Portugaliae Acta Biologica - 1949
A Cytophysiological Theory of the Gene, Gene Mutation
and Position Effect

J. A. Serra

Secção de Zoologia-Anthropologia, Faculdade de Ciências. Universidade, Coimbra

2. THE BASES OF THE MODERN GENE CONCEPT

The modern concept of the gene as an hereditary unit is based both upon the abstract factors of Mendel and formal Genetics, and the determinants of Weismann and the corresponding material units of the cytologists. The mendelian factors are in fact a kind of mathematical abstraction corresponding to one or more physiological differences which, acting during some period of ontogenesis, shall result in an observable alteration of the phenotypic appearance or of the functioning of the individual. What demonstrates the existence of a factor is the appearing of a mutated factor, the existence of a variant which, by physiological happenings in the ontogeny, manifests in the phenotype. That is, the roots of the concept of factor are in fact physiological and, due to the method of analysis, the demonstration of a factor depends on the existence of at least two alternative modes of reaction.

On the other side, the determinants or those materials which under the form of discrete units must exist in the sex cells and determine the physiological differences corresponding to the factors are essentially a cytological concept. Modern Genetics has tried to articulate this cytological concept with the physiological concept of factor. However, the particulate genes usually accepted to-day as the basis of heredity remained essentially a physiological concept and only in a very restricted number of cases has a morphological basis been sufficiently worked out to warrant a true articulation of the two bases, the physiological of the "character" and the cytological of the "elementary chromomere". As we have already stressed in former work (Serra 1944) the kind of hybrid origin of the modern gene concept explains in part its weakness.

THE PHYSIOLOGICAL UNITS

In order that a particulate gene concept has a sound basis, it is necessary that the physiological unitary differences and the cytological well delimited entities be demonstrated to correspond to one anothers. These discrete units may be called, in a more general way, discontinuities. Mutation, which is the way in which an alternative factor reveals, should in typical cases correspond to a difference in one reaction of a reaction chain, or to a single morphogenetic process when the phenotype manifests morphologically; that is, the primary action of the gene should be unitary.

This question of the unitary action of the genes is related to the question of pleiotropism. In the best analysed cases of biochemical characters it has been shown that each mutation apparently corresponds to the alteration of a single step in a reaction chain. Beautiful examples of these characters have been studies in Neurospora by Beadle and co-workers and the existence of recent reviews on this subject excuses us from detailing here any example (Beadle 1945, 1948 and later.). Other cases are the metabolism of phenylalanine in Man, the formation of the brown pigment in the eyes of Insects, certain biochemical characters in the rabbit, Drosophila, etc. (refs. and examples: Haldane 1941, Beadle 1945, Serra 1949). Another case is that of melanin pigmentation (Serra 1945, 1949 and bibliogr.). The conclusion in all the cases sufficiently analysed is that biochemical characters express themselves primarily by means of a single alteration in a reaction chain.

The phenogenesis of form is more complicated and therefore it is expected that only with difficulty can mutations be demonstrated to correspond to a single alteration in a morphogenetic process. This, however, has been shown very probably to occur in the case of pleiotropic factors and in certain morphogenetic chains. As this point of the existence of unitary differences in the realization of form under the action of the genes is less understood than the corresponding case of the biochemical characters, we propose to discuss it here, if only briefly.

The possibility of tracing back the realization of complicated morphological characters to a single primary action of a gene gave rise to the admission, in principle, of a genuine or real pleiotropism and a false or spurious one (Grüneberg 1943). Real pleiotropism would be the consequence of the primary action of the gene being plural, exerted at the same time upon several steps of a reaction chain or upon several distinct reaction chains. As yet no such cases have been demonstrated and indeed, cases which apparently were of real pleiotropism, with a more detailed analysis turned out to be only of spurious pleiotropism. Typical examples of apparent pleiotropism are those of hydrocephalus in the mouse and the disease of cartilages in the rat (Haldane 1943). In both cases, an anomaly during the formation of certain cartilages in the embryo brings about a series of consequences which culminate respectively in hydrocephaly and in death by inanition or by respiratory and circulatory failure.

Interesting are also the cases of brachyury in the mouse, which can be traced back to irregularities in the formation of the notochord from the mesoderm, at the 8.5-9 days of development (Dunn and Caspari 1945). Similar cases of anomalies in the tail vertebra of the fowl have been shown to be due either to the action of hereditary factors or to mechanical or chemical injury to the early developing embryo (Landauer 1945). In reality, it seems that several actions which are not lethal and act upon the early embryo, when the mesoderm somites are in process of differentiating, may bring about tail anomalies, not only in the fowl as also in the mouse (Serra 1947). These cases illustrate another interesting point in the phenogenesis of form, namely the non-specificity of many of the actions which may alter the pattern of development of morphological characters. Although the tail characters probably constitute an extreme with respect to the lability of their causation, nevertheless the possibility exists that non-specific actions may also be operative in other cases of morphogenesis and indeed it seems probable that this principle applies to several other examples, for instance to polydactily in the fowl (Landauer 1948). This is to be expected especially in the case of characters more strongly dependent upon the pattern of early embryonic development, when a given area with great prospective potence may give rise to several organs or systems of the adult. It is in these cases of manifestation in early embryonic life that the more typical examples of apparent pleiotropism are expected to occur, and also that a full analysis of development must demonstrate interesting morphogenetic reaction chains of what may be called "characters with outstanding morphological effects".

The principle of unitary primary action may also be demonstrated to be operative in the case of morphogenetic reaction chains. Although the example of wing development should perhaps prove to be more complete, due to consideration of space we reproduce here only a scheme of the development of the eyes in Drosophila melanogaster, extracted from Serra (1949), based chiefly on work of Waddington, Pilkington and Steinberg (1941-45)—Fig. 1.

FIG. 1. Phenogenetic scheme of the development of the eyes in Drosophila melanogaster. The arrows indicate the points where it is presumed the genes act.

In this scheme only the general lines of the development have been shown and some incertitude as to the true points where some of the mutated factors act exists as yet, especially in what concerns the early development, subject to controversial interpretation (comp. Waddington and Pilkington with Steinberg). Bar, eyeless and Lobe all act at an early stage, sooner than, or at most contemporary to the differentiation of the ocular disc from the cephalic complex; while the cephalic complex of eyeless and Lobe already shows, at the end of the first day of development, a well apparent difference from the wild, the Bar mutant by this time has an almost normal (Steinberg 1943) and therefore must act afterwards. The time of action of the other mutants shown in the scheme is also subject to correction by future work, but it seems that the processes upon which they act must not be very different from those pointed out in the scheme.

In what consists the primary action of these factors is yet subject to a greater conjecture than their timing. Only the case of Bar and some of the others have been investigated in greater detail. So long as the factors act autonomously in explants, there is no way of demonstrating active substances or development hormones. All these mutants seem to act autonomously and this greatly difficults their analysis. Bar acts upon the process of ommatidia formation but little influences the size of the ocular disk area. Bar larvae shall have their ommatidia limited to an area along the greater axis of the ellipsoid surface of the normal eye, but the final number ommatidia is fixed during the subsequent course of development, according to the rest of the genotype and a series of ambiental factors. Bar determines the approximate limit, not the exact one, of ommatidia formation. Its action may consist either: 1. in agenesis of ommatidia from precursor cells or 2. a destruction of ommatidia already differentiated, or in process of formation, or else 3. the stopping of growth of ommatidia in process of differentiation (Goldschmidt 1945). This latter type of action seems to be excluded by the fact that the ommatidia of the Bar eye are practically of the same size as those of the wild fly. The agenesis hypothesis seems to be the most consistent with the observations already made on the development of the Bar cephalic complex. It seems that the action of the Bar mutant consists in depriving the differentiating cells of the ocular disc of a substance, or a condition, necessary for the differentiation of the ommatidia, except in a small area along the long axis of the eye. This substance or condition, while probably acting like a vitamin or hormone, seems to be non-diffusable from the cells, although its formation within the cells may be influenced by external factors. In the limit of the area of action, the amount of substance, due to external or internal factors, is so small that it easily remains below a threshold.

Similar modes of action must have eyeless and Lobe; only their point of action is located earlier and probably this accounts for the difference in the localization of the ommatidia which are allowed to develop. On the other factors, we will refer here only to ophtalmopedia (data on the others may be found in Waddington 1943, Pilkington 1942 and Serra 1949). The apparent action of this factor consists in an overgrowth of a more or less central area of the eye disc, immediately after the cells of this latter begin to differentiate into three layers. This overgrowth hinders the normal differentiation and instead, a palpus like structure develops, this being a mere consequence of a greater growth rate; any overgrowth in the cephalic complex tends to give a kind of finger like structure, similar to a palpus or small antenna or leg. The eye mutants as a rule act upon the growth of the eye disc or of certain of its structures and it may well be postulated that some substance is responsible for their primary action. With the limited knowledge we have at present on the growth hormones of Insects, it is not possible to draw a final conclusion on the action of these mutants. Nevertheless, from the examples known in Vertebrates of the profound morphological action of the pituitary, thyroid and sex hormones, it must be inferred that also in Insects the morphogenesis may be affected by developmental factors of a chemical nature, though here they may have in intra-cellular, non-diffusible component.

The conclusion to be drawn from the more thoroughly investigated cases is that in the phenogenesis of form the postulate of the existence of an unitary primary gene action seems legitimate in view of the already known facts and is only a kind of morphogenetic counterpart of the better known biochemical characters. As in the chemical reaction chains, the action of the genes acting upon the morphogenetic reaction chains seems to be primarily unitary.

Having reached this point, it is perhaps profitable to examine the other side of the question, that is if other ways of logically explaining the action of the genes in phenogenesis of biochemical and morphological characters are not possible. Let us suppose that the development is a kind of continuum in what concerns the action of the hereditary materials and that the apparent discontinuities of the phenotype, for instance the production of a Bar eye, are no more than an index or a more apparent and localized change, of a more general action pertaining to the organism as a whole. Then the action of each mutant should consist in the alteration of the whole organism by means of differences in several functions, which usually should express in a lowered viability.

*It must be remarked that this hypothesis of a plural or generalized action of every factor is different from "blending inheritance", which supposes a final intermediary action. If in fact the final action expresses in one character, the phenotype is apparently discontinuous; therefore, the well known argument of the constancy of the variability in F2 and subsequent generations of mendelian populations, which excludes the hypothesis of a blending inheritance, does not apply to the present case.
The characteristic or "visible" effect could in this case be a mere consequence of the lowering in viability being more marked, or attaining first a threshold value, when the structure or character affected was in a sensitive phase of development; that is, according to this hypothesis the apparent unitary action of the factors should result from the fact of them lowering the viability—in itself the result of many physiological reactions—first, or more markedly, at a certain period of ontogenesis or of life.*

The adoption of this hypothesis would correspond to the rejection of the unitary factor concept. It is known that in Drosophila almost every mutant shows a viability different from that of the wild type standard and, as far as has been investigated, the same is valid for other animal and plant species. The hypothesis of the existence of a kind of hereditary "continuum", or more precisely, of a plural action of each mutation, would immediately explain these differences in viability, while the unitary hypothesis, at least at first sight, encounters some difficulties when attempting to give an explanation of the phenomenon. In fact, if in certain cases, for instance when the eye of Drosophila is deprived of pigments or is drastically reduced to a few ommatidia, the lowering of the viability of the adult may be explained simply by the anomaly of the chief character, the same does not apply to the larva, in which it seems difficult to realize how a loss of pigment in immaginal buds and internal organs affects the viability. At first sight it seems simpler to admit that this lack of pigment is only a visible index of more profound changes which, inter alia, bring about a disturbance of the enzymatic system capable of oxidizing tryptophane and the precursors of the red pigments, or of the structures necessary to bind the pigments in the pigmentary cells. However, the unitary theory also explains the differences in viability, though this explanation is as yet entirely hypothetical. According to this hypothesis, in the above case of the pigments of the Insect's eye the lack of the oxidases, or of the pigmentary structures, should act upon every organ in which these enzymes of the materials necessary for the building of the pigmentary structures are also utilized in reactions affecting the larva vigour and viability. Somewhat different is the case of Bar, for instance; although in this case it may also be postulated that the lack of the condition responsible for the non-differentiation of ommatidia or the non-multiplication of precursor cells acts also upon the general viability, another complication arises from the fact that Bar is a small duplication and this may bring about an unbalance in the genotype, simply by changing the proportion of genes in relation to one anothers, but in reality this effect is small in comparison to the position effect caused by the duplication. This particular case of Bar is interesting also because it may furnish an argument in favour of the architectural theories. According to these theories, it is the very fact of a change in the structure—or better, as this term may bring about confusion with the hypotheses considering an atomic or molecular structure of the particulate hereditary units as relevant—in the "architecture" of the chromosome, or more generally of the hereditary materials, that causes the mutated phenotype and the changes in viability, both being no more than the two sides of the same basic happening. In the last analysis, these architectural theories tend in the limit to the hypothesis of an hereditary continuum and their criticism may be jointly attempted.

Although in the cases of morphogenesis there is yet room to admit the hypothesis of the generalized viability differences as primary and the particular and more apparent action as a mere corollary, it would be very difficult to adapt these hypotheses to fit in the case of biochemical characters such as those of Neurospora, in which it suffices to supply a certain chemical in order to restore growth, or in the case of the brown eye pigment of Insects, in which the mere supply of tryptophane oxidation products brings about the "healing" of the condition. For the biochemical characters and for morphological characters depending upon diffusible factors, the unitary theory of the primary gene action is the only which explains the facts with a minimum recourse to supplementary hypotheses, and as the number of cases explained by this theory is always increasing at the measure the investigations are continued—it seems perfectly reasonable to generalize in this domain and to admit that, at least in the simplest mutations, when only one hereditary factor is altered without at the same time bringing about quantitative genetic changes of the unbalance or of the position effect types, mutation corresponds to a primary action exerted through a single change in a certain step of a biochemical or morphogenetic reaction chain. This conclusion is the logical extension of the atomic concept of factor and is in accord with the existence of physiological discontinuities in the phenogenetic realization of hereditary characters.

THE CYTOLOGICAL DISCONTINUITIES

The other aspect of the gene concept, the morphological, is especially concerned with the articulation of the essentially physiological concept of factor with the particulate material counterpart in the reproductive cells. Cytogenetics has demonstrated beyond any doubt that the chromosomes and their mitotic and meiotic divisions furnish the only possible support and mechanisms, respectively, for the localization of the hereditary materials and their orderly distribution to the cells of the organism and its gametes. In reality, it should be contrary to the economy of nature that such complicated processes as mitosis and meiosis, which must require an high amount of energy for the changes they imply in the cell, were not connected with fundamental processes. The proofs of the localization of the typical mendelian factors in the chromosomes are so extensive and complete that it is not worth while to consider here some sweeping assertions against them, rather recently made from certain quarters. This, of course, does not preclude the existence of hereditary materials outside the chromosomes, and in fact such materials must exist in the cytoplasm and its inclusions, as every geneticist well knows; however, due to considerations of space, in this paper we will not deal with the question of plasmagenes and cytoplasmic inheritance but only with the classical chromosome genes.

The interkynesis nucleus generally shows some points with thymonucleic acid, but usually it is not possible to define an orderly arrangement of them. Metaphasic chromosomes, with the exception of primary and secondary constrictions, do not show, also, any discontinuities. These appear during the prophase of meiosis and in the parallel case of Dipteran salivary chromosomes (and also chromosomes of other larval tissues) and to a lesser extent, although always little clearly and only in some species, in the prophase and telophase of mitosis. As is well known, the discontinuities in the case of the meiotic leptotene-pachytene chromosomes are between bead-like chromomeres and thread-like interchromomeres, while in salivaries the alternative is between chromatic bands and achromatic interbands. Naturally, there is a tendency to homologize the bands with chromomeres and the interbands with interchromomeres, but how far this assumption is correct is a matter for discussion and will be considered in more detail below.

Belling was the first to succeed in comparing the number of pachytene chromomeres with the number of genes. In Liliaceae, the bead like chromomeres are of the order of 2,000 which, according to the knowledge gained in Drosophila, seems also to be a not too unreasonable estimate of the number of genes. In view of the incertitudes in such a comparison, however, it does not seem to give much weight to the homology of genes with pachytene chromomeres, even in the particular case of the plants used for the computation. What is really important in these cases is that the pattern of the bead like chromomeres of the chromosomes is constant for a given plant and, within the limits of chromosome structural changes, also for a certain species. Using again the argument of the economy of nature, it seems highly probable that this must correspond to an important property of the hereditary materials located in the chromosomes. In the case of maize it has been possible to go further in the direction of assigning to certain chromomeres the correspondence with certain hereditary factors and the facts hitherto discovered are consistent with the hypothesis of each chromomere corresponding in some cases to a single factor, while in others one chromomere contains two or more factors.

This point of the homology between chromomeres and genes requires more thorough consideration, as it touches the very roots of the gene concept, and therefore we propose to deal with it in the following section. Here it suffices to point out that in principle it is not necessarily implied in the hypothesis of the homology that the genes are in fact located in the chromomeres and not in the interchromomeres. Indeed, it has already been claimed that this might be the case (Kostoff 1940). The results obtained in the extreme of the short arm of the 9 chromosome in maize (McClintock 1944) are not inconsistent with such an hypothesis. In this case, a pale yellow seedling (pyd) phenotype appeared when an homozygous deficiency of the terminal heterochromatic knob plus the thin adjacent strand joining the knob with the first visible chromomere was present, while the homozygous loss of the knob only had no visible effect. It must be remarked that in the same experiments the homozygous deficiency of the terminal thin strand plus a part, about a half, of the adjacent first chromomere produced a white seedling (wd) phenotype. McClintock estimates as probable that wd may also be caused by the loss of this half chromomere alone, without the cumulative loss of the adjacent thin strand.

If these results can be taken at their apparently simplest meaning, the conclusion would be reached that pyd is located in the "thin strand" or interchromomere. However, it is also possible, as McClintock states, that together with the thin strand a small imperceptible part of the adjoining first chromomere is also lost when the pyd phenotype appears. Another possibility is that the thin strand has a minute chromomere not visible in pachytene, or that this chromomere is immediately adjacent to, and indistinguishable of, the first chromomere. There is yet another possible explanation, namely that pyd is no more than a position effect caused by the loss of the interchromomere adjoining the first chromomere. The most probable conclusion, however, seems yet to be that the loss of the thin thread brings about with it the loss of the adjoining simple chromomere, which is only a part of the evidently multiple or composed first chromomere, without any doubt formed of several unitary chromomeres.

According to the best evidence, to date there is no proof of the hereditary factors being localised in the (pachytene) interchromomeres, as against such a location in the chromomeres. But on the other side, it is not proved, also, that the factors correspond exclusively to pachytene chromomeres and not, as seems more probable, to the chromomeres plus the adjoining interchromomeres, or less probably, to the zones of contact between chromomeres and interchromomeres. This is an important point which will be further considered when dealing with the chemical composition of the gene.

Another question concerns the reality of the discontinuities in the chromosomes. Contrary to the usual interpretation of the existence along the pachytene chromosomes of bead like chromomeres, attempts have been made to explain the aspect of beads as being due to a closer helicoidal coiling at these points, giving by an optical effect the appearance of a bead (Huskins 1941, Nebel 1941, Ris 1945). The partisans of the "spiralization" hypothesis endeavour to explain the particularities of the chromosomes by a more or less close coiling, but it rests to be explained why the coiling is more close at such points; since there is a constant pattern of the position and size (within the observational variations, of course) of the beads, these must have a strict causality and some well defined cause or causes shall be responsible for the differences in coiling along the chromosome threads. The spiralisation hypothesis, while perhaps relevant to the interpretation of the chemical composition of chromonemata and genes, does not disprove the existence of discontinuities along the chromosomes and even if it should prove to hold true, could not invalidate the particulate theories of the hereditary materials located in the chromosomes.

The same may be said about a correspondent interpretation given to the salivary chromosomes of Diptera (Goldschmidt and Kodani 1942, Kodani 1942), according to which the aspect of bands is due to the existence of closer helix turns at these points, upon which bulbs of thymonucleic acid would deposit, giving rise by an optical effect to the aspect of a band. A criticism of these interpretations has been made in a former work (Serra 1945, 1947) and in what concerns the possibility of they contradicting the significance of the bands, the answer is the same as that given about the pachytene chromosomes: in principle, what is relevant is the constancy of the pattern of a certain discontinuity of zones with different properties. Even if the spiralisation hypotheses would prove to hold true, this should not invalidate the attempts to find in the chromomeres the particulate equivalents to the unitary factors, as the necessary (but not sufficient) requisites for this are, at the cytological level: 1) the existence of a discontinuity and a constant pattern of this latter, and 2) that separate units in the chromosomes may be demonstrated. This second point will be considered in the following.

THE SEPARATE UNITS OF THE CHROMOSOMES

The demonstration of separate units in the chromosomes is based on two orders of proofs: crossing-over and chromosome rearrangements. I reality, both proofs depend upon the breakage of the chromosomes at certain points and not at others. The existence of discontinuities in what concerns the breakability along the chromosomes is in accord with the definition of two elementary entities, the minimum crossing-over segment and the minimum breakable segment. The first of these segments corresponds approximately to the gene and is defined by the absence of recombinations in a sufficiently great descendency, which in fact is only a genetico-statistical proof based on the factor concept.

* We will not discuss here hypotheses on the formation of recombination classes, other than the crossing-over theory. According to the actually known facts, the conversion theory of Winkler and the mutation theory seem improbable.
On the other hand, the minimum breakable segment of chromosome as a rule can be defined only indirectly, by observing the rearrangements and not the fragments themselves. We will examine briefly the relevant facts on these two orders of proofs of the existence of separable chromosome segments.*

Generally it is not possible to demonstrate crossing-over in a small segment by cytological observation and therefore the final proof lies in the obtention of recombinations. When two factors are located near one another, great descendencies may be necessary in order to exclude the possibility of crossing-over. For instance, if the expected recombination fraction is 0.1% about 753 descendants are necessary to get at least one individual of the combination classes with a chance in two (50% probability); or 2994 descendants for a probability of 95%. With a recombination class of 0.01% about 6936 descendants are necessary to obtain at least one of the expected fraction with a chance of 50%. It is not frequent to analyze so great offsprings and if this was done, not always is it possible to distinguish between the phenotypes of neighbouring factors. Another complication is brought about by the possible existence of a local hindering of the crossing-over by partial non-homology or other causes.

That the difficulty of differentiating between the phenotypes of closely linked factors is more than a possibility is shown for instance by the case of Star and asteroid in Drosophila melanogaster, which are 0.02 cross-over units apart and behave like alleles if carried by homologous chromosomes (Lewis 1945). Parallel cases are also known for lozenge and for the facet region. These complications are also in part a consequence of rearrangements in these regions. On theoretical grounds, it is to be expected that in some cases neighboring loci may show similar phenotypic effects and in consequence a poor discrimination of the recombinations occurs. Clearly, the definition of the least cross-over segment by recombination is only a genetical basis, not a cytological criterium. The very nature of the homologous segments, with their microscopical structure exactly alike if crossing-over is habitually to occur, precluded of such a demonstration and only indirect inference, especially by comparison with the rearrangements, may be drawn about the cytological entities which correspond to the minimum crossing-over segment.

The rearrangements are the only really cytological proof of the existence of a minimum chromosome segment which behaves as one unit. We will call the minimum segment of the chromonema which may not be breaked the nemamere (nema, thread, and mere, part). Unfortunately, in ordinary chromosomes it is not easy to observe the minutest fragments and the knowledge of the pachytene chromosomes in genetically analysed species is yet poor. In the above mentioned example of pyd and wd in maize a particularly accurate location has been possible grace to the terminal position of the loci. Other small deficiences in maize have also been demonstrated and it is highly desirable that such close locations be worked out in as many species as possible with pachytene chromosomes suitable for observation. Improved methods of chromosome observation in this phase may be of help (Lima de Faria 1948).

The pyd and wd case demonstrates that a pachytene chromomere as that following the thin strand and knob of the maize chromosome 9 is not an ultimate unit, since it may be broken. As a general principle, it may be assumed that several chromomeres may coalesce into one composite chromomere body, by fusion of the respective matricial nucleoproteins or by differential coiling (spiralisation). This is to be expected especially in heterochromatic regions and in their neighborhood. The terminal part of maize chromosomes provided with heterochromatic knobs must be liable to junction of the chromomeres into composite bodies. It is possible that in such cases as the Liliaceae, and particularly Aloe, where heterochromatin is either absent or exists only in a little amount not easily forming chromocentres or similar bodies, pachytene chromomeres are in a great measure simple structures. Even in this case of the existence of little or no heterochromatin, it is to be expected that adjacent chromomeres may show some degree of coalescence by their peripheric nucleoproteins.

If the least chromomere which may be individualized as such is designated as the elementary chromomere, the existence of this cytological entity at present is a rather logical postulate than an observational fact. By stretching pachytene (early pachytene, of course, not late pachytene) chromosomes it is possible to distinguish small chromomeres. In every case these chromomeres must give a positive Feulgen reaction. However, only in genetically well known species may an homology between the minutest chromomeres and the mendelian factors be attempted. This remains to be done almost completely and indeed is very difficult from the observational point of view. Until sufficient data on this important are available, a considerable incertitude shall continue to reign about such an homology, and hasty conclusions must be avoided.

When, instead of pachytene chromosomes, salivary ones are observed, a closer homology, in principle, is possible. Unfortunately, in view of the smallness and the lack of detailed marking structures of Dipteran pachytene chromosomes, when an homology is established between a factor and a certain salivary segment, it can not be applied also to the pachytene chromomeres and interchromomeres; in Diptera, the visible pachytene chromomeres are so few that without any doubt they are of a composite nature.

Examples of closer location of factors on salivary chromosomes of Drosophila melanogaster are for instance those of Demerec and colls. (Demerec 1940), Slyzinska (1938) for the 2D-3E region of the X chromosome and Sutton (1940, 1943) for the tip of the same chromosome. In these cases it has been possible to locate some factors apparently with a precision of one band, while for others two or more bands is the correspondent salivary locus; or conversely, several factors correspond to one band. For instance, rst (roughest, 1.7) should be located at 3C4, a thin band between two doublets, while y (yellow, 0.0) and ac (achaete, 0.0+) are within the 1A5-8 bands and sc (scute, 0.0++) is at the doublet 1B3-4; and fa, spl, Ax (facet, split, Abruptex, 3.0) were all placed at 3C7 by Slizynska.

However detailed and accurate, these locations seem to be always subject to revision. For instance, w has been located at 3C1 or 3C2, at 3C2, at 3C2-3 and at 3C3 respectively by Slizynska, Schultz, Demerec and Sutton, and Prokofyeva-Belgovskaya (refs. Bridges and Brehme 1944). Also, for the three factors fa, spl and Ax formerly located at 3C7, it is now assumed that Ax is a small duplication of this thick band, fa itself being probably a small deficiency according to Oliver (Goldschmidt 1944) and possibly split will yet be dissociated from this complex. The principle implicitly followed in these studies of location is that, given enough deficiencies and a suitable salivary region, in the end it will be possible to locate precisely every factor in one band. According to this principle, the circumstances which interfere with the attaining of such a goal are the lack of favourable deficiencies, and above all, the disturbances caused by position effects.

For the tip of the X and other chromosomes, pseudo-deficiencies may also be embarrassing (Goldschmidt and Kodani 1943) due to the well known property of the salivaries forming a kind of spireme by fusion of their, probably heterochromatic, ends with one anothers in the intact, non smeared, nucleus: and also, yet more embarrassing, there is the possibility that some bands do not differentiate from a complex, this being a remarkable characteristic of some stocks and not of others (Kodani 1947). This latter observation very probably signifies that, within the limits of current observation methods (acetic-orcein and acetic-carmine) there is always the possibility of some bands non-resolving from a complex during the growth of the salivary chromosomes. While other interpretations, namely that the bands in question really are lacking and not fused with others, are also possible, the more probable interpretation is that the growth of every region of salivary chromosomes is, within certain limits, controlled by the genetic constitution of the stock, especially the heterochromatin present, and some ambient conditions; accordingly, the details of structure are also different, one band in one case corresponding to two or several thinner bands in others.

This brings us directly to the problem of the derivation of the salivary structures from those of ordinary chromosomes. It is assumed by all workers in this field that the structures seen in the salivaries are already present en potence in the mitotic chromosomes of the early larva salivary cells. This seems a sound assumption and only rarely has it been postulated that salivaries may represent something different: for instance, Caspersson (1941) has supposed that the chromosomes may reduce their chromomeres in the contracted stages and the interchromomeres, corresponding to the salivary interbands, would be produced by the chromomeres, at both sides, during telophase or the development of the salivaries. The salivaries have thus been homologized to interkynetic nuclei. This assumption, while not incompatible with what is known about the chromosomes in general, actually seems to be unnecessary since the possibilities of explaining the shortening and the subsequent elongation of the chromosomes during mitosis and meiosis not only have not yet been exhausted (Serra 1942, 1947) as also they seem perfectly to account for the known phenomena.

According to the actually known facts, it can not be concluded that the salivaries represent the ultimate possibility of the chromosomes unraveling their structure. In fact, this has been admitted by every worker in this domain and the photography in the ultraviolet following partial digestion and nucleic acid precipitation (Caspersson 1936) revealed bands of about 1µ thickness. Modern electron microscope studies (Pease and Baker 1949, Schultz, Duffee and Anderson 1944 and refs.) and diffraction spectra (Buck and Melland, 1942) also reveal, as was to be expected, a finer structure of salivaries than it is possible to observe at the ordinary microscope. Nevertheless, it has not yet been possible to attempt any kind of homology between finer structures and genetic factors.

Returning now to the question of the location of the factors in the ordinary salivary bands of full grown larvae: The greater obstacle to a precise location is the phenomenon of position effect; under this name are grouped any causes which bring about the effect of a mutated gene when a break has occurred near that one band which is presumed to correspond to the factor, and no gain or loss of chromonema material is observed. Clearly in this assumption there is something of a circular argument since it has not been proved that that same deficiency, which finally permits the one-band location of the factor, is not itself a position effect due to the next band which was not deleted. In reality, even in cases where this does not apply, the existence of position effects causes a considerable incertitude in the location of the factors and this is the strongest argument in favour of the architectural theories.

If we try to sum up the examples of more precise location of factors on the salivaries of Drosophila, it seems unescapable to conclude that it is not yet proved that any factor corresponds to a single elementary band, if this is taken as the ultimate band not decomposable in thinner (visible at the ordinary microscope, that is with a thickness greater than 0.1-0.2µ). It seems, therefore, that on the basis of the known facts what may be concluded, at least provisorily and as long as a greater deal of data on location are obtained, is that each factor may correspond to several visibly separable (at the ordinary microscope) bands of the salivary chromosomes and that in case any factor should correspond to one band only, this band can be subdivided in smaller bands. That is, the gene, in what concerns the salivaries, is a composite structure and these chromosomes have in certain cases stretched to a size where the elementary chromomeres (logically postulated to be equivalent each to one factor) have decomposed into their components; the elementary chromomeres could correspond to only one or, on the contrary, to several visibly separated bands of the salivaries, according to the considered region and the growth of the chromosomes.

The same conclusion may be drawn from a consideration of the number of mutant loci compared to the number of bands. For the X chromosome there are about 800-1000 bands and the number of genes could perhaps at the first sight be supposed to be of the same order of magnitude. However, if position effects and effects of rearrangements are excluded, according to Goldschmidt only about 100 authentic mutant loci remain. This number may perhaps be doubled or trebled in future, if other loci are demonstrated by mutation and, above all, by taking into account the factors with a little marked action, especially those of physiological and metrical characters (these factors being also themselves in many cases effects or rearrangements). Even so, the number of loci would remain about 1/3 - 1/5 of that of visibly separable bands and this should signify that each factor corresponds in mean to 3-5 bands—see Fig. 2.

Of course, this sort of calculation has no more merit than many other a similar computation involving several incertitudes in the basic assumptions; in this case the number of "authentic" or "good" loci and the frequency of factors with a small action, of the quantitative "polygenic" type, are particularly debatable. Nevertheless, in conjunction with the other arguments, it adds weight to the conclusion that there is no simple correspondence between the phenogenetically postulated factor on one side and the elementary chromomere and a single band of the salivaries, on the other. It is also interesting that 3-5 is the number of bands normally reached by position effect of the euchromatic-euchromatic type; this is taken into due account in the interpretation of the position effect (see the following chapter).


FIG. 2. Correspondence between phenogenetic units (a), pachytene chromomeres (b) and salivaries (c). In b each chromomere is marked by a more close helix turning and the deposition of matrix nucleoproteins (dotted).

Summarising this discussion on the bases of the modern gene concept, it may be said that the weakest side of the concept is the cytological: while the view that the factors (of course, not the visible "characters") have a unitary primary action is well grounded, especially in the case of biochemical characters,—the unitary individuality of the particles which in the chromosomes are the cytological counterparts of the factors is rather a logical postulate than a demonstrated fact. That is, as yet no irrefutable proof has been adduced demonstrating that the postulated particulate equivalent of the factor (called elementary chromomere for the sake of definition) is indeed a real pachytene chromomere in any organism, or only one band in the salivaries of the Diptera.

Instead of such a simple correspondence, the actually known facts rather point out that a more complicated relation exists between pachytene chromomeres, salivary bands and single genes—see Fig. 2. What is known in the case of maize is in favour of concluding that a pachytene chromomere may correspond to more than one factor; and what is known of the salivaries of Diptera points to a conclusion in the opposite direction, namely that as a rule each factor corresponds to more than one visible band in the chromosomes of full grown larvae. It seems that if real progress in this domain is to be attained the simple, not proven, postulate of a straight correspondence between gene and chromomere, or between gene and salivary band, must be considered just what it is—a postulate of a simplist logic and not a proved fact. An open mind is also necessary in not supposing that this means the end of the true gene concept: this concept has indeed been oversimplified in its cytological context and the reality may or may not be more complex. It is for future cytogenetic research to obtain the cytological equivalents, in each case, of the phenogenetical units or factors. This must be directly studied in the chromosomes and a great deal of detailed observation on meiotic and salivary chromosomes is necessary.

In the following we present an attempt to solve some of the apparent contradictions which have resulted from the lack of correspondence between the concept of the unitary phenogenetic unit or factor and an apparently complicated cytological equivalent. This is tried with basis on the physiological properties of the genes and the structural, physical and chemical properties of the chromosomes. A detailed discussion of position effect will be attempted as an indispensable preliminary for the comprehension of the nature of the gene.