the inner workings of the cell, appeared to satisfy not only Muller, but also his critics:

"The compensation mechanism must be concerned with the equalization of exceedingly minute differences. Dosage compensation has in fact become established because of its advantage in regulating more precisely the grade of characters whose variations in grade, even without it, would be exceedingly minute" (Muller's italics). "The selective forces that established it must depend on such minute advantages".

    No less paradoxical were subsequent studies in other species (Ohno, 1967). Some X-linked genes encode enzymes. While several enzymes may contribute to a metabolic pathway, only one of these is normally rate-limiting (Forsdyke, 1994). This rate-limiting enzyme is subject to numerous fine controls, such as end-product inhibition, resulting in large variations in activity (e.g. 10-fold). Even if an X-linked enzyme were normally rate limiting, it is unlikely that a two-fold change in enzyme concentration would affect flow along the metabolic pathway. 

    Indeed, it is a common observation that heterozygotic carriers of various traits are often no worse off than the corresponding homozygotes (Ohno, 1973). Most deleterious mutations show a high degree of recessivity. Thus, on the basis of these studies also, phenotypic differences upon which the selective forces of evolution could have acted are unlikely to have been significant. Along these lines, Chandra (1985) has argued that X-chromosome inactivation is primarily a sex-determining device and that any compensating effects are incidental.

4. Fine-tuning of Intracellular Protein Concentrations

    A consideration of the fine-tuning of the concentrations of individual proteins within cells, discussed fully elsewhere (Forsdyke, 1995; click here), provides a possible solution to Muller's paradox. Over a series of generations, a Y-chromosome and its descendants exist in a succession of male cells. This path may be expressed as:

M M M M M MM M M M M M M M..…

Over evolutionary time, factors such as the transcription rates of genes on the Y-chromosome and the stabilities of their products (mRNAs and proteins) could have become fine-tuned to the needs of this relatively stable intracellular environment. A relatively constant constitutive concentration of each gene product could have become established. On the other hand, the X-chromosomes and the autosomes and their descendants alternate between male and female cells. A typical path might be:

M F M F F MF M F M F F M M..…

Over evolutionary time it would be difficult to fine-tune both for cells containing a second X-chromosome and for cells containing a solitary X-chromosome. By inactivating one X whenever two are present (the mammalian model), or hyperactivating the X whenever one is present (the fruit fly model), the intracellular environment would be stabilized and fine-tuning could occur.


    darw26b.gif (750791 bytes)

FIG. 1. Fine tuning of protein concentration is not possible without X-chromosome dosage compensation

    Passage of Y-chromosomes and X-chromosomes through the generations occurs either in male (M) or female (F) cells. Contributions of chromosomes to the cytosolic protein concentration are shown for autosomes, for X-chromosomes (grey), and for Y-chromosomes (vertical stripes). The contribution of the latter is actually much less than shown,  so that halving the contribution of the X-chromosome ("dosage compensation") in female generations would keep the cytosolic protein concentration essentially independent of the sex of the host cell. [This figure was not included in the original 1994 paper.]


   What is it about the intracellular environment that has to be stabilized? Why is fine-tuning important? A possible answer was given previously when addressing another paradox (Forsdyke, 1985, 1991, 1992). Cytotoxic T cells recognize surface complexes of MHC class I proteins in association with peptides derived from proteins synthesized within the same cell. Since mechanisms of self/not-self discrimination appear to be extracellular, cells are held to load MHC proteins intracellularly with peptides from both self and not-self proteins for display at the cell surface. 

    Some MHC-self-peptide complexes are indeed found (Rotzschke & Falk, 1991). However, the virtue of an obligatory display of self peptides is not readily apparent; it requires the prior deletion or inactivation of all T cells specific for MHC-self-peptide complexes, thus generating extensive "holes" in the T-cell repertoire (Du Pasquier & Blomberg, 1982; Vidovik & Matzinger, 1988; Schild et al., 1990; Ohno, 1991). A mechanism permitting some intracellular discrimination between self proteins and not-self proteins would allow the preferential loading of MHC class I proteins with peptides derived from not-self proteins, thus avoiding the logistic problem of competition with a myriad of peptides derived from self proteins.

     For a cell to distinguish intracellularly between a self protein and a not-self protein (encoded by an intracellular pathogen), would appear to be a formidable problem. Both classes of protein might, after all, be synthesized on host ribosomes and might be released into similar cytosolic compartments. However, there are two key differences which might be exploited by an appropriate surveillance mechanism.

  • The first of these is that, relative to not-self protein encoding genes, each self protein-encoding gene has had more evolutionary time to fine-tune its product concentration to the intracellular environment created by the other self genes with which it has been travelling through the generations.

  • The second is that, whereas self protein-encoding genes are essentially at peace with the concentrations at which their products have arrived, the raison d'etre of most foreign pathogens is, at some time within the lifespan of their host, to increase in number. This may imply synthesizing specific proteins at rates resulting in unacceptably high cytosolic concentrations.

If a pathogen with a short generation time and high mutation rate could gain a foothold, it would readily accommodate to the first difference. However, the second difference, combined with the heat-shock response (discussed elsewhere; Forsdyke, 1994; 1995), could be decisive.

5. Differential Aggregation of Foreign Proteins

    How would an "unacceptably high" cytosolic concentration be detected? When macromolecules in solution reach a critical concentration it becomes energetically more favourable for them to aggregate, like-with-like, than to remain in simple solution. The aggregation involves a liberation of bound water and an increase in entropy. Being primarily entropy driven, the aggregation is promoted by an increase in temperature (Lauffer, 1975, 1989; Leikin & Parsegian, 1992).

    The crowded cytosol constitutes an environment which readily drives proteins out of solution when they exceed individual concentration thresholds, This is well recognized from the difficulties encountered when trying to over-express proteins within foreign cytosols using expression vectors. In such systems the formation of insoluble aggregates is greatly enhanced by increasing temperature over a physiological range. This has been shown very clearly in a recent study of foreign protein expression in mouse cells (Nguyen et al., 1989).

    The proposal, then, is that not-self proteins more readily "trip" the intracellular surveillance system because their concentrations are not so fine-tuned to the phase-separating proclivities of the crowded host cytosol as are the concentrations of self proteins (Fig. 2). In an organism without cytotoxic T cells, or their equivalent, this would trigger cell death (Forsdyke, 1995). In an organism with T cells the aggregates would be directed to a site for degradation to peptides. Specific peptides would then be displayed in association with MHC class I proteins at the cell surface (Rotzschke & Falk, 1991). A cell would thus become labelled for destruction by cytotoxic T cells (Forsdyke, 1995). An advantage of this scheme is that it leaves open for an organism the option of declaring one of its own self proteins as "foreign" should its synthesis or turnover become disordered.


paradox2.gif (54902 bytes)

FIG. 2. Theoretical curves showing that most gene products only limit phenotype at low concentrations, and that aggregation occurs at concentrations exceeding that corresponding to two autosomal allelic genes or one mammalian X-linked gene.

    Although gene dosages are normally discrete, gene product (protein) concentrations (circles) and phenotypes (triangles) are shown as continuous functions. The situation prevailing in a normal cytosol is indicated by the vertical arrow. In the absence of aggregation, the concentration of gene product is taken to be a rectilinear function of the dosage (copy number) of a constitutive gene (filled circles). Normally, a particular gene product only limits the phenotypic character to which it contributes at low concentrations. As the normal cytosolic concentration is approached other gene products become limiting (plateau in values of phenotypic character assayed; filled triangles). 

    Thus, decreasing gene product concentration by half may not change the phenotype. At above normal gene dosages, gene product aggregation occurs and there is a fall in the quantity of gene product present in non-aggregated form (loss of rectilinearity; open circles). These changes may have some effect on phenotype (fall in phenotypic parameter assayed; open triangles), but the major effect will be the "tripping" of the intracellular not-self surveillance system by aggregates.


6. Role of X Chromosome Inactivation

    Without dosage compensation there would be a fluctuation in the total intracellular protein concentration between male and female generations. This would imply a fluctuation in the pressure to drive individual proteins out of simple solution when their concentrations exceeded specific concentration thresholds. An increase in the activity of the X chromosome in male generations (Muller, 1948; Kuroda et al., 1991), or inactivation of one X chromosome in female generations (Lyon, 1992), would stabilize the pressure and favour fine-tuning of gene-product concentrations over evolutionary time. 

    A corollary of this is that, in addition to being under evolutionary constraint to preserve specific function, genes encoding proteins are also under evolutionary constraint both to maintain the collective pressure to drive individual proteins from solution and to maintain individual protein solubilities in the face of that collective pressure (Forsdyke, 1994b).

    A disturbance of phenotype resulting from hemizygosity enforced by deterioration of the Y chromosome could involve the specific functions of sex chromosome-encoded proteins and/or their collective functions as proteins per se. While the concentration of a protein in a heterozygote might decrease to a level insufficient to affect specific function (i.e. the concentration of the protein would still correspond to a point on the plateau of the dose-response curve; Forsdyke, 1994), the decrease in concentration of the protein might itself be sufficient to affect genetic fitness due to an "exceedingly minute", but real, influence on some concentration-dependent collective protein function, such as intracellular self/not-self discrimination. This would have driven the evolution of dosage compensation and would appear to resolve Muller's paradox (Muller, 1948). Collective functions less likely to be involved would include effects on the polymerization of tubulin and actin, and the binding of ions (Donnan equilibrium).

7. Two Active X Chromosomes in Oocytes and Embyros

    No dosage compensation is evident in mammalian oocytes or in female preblastula embryos (Lyon, 1992). The prolonged phase of X-chromosome decondensation in meiotic oocytes probably relates to the need to reactivate the inactive X-chromosome (resetting methylation patterns), and to correct DNA damage (Bernstein & Bernstein, 1991). This is obviously a very special case for which there should be mechanisms to prevent inadvertent aggregation of X-encoded proteins. 

    The preblastula embryo is also a special case. Even if some intracellular aggregation were to occur and peptide presentation were possible in a preblastula embryo, at this stage of development the differentiation of cytotoxic T cells would not have occurred. It is possible that aggregation of an X-encoded regulatory protein in a female preblastula embryo would play a critical role in switching off one of the X-chromosomes (Forsdyke, 1995).

8. Compensation in Z/W Chromosome Systems

    In some species (e.g. birds, butterflies, snakes), the female is the heterogametic sex with Z and W chromosomes and the male is homogametic (two Z chromosomes). Whereas in eutherian mammals the X-chromosome is about 5% of the genome, the avian Z-chromosome is about 10%, which approaches the value for the fruit fly. Thus, if compensation is good for mammals it would seem even more appropriate that compensation exist in birds. 

    Dosage compensation in avian systems does not seem to follow the mammalian model; there is no equivalent of the Barr body and both Z chromosomes replicate together in males (Ohno, 1967). On the basis of very limited studies it has been argued that species with Z/W chromosome systems are dosage tolerant (Johnson & Turner, 1979; Baverstock et al., 1982). However, these studies measured protein activities, not actual protein concentrations. 

    It has been found that, even in the case of compensated genes, product activity levels may vary between the sexes in different tissues, presumably in response to hormonal influences (Steel & Midgeon, 1973). It is predicted that dosage compensation will be found in Z/W chromosome-bearing species, possibly following the fruit fly model with increased expression of the Z chromosome when in the heterogametic sex. The reason for this is that intracellular self/not-self discrimination is a quite fundamental process conferring resistance to infection and is likely to have evolved well before the evolution of sex chromosomes (Forsdyke, 1985, 1991, 1992). Other predictions of the hypothesis are presented elsewhere (Forsdyke, 1995).

The author thanks Dr Susamo Ohno for review of an early version of the manuscript and the Medical Research Council of Canada and the Leukaemia Research Fund of Toronto for support.

[ADDED NOTE: 2001. Strong evidence for avian dosage compensation was reported by McQueen, H., McBride, D., Miele, G., Bird, A. P., & Clinton, M. (2001) Dosage compensation in birds. Current Biology 11, 253-257.]

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Fine Tuning of Intracellular Protein Concentrations, a Collective Protein Function Involved in Aneuploid Lethality, Sex-Determination and Speciation?

Journal of Theoretical Biology (1995) 172, 335-345.
(With copyright permission from Academic Press)
(Received on 26 January 1994, Accepted in revised form on 24 August 1994)

Abstract

1. Introduction

2. Aneuploid Lethality

2.1. CUMULATIVE LOSS/GAIN OF MANY SEGMENTS REQUIRED FOR LETHALITY

2.2. MANY CHROMOSOMAL ELEMENTS DETERMINE SEX AND DOSAGE COMPENSATION

2.3. X-CHROMOSOME DOSAGE COMPENSATION

3. Aggregation Hypothesis

3.1. SOME MUTATIONS AFFECT SPECIFIC PROTEIN CONCENTRATION, NOT FUNCTION

3.2. FINE-TUNING OF INTRACELLULAR PROTEIN CONCENTRATIONS TO A MAXIMUM        CONSISTENT WITH AVOIDING SELF-AGGREGATION

3.3. DOSAGE COMPENSATION NECESSARY FOR FINE-TUNING

3.4. CELL VOLUME AND FINE-TUNING IN DIFFERENT TISSUES

4. Implications of Hypothesis

4. 1. TEMPERATURE SELECTIVELY DESTROYS MALE INTER-SEX CELLS

4.2. DEPENDENCE ON X/A RATIO MEANS DEPENDENCE ON CONCENTRATION OF        X-ENCODED PRODUCTS

4.3. LESS AGGREGATION PRESSURE IN MALE ZYGOTES SWITCHES ON X HYPERTRANSCRIPTION

4.4. SPECIATION AS A COLLECTIVE GENE FUNCTION. HALDANE'S RULE

4.5. RECOMBINATION HELPS GENES PREDICT THEIR FUTURE ENVIRONMENT

4.6. FINE-TUNING IN ASEXUAL SPECIES


Abstract. The assertion that sex-chromosome dosage compensation arose because aneuploidy for an entire chromosome is lethal, begs the question of why aneuploidy is lethal. It has been proposed that aneuploid lethality results from impairment of a collective protein function (Forsdyke, 1994, J. theor. Biol. 167, 7-12). 

    Cytosolic proteins, by virtue of their concentrations, exert a pressure tending to drive members of individual protein species into self-aggregates. Other evolutionary time, each gene has fine-tuned the concentration of its product to a maximum consistent with avoiding self-aggregation in the crowded cytosol. 

    Because of this aggregation pressure and the imprecision of their own fine-tuning, the proteins of members of other species, the corresponding genes of which may have been transported to a cell as viruses (or gametes), are specifically aggregated. The death of the cell and its enclosed virus results. Aneuploidy impairs this process, with lethal consequences for the organism. 

    The hypothesis leads to explanations for a variety of phenomena. 

  • On the assumption that the concentration of autosomal products determines cell volume, the observed dependence of sex-determination on the ratio of X-chromosomes to autosomes is shown merely to be a dependence on the concentration of the products of one X-chromosome. 


  • The inviability of the heterogametic sex among the offspring of an interspecies cross (Haldane's rule), follows from the species-specific fine-tuning of the concentrations of X-chromosome encoded products, relative to the concentration of autosomally encoded products.   

 

  • Species may initially have evolved as a barrier behind which fine-tuning could occur to protect cells against intracellular pathogens.

1. Introduction

    In species with sex chromosomes, one sex is usually haploid with respect to one of the sex chromosomes. Thus, in humans and fruit flies, males have only one copy of the X-chromosome (Jablonka & Lamb, 1990; Charlesworth, 1991). Since the concentration of a final gene product (usually a protein) is often directly related to the number of gene copies, the concentration of X-chromosome gene products in males should be half that in females. The process known as dosage compensation ensures that this does not occur. In humans dosage compensation is achieved by inactivating one of the X-chromosomes in females (Lyon, 1992). In the fruit fly Drosophila melanogaster the transcriptional activity of the single male X-chromosome is increased (Muller, 1948; Stern, 1960).

    Muller's choice of the value-laden term "compensation" to describe the adjustment of dosage implies that the process is advantageous. What adaptive advantage could dosage compensation confer? The standard answer to this question begins by noting that, whichever chromosome is affected, aneuploidy for more than 3% of a genome is usually lethal. Since the human X-chromosome is approximately 5% of the genome and the fruit fly X-chromosome is approximately 20% of the genome, then without dosage compensation there might be an intolerable degree of aneuploidy. 

    This answer was given by Muller & Kaplan (1966), and by Ohno (1967), and has been repeated in most subsequent reviews (e.g. Baker & Belote, 1983; Jaffe & Laird, 1986). Certainly, mutations in genes affecting dosage compensation are usually lethal (Lucchesi & Manning, 1987; Hodgkin, 1990). This implies that aneuploidy for the X-chromosome would indeed be lethal. However, why aneuploid states are lethal is not explained.

    If one accepts the deduction that X-chromosome aneuploid lethality is just one example of the general problem of aneuploid lethality, then two inductions can be made:

  • (i) Since X-chromosome aneuploidy in male fruit flies and humans can be compensated by adjusting the concentrations of X-encoded gene products, then aneuploid states, in general, may be lethal because of disturbances at the gene product level.

  • (ii) Since most of the X-chromosome is the subject of compensation, then aneuploid states, in general, may be lethal because of a collective disturbance of many gene products. Thus, aneuploid lethality is unlikely to occur because there is a small subset of genes which are aneuploid sensitive.

   In this paper I first review evidence supporting these inductions, derived from studies in three areas; 

  • (i) segmental aneuploidy, 

  • (ii) determination of sex by the ratio of X-chromosomes to autosomes (X/A ratio) and 

  • (iii) the evolution of dosage compensation in the apparent absence of any selective advantage (Forsdyke, 1994b).

I then review a recently proposed mechanism by which gene products could act collectively to produce lethal effects (Forsdyke, 1995). Finally, some implications for the chromosomal determination of sex, and interspecies incompatibility at the intracellular level, are discussed.

 

2. Aneuploid Lethality

2.1. CUMULATIVE LOSS/GAIN OF MANY SEGMENTS REQUIRED FOR LETHALITY

   Lindsley and co-workers (1972) studied segmental aneuploidy in fruit flies. Small chromosome segments were either deleted to make a region haploid, or duplicated to make the region triploid. Aneuploidy for only one locus was found to be lethal. This was the triplolethal locus, which appears to encode an RNA helicase (Dorer et al., 1990). In this case, neither the haploid state nor the triploid state was tolerated. This would explain why the loss or gain of a DNA segment containing triplolethal would not be accepted, but it would not explain aneuploidy as a general phenomenon. In general, segments could be removed or duplicated with no serious impairment of function. A collective loss/gain of many segments was necessary for lethality. Lindsley and co-workers concluded that:

"The deleterious effects of aneuploidy are, in the main, caused by the additive effects of genes that slightly reduce viability, and not by the individual effects of a few aneuploid-lethal genes among a large array of dosage-insensitive loci" (my italics).

2.2. MANY CHROMOSOMAL ELEMENTS DETERMINE SEX AND DOSAGE COMPENSATION

    Another line of evidence in support of the above inductions derives from studies of the determination of sex and dosage compensation in certain polyploid variant organisms. These studies show that sex chromosome dosage compensation depends, not on the absolute number of X-chromosomes, but on the X/A ratio. In fruit flies, both sex and dosage compensation are determined by the X/A ratio. In humans, only dosage compensation is determined by the X/A ratio (Book & Santesson, 1961; Harnden, 1961; Mittwoch, 1973; Webb et al., 1992).

    In 1983 Baker & Belote concluded that the X/A ratio "remains the most enigmatic aspect of the hierarchy controlling sex and dosage compensation". In spite of dramatic advances in our understanding of the molecular basis of the implementation of the information provided by the X/A ratio, a decade later the enigma remains. It has been argued that a limited number of X-encoded proteins, (such as sis-a, runt and the helix-loop-helix protein sis-b), may be sufficient to act as numerators, and that a limited number of autosomally encoded proteins (such as the helix-loop-helix protein deadpan) may be sufficient to act as denominators (reviewed by McKeown & Madigan, 1992). These arguments do not take into account the historical evidence for a great multiplicity of numerator/denominator elements.

    Dependence of sex determination on the X/A ratio has been described for the fruit fly (Bridges, 1925). 

  • A ratio equal to or less than 0.5 generates a male (chromosome complement designated X,2A if the Y-chromosome is ignored). 

  • A ratio equal to or more than 1.0 generates a female (2X,2A). 

  • A triploid individual (3X,3A) has a ratio of 1.0 and is female. 

  • A triploid individual with only one X-chromosome (1X,3A) has a ratio of 0.33 and is male. 

  • A triploid individual with two X-chromosomes (2X,3A), however, has a ratio of 0.67. Such "intersexes" have varying proportions of male and female cells, each cell type being found in course patches indicating an early random commitment of individual cells to become clones of a particular sex (Sanchez & Nthiger, 1983).

2.2.1. Numerator elements

   The addition of small segments of the X-chromosome to 2X,3A zygotes, prior to the time the male/female decision is made, should increase the proportion of female cells. This was indeed found (Dobzhansky & Schultz, 1934; Baker & Belote, 1983). Two remarkable features of these experiments were that:

  • (i) Most small segments of the X-chromosome were equal with respect to their abilities to increase the proportion of female cells.

  • (ii) Increasing the length of the segment increased the proportion of female cells.

Thus the numerator element in the X/A ratio appeared as a collective function of X-chromosome segments and, by implication, of their genes and gene products.

2.2.2. Denominator elements

   Baker & Belote (1983) were able to conclude only that:

"The autosomal [denominator] component(s) whose dosage is important for the primary determination of sex and/or dosage compensation has eluded detection".

Little progress has been made in subsequent fruit fly studies (Younger-Shepherd et al., 1992). However, studies in human trisomies provide some evidence that the denominator element in the X/A ratio determining dosage compensation might also be a collective function (Jacobs & Midgeon, 1989).

    In normal human males (designated X,2A if we ignore the Y-chromosome), there is one active X-chromosome which [for convenience] can be regarded, by analogy with the fruit fly, to be permanently "hypertranscribed" [relative to those of the female Xs if the latter were fruit fly Xs], so that its products "balance" those of the autosomes. Thus, the ratio of X-encoded products to autosome-encoded products can be designated as 1.0

    In normal human females (2X,2A) one X is inactive and replicates late in S-phase. The other X is "hypertranscribed" as in the male (giving a product ratio of 1.0). In tetraploid females (4X,4A) there are two late-replicating X-chromosomes and two "hypertranscribed" X-chromosomes whose products would thus balance those of the four sets of autosomes (product ratio again 1.0).

    In triploids (3X,3A) there are two cell populations reflecting two options, neither of them perfect. If there are two "hyperactive" X-chromosomes (and one inactive) then the level of X-chromosome products is set to the level required to balance the products of four, not three autosomes (predicted product ratio 1.33). If there is one "hyperactive" X-chromosome (and two inactive) then the level of X-chromosome products is set to the level required to balance the products of two, not three autosomes (predicted product ratio 0.67).

    Jacobs and Midgeon asked whether there is one powerful denominator autosome, or whether the denominator function is a collective function of more than one autosome. They found, in individual human females, each trisomic for a particular autosome, no cells in which the presence of the extra autosome would allow both X-chromosomes to remain active. The study was unsatisfactory for various reasons, particularly the fact that spontaneous trisomies for four of the 22 possible autosomes were not found. Nevertheless, the data were consistent with the hypothesis that the denominator function is collective. The authors wondered whether compensation involves:

". . the action of a specific autosomal gene(s), or does it depend on a more indirect attribute, such as an increase in nuclear volume ... ?" (my italics).

In the same vein, Dobzhansky and Schulz wrote in 1934:

"One may, perhaps, account for the sex-determining function of the autosomes even without assuming any autosomal sex genes. Sex may be determined by the ratio between the number of X-chromosomes present in the cell and the size of the cell. Cell size in most organisms (including Drosophila), is positively correlated with the volume of chromosomal material contained in the nucleus."

It should be noted that hyperploidy is usually associated with an increase in cell volume (Dobzhansky, 1929; Tal, 1980; Lucchesi & Manning, 1987), and hypoploidy with a decrease in cell volume (Bridges, 1939). This would appear to reflect the general dependence of protein quantity on gene dosage. A change in volume would accommodate a change in the quantity of an intracellular protein while maintaining its concentration constant (Colclasure & Parker, 1991). This is an important point to which we will be returning.

2.3. X-CHROMOSOME DOSAGE COMPENSATION

   Many genes encode proteins with dosage sensitive functions, such as enzymes. Typically, with increasing enzyme concentration there is a progressive increase in the quantity of enzyme product formed. The quantity of this product may be reflected in the phenotype. However, above a certain enzyme concentration some other factor becomes rate-limiting (e.g. substrate availability); curves plotting the rate of product formation as a function of enzyme concentration then tend to plateau.

2.3. 1. Dosage imbalance does not impair specific protein functions

    Muller (1948) was struck by the fact that the concentrations of most enzymes found within diploid cells correspond to a point well along the plateau of the dose-response curve. In most cases, halving the concentration of the enzyme would still generate a product level corresponding to the plateau of the dose-response curve (Forsdyke, 1994a). Thus, there would be no phenotypic difference between a female fruit fly cell containing two active X-chromosomes and a male fruit fly cell containing one uncompensated X-chromosome. Muller's paradox is that dosage compensation has evolved when there would appear to be only, in his words, "exceedingly minute differences" between compensated and uncompensated phenotypes.

2.3.2 Dosage imbalance impairs collective protein function?

    It has been proposed that the paradox would be resolved if the pressure to evolve dosage compensation involved selection acting, not on the unique individual phenotypes characteristic of each individual gene product, but on a phenotype generated collectively by all X-encoded gene products (Forsdyke, 1994b). 

    The Donnan equilibrium is an example of a collective protein function. Intracellular proteins at physiological pH are usually charged molecules which can influence the equilibrium distribution of ions across protein-impermeable membranes (Hitchcock, 1924). Over the range of protein concentrations found in cells, the strength of the Donnan effect is directly proportional to the quantity of intracellular protein. There is not an intracellular protein concentration beyond which the effect no longer increases. If the X-chromosome (20% of the genome) were not hyperactivated in the male fruit fly, then male cytoplasm would have approximately 90% of the protein concentration of the female cytoplasm. A difference of this magnitude is not "exceedingly minute". It might adversely affect the Donnan equilibrium and hence negatively influence phenotype.

    However, the Donnan effect would appear to be volume-sensitive. Adjusting cell volume should correct a concentration difference between proteins in male and female cells. That this may not be feasible becomes apparent on considering a novel collective function of intracellular proteins -- this is discussed below.

 

3. Aggregation Hypothesis

3.1. SOME MUTATIONS AFFECT SPECIFIC PROTEIN CONCENTRATION, NOT FUNCTION

    Some gene mutations affect the functions of unique gene products. Other mutations affect the concentrations of those products, often without influencing function per protein molecule. For example, the concentration of a protein might be changed if the concentration of its mRNA were changed, either by a promoter mutation affecting the transcription rate, or by a mutation in the 3' non-coding region affecting mRNA stability. Similarly, a mutation in a protein encoding region might affect protein stability, but have no effect on function per protein molecule.

    As discussed above, within cells the intracellular concentration of most proteins corresponds to a point well along the plateau of the plot of activity (phenotype) against protein concentration. When mutations occur resulting in a specific decrease in concentration of a protein, the protein concentration will move to the left, corresponding to points on the plateau of the dose-response curve. 

    Eventually a point will be reached where specific function will begin to be impaired. There will then be an effect on phenotype and a decrease in fitness. A detailed discussion of this, in the context of the evolution of genetic dominance, is presented elsewhere (Forsdyke, 1994a). The basic point being made here is that there is a lower limit below which a mutation-driven decrease in concentration of a gene product cannot occur without affecting specific function.

    By the same token, when mutations which specifically increase the concentration of a particular protein occur, the protein concentration will move to the right corresponding to points on the plateau of the dose-response curve. In this case, there is no obvious change in specific phenotype. For a given intracellular protein, does the actual concentration found among members of a biological species fluctuate between the lower limit, defined above, and an upper limit set simply by the solution limit? Alternatively, are there further selective forces which determine a precise concentration setting for each protein?

3.2. FINE-TUNING OF INTRACELLULAR PROTEIN CONCENTRATIONS TO A MAXIMUM CONSISTENT WITH AVOIDING SELF-AGGREGATION

    Generally, it is easier for a protein to remain soluble in a salt solution at physiological pH than to remain soluble in the "crowded cytosol" (Fulton, 1982). The solubility of a particular protein species is determined by the concentrations of the other proteins that accompany it (Lauffer, 1975). It has been proposed that, over evolutionary time, each individual gene fine-tunes the concentration of its product to the concentrations of the products of the other genes with which it has been travelling through the generations (Forsdyke, 1995). 

    Irrespective of its specific function, the "aim" of each gene is to maximize the concentration of its own products. Collectively, the products of all genes exert a pressure tending to drive individual protein species into aggregates if they exceed their solubility limit. Each individual protein species both contributes to the pressure and is acted upon by the pressure (Fig. 1).


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FIG. 1. What point on the dose-response curve corresponds to the normal concentration of a gene product within a cell? 

    This hypothetical in vivo dose-response curve shows some quantitative measure of phenotype (e.g. rate of formation of enzyme product) as a function of the concentration of a gene product which contributes to that phenotype (such as the concentration of an enzyme). The phenotypic parameter increases with gene dosage until point A when some other factor (e.g. substrate availability) becomes rate-limiting. The curve then plateaus. B corresponds to the minimum concentration of gene product required for a "margin of safety", so that heterozygote function is not impaired (Forsdyke, 1994a). 

    E corresponds to the concentration at which the gene product would still be soluble if no other proteins were present. Above this concentration, the protein would self-aggregate and the phenotypic parameter would decrease. D corresponds to the concentration at which aggregation would occur in the presence of cytosolic proteins (Forsdyke, 1995). The horizontal arrows symbolize the aggregation pressure exerted collectively by cytosolic proteins, which tends to push the descending limb of the dose-response curve to the left. 

    Thus, it would seem that the concentration of a protein in cells of different members of a species could fluctuate between points B and D with no effect on phenotype. It is proposed, however, that over evolutionary time genes have "fine-tuned" the concentrations of their products to a maximum consistent with avoiding self-aggregation. This point might correspond to C (marked by a vertical arrow), which is slightly to the left of D, thus providing a small "margin of safety" against inadvertent self-aggregation.


   The selective force leading to the evolution of this pressure involves the invasion of the intracellular space by a member of another species of organism (e.g. a virus), which has not had the opportunity to fine-tune so precisely the concentrations of its gene products. As discussed elsewhere (Forsdyke, 1985, 1991, 1995), by mechanisms involving heat-shock proteins, proteins encoded by virus genes would tend to be specifically aggregated. Irrespective of any effect on specific function, this aggregation would trip an output system which, depending on the organism, would involve cell death inflicted either directly, by an intracellular mechanism (e.g. apoptosis), or indirectly, by cytotoxic T cells. A key feature of the aggregation is that it is primarily entropy-driven and thus is favoured by a small increase in temperature (Lauffer, 1975). The death of the cell and the pathogen it contains would usually be beneficial to the organism. In some cases aggregation alone might suffice to limit the spread of the pathogen.

3.3. DOSAGE COMPENSATION NECESSARY FOR FINE-TUNING

    The need for fine-tuning over evolutionary time provides a clear rationale for the evolution of dosage compensation (Forsdyke, 1994b). Without dosage compensation, the cytosolic protein concentration would fluctuate between male and female generations. Fine-tuning would be possible only for genes encoded by Y-chromosomes, which are eternally locked in male generations. Failure to fine-tune would impair intracellular self/not-self discrimination and thus increase the vulnerability of an organism to intracellular pathogens.


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FIG. 2. Evolutionary fine-tuning of intracellular protein concentrations as an explanation for X-chromosome dosage compensation. 

    Rectangles refer to maternal (M) and paternal (P) haploid chromosome complements which combine to form either (a) a female zygote, (b) an uncompensated male zygote, or (c) a compensated male zygote. Gene products (proteins) are A, B and C (for the X-chromosome), D, E and F (for autosome A1), and G and H (for autosome A2). The specific function of these proteins is not relevant to the primary establishment of the X/A signal governing the initiation of X hypertranscription in male fruit fly (dosage compensation). 

    The numbers below each protein indicate its quantity, which has been fine-tuned over evolutionary time to a maximum consistent with remaining in solution in the crowded cytosol. Numbers in brackets are the quantities of products contributed by the maternal X-chromosome, which balance the products of the paternal autosomes.

     The proteins, by virtue of their concentrations, exert a collective pressure (which can be expressed as the sum of individual protein concentrations), tending to drive individual protein species into self-aggregates. For female zygotes and compensated male zygotes the net aggregation pressure might be 40 units per cell. For an uncompensated male zygote the net aggregation pressure would be only 34 units/cell. 

    Cell volume cannot be decreased by a factor of 34/40 in male generations to maintain the aggregation pressure because the quantities of autosomal products (D-H) are already fine-tuned to a maximum consistent with remaining soluble in female generations. Since a high aggregation pressure is needed for the detection of proteins encoded by intracellular pathogens, the viability of uncompensated phenotypes will be decreased.


    Figure 2 shows a model of how this might work. It is postulated that, without dosage compensation, the discrepancy in cytosolic protein concentrations between male and female generations would not readily be corrected by an intergenerational adjustment in cell volume. Reducing cell volume in male generations [by a factor of 34/40 in Fig. 2(b)], would slightly concentrate the products of autosomal genes. Since these had already been fine-tuned in female generations to maximize the solubility of each individual protein species, a further concentration might trigger their aggregation. Intergenerational volume adjustment would work only by compromising the degree of fine-tuning in female generations.

    It can be argued that the lower concentration of X-encoded products would slightly decrease the net pressure tending to drive the autosomally encoded proteins into aggregates, and this might make it possible to accommodate autosomal proteins in a smaller volume without triggering their aggregation. However, it is postulated here that this would not be a feasible alternative. Cell volume is autosomally determined. Experimental evidence to the contrary would falsify this aspect of the hypothesis.

3.4. CELL VOLUME AND FINE-TUNING IN DIFFERENT TISSUES

    It is apparent that the general setting of cell volume as a function of the number of chromosomes (see Section 2.2.2) is subject to tissue-specific modification. The major cell of vertebrate lymphoid tissues, the small lymphocyte, is an extreme example. The ratio of cytoplasm to nucleus is very low. This can be regarded as an adaptation for circulating in the lymphatic and vascular systems from which lymphocytes migrate to regions of inflammation. It can be assumed that the concentration of cytoplasmic proteins is no less than in a cell with more cytoplasm.

    It should also be noted that the differential gene expression associated with cell specialization requires fine-tuning to a particular subset of genes in each tissue, as well as to the more generally expressed "house-keeping" genes. This might require different final concentrations of the same gene product in different tissues. In some cases this could have been achieved by gene duplication so that the same protein could be expressed under the influence of different promoters in different tissues.

    As discussed elsewhere (Forsdyke, 1995), it is envisaged that, in the case of proteins whose concentrations fluctuate as part of physiological processes, concentration fine-tuning would have been with respect to the maximum concentration attained. Similarly, in the case of proteins that form stoichiometric complexes with other proteins, the whole complex would have been treated as a unit during evolutionary fine-tuning. In the case of reversibly modified proteins (phosphorylation, glycosylation), concentration fine-tuning would be with respect to the least soluble form of the protein.

 

4. Implications of Hypothesis

4. 1. TEMPERATURE SELECTIVELY DESTROYS MALE INTER-SEX CELLS

    The aggregation hypothesis makes numerous explicit experimental predictions and reinterpretations of previous data (Forsdyke, 1995). One of these is that male cells in fruit fly intersexes should be more vulnerable to high temperatures than female cells in fruit fly intersexes. Cells of a triploid intersex fruit fly (2X,3A) can have their X-chromosome transcribed either in the male mode (giving a protein level which would balance that corresponding to four autosomes), or in the female mode (giving a protein level which would balance that corresponding to two autosomes). The relative X/A product ratios in each actual case would be 1.33 (male cell) and 0.67 (female cell). 

    The excess of X-encoded products in male cells would tend to aggregate, leading to the selective death of male cells. Since aggregation is promoted by a small increase in temperature (Lauffer, 1975; Forsdyke, 1994a), incubating triploid intersexes at high temperatures would enhance the death of male cells, while having little effect on female cells in which the pressure to aggregate would be below normal (normal corresponding to a relative product ratio of 1.0). Evidence in support of this has been presented by Dobzhansky (1930) and Lauge (1980).

4.2. DEPENDENCE ON X/A RATIO MEANS DEPENDENCE ON CONCENTRATION OF X-ENCODED PRODUCTS

    It was proposed above (Section 3.3) that the volume of a cell is directly related to the quantity of autosomal products it must contain. Assuming, for simplicity, a rectilinear relationship, cell volume (V) is equal to kAp, where Ap is the quantity of autosomal products and k is a constant. It follows that the concentration of X-encoded products (Xp /V where Xp is the quantity of X-chromosome-encoded products), is proportional to the ratio of the quantity of X-chromosome encoded products to the quantity of autosomally encoded products. Thus:

        Xp/V = Xp/kAp = (1/k)Xp/Ap.

In that the quantities of the products are proportional to the relative gene dosages, it follows that the concentration of X-encoded products is proportional to the X/A ratio (defined as the ratio of the number of X-chromosomes to the number of autosomes). From this it can be concluded that the statement that the determination of sex and/or dosage compensation is dependent on the X/A ratio (Bridges, 1925), may merely be acknowledging a dependence on the concentration of X-encoded products (Xp/V).

4.3. LESS AGGREGATION PRESSURE IN MALE ZYGOTES SWITCHES ON X HYPERTRANSCRIPTION

    Prior to the onset of dosage compensation, male fruit-fly zygotes would experience a decline in aggregation pressure as X-encoded maternal gene products decreased to be replaced by a zygote's own single X-encoded products (Mahowold & Kambysellis, 1980). In female fruit fly zygotes the aggregation pressure would be sustained because decreasing maternal X-encoded products would be replaced by the products of both X-chromosomes of the zygote. The aggregation pressure in female cells (Pf) can be regarded as equal to k'[2Xc+Ac], where Ac is the concentration of autosomal products, Xc is the contribution to the concentration of X-chromosome encoded products of one X-chromosome and k' is a constant.

    Similarly, the aggregation pressure in uncompensated male cells (Pm) would be equal to k'[Xc + Ac]. Then the difference in aggregation pressure between males and females (Pf - Pm) would be equal to k'Xc. The latter includes the concentration of the extra set of X-chromosome encoded products which, as argued in Section 3.3, would depend on the X/A ratio (since cell volume is held to be autosomally determined). Thus, the switch to X hypertranscription would depend primarily on the collective functioning of all X-chromosome encoded genes.

    A secondary effect of the greater pressure in female cells could be that the homo- or hetero-dimerization of helix-loop-helix sex-determining proteins, such as daughterless (maternally derived) and sis-b (zygotically-derived), would be promoted. The regulatory activity of helix-loop-helix proteins is usually greatly affected by such aggregation (Thayer & Weintraub, 1993). The helix-loop-helix protein aggregates might activate the zygotic sex-lethal gene early promoter (carried by both X-chromosomes) thus switching to the female pattern of development (Keyes et al., 1992). In male zygotes, the aggregation pressure would be less and helix-loop-helix protein aggregates would tend to dissociate so that the single sex-lethal gene would be inactive. This would remove the negative influence of the sex-lethal product on the autosomally encoded genes which initiate hypertranscription of the male X-chromosome (Gorman et al., 1993).

4.4. SPECIATION AS A COLLECTIVE GENE FUNCTION. HALDANE'S RULE

   A biological species can be viewed as having set up intracellular barriers against members of other species, be they intracellular pathogens, or gametes of a species related enough to show no prezygotic isolation (Templeton, 1989; Coyne, 1992). The fine-tuning of cytosolic protein concentrations, continuing through the generations and communicated to all interbreeding members of the species, would have created an intracellular environment hostile to the gene products both of foreign pathogens, and of the gametes of other species (Forsdyke, 1995).

    A failure of members of a species to interbreed, owing to some form of allopatric or sympatric isolation, would provide an opportunity for mutational differences to arise between alleles that might affect the abilities of their products to contribute to the cytosolic aggregation pressure. This might occur before any allele-specific differences in phenotype could arise. Fusion of gametes from such reproductively isolated lines would expose a cytoplasmic incompatibility owing to differences in the fine-tuning of cytosolic protein concentrations. Thus, the list of potential cytoplasmic incompatibilities between organisms should include "warfare", not only between organelles, as is widely recognized (Hurst, 1993), but also between cytosolic proteins (Forsdyke, 1995). The latter might have played a role in the origin of species.

    The particular vulnerability of members of the heterogametic sex among the offspring of interspecies hybridizations (Haldane's rule), may serve to illustrate the latter point. The cytosolic concentrations of proteins encoded by one X-chromosome are uniquely fine-tuned to the concentrations of proteins encoded by its haploid autosomal complement. In female fruit fly, each X is accompanied by at least one compatible autosome complement. In male fruit fly, the solitary X is hypertranscribed and only one X is accompanied by a compatible autosomal complement. 

    This could result in changes in net aggregation pressure with adverse consequences for the organism. Figure 3 shows a simple model of how this might work. Any X-linked gene which mutated so that the concentration of its product changed would suffice to begin the isolation process. There would be no distinct "speciation genes".


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FIG. 3. Evolutionary fine-tuning of gene product concentrations as an explanation for decreased viability of the heterogametic sex among the offspring of interspecies crosses (Haldane's rule). 

    Following the convention of Fig. 2, at the top are the genomes of two wild-type members of a species. During gametogenesis, one produces a concentration deficiency mutant (-1 unit) for an X-chromosome encoded gene (mutant 1). The potential contribution to the net aggregation pressure thus falls from 20 to 19 units. This decrease in aggregation pressure might be corrected in female zygotes in one step if a second gamete happened to have a perfectly matching concentration-excess mutant (+1 unit). The figure shows correction in two steps. 

    The partially compensated decrease in aggregation pressure (39.5 units) in the organism formed by mutants 1 and 2, is tolerated for a sufficient number of generations for a second concentration excess mutant to arise (mutant 3) to finally rebalance the genome. However, the relative balance between X-chromosome encoded products and autosomally encoded products would have been changed. A back-cross with the original wild type would generate females with normal aggregation pressures, but, owing to hypertranscription of the solitary X-chromosome in the heterogametic sex, there would be an imbalance in males.


    The idea that Haldane's rule reflects an X-chromosome/autosome incompatibility is an old one (Dobzhansky, 1937). However, studies of hybrids between different fruit fly species have been interpreted by Coyne (1985) as supporting an alternative X-chromosome/ Y-chromosome incompatibility theory. Wu (1992) noted that Coyne only considered hybrid sterility. Recently, Orr (1993) provided evidence that hybrid non-viability reflects an X-chromosome/autosome incompatibility, in keeping with the arguments presented here.

    The founders of the "modern synthesis" proposed that speciation required the divergence of many "ordinary" genes. Thus, speciation is a collective gene function (Dobzhansky, 1937; Muller, 1942). Although there are some special cases, in general, subsequent work has supported the hypothesis (Coyne, 1992). For example, at least 150 genes are implicated in the inviability of hybrids between various grasshopper races which are believed to have arisen quite recently (Barton & Hewitt, 1981). The role of a possible "speciation gene" involved in hybrid male sterility in fruit fly (Orr, 1992), remains to be determined.

    I present evidence elsewhere that fine-tuning occurring at the DNA level ("GC/AT pressure"), may also contribute to speciation (Forsdyke, 1994c).

4.5. RECOMBINATION HELPS GENES PREDICT THEIR FUTURE ENVIRONMENT

    A major theme of this paper is that alleles compete to get into the next generation, not only on the basis of differences in the specific functions that they encode, but on the basis of their abilities to be "good mixers" and fit in with other genes in contributing to collective functions, particularly the function concerned with generating a cytosolic aggregation pressure. Recombination cross-over between genes serves this purpose in that it separates emerging gene coalitions, thus effectively homogenizing the genome and ensuring that each allele "knows" with some certainty what aggregation pressure its products can expect to encounter in the next generation (Dawkins, 1990).

4.6. FINE-TUNING IN ASEXUAL SPECIES

    In both sexual and asexual species an ability to aggregate specifically proteins encoded by an intracellular pathogen would appear advantageous. The effectiveness of this would depend on the effectiveness of the fine-tuning of intracellular protein concentrations. Since the pressure to aggregate would be greatly affected by ambient temperature (Lauffer, 1975), homeothermic species would be able to fine-tune more precisely than poikilothermic species. Homeothermy is encountered in sexual species, but many sexual species are also poikilothermic.

    In sexual species, because of the "horizontal" mingling of genomes, mutations affecting the concentration of a protein can be compensated by other mutations and can spread (Fig. 3). In asexual species, a genomic response to an adverse mutation requires a transmission "vertically" through the generations until compensating mutations can occur (Muller, 1932). Thus, fine-tuning in asexual organisms might depend more on the direct elimination of concentration mutants, rather than on the emergence of compensating mutants.

This work was supported by the Leukaemia Research Fund (Toronto), the Canadian Medical Research Council and the School of Graduate Studies of Queen's University. I thank Mr. H. Metz for assistance with the figures.

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Last edited 29 Oct 2003 by Donald R. Forsdyke