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Chapter XVIII Plasmagenes
In recent years a considerable body of evidence has accumulated for the existence in the cytoplasm of bodies of a more or less gene-like nature. Geneticists have been particularly active in investigating them and have referred to them by a variety of names, such as plasmagenes, cytogenes, blastogenes, proviruses, etc. The first has been the most commonly accepted, and will be used here in a rather wide sense, to cover several rather different types of gene-like entities.
Broadly speaking, plasmagenes are revealed by two different kinds of evidence. On the one hand, breeding experiments may demonstrate that certain characters are inherited through the cytoplasm and not throuzh the nucleus, and thus provide evidence of the existence of cytoplasmic hereditary determinants. Evidence of this kind can be of two grades. We may find that, throughout a number of generations, a certain character follows the transmission of cytoplasm, even when the whole set of chromosomal genes is removed by crossing. For instance, if a female of type A is crossed to a B male, and her female offspring again backcrossed to B males, and so on for several generations, the A chromosomes will gradually be replaced by B ones, and if any characteristics of A still remain in the offspring after several generations, one may conclude that they are dependent on hereditary factors transmitted through the egg cytoplasm and capable of continued multiplication in the absence of their corresponding A genes. On the other hand, it may be found that although a character is transmitted only by a parent which contributes cytoplasm to the offspring, and is thus directly dependent on a cytoplasmic determinant, nevertheless this determinant can persist only in an organism provided with the appropriate gene. In such cases (a good example is the ‘killer’ character in Paramecium) we have to do with a gene-dependent plasmagene.
A different type of evidence for the existence of plasmagenes appears when it can be shown that a character can be transmitted from cell to cell by inoculation or other treatment with extracts which do not contain functional chromosomes; we may then conclude that we are confronted with a determinant, presumably derived from the cytoplasm, which can persist and impress some definite character onto the living cells into which it is introduced. The classical examples of such types of behaviour are the transmissable viruses. When from such evidence we deduce the existence of a plasmagene, it is presumably implied that the cytoplasmic determinant is a fairly complex body, probably of the order of magnitude of a virus particle or a gene. Considerable caution should be exercised in making such deductions. Many years ago, in the early years of the investigation of cancer-producing viruses, it was pointed out that, given a tissue which has an appropriate competence, a particular type of cellular differentiation could be transmitted through an indefinite series of inoculations by means of cell-free extracts whose operative factors, however, were quite simple molecules which acted as evocators (cf. Needham 1936). One knows now that the effective molecules might be even simpler than was realised at that time. It would be quite possible to carry on an indefinite series of transformations of gastrula ectoderm into neural tissue by means of inoculations of cell-free extracts, provided only that these extracts were sufficiently acid. Moreover, one might easily obtain phenomena which simulate a mutation of the virus. If the extracts came to contain free ammonia they would transform the gastrula ectoderm not into neural tissue but into derivatives of the axial mesoderm.
More recently Lederberg (1952) has drawn attention to the same source of possible error. Again, Pollock (1953) has suggested a mechanism for the operation of self-perpetuating and even growing systems which might very easily be confused with plasmagenes (Fig.18.1). In the form he advances it, the idea depends on the phenomenon of enzymatic adaptation (p. 400); but somewhat similar systems might be produced in other ways. Pollock’s suggestion is this; suppose that a substance A, supplied to a cell, causes it to synthesise an enzyme a which converts A to B; then suppose that B induces the formation of enzyme b, which converts B (perhaps in combination with other substances already in the cell) into C; then that similarly C is converted into something else, and that finally a product is produced which is converted again into A. Such a system will be set going by the addition of an initial quantity of A and will then carry on indefinitely. In fact if the system absorbs energy, more A may be formed at the end of a cycle than entered it at the beginning, and the system will be able to grow. Such a system clearly has many of the properties attributed to plasmagenes; but it need not be incorporated in a particle. Thus to be justified in using grafting or inoculation experiments to postulate the existence of a plasmagene, one needs evidence not only that the character can be transmitted by cell-free extracts but that the effective factor in the extracts is a particle of the right order of complexity.
Discussions which on the whole favour the importance of plasmagenes in development will be found in Darlington 1944, Darlington and Mather 1949, Spiegelman 1948, Medawar 1947, Ephrussi 1953; 2 somewhat more reserved attitude is taken by Brachet 19526, Beadle 1949, Waddington 1948), Sonneborn 19514, b, and Haldane 1954. General reviews of the phenomena, less concerned with development, are Lederberg 1952, Caspari 1948, Cold Spring Harbor Symposia Nos. 11 and 16, 1948 and 1951, and the symposium published as Unités biologiques douées de continuité génétique (Paris 1949).
A self-reproducing cyclic system. It is supposed that when A is ‘added to the cell, it induces the formation of enzyme a, which converts it, together
with m from the rest of the cell, into B. After several such steps, during which raw materials (e.g. 1) are used, and other substances (e.g. p, q) pro duced, the system finally absorbs energy (Z) and results in the formation of more A than was originally supplied. This restarts the original cycle, and also brings into being a new cycle. (After Pollock 1953.)
From the point of view of their possible importance in differentiation, plasmagenes may be considered under the following headings (cf. Fig. 18.2).
1. Exogenous plasmagenes
Many viruses, such as those producing disease, are clearly not essential constituents of the animal or plant and are introduced into the cell from outside. There is considerable variation in the ease with which this introduction can take place. Some of the bodies which were orginally thought of as true plasmagenes should be regarded as essentially exogenous factors for which infection is rather difficult. This probably applies to the kappa particles in Paramecium (Sonneborn 1947, 19514, 6). In these Protozoa, some strains, known as ‘killers’, produce and secrete into the medium a substance which kills certain other strains, known as ‘sensitives’. The production of the killer substance is controlled by cytoplasmic particles known as kappa, and these in turn depend on the nuclear alleles K and k. The importance of the cytoplasmic particles is demonstrated by the experiment summarised in Fig. 18.3; if KK killers are crossed with kk sensitives, all the offspring will have the genic constitution Kk, but only those which derive their cytoplasm from the killer parent will contain kappa and behave as killers. If, in such a cross, the period of union of the two Protozoa is unduly prolonged, some kappa-containing cytoplasm may pass into the offspring derived mainly from the sensitive parent, and provided these offspring contain at least one K gene, the kappa particles multiply (Fig. 18.4). On the other hand, in cells containing only the recessive k, kappa particles which may originally be present fail to multiply and gradually become diluted out of existence as the strain of Protozoa proliferates.
Types of plasmagenc. The diagram shows a cell containing a nucleus, and the following types of plasmagene: E, exogenous, originating from outside the cell; can usually multiply only with the co-operation of the nucleus, which may also influence its physiological effect a; P, a true plasmagene, independent of the nucleus both in multiplication and in effect b; V, a visible cytoplasmic particle; can usually multiply independently of nucleus, but may affect the latter (cf. Stentor), while the nucleus may influence its physiological effect c; G, a gene-initiated plasmagene, originating under the influence of the nucleus, but multiplying and being physiologically active in relative, though not complete, independence of it.
Thus the persistence of kappa is strictly gene-dependent. The particles can be seen in microscopical preparations, following suitable staining. Unlike most normal cytoplasmic particles, they contain DNA, and it seems likely that they should be regarded as invasive exogenous organisms, perhaps comparable to large viruses or rickettsias, rather than as normal parts of the Paramecium.
A rather similar case, and this time in a higher organism, is the socalled CO, genoid in Drosophila (L’Heretier 1951). This is a cytoplasmic factor which causes the individuals carrying it to be highly sensitive to CO,, which produces in them an irreversible anaesthesia at a concentration which has little effect on normal flics. The agent is easily transmitted by the cytoplasm of the eggs, and can also pass through the sperm, although its passage through the male is very irregular, presumably because of the small quantity of cytoplasm carried by male gametes. There are no genes known with a clear-cut effect on its propagation, but it certainly multiplies more easily in some genetic stocks than others, so that it too may be considered as gene-dependent. There seems no reason to think of the Drosophila genoid as anything other than an exogenous virus. For many other viruses diseases, there is as yet little evidence of nuclear control of susceptibility or resistance. On general grounds, however, it is probable that there is always some variation in this respect and this will probably be under the control of numerous nuclear genes, each of small effect. Again, the physiological result of infection with the exogenous particle in some cases clearly depends on the nuclear constitution of the individual involved. The classical example is the virus-like particle in the King Edward race of potatoes, which has little effect in that stock but which, when transferred to other races of potato by grafting, produces the symptoms of a severe virus disease (Salaman and Le Pelley 1930). In other cases such variation in effect is less in evidence, but it seems likely that careful search would always reveal some degree of variability of this kind.
FIGURE 18.3 Cytoplasmic inheritance of kappa. In the figure on the left, conjugation is occurring between a killer (with genotype KK) and a sensitive (with genotype kk). In the third figure they are exchanging nuclei and in the fourth and fifth the macronuclei are being reconstructed. Both daughters (now ready to divide) have the genotype Kk, but only that with the original kappa cytoplasm will be a killer. (From Beadle 1949, after Sonneborn and others.)
2. True plasmagenes
One can pass by more or less insensible gradations from cases in which infection is easy and the infecting particle obviously foreign, to the other end of the range at which infection cannot be definitely demonstrated to occur, and the plasmagenes appear to be normal constituents of the organism. Particles coming at the latter end of the range may be considered as true plasmagenes. Most of the cases known are from the plant world (reviewed in Caspari 1948). Perhaps the best investigated are the factors which are clearly inherited through the cytoplasm in crosses between different races of the willow herb, Epilobium (Michaelis 1951). Another example is provided by the cytoplasmic factors causing male sterility in crosses between races of a number of different species of plants (e.g. flax, maize, etc.).
Transfer of kappa when conjugation lasts abnormally long. The rate of multiplication of the transferred kappa has been exaggerated for diagrammatic purposes. (From Beadle 1948, after Sonneborn and others.)
Evidence for such factors in higher animals is exceedingly rare. The case which perhaps seems most likely to fall into the category is that desctibed by Laven (1953) in mosquitoes of the genus Culex. He made reciprocal crosses between a number of races of the species-group Culex pipens, and found that they fell into three groups; between the members of a group, crossing led to offspring whichever race provided the female, while when the crosses were made between races of different groups, they only succeeded in one of the two possible ways. Thus the cross between the O and H races gave offspring only when the female belonged to the latter. Further analysis showed that the infertility of the opposite cross is due to a factor carried in the H sperm, which inhibits the development of the embryo when it gets into an egg with O cytoplasm. By backcrossing of the hybrid females to O males for several generations, one obtains males which will contain an almost completely O genotype; nevertheless they continue to give sperm which lead to a failure of development of the O eggs. It seems that this must be due to a cytoplasmic factor which came from the H female in the original interracial cross and which has been carried on by the eggs through subsequent genera~ tions, without being influenced by the increasing number of O genes. The nature of the factor is still obscure. It may be similar to the plasmagenes described in plants such as Epilobium. But its distribution is worthy of note. In Culex it distinguishes various geographical races; and within a race which has it, it is quite undetectable until an attempt is made at an interracial cross to a strain which lacks it. In another related genus, a similar factor, or perhaps the same, distinguishes the two species Aedes aegypti and A. albopictus; and in Aedes scutellaris there is a distinction between two local races similar to that in Culex. This suggests that we are dealing with a phenomenon which might be compared to the acclimatisation of some local races to a virus, rather than with a situation which can assist us to understand the origin of tissue differentiation. There are in the literature a few other cases of persistent cytoplasmic differences between local races (cf. Goldschmidt 1938 on Lymautria) but these require further investigation.
None of the entities in this category of true plasmagenes can yet be seen and there is no direct evidence as to the size of particle involved. The indirect evidence, chiefly from the type of physiological effect which they produce, is usually held to suggest that they are bodies of a gene-like order of complexity. It is not impossible, however, that in the future some of them may turn out to be simpler than has been previously thought.
3. Visible cytoplasmic particles with genetic continuity
In many Protozoa, self-duplicating cytoplasmic particles can be seen fairly easily in microscopical preparations stained in the appropriate way (by silver impregnation, for example). There are, for instance, the granules which lie at the base of the cilia with which the body of ciliate is covered (Fauré-Fremiet 1948, Lwoff 1949, 1950, Weisz 1951). These so-called kinetosomes lie in rows, and along the side of each row is a thread, the kinetodesma, the whole complex being known as a kinety (Fig. 18.5). It can be shown both by experiment and observation that kineties develop only out of pre-existing parts of kineties and they therefore possess at least one type of genetic continuity.
These kineties are of undoubted morphogenetic importance. In fact the structure of a ciliate, which may be quite complicated, is in the main a matter of local differentiation of the cuticular layer or ectoplasm, and this can be shown to depend on the activities of the kineties. The evidence for this comes largely from observation of the process by which a single individual becomes reorganised into two at the time of cell-division, and from studies of the processes of regeneration of a whole individual from fragments. In both cases it is clear that the kineties are reorganised first, and only later produce the more obvious morphological structures such as the gullet, flagellae, cirri, trichocysts, etc. The kineties associated with these various organs do not, however, become fully determined, so as to possess only one specific character; thus a kinety originally associated with a single cilium may, during regeneration, take part in the formation of a gullet or contractile vacuole or any other organ. The kineties are in fact organised in relation to one another in a ‘gradient-field’, somewhat reminiscent of that seen in Platyhelminthes, for instance. Thus there is a leading kinety (associated usually with the mouth), to which the rest are subordinated as the hind parts of a flatworm are subordinated to the head.
On the left, a drawing of Stentor: mac.n., the nodular macronucleus; pb, peristome band; cv, contractile vacuole; ap, the adoral zone, from which the new peristome band originates in regeneration; ft, foot; [bs and rbs left and right sides of the ramifying zone, from which new kineties are formed. (Modified from Weisz 1951.) On the right, diagram of a ciliate, showing one kinety (composed of kinetodesma and kinetosomes) on the near side, and a similar one seen by transparency on the far side. (After Faure-Fremiet 1950.)
The fact that one and the same kinety may, under the conditions of regeneration or reorganisation, produce different structures, must mean that the effect of the kinety depends on the properties of the cytoplasm with which it is in contact. The situation is comparable with that which we have discussed in relation to the nuclear genes; the kinetosome may continue to duplicate itself identically but at the same time interact with the local cytoplasm, producing different active products according to the raw materials or other substances available. The organisation of the kineties into a gradient field is presumably brought about by the leading kineties affecting the cytoplasm in some way which diffuses outwards and influences the activities of the subordinated kineties.
Besides these mutual interactions between the various cytoplasmic particles, there are very interesting relations between the particles and the nuclei. Weisz (1951) has studied the matter in the particularly favourable case of Stentor. This is a large sessile ciliate, which possesses not only a micronucleus but also a large macronucleus which consists of a string of swollen nodes connected by a much thinner strand. The micronucleus seems to be concerned solely with sexual reproduction, and a Stentor from which it is missing can live for many generations of vegetative fission, and regenerate quite adequately (in some other ciliates the micronucleus is necessary for regeneration). These processes are, in fact, under the control of the macronucleus. The genetic constitution of this is not known with any certainty, but it seems likely that it is to be considered as a highly polyploid nucleus in which the original set of chromosomes has been multiplied many times; probably each node contains at least one diploid set of chromosomes and possibly more (Fig. 18.5).
During fission of a Stentor into two daughter individuals, the macronucleus also undergoes reorganisation, and in the period shortly after this it is found that all the nodes are equivalent, in the sense that any one of them is sufficient to make possible the regeneration of a whole individual. Later in the life-cycle, as the time approaches for the next fission, this is no longer the case. If at this stage a fragment is cut off a Stentor in such a way as to include only a posterior node, regeneration is not complete and a mouth is not formed. This is so even if the cytoplasm comes from an anterior region and is known to be capable of carrying out a complete regeneration when provided with an anterior node. Further, if all but the posterior node is removed from an intact individual, the mouth and other anterior parts degenerate and disappear.
There is, then, a gradual loss by the posterior nodes of ‘potency’ to mediate regeneration or to support differentiated structures. This loss appears to be caused by the kineties of this region of the body, since if anterior nodes are forced into the posterior they soon lose their power to support full regeneration. We are therefore confronted with a two-way interaction between the cytoplasmic particles and the macronucleus; (1) the kineties influence the nearby parts of the nucleus, causing the posterior nodes to lose ‘potency’; (2) the macronucleus controls the morphogenetic activity of the kineties, so that the mouth and other anterior organs cannot be formed in the absence of a fully potent nuclear node. It would seem, therefore, that the kineties cannot maintain their specific character, or at least cannot continue to produce their specific effect, without the collaboration of nuclear factors; their autonomy over against the nucleus is by no means complete.
4. Gene-initiated plasmagenes
In contrast to the preceding categories there are a group of factors» which are also often considered to be plasmagenes, and which are characterised by the fact that they can arise anew within cells from which they were originally absent. Their initiation seems in all cases to depend on the functioning of corresponding genes in the nucleus and is impossible if the effective gene is absent. Other conditions of an environmental kind are usually necessary to bring the gene into play and cause it to produce the cytoplasmic factor.
In Paramecium, besides the kappa particles which have already been described, there are cytoplasmic determinants of certain antigenic properties (Sonneborn and Beale 1949, Beale 1951, 1952, 1954). We have already (p. 359) referred to these in connection with the cytoplasmic control of gene activity and they also illustrate other points which may be relevant. Thus the cytoplasmic determinants (which are presumably particles, though they have not yet been scen) are under close control by genes. A given type of determinant cannot persist indefinitely in a cell from which the corresponding gene is absent; and in this their behaviour is perhaps similar to that of the Stentor kineties. Further, the genes in the Paramecium nucleus appear to be able to bring into existence the cytoplasmic determinants which correspond to them, even if there were previously no representatives of this particular type in the cell. Thus if Paramecia are kept at 29-33° C. they will develop one of the D antigens (depending on which of the D alleles is present in the nucleus). If the strain is then transferred to 18°, the D antigen will eventually disappear, and an S antigen (corresponding to the S allele present) will take its place. It is, of course, difficult to be certain that there was absolutely no S antigen in the cells when they were originally kept at 29-33°, but it certainly seems probable that this was the case, and that the cytoplasmic determinants producing the S antigens have been formed anew under the influence of the S gene when it became activated by the effect of the lower temperature. These antigen-producing determinants therefore seem to provide examples of ‘gene-initiated plasmagenes’.
Another important case is that described by Billingham and Medawar (1948, 1950, Medawar 1947, Fig. 18.6). If a patch of black skin from a spotted guinea-pig is transplanted into a white area, it is found that pigmentation spreads out from the graft into the surrounding area, forming a sort of halo. In mammalian skin, pigment is formed as granules in the cytoplasm of a system of highly branched dendritic cells, the melanocytes, which are present even in white areas, although there the pigmentforming system is inoperative. Billingham and Medawar argue that the spreading of pigmentation which they have studied is duc to the ‘infection’ of the melanocytes surrounding the graft by a virus- or plasmagene-like particle from the pigmented cells of the transplant. This particle must be capable both of self-duplication and of causing the host cell to produce pigment; possibly it may be the pigment-forming particle itself, in which case these two actions would amount to the same thing.
On the left, a shaved area of skin on a guinea-pig, into which a graft of black skin had been made $0 days previously: the black patch is due to induced pigmentation, most if not all of the grafted cells having died. On the right is shown the condition 20 days after an immunising graft from the same original donor was made; the pigmentation is very largely bleached away, although some traces remain in the centre of the area. (After Billingham and Medawar 1950.)
There are two alternative hypotheses to be eliminated before this theory can be accepted. On the one hand, the spreading of dark pigmentation around a graft might be due to the migration of actual pigmentary cells rather than of intra-cellular particles. Such migration certainly occurs in embryonic grafts of pigmentary cells in birds, but Billingham and Medawar argue that it does not account for all the phenomena observed in mammals, if indeed it occurs at all. In particular, it is difficult to reconcile with the fact that if a graft of, say, black skin from the ear is grafted onto the sole of the foot, and pigmentation spreads around it, this blackened area will have the characteristics usually found in sole-of-foot epidermis and not that of the ear-skin which initiated it. But although this makes it difficult to suppose that the spread of pigmentation is due to the migration of actual ear-skin cells, it suggests the other of the alternatives to the plasmagene hypothesis, namely that we are dealing merely with a serial evocation, in which some substance of low molecular weight diffuses from a dark cell to the neighbouring unpigmented one and sets going within it an already present pigment-producing system. There are, however, phenomena which this possibility cannot easily explain. For instance, if a graft is made of black skin from guinea-pig A onto guinea-pig B, the halo of pigment formed round it retains the immunological specificity of individual A. If another graft of A’s skin is made onto B, it will set up an immunological reaction and as a consequence of this the pigment particles disappear from the zone of pigment spread around the original graft, although the dendritic cells themselves do not seem to be adversely affected. Billingham and Medawar, on these and other grounds, argue that we must be dealing with a particle large and complex enough to carry the immunological specificity of the individual from which it originated, and capable both of spreading from cell to cell of the dendritic system and of identical self-duplication.
This case is of great interest and theoretical importance, since it is one of the very few examples of an alleged plasmagene in a higher animal which has been thoroughly studied in recent years, and in which there is no question of our being deceived by an extraneous virus. There are several further points to notice about it. First is the rather surprising fact that the ‘plasmagene’ carries the immunological specificity of the individual in which it arises, which makes it extremely difficult to transmit from individual to individual (although this can be achieved if the graft is small enough not to provoke an intense anti-body formation by the host). In this it is very different from most viruses. Secondly, the plasmagene does not possess complete genetic continuity, but must arise anew during the development of the melanocytes from the neural crest; presumably the initiation is gene-controlled, although the difficulty of interindividual transplantation makes it difficult to prove this formally. Again, one must remember that the pigment-forming cells are peculiarly adapted to make contact with their neighbours, and are known to pass pigment granules into cells, e.g. in the hair follicles, which are themselves incapable of manufacturing pigment. Thus the situation which enables us to recognise the existence of these plasmagenes by their transmission from cell to cell is a very unusual one. One might deduce from this that many such particles might exist in other cells, without our being able to detect them; equally one might argue that it is only because the melanocytes provide this peculiar mechanism of passing granules into contiguous cells that particles which can multiply and preserve their genetic character have been evolved to take advantage of it. Finally, the relations between the particles and the genetic factors in the nucleus remain obscure because we do not understand what genetic difference, if any, exists between a white and a dark area in a piebald guinea-pig; both areas are, of course, derived from a single fertilised egg, and probably both contain exactly the same genes, although some authors have suggested that the picbald pattern is due to mutation of a colour-controlling gene in some of the somatic cells. It remains obscure, therefore, whether, when the pigment-forming plasmagene is passed into a white cell and proceeds to multiply there, it is aided in doing so by the gene which initiated it, or whether that gene has mutated to an inactive form and the plasmagene is wholly responsible for the maintenance of its genetic character.
Some suggestive but indirect evidence for the existence of cytoplasmic plasmagene-like particles in normal cells can be found in some of the studies which have been made on tumour-inducing ‘agents’ of the kind which are often classed as viruses. These agents seem to be particulate in nature, and when suspensions of the particles are injected into appropriate healthy cells, they can multiply and cause the cells to develop into tumours. One important point in the present connection is that particles extremely similar to those of the tumour-virus can also be found in normal healthy cells; they are in fact the microsomes (Claude 1940). Further, when normal cells are acted on by certain carcinogenic chemical substances, the tumours which are induced are found to contain cytoplasmic particles capable of acting as tumour-viruses; and it is certainly simplest to suppose that these have originated by a comparatively slight alteration in the normal cell particles, to which one would therefore have to attribute a capacity for self-duplication. Finally, the tumour-viruses sometimes show extreme specificity in the type of tissue they can infect; indeed Rose (19526) has shown that a frog virus may be so specific that it travels through the body and settles in only one particular region of the skeleton (cf. the reference to regional specificity on p. 308). Now Rose has found that if the agent can be caused to grow in an unusual tissue, it may acquire a new specificity from it; its original specificity is sometimes retained, sometimes lost. He suggests that the new character may be picked up by an interchange process, akin to bacterial ‘crossing-over’ or ‘transformation’, with cytoplasmic constituents of the normal cells which are essentially similar to the virus particles (but cf. Luria 1953). The suggestion is interesting, and considerable developments may be expected in this field; but at present the whole of this group of phenomena is so little understood that it seems dangerous to use it as a basis for a general theory of differentiation. For instance, according to Rose these tumour-inducing agents seem, after passage through a number of different hosts, to lose nearly all their tissue or even species specificity, which makes one wonder whether they may not be damaged forms of the normal cell microsomes, the damage being of such a kind as to render them insensitive to the normal control of the nucleus; if this were so, they could not be taken as evidence that the cytoplasm of a healthy cell contains particles which are independent of the nucleus.
Another type of phenomenon, which may involve the activity of plasmagenes, is that known as enzymatic adaptation (Reviews: Monod 1947, 1950, Monod and Cohn 1952, Spiegelman 1950, Gale and Davies 1953). There is abundant evidence which strongly suggests (though perhaps it does not completely prove) that in bacteria, yeasts and other lowly organisms suitable for such studies, the formation of most enzymes attacking exogenous substrates is specifically increased by the presence of the substrate and in fact hardly occurs at all in its absence. The formation of the adaptative enzymes involves the synthesis of protein, and the physiology of the process has been studied by several authors (e.g. Spiegelman and Sussman 1952). The formation of an adaptive enzyme in the presence of any particular substrate requires the activity of a corresponding nuclear gene, and we therefore have here a good example of the influence of substrates on the activities of genes. But it has been suggested that something more is involved. Lindegren and Spiegelman (Cold Spring Harbor Symp. 1946) originally put forward the hypothesis that the production of the adaptive enzyme was a property of a cytoplasmic plasmagene, which, in the presence of the substrate, could persist indefinitely even if the corresponding gene had been removed from the cell by crossing. Their original evidence, obtained in yeasts, was later shown to be inadequate, but there still remains some evidence which may indicate that such plasmagenes exist (Spiegelman 1951).
Ephrussi (1953), (Ephrussi and Hottinguer 1951) has also found evidence which suggests the existence of cytoplasmic self-duplicating particles concerned with enzymatic adaptation. In his case, yeast cells cultivated in the presence of the nuclear poison acriflavine are shown often to give rise to colonies which grow abnormally slowly and in which the cells have lost certain respiratory enzymes and have an abnormal cytochrome system. The change is quite stable through many generations of vegetative division. Crossing experiments show that it is immediately dependent on a cytoplasmic, not on a nuclear, factor; but again further investigation has demonstrated that the plasmagene concerned is itself under the eventual control of a gene. The enzyme changes induced by the acriflavine are not themselves adaptive, but they are closely similar to undoubtedly adaptive changes which yeast cells exhibit when cultivated in the absence of oxygen.
It is questionable whether it is really appropriate to employ the word plasmagenes for any of the gene-initiated factors considered in this section. The character they share with the true plasmagenes is a certain ability to multiply in the cytoplasm. It is not clear, however, that any case is known in which a gene-initiated cytoplasmic factor acquires complete autonomy in its powers of reproduction. Certainly the Paramecium antigen determinants can only persist for a very limited period after the removal of the gene. The situation of the factors studied by Billingham and Medawar is obscure, since in the piebald guinea-pigs they studied, the originally colourless cells into which the factor passes probably possess the same genotypic constitution as the coloured cells out of which it comes, the difference be ‘tween the cells being one which arises during differentiation rather than of a truly genetic nature. Beale (1954), who has studied these phenomena as closely as anyone, has recently expressed a lack of satisfaction with the term plasmagene for such factors. Haldane (1954) is apparently of a similar opinion and has suggested calling them mnemons. For convenience in the present discussion, however, I shall continue to refer to them as gene-initiated plasmagenes.
5. The role of plasmagenes in differentiation
It will be noticed that the overwhelming majority of the evidence for the existence of plasmagenes come from studies on micro-organisms. It might be, however, that this is caused not by their rarity in other forms but by factors which make their detection particularly difficult. It is clear, for instance, that if plasmagenes were to play an important part in the differentiation of multi-cellular organisms, they could not in general be capable of easy infective transmission from one cell to another, since that would lead to an intermingling of different organs or types of tissue which should remain separate. Thus we cannot expect to find many cases similar to that of Billingham and Medawar, even if factors of an essentially similar nature are widespread. It is necessary, therefore, to approach the matter to some extent from an a priori point of view to try to determine how far plasmagene-like factors could fit in to the mechanisms of differentiation in so far as we understand them at present.
It is clear that the exogenous factors mentioned under group (1) above do not come into the question. In the examples of the true plasmagenes mentioned in group (2), the cytoplasmic determinant is a part of the general genetic constitution of the organism and no more directly related to the regionalisation of its various parts than are the nuclear genes. It is, however, possible to imagine that the cytoplasm of the egg of a given species might contain a number of different true plasmagenes localised in various regions. Each region of the egg would then contain characteristic cytoplasmic factors endowed with genetic continuity which might determine the nature of the organs which develop out of it. Such localised plasmagenes would, in fact, be the same thing as used to be referred to at the beginning of this century as organ-forming substances. Now there is no doubt that in many eggs different regions of the cytoplasm have different properties. The regions concerned are nowadays referred to as ooplasms, and opinion has rather moved against attributing their properties to the presence of substances which are autonomous over against the nucleus.
The arguments which have swayed opinion against the old idea of organ-forming substances are numerous. One is that the evidence suggests that the ooplasms are only effective when they are able to interact with the nuclei. For instance, the cytoplasmic formation centre in the posterior of an insect egg orily becomes active when nuclei reach it (p. 125); the same is true of the grey crescent ooplasm of the amphibian egg (p. 149). Again, differentiation from the egg to the final form takes place in a series of steps. It does not look as though we are dealing merely with the sorting out of a number of factors which from the beginning preserve their character unchanged, but rather as if development consists of a series of reactions during which the constituents of the system change continuously until the final condition is gradually built up. We are already faced with the difficulty of accounting for this progressive series of changes in a system one of whose major components consists of genes which we believe to retain their identity throughout. The difficulty is only made the greater if we have to suppose that the major factors in the cytoplasm also retain their identity.
As a third argument, one may point to the fact that the localisation of different organs within the developing body may often be altered by factors which operate after the segregation of plasmagenes in the egg cytoplasm must have been completed. For instance, one might be tempted to attribute the localisation of the organs in a developing Drosophila to the segregation of organ-forming substances or plasmagenes in the eggs, which are known to belong to the mosaic type; yet we have seen (p. 141) that environmental treatments applied many hours after fertilisation can divert the differentiation of particular regions into abnormal paths.
The mere fact that a gene, like aristopaedia, can cause a mass of tissue which should normally develop into an antenna to develop into a leg instead, shows that even if we try to attribute the major process of differentiation to plasmagene-like bodies, these cannot be autonomous in their properties but must be highly susceptible to modifications caused by interaction with genes. Finally, it would be still more difficult for a hypothesis which attributed differentiation to be activities of autonomous plasmagenes to account for metaplasia, which, though rare, does seem to occur (p. 308).
It appears, therefore, that the postulation of true plasmagenes as organforming substances in the cytoplasm of the egg does not materially simplify the theoretical task of accounting for the phenomena of differentiation. That does not necessarily mean, of course, that such bodies do not or cannot exist; we should have to take account of them if there was unequivocal evidence for the existence in the eggs of multi-cellular animals of cytoplasmic factors which had genetic continuity independently of the nucleus, As yet there seems to be no compelling evidence to this effect. Indeed, attempts to assess the autonomy of cytoplasmic factors in the egg over against the nucleus have been few and far between. The studies with hybrid merogons in Amphibia (p. 358) are perhaps the most promising. The facts there can probably all be accounted for in terms of a mere persistence of cytoplasmic character, without the need to postulate that the cytoplasmic factors can reproduce while retaining their specific nature. The case which argues most strongly in the opposite direction is perhaps that of Hadorn (1936), who found that epidermis derived from Triturus palmatus cytoplasm fertilised by T. cristatus sperm developed the typical characteristics of palimatus as late as after metamorphosis. This may indeed be evidence of the existence of a true plasmagene, but it might equally be the result of a plasmagene initiated in the egg cytoplasm by the maternal genes during the maturation of the egg. One may conclude that there is hardly any evidence that plasmagenes with complete autonomous genetic continuity exist in metazoan eggs, and that it seems most improbable that the major phenomena of differentiation can be attributed to them.
The same conclusion applies even more forcibly to the plasmagenes of category (3), namely microscopically visible cytoplasmic particles with genetic continuity. These certainly occur in certain special cases, as for instance in ciliates, but in general the histological evidence makes it clear that differentiation does not consist to any large extent of the mere sorting out of the already existing visible particles in the egg cytoplasm. Such particles probably play an important part in development, but not by the mere retention of their original characteristics.
The situation is rather different when we turn to the fourth category, that of gene-initiated plasmagenes. If these were to play an important part in development we should have to imagine that the various ooplasms of the egg differentially excite the nuclei which enter them; that the particular genes which are activated in a given region then cause the appearance of cytoplasmic factors, and that these factors, when they have appeared, show a certain degree of autonomy, being able to reproduce for a short time with repetition of their character even if the nucleus is removed or changed. If one supposes that, once they have been formed, the autonomy of the plasmagenes is complete, this suggestion would come up against the same difficulties as confronted the hypothesis of organ-forming substances in accounting for the sequential character of differentiation and phenomena such as the metaplasia of retinal cells into lens in Wolffian regeneration. We have seen, however, that in the best-studied examples of gene-initiated plasmagenes the autonomy is by no means complete. If one waters it down sufficiently, the difficulties which have just been mentioned could be overcome.
The hypothesis would then amount to the suggestion that during differentiation the genes cause the appearance in the cytoplasm of bodies with a certain limited amount of autonomy. There seems nothing impossible, or even very difficult, in such a suggestion. As was pointed out earlier (p. 212), Brachet (1944, 19522) has argued with considerable persuasiveness for the importance of the ultra-centrifugable ribose-nucleic-acid-containing microsomes, and he does not hesitate to refer to these as plasmagene-like in character. The problem that still remains at issue is how far these particles, once their character has been determined, become independent of the nucleus. Only the transplantation either of the nuclei or of the particles from one type of differentiating cell to another can settle the matter conclusively. There would be nothing surprising if experiment eventually showed that, in cells which are more or less completely determined and are in process of producing their final cytoplasmic constituents, the cytoplasm is able to carry on synthesising these nearly independently of the nucleus. However, such a fact does not yet seem to have been demonstrated. Whether, if it were, we should be justified in speaking of the effectiveness of plasmagenes in differentiation would be largely a matter of definition; it would depend on whether we are satisfied that such geneinitiated cytoplasmic factors of limited autonomy are comparable to plasmagenes of the more completely autonomous kind.
Darlington 1944, Ephrussi 1953, Haldane 1954, Chapter 7, Medawar 1947, Monod 1947, Sonneborn 19514 or b, Weisz 1951.
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