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Nuclear Reproduction*
Nuclear Reproduction* C. LEONARD HUSKINS Department of Botany, University of Wiscoiisin, Madison, Wiscowin. CONTENTS
I. 11. 111. IV. V. VI. VII.
Introduction ......................................................... Nuclear or Cell Division ............................................. Chromosome Reproductioii ............................................ Variation in Nuclear Size ............................................. Reduction in Chromosome Number ..................................... Conclusions .......................................................... References ...........................................................
Pllgc
9 10 12 15 18 21 24
I. INTRODUCTION I t is difficult to find anything new to say about morphological aspects of nuclear reproduction, but there are cytochemical and genetic data which should be correlated with the descriptive in any evaluation of the problem. It may also be useful to emphasize the distinct aspects of some of the processes that conlnlonly occur in association. Any correlated analysis or evaluation must at the present time contain many speculations, but I do not think that harmful, provided facts and ideas are plainly differentiated. Everyone knows that reproduction of the elements within the nucleus is not the same thing as reproduction of the nucleus itself, yet a number of geneticists, for example, have failed to make this distinction explicit in their consideration of problems of gene action. Usually this makes no difference to the argument, but it can do so when as in some recent papers on the possible role of heterochromatin in differentiation, conclusions as diverse as that of Caspersson (1939) and others, that heterochromatin is concerned with the division of the chromosomes is cited along, for instance, with Darlington and Thomas’ (1941) conclusion that it is responsible for supernumerary divisions of the pollen cell in Sorghum, and so on, without any indication being given that these are very different processes or that the evidence for the conclusions has been obtained from very different observational levels. Though chromosome and nuclear reproduction are both normally antecedent to reproduction of the cell, any one of the three processes can, of course, occur without either one of the others.
* Presented at the Seventh International Congress of Cell Eiology, Yal: University, September 4-8, 1950. 9
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If the geneticist sometimes errs by implying nuclear and cell division when his evidence relates only to gene or chromosome reproduction, the experimental cytologist in the past often paid too little attention to the chromosome and genes. Total disregard is not possible today, especially when once widely divergent disciplines are brought together in a congress such as this, but there is still evident need, even in the abstracts of our program, for all of us to make more extended use of each other’s data.
11. NUCLEAR OR CELLDIVISION Professor Heilbrunn will show later in this program that “cell division is not necessarily initiated by an increase in cell permeability, nor is it always accompanied by an overall increase in cell permeability,” and will present evidence on the gelatin-liquefaction cycle associated with mitosis. H e maintains that : “The colloid-chemical theory offers a logical explanation of all the known facts” of cell division. It is evident that in his argument he is including both karyokinesis and cytokinesis but not chromosome reproduction. I shall therefore concentrate chiefly on chromosomes, especially since it is the field with which our research group is predominantly concerned. As for the correlation between karyokinesis and cytokinesis, we may say that the concept of nucleoplasmic ratio determining nuclear and cell division, which was derived predominantly from morphological observations, can no longer be considered seriously in its original form, but that the data it subsumed must still be taken into consideration and that the concept itself is not wholly invalid. As for morphogenesis, while fully realizing that nothing should be regarded as unimportant in the present embryonic state of our knowledge, I shall for today also assume that we need pay little attention to nuclear or cell division or size as such in this connection. W e know, e.g., Weisz (1947), that cell mass may influence the course of differentiation and also that nuclear and cell size may have striking effects in some cases. The latter is evidenced by the differences, both morphological and physiological, sometimes found between diploids and their autopolyploids. But in some cases an increase of the chromosome number appreciably increases neither nuclear size nor any other characteristic. Correlatively, differences in the number of nuclei per cell do not appear to be causally related to differentiation. I n Acetabularia, for instance, Hammerling ( 1946) has found that nuclear divisions normally begin only after niorphogenesis is completed, However, a young nucleus will divide when transplanted into an old system, which, since the nucleus controls the differentiation, can be taken to indicate that nuclear division is ultimately regulated
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through the mediation of its own products. Schulze (1939) dismissed the possibility that increase of chromosome material, either by polyteny or polyploidy, is involved in the increase of nuclear size which accompanies differentiation in this alga, but the material does not seem sufficiently favorable for this to be ruled out by a descriptive cytological analysis. DNA measurements should give a more decisive answer. In dikaryotic fungi a gene in one nucleus can dominate in its effect over a gene in the other, just as one allele over another in a diploid or polyploid nucleus. Heterosis also is exhibited in heterokaryonts. If we look for functional significance of the multinucleate condition it seems to be found not in anything connected with differentiation but in the occurrence of nuclear competition, whereby nuclei containing disadvantageous genes are apparently subject to adverse selection, as found in Neurospora. The reported stability of the polykaryotic Mucor species after an initial adaptive period when first placed on artificial media (Hesseltine, unpublished thesis, University of Wisconsin) could also have this explanation. Fankhauser (1948) has shown that while a polynucleate condition causes abnormal cleavage in frogs and toads, it does not do so in most Urodeles. I n these, fertilization is normally polyspermic but at the critical stage in development the principal sperm nucleus unites with the egg nucleus, and the accessory nuclei begin to degenerate. The latter may go through prophase, and hence doubtless through chromosome reproduction, but not through mitosis. Barber (1942) found that orchid pollen grains with sub-haploid nuclei could divide normally if separated by only a thin cell wall from grains with a full haploid chromosome complement. As shown by Clark (1942) even fragmentation of the nucleus has no necessary effect on development and germination of corn pollen so long as all the fragments remain in the cell. To conclude this section of our discussion on the limited significance of nuclear or cell division for differentiation and development, brief reference may be made to a few data on the time and scope of gene action relative to mitosis and cytokinesis. Berrill and Huskins (1936) stimulated discussion of this issue by proposing that “energic” replace the term “resting” nucleus. C. Stern (1938) showed that in a number of decisive cases specific genes interact with the cytoplasm during the energic state of the nucleus. He pointed out that in some cases it is possible, as in some examples of pollen dimorphism, that gene-controlled substances exert visible effects only after the breakdown of the nuclear membrane, but decisive evidence of this was, at that time lacking. I am not aware of any more definitive evidence having been advanced since then. H. Stern
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(1946) showed that there is an increased permeability to sucrose of the plasma membrane during meiosis and postmeiotic mitosis in Trillium pollen and that it begins to rise before breakdown of the nuclear membrane. Data on permeability during endomitosis are lacking and should be sought, but sugar intake need, of course, bear little or no relation to diffusion of nuclear products, Jones (1947) shows that changes in the nucleus may have visible effects in the cytoplasm and that gene-determined pigments in corn may be either cell-limited or diffusible over a considerable area of tissue. Commoner (1949), from further analyses of somatic mutations at the A locus, suggests that genetic determination of the anthocyanin content occurs before cell enlargement and that the specific action of the A gene is based on initiation of production of a precursor. The radial pattern suggests to him distribution of the gene product during formation of constituent cells ; evidence on the issue of influence during mitosis might be found here. 111. CHROMOSOME REPRODUCTION Let us turn to the problem of chromosome reproduction without nuclear division. Almost without exception, biology textbooks teach that the chromosome number is constant in all somatic cells of a multicellular organism. A brief sketch of the development of this concept may be of interest and not without value for future work and concept formulation. Weismann (1893) wrote: “With certain exceptions . . , the number of chromosomes is constant for each species.” Wilson (1900) went further : “The remarkable fact has now been established with high probability that every species of plant or animal has a fixed and characteristic number of chromosomes which regularly recurs in the division of all its cells; and in all forms arising by sexual reproduction, the number is even.” I t remained for 0. Hertwig (1918) to formulate in detail the “law of constancy of chromosome numbers”: “This law tells us that the number of chromosomes in all cdls of a plant or animal species, with the occurrence of nuclear division, is always exactly the same whether we are dealing with epidermis, cartilage, muscle, or glandular cells, etc. However, . . the egg and sperm contain one half the number of chromosomes of the somatic cells. This also is a lawful phenomenon.” One embryologist later extrapolated the law to the extent of writing: “All cells whether they continue to divide or not ultimately contain the same genetic proteins in equal quantities.” There were many factors involved in the gradual consolidation of the law of constancy of chromosome numbers. Nemec in 1904 and 1910 had
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discovered tetraploid nuclei in chloralized roots, but Strasburger ( 1907) was convinced that they could not persist and therefore were of no significance. H e even explained away as obviously due to some “disturbing influences, such as wounding by small animals, etc.,” his own discovery of rows of tetraploid cells in control roots. Of this Winkler (1916) wrote : [trans.] “This last remark of Strasburger is extraordinarily characteristic. I t shows that the conviction that in normal somatic tissue only diploid cells could occur has become a dogma under the influence of which the best plant cytologist comes at once to the opinion that the occurrence of tetraploid cells found therein must be pathological, without even considering any other possibilities.” Winkler himself, after establishing with certainty the occurrence of polyploid cells and tissue in Solanum species and of the production of polyploid plants from the callus of grafts, concludes : “since the germ cells always arise directly from embryonic tissues they will always have the typical chromosome number and hand it down to the next generation. The constancy of chromosome number is safeguarded even when there is vegetative reproduction, since plants grow with their growing points which, by definition, are always embryonal. . . . W e therefore come to the view that the regular occurrence of polyploid cells in the somatic tissue of higher plants by no means refutes the laws of constancy of chromosome number but must be expected in view of the importance of the chromosome number for cell size.” Except possibly for the last clause, with its teleological flavor, this is as clear and acceptable a statement on polysomaty as could be made today. Why has it so very generally been ignored, not only by textbook writers but also by most research workers ? It must be remembered that from the time the correlation of chromosome behavior in meiosis and of Mendelian factors in segregation and recombination was first clearly enunciated it took about a quarter of a century to establish the “chromosome theory of Keredity” to the satisfaction of the overwhelming majority of biologists. As late as the 1920’s there were some who still considered the chromosome complement as a variable characteristic which was no more and no less a part of the plant phenotype than, say, the number and shapes of the leaves. The important general concepts involved in the chromosome theory of heredity were most conclusively established by showing that in exceptional organisms with chromosome numbers or arrangements of their parts that deviated from the norm of the species, such as haplo-IV Drosophila, trisomic Oenothera, tetraploid Datura and translocation stocks of Drosophila, maize, etc., the genetic behavior was altered correlatively.
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By 1937 it was realized that colchicine produces polyploid cells by inhibiting spindle action, and in view) of the importance of polyploidy in plant breeding there was immediately a wide search for other chemical agencies that would induce it. I t is not surprising, therefore, that when polyploid divisions were found in mature plant tissues that had been stimulated by “growth substances” it was assumed that the polyploidy was caused by the treatment. It seemed to me, however, that the effect of “auxins” was more probably stimulation of divisions in already polyploid nuclei. Geitler (1948) apparently reached this conclusion at about the same time and during the war he effectively established that “polysomaty” is common in the leaves and stems of a large series of plants, as he had earlier found it is in many insects. He had stimulated divisions by wounding. Unaware of his work, we had initiated complementary studies on roots with indole acetic acid in dosages that gave results quickly enough for us to determine that at least the higher polyploid nuclei had not been produced after initiation of the treatment (Huskins and Steinitz,
1948). Since polysomaty has not been considered to have any effect on genetic behavior, it was of no significance in that stage of the rapidly developing science of genetics where establishment of rules of transmission was the major goal. The question today is whether or not it is significant in developmental or physiological genetics. Let us first consider from this point of view some of the recent work, including that of members of our own group, on polysomaty. Incidentally, there is much confusion due to unsatisfactory terminology in many discussions of nuclear reproduction. I have been accused of confusing endomitosis, endodivision, polysomaty, and polyteny because I have not always in all contexts differentiated sharply between them. I risk this charge again and for the same reason as previously, namely, that I think the distinctions unimportant at the present time in discussions of possible functions. They are not unimportant in descriptive cytology, and I do not ignore them in that context. Further, since I have been so widely misunderstood on another point, despite two separate and specific warnings in my original speculative discussion on the possible significance of polysomaty ( Huskins, 1947), let me here emphasize that polysomaty as suck cannot possibly be of any great general significance in differentiation. It may be in special cases; in all cases it docs prove that chromosome, and therefore gene, reproduction continues after nuclear or cell division ceases and thereby opens the way to coilsideration of the possibility that gene action i s correlated zcuWa gene reproduction. The cyto-
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logical evidence, now generally accepted, that the chromatid is not a transversely unitary structure, shows that Mendelian segregation involves units at a higher level of integration than those resolvable by even such a relatively crude analyzer as the light microscope. We may therefore justifiably consider the possibility that the ultimate units which may be effective in differentiation could be at a very much lower level. There is considerable evidence that, at the microscopic level, reproduction of the component strands of a chromosome is not uniform throughout its length. The possibility that reproduction of the materials making up a Mendelian gene may be differential for different genes in different tissues therefore becomes almost an a priori probability. The problem is to devise methods for testing i t ; such are, of course, appearing as soon as the problem is envisaged. IV. VARIATION IN NLTCLEAR SIZE To return to simpler levels of discussion: Huskins and Steinitz (1948) attempted to analyze the great variation in nuclear size in differentiated regions of Rhoeo roots. Evidence obtained by counting the number of heterochromatic bodies (which was the method devised by Geitler for insect tissues) and by treatment with indole acetic acid, coincided in showing the variation to be correlated with degree of polysomaty. Similar results were obtained with barley (Leonard-Bennett, unpublished). Duncan and Ross (1950) have shown that in nuclei of niaize endosperm undergoing mitosis the normal triploid number of chromosomes is usually present. However, in giant energic nuclei a high degree of polyteny is observed in regions of chromosomes marked, for observational purposes, by heterochroinatic “knobs.” They have, further, shown a different range in nuclear size in different areas of the endosperm. An apparent reduction in the size of endosperm nuclei adjacent to the embroyo as its development proceeds is of special interest and is currently being studied further. Nuclear and cell volume, chromosome and chromatid number in pith cells of Nicotium towentosu are being studied by Dr. Muriel Bradley. Her data show, iiiter ulia that in this material nuclear volume is related directly to chromatid and not to chromosome number. I t seems to make little or no difference whether 8n chromatids are present as the 4n number of ordinary two-chromatid chromosomes or as 2n chromosomes each having four chromatids. The data show also that following the halving of the total cell volume after telophase there is within each of the polyploid classes a relatively slight increase in nuclear volume which is followed by something like a doubling before the next prophase. These morphological data accord with cytochemical studies. They support indirectly the as-
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sumption made from microchemical analysis by Boivin, Vendrely and Vendrely (1948), Mirsky and Ris (1949), and others that with certain exceptions the DNA content is the same in all somatic cells. Certain variations in DNA content have been related, obviously correctly, to reproduction of the chromosomes and to polyploidy. Relating the constancy, apart from polysomaty, to the concept that the genotype is the same in all cells, it is usually concluded that DNA is an essential, and probably constant, component of the gene. The most recent published work along these lines is that of Swift (1950) who has shown, by absorption spectrophotometry following Feulgen staining, that the nuclei of ten different somatic tissues of young and adult mice all show approximately the same amount of DNA except for some of those in the liver, pancreas, thymus, blood lymphocytes, and Sertoli cells which contained two or four times the common amount. Occasional rare intermediate values were found and presumed to be associated with mitosis. Mouse spermatid nuclei had half the DNA of the common somatic nuclei. Primary spermatocytes had four times and secondary spermatocytes twice the spermatid value. Some premeiotic sex cells had the somatic amount and some twice as much. The former predominate in testes of 1- to 10day old mice and the latter are commoner at maturity. Nuclei of six tissues of adult frogs all had approximately the same amount of D N A (excepting for a few liver nuclei which had twice as much), and this was slightly less than twice that characteristic of the mouse. From studies of embryonic mouse liver and Amblystoma larvae, it was shown that DNA content “builds up in the interphase nucleus before the visible onset of prophase” and that “during the visible stages of mitosis no DNA is synthesized.” I n the Malpighian tuhule nuclei of a grasshopper four classes of DNA content with the ratio 1 :2:4:8 were found. In our laboratory (Bloch and Patau, unpublished) the relative DNA content of mouse liver nuclei has been determined, following Feulgen staining, with both an electro photometric and a visual microphotometer, the latter instrument having been designed by Dr. K. Patau. The results with liver of adult mice of pure lines and their hybrids agree with those of Swift and confirm that the step from one class to the next higher is a very accurate doubling of the DNA content accompanied by a doubling of nuclear size. In embryonic liver the DNA content ranges from 1 :2. It doubtless is reflecting the synthesis of DNA in nuclei preparing to undergo mitosis. Intermediate values between higher classes may be reflecting chromosome reproduction preparatory to either mitosis or endomitosis. Schrader and Leuchtenberger ( 1949) have stressed the
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variation “which may be due to different degrees of polyteny” in different tissues of Tradescantia. Since oral presentation of this article, Swift (1950) has reported findings in maize and Tradescantia. In our laboratory, measurements are being made on Allium and Tradescantia tissues (Nelson, unpublished). For the roots the results are similar to those of Swift, excepting that he reports none of the lowest class in the elongation region of maize roots but several higher multiples, whereas in the same region of Allium roots, Nelson finds large numbers of the lowest and second classes, very few of the third, and none higher. Interesting additional findings by Nelson are : (1) that the doubling which precedes mitosis occurs early in the interphase period (unpublished data of Dr. Alma Howard, Radiotherapeutic Research Unit, Hammersmith, on uptake of radioactive phosphorus, are in accord) ; ( 2 ) that the guard cells of stomata have constantly the lowest amount of DNA normal for diploid cells, while other cells of the epidermis have double this amount ; (3) immediately after the first division of the microspore nucleus the resulting “vegetative” and “generative” nuclei have the same (haploid) amount of DNA but as the pollen grain “ripens” the generative nucleus doubles its content - in readiness for its division - while the content of the vegetative remains constant, contrary to the opinion of the many descriptive cytologists who have noted its fainter staining. In all of the foregoing, the findings confirm the ideas of Jacobj (1925) and many subsequent workers that increase of nuclear size may be caused by geometric increase of chromosome number, i.e., by polysomaty. It has, however, also long been clear that nuclear size can increase greatly without change in chromosome number. In some cases it is now evident that this may be correlated with increase in degree of polyteny, as shown by Duncan and Ross in maize endosperm. In yet others it may have little or no relation to the “chromatin” content. Schrader and Leuchtenberger ( 1950) have shown cytochemically that in the very different-sized nuclei which characterize different lobes of the testes of Armelitis albopunctatus, a hemipteran insect, the DNA content is approximately the same; it is the total protein and the RNA content that are correlated with nuclear, nucleolar and cytoplasmic volume. Apart from internally regulated “permanent” changes in nuclear volume, there are, of course, changes which appear to be correlated with developmental or physiological factors. Metz and his students (Buck and Roche, 1938) have shown that osmotic and mechanical pressure changes may cause reversable increase or decrease in nuclear and chromosome size as great as 25 per cent. The diverse effects of various fixatives on nuclear size are also, of course, well recognized.
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To summarize this discussion of polysomaty we may safely say: (1) The concept of genic and chromosome identity of all cells obviously needs both amplification and circumscription; (2) Many more data on its occurrence are needed before we can even decide whether or not it, as stich, is at all likely to be found of any general significance in differentiation and development. (Regular and characteristic differences in number of chromosomes in certain tissues such as Geitler and others have found in various insects and in DNA content such as Nelson is finding in plant epidermis seem to point affirmatively, whereas the difference in these regards between morphologically similar regions of maize and onion roots and many other data point negatively.) ; (3) The newer techniques if used on specially chosen materials, particularly if these permit a concomitant genetic analysis, are capable of giving us answers to problems that were unassailable by the methods of cytological study available until very recently; (4) Whatever the relationship of D N A to ultimate gene structure, it seems certain that it is precisely related to the reproduction of the chromatid, which is the subdivision of the chromosome that is significant for Mendelian heredity-so far there is no evidence of a chromosome’s being able to divide without first doubling its DNA content, but, of course, very few studies of this question have yet been made: ( 5 ) Though chromosome reproduction must “normally” precede nuclear division, either of the two processes can proceed independently of the other -the relationship is parallel to that of cytokinesis to karyokinesis.
v.
REDUCTION IN
CHROMOSOME
NUMBER
We may next consider the process of reduction of chromosome number. To the long-established account of the process in germ cells the only important item to be added from current studies with the newer techniques seems to be the discovery (Swift, Nelson, and probably many others unpublished) that the D N A content reaches the same heightened level by pachytene of the first meiotic division as that typical for early mitotic prophase. Therefore, two divisions without an intervening increase during their interkinesis are required to restore the normal relationship between number of chromosomes and nuclear D N A content since the former has been halved. W e have moved far from the concept held by many cytologists not so long ago that the first division was the reduction division, and the second an unexplained concomitant ; genetics has long shown that both divisions are essential to genetic reduction, the occurrence at first prophase of four chromatids in each bivalent indicated the same from the descriptive cytological aspect, and now we see that they are needed to restore cytochemical balance also.
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Somatic reduction and segregation have long been known but little investigated until recently. Many textbooks list Winkler’s ( 1910) Solununz danoriniaiticm as having originated by somatic reduction, but none that I know of list his 1916 cases. I believe (see Huskins, 1948) the former to be an erroneous interpretation while the latter are clearly valid. Winkler’s 1916 remarks warrant quotation as a basis for discussion of current studies. [translation] To obtain such reversions to the diploid normal form from a tetraploid gigas type, the cells which gave rise to the atavistic tissue complex must have undergone a reduction-division. We may therefore not doubt the possibility that reductiondivisions occur in somatic cells. What stimulates them has to be left undecided. That the tetraploid condition of the nucleus by itself should have caused a tendency for the reduction of the increased chromosome number cannot be assumed, since tetraploid types exist and in general persist. It will also have to remain undecided for the time being whether the halving of the chromosome number occurs by typical reduction-divisions or otherwise. As a matter of fact it will be very difficult to find such a reduction-division since the reversions, at least so far, have occurred rarely and quite irregularly, that is in places which could not be predicted.
Two cases are reported in which somatic reduction appears to be a regular process: (1) prior to meiosis in the hermaphrodite gonad of a coccid (Hughes-Schrader, 1927) and (2) in the ileum of mosquito larvae (Berger, 1941 ; Grell, 1946). Somatic pairing and segregation are, of course, well known in the Diptera, but adequate data are lacking on reduction, though it was early reported by Bridges. Bateson (1926) insisted that there was much genetic evidence for somatic segregation in various tissues, but most such cases investigated in the past quarter century have been interpreted as somatic mutation, which he warned against as likely to obscure the issue. Recent evidence indicates that in any specific case both must be considered as possible explanations until the one is ruled out. The sporadic occurrence of chromosome pairing and/or reduction in somatic tissues has frequently been recorded. For example, Gates (1912) observed it in the nucellus of Oenotheru Zutu; Ludford (1935) and others have recorded it in tumors and tissue cultures of tumors; Metz (1942) found in a Sciara hybrid a salivary gland nucleus containing chromosomes from only one of the parents; Love (1936) found pollen mother cells that had undergone reduction prior to meiosis. East ( 1934), Nishiyama (1933), Kiellander (1941), Sparrow (1941), and Vaarama (1949) obtained plants with reduced, ancestral, chromosome numbers among the progeny of polyploid strawberries, oats, Poa, wheat, and Ribes, respectively. Brown (1947) found a reduced sector in an unbalanced poly-
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ploid cotton plant. Upcott ( 1939) observed irregularly reduced chroniosome numbers in tetraploid Primnula kewensis; it is possible that by attributing these to “split spindles” she may have diverted attention from the problem as Strasburger, in Winkler’s opinion, quoted earlier lierein (1916), diverted attention from polysoniaty by assuming that it must be pathological. Following sporadic discovery of haploid cells in various plant roots, consideration of Caspersson’s ( 1939) suggestion that nucleic acid plays a role in synapsis and chromosome division led to a search for somatic reduction in preparations of Allium root tips treated by Dr. M. Kodani (1948) with sodium nucleate. Many cells with two reduced groups of chromosomes were found (Huskins, 1948). They were later found also in roots grown in solutions rich in phosphates (Galinsky, 1949). More recently it has been found (Huskins and Cheng, 1950, and unpublished) that prolonged low temperature treatment also increases the frequency with which “reductional grouping” occurs. There is also evidence that genetic factors affect the frequency with which reduced tissues or organs occur. In one strain of tetraploid Rhoeo we have obtained diploid and triploid roots and shoots with and without treatment. In another strain, treatments increase the frequency of reductional groupings, but no reduced tissues have yet been obtained. Allen, Wilson, and Powell (1950) have recently compared the sodium nucleate results with the chromosome groupings that occur after colchicine treatment. An extensive study has been made by Patau and Steinitz (1951) on the origin of reductional groupings and of reduced cells (see also, Patau, 1950). It is clear that somatic reduction is of not infrequent spontaneous occurrence, that its frequency can be increased by various treatments and “natural” conditions such as low temperatures, and therefore that polyploidy is a reversible evolutionary process. Battaglia ( 1948) has recently reported that somatic reduction occurs regularly in the basal portion of the style of Sambucus, and Christoff and Christoff (1948) report it in the integumental cells of Hieracium. If these and the cases in insects mentioned earlier are confirmed and extended, the “law of constancy of chromosome numbers” and our concepts of nuclear reproduction will have to be extended in this direction also. Besides polysomaty, polyteny, and reduction, which affect the total chromosome complement, there is also aneusomaty, i.e., the occurrence of cells with variable numbers of individual chromosonles, to be taken into account. It appears probable that aneusomaty (not to be confused with aneuploidy which refers to deviations between, not within, organisms)
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most commonly involves chromosomes that are wholly or in large part heterochromatic. Too little carefully controlled work has yet been done on aneusomaty to warrant any conclusions on either the mechanism of its occurrence or its significance. Duncan (1945) concluded that in the root tips of an orchid the occurrence of variable numbers of chromosomes was due to a differential rate of reproduction of euchromatic and heterochromatic chromosomes. Darlington and Thomas ( 1941) attributed similar variable numbers in Sorghum to selective elimination in the roots but not in the shoots. Randolph (1941) presents numerous cytogenetic data which show that in maize the problem of B chromosome function and behavior is very complex. In Cimex, Darlington (1939) found from 0 to 12 extra X chromosomes in the males, with the average number higher in natural populations (9.0) than in mass cultures (4.3). H e correlates cycles of chromosome and centromere division with the “differential precocity” of autosomes, M chromosomes, and sex chromosomes and functionally relates the various changes observed to “adaptive balance” in sex-determining mechanisms. The mechanism of variability is related to “the state or precocity” of the centromere and the relative size of the chromosomes. These issues would carry us far beyond the scope of the present review, but they lead up (as Darlington points out in his Appendix 11) to the problem of preferential segregation, which is also beyond present scope except that it must be pointed out that special spindle mechanisms exist which provide for elimination of whole sets of chromosomes. The best analyzed of such cases is probably Sciara, for which both genetic and cytological data are available. It seems probable that such elimination is functionally related to chromatin diminution that involves only parts of chromosomes and that some form of differential reproduction may be basic to all such. Investigation with the newer techniques and with wider concepts in mind than those which guided earlier descriptive studies may lead to the establishment of some of the generalizations which at present are almost entirely speculative.
VI. CONCLUSIONS To conclude we may summarize a few of the data which, though none alone may be conclusive, together suggest a need for revision and extension of some of our more orthodox concepts on various aspects of the reproduction of the nucleus and its components and of the role of the nucleus in differentiation and development. First, against the simplest unitary concept of the gene there is, to repeat, the cytological evidence that there are, frequently at least, more microscopically separable strands in both the
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mitotic and meiotic chromosomes than there are chromatids, which are the unitary gene strings of Mendelian segregation. The concept of the Mendelian gene as made up of identical “lamellae” would fit this. (The term “lamella” must not be taken to connote an undue simplicity in either the concept or the writer ; it may at our present stage of concept-forming serve as the equivalent of the beads-on-a-string model which was useful when the linear order of the genes was the issue.) Against it are the onehit radiation data, together with the occurrence of reverse mutations. It would be easy to imagine a change in one “lamella” being transmitted to all the others if the process went only one way, but not if it goes either way with anything like the same frequency, as some few gene mutations do. However, the one-hit hypothesis is not unassailable. For recent discussions of it see Muller (1950) and Opatowski (1950). Secondly, against the concept of gene identity of all cells (leaving aside whole chromosome changes for the moment) there are observations which are taken to indicate : (1) that heterochromatic and euchromatic parts of chromosomes may reproduce at different rates (Schultz, 1941) ; (2) that the banding pattern and length and breadth is visibly different in the same giant chromosomes from different tissues (see Kosswig, 1948) ; (3) that “specific chromosome loci [produce] lateral loops” (Duryee, 1950) ; and (4) that different parts of the salivary gland nuclei give strikingly unequal phosphatase reaction (see Brachet and Jeener, 1948), which suggests the possibility of variation from one gene to another in speed of renewal of phosphorus in the DNA. This would leave open the alternatives of phosphorus renewal in DNA playing a role in synthesis of proteins concerned with growth or of differential reproduction of chromosome regions. Differential reproduction of gene lamellae would provide a mechanism for differentiation not envisaged by Goldschmidt and probably not compatible with his present ideas on the gene, but it can be related conceptually to his early theory of timed, sequential physiological activity of the genes. Thirdly, it now seems fairly certain that the DNA content of nuclei from different tissues is constant, or, more strictly speaking, that it is constant relative to the total number of chromatids per nucleus, whatever the tissue. However, the R N A and protein content are both variable in different tissues. Chargaff (1950) reports that the D N A from different species differs in chemical composition and puts out the interesting suggestion: “It would be gratifying if one could say-but this is for the moment no more than an unfounded speculation-that just as the desoxy-pentose nucleic acids of the nucleus are species-specific and con-
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cerned with the maintenance of the species, the pentose nucleic acids of the cytoplasm are organ-specific and involved in the important task of differentiation.” Daly, Allfrey, and Mirsky (1950), however, dispute his findings. As for the possible role of the nucleus in differentiation, it is pointed out by Dunn ( 1949), Gluecksohn-Schoenheimer (1949), and others that the developmental reactions controlled by mutated genes are in some cases very like those occurring in normal differentiation, and that they are possibly “more fundamental and perhaps much closer to gene action than we suspect now.” Weiss (1950) emphasizes that the term differentiation has been very loosely used and that morphological criteria have played too large a part in the classification of cellular changes. H e classes as “modulations” those changes which are reversible and stresses the fact that many specialized cells cannot “dedifferentiate.” It is, of course, generally agreed that though permanent changes take place in many cells, the early crude concepts of differentiation always being determined by segregation of particles during early segmentation of animal eggs, or of its determination by any simple type of regulated gene segregation or mutation, are quite untenable. Further, the concept of genic identity of all cells was an essential step in the development of our understanding of Mendelian heredity and of the essential differences between asexual and sexual reproduction for which the basic mechanisms are mitosis and meiosis. Have we now reached the stage when we can profitably consider the possibility that our concept of the gene of hereditary transmission subsumes the “gene of differentiation and development” ( 1947) and that the two must now be distinguished? So long as we assume the chromosomal genes to be the same in all cells we are forced either to consider the cytoplasm as the seat of the primary differentiating materials on which the genes act or as (see Schultz, 1950) the variable member of the reciprocally interacting units-these are analytically the same, though the latter is conceptually more satisfying. The assumption that there must be units in the cytoplasm that determine differentiation has led to the very fruitful discoveries of the entities that currently are most frequently referred to as plasmagenes though the concepts this term implies cause its rejection by many (see Schultz, 1950). Plasmagenes that are found to be dependent on chromosome genes in their function, even though autonomous in reproduction (Ephrussi, 1950) do not of course conflict with the orthodox concept of gene identity of all cells. If, however, we should be forced to the conclusion maintained by Darlington (1949) that the only difference between nuclear genes and plasmagenes is that the latter “have been denied . . . the gift . . . of coordinated seg-
24
C. LEONARD HUSKINS
regation at meiosis,” we run into logical, or at least semantic difficulties. Though nucleus and cytoplasm are probably always interacting, the Acetabularia and other evidence seem to me to indicate that the nucleus primarily controls differentiation. But if cellular differentiation should in other cases be determined primarily by fully autonomous cytogenes which arise during ontogeny, as Sonneborn (1949) and others have suggested, then such cells are by definition genetically different even though we call their new “genes” plasmagenes. These cells should show different hereditary capacities not only in vegetative reproduction but also in the sexual reproduction of plants, if such differentiated cells can ever give rise, however remotely and indirectly, to female ,gametes. This latter would demonstrate definitely that the plasmagenes really are genes in the accepted meaning of the term, but it is the concept of chromosome identity, not of genic identity, of all cells that can be saved by the concept of two sorts of genes differing only in their location. This with the data on constant DNA content appeals to those of us who picture the chromosomes, but not necessarily the genes, as characterized by DNA at least during their period of reproduction and also of “division.” W e have, however, seen the limitations of “the law of constancy of chromosome numbers” and the constancy of chromosome parts seems very likely to prove even more limited. Which brings us back to nuclear reproduction: it is a very complex process which normally comprises many subsidiary processes which for the present we can safely classify into only two, viz., chromosome reproduction and chromosome separation. Various types of separation are accomplished by mitosis, endomitosis, and meiosis, each with many variants, but in each there is an essential basic uniformity. To these processes we must add the very incompletely known mechanisms of somatic reduction and of aneusomaty and differential elimination of parts of chromosomes, whole chromosomes of special types, and of genomes or sets of chromosomes. W e do not have to consider amitosis, for most of the descriptive work on it has long ago been shown to include errors of interpretation and the evidence of genetics shows clearly that it cannot be a normal process of nuclear reproduction, if by normal we mean the production of nuclei continuing to have potentially unlimited capacity for further reproduction. VII. REFERENCES Allen, N. S., Wilson, G. B., and Powell, S. (1950) J. Hered. 41, 159. Barber, H. N. (1942) J. Getwt., 48, 97. Bateson, W. (1926) 1. Genet., 16, 201: Battaglia, Emilio (1948) Nuovo G. bot. it&., 65, I.
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Berger, C. A. (1941) Cold Spr. Harb. Symp. quant. Biol., 9, 191. Berrill, N. J., and Huskins, C. L. (1936) Amer. Nut., 70, 258. Boivin, A., Vendrely, R., and Vandrely, C. (1948) C. R. Acad. Sci., 226, 1061. Brachet, J., and Jeener, R. (1948) Biochim. Biophys. Acta, 2, 423. Bridges, C. B. (1930) Science, 72, 405. Brown, Meta S. (1947) Amer. J . Bot., !M, 384. Buck, John B., and Boche, Robert D. (1938) Coll. Net., 19. Caspersson, T. (1939) Arch. exp. Zellforsch., 22, 655. Chargaff, E. (1950) Experentia, 6, 201. Christoff, M., and Christoff, M. A., (1948) Genetics, 53, 36. Clark, F. J. (1942) Genetics, 27, 137. Commoner, B. (1949) Abst. Amer. J. Bot., 86, 822. Daly, M. M., Allfrey, V. G., and Mirsky, A. E. (1950) J . yen. Phjsiol., SS, 497. Darlington, C. D. (1939) J. Genet., 39, 101. Darlington, C. D. (1949) Hereditas Suppl., 1949, 189. Darlington, C. D., and Thomas, P. T. (1941) Proc. roy. Soc., B190, 127. Duncan, R. E. (1945) Amer. J. Bot., 32, 506. Duncan, R. E., and Ross, J. G. (1950) J . Hered., 41, 259. Dunn, L. C. (1949) Anniversary Symp., Jackson Laboratory, Ear Harbor, Maine. Duryee, William R. (1950) Ann. N . Y. Acad. Sci., 60, 920. East, E. M. (1934) Genetics, 19, 167. Ephrussi, Boris (1950) VIIth Int. Congr. Cell Biology, Yale University, p. 24. Fankhauser, G. (1948) Ann. N. Y . Acad. Sci., 49, 684. Galinsky, Irving (1949) J. Hered., 40, 289. Gates, R. R. (1912) Ann. Bot., 26, 993. Geitler, Lothar 1948) Ost. bot. Z., 9, 277. Gluecksohn-Schoenheimer, S. (1949) Growth, 9, 163. Grell, Sister Mary (1946) Gewtics, 31, 60-76, 77-94. Hammerling, J. (1946) Natzwwissenschaften, SS, 337. Heilbrunn, L. V. (1950) VIIth Int. Congr. Cell Biology, Yale University, p. 36. Hertwig, 0. (1918) Das Werden der Organismen. Fischer, Jena. Hughes-Schrader, S. (1927) 2. Zellforsch., 6, 509. Huskins, C. L. (1947) Amer. Nut., 81, 401. Huskins, C. L. (1948) J . Hered., 99, 311. Huskins, C. L. (1950) I. Hered., 41, 13. Huskins, C. L., and Cheng, K. C. (1950) J . Hered., 41, 13-18. Huskins, C. L., and Steinitz, L. M. (1948a) J. Hered., 99, 34. Huskins, C. L., and Steinitz, L. M. (1948b) J. Hered., 99, 66. Jacobj, W. (1925) Arch. EnfzuMech. Org., lU6, 124. Jones, D. F. (1947) Proc. eat. Acad. Sci., Wash., 99, 363. Kiellander, C. I. (1941) Svemk bot. Tidskr., 96, 321. Kodani, Masuo (1948) I. Hered., 83, 115. Kosswig, C. (1948) Proc. 8th Int. Congr. Genet., Stockholm. Love, R. M. (1936) Nature, Lond.,1S8, 589. Ludford, R. J. (1935) Arch. exp. Zellforsch., 17, 411. Metz, G. W. (1942) Amer. Nut., 88, 623. Mirsky, A. E., and Ris, Hans (1949) Nature, Lond., la,666. Muller, H. J. (1950) I . cell. comp. PhySioJ., 35, 9.
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Nemec, B. (1904) Jb. z k s . Bot., 89, 645. Nemec, B. (1910) Das Problem der Befruchtungsfrage. Berlin. Nishiyama, I. (1933) Jap. 1. Genet., 8, 107. Opatowski, I. (1950) Genetics, I, 56. Patau, Klaus (1950) Genetics, MI 128. Patau, Klaus, and Steinitz, L. M. (1951) (In press.) Randolph, L. F. (1941) Genetics M, 608. Schrader, F., and Leuchtenberger, Cecilie (1949) Proc. nat. Acad. Sci., Wash, 86, 464. Schrader, F., and Leuchtenberger, Cecilie (1950) Ezp. Cell Res., 1, 421. Schultz, Jack (1941) Cold Spr. Harb. Symp. qzcant. Biol., 9, 55. Schultz, Jack (1950) Science, 111, 403. Schulze, K. L. (1939) Arch. Protistertk., 92, 167. Sonneborn, T. M. (1949) AMY. Scient., 87, 33. Stern, C. (1938) Amer. Nat., 7!2, 350. Stern, Herbert (1946) Philos. Tram., 40, 141. Strasburger, E. (1907) Jb. w'ss. Bot., 44, 482. Sparrow, A. H., Huskins, C. L., and Wilson, G. B. (1941) Canud. J. Res., 19, 323. Swift, H. H, (1950a) Proc. nut. Acad. Sci., Wash., SS, 643. Swift, H. H. (1950b) Physiol. ZoGI., 2S, 169. Upcott, M. (1939) J. Genet., 89, 79. Vaarama, A. (1949) Hereditas I,136. Weismann, August (1893) The Germ Plasm. W. Scott Ltd. London. Weiss, P. A. (1950) Quart. Rev. Biol., I,177. Weisz, P. B. (1947) J . Morph., 81, 45. Wilson, E. B. (1900) The Cell. Macmillan Co., N. Y. Winkler, Hans (1910) Ber. dfsch. bot. Ges., 28, 116. Winkler, Hans (1916) 2. Bot., 8, 417.
I. 11. 111. IV. V. VI. VII.
Introduction ......................................................... Nuclear or Cell Division ............................................. Chromosome Reproductioii ............................................ Variation in Nuclear Size ............................................. Reduction in Chromosome Number ..................................... Conclusions .......................................................... References ...........................................................
Pllgc
9 10 12 15 18 21 24
I. INTRODUCTION I t is difficult to find anything new to say about morphological aspects of nuclear reproduction, but there are cytochemical and genetic data which should be correlated with the descriptive in any evaluation of the problem. It may also be useful to emphasize the distinct aspects of some of the processes that conlnlonly occur in association. Any correlated analysis or evaluation must at the present time contain many speculations, but I do not think that harmful, provided facts and ideas are plainly differentiated. Everyone knows that reproduction of the elements within the nucleus is not the same thing as reproduction of the nucleus itself, yet a number of geneticists, for example, have failed to make this distinction explicit in their consideration of problems of gene action. Usually this makes no difference to the argument, but it can do so when as in some recent papers on the possible role of heterochromatin in differentiation, conclusions as diverse as that of Caspersson (1939) and others, that heterochromatin is concerned with the division of the chromosomes is cited along, for instance, with Darlington and Thomas’ (1941) conclusion that it is responsible for supernumerary divisions of the pollen cell in Sorghum, and so on, without any indication being given that these are very different processes or that the evidence for the conclusions has been obtained from very different observational levels. Though chromosome and nuclear reproduction are both normally antecedent to reproduction of the cell, any one of the three processes can, of course, occur without either one of the others.
* Presented at the Seventh International Congress of Cell Eiology, Yal: University, September 4-8, 1950. 9
10
C. LEONARD H U S K I N S
If the geneticist sometimes errs by implying nuclear and cell division when his evidence relates only to gene or chromosome reproduction, the experimental cytologist in the past often paid too little attention to the chromosome and genes. Total disregard is not possible today, especially when once widely divergent disciplines are brought together in a congress such as this, but there is still evident need, even in the abstracts of our program, for all of us to make more extended use of each other’s data.
11. NUCLEAR OR CELLDIVISION Professor Heilbrunn will show later in this program that “cell division is not necessarily initiated by an increase in cell permeability, nor is it always accompanied by an overall increase in cell permeability,” and will present evidence on the gelatin-liquefaction cycle associated with mitosis. H e maintains that : “The colloid-chemical theory offers a logical explanation of all the known facts” of cell division. It is evident that in his argument he is including both karyokinesis and cytokinesis but not chromosome reproduction. I shall therefore concentrate chiefly on chromosomes, especially since it is the field with which our research group is predominantly concerned. As for the correlation between karyokinesis and cytokinesis, we may say that the concept of nucleoplasmic ratio determining nuclear and cell division, which was derived predominantly from morphological observations, can no longer be considered seriously in its original form, but that the data it subsumed must still be taken into consideration and that the concept itself is not wholly invalid. As for morphogenesis, while fully realizing that nothing should be regarded as unimportant in the present embryonic state of our knowledge, I shall for today also assume that we need pay little attention to nuclear or cell division or size as such in this connection. W e know, e.g., Weisz (1947), that cell mass may influence the course of differentiation and also that nuclear and cell size may have striking effects in some cases. The latter is evidenced by the differences, both morphological and physiological, sometimes found between diploids and their autopolyploids. But in some cases an increase of the chromosome number appreciably increases neither nuclear size nor any other characteristic. Correlatively, differences in the number of nuclei per cell do not appear to be causally related to differentiation. I n Acetabularia, for instance, Hammerling ( 1946) has found that nuclear divisions normally begin only after niorphogenesis is completed, However, a young nucleus will divide when transplanted into an old system, which, since the nucleus controls the differentiation, can be taken to indicate that nuclear division is ultimately regulated
NUCLEAR REPRODUCTION
11
through the mediation of its own products. Schulze (1939) dismissed the possibility that increase of chromosome material, either by polyteny or polyploidy, is involved in the increase of nuclear size which accompanies differentiation in this alga, but the material does not seem sufficiently favorable for this to be ruled out by a descriptive cytological analysis. DNA measurements should give a more decisive answer. In dikaryotic fungi a gene in one nucleus can dominate in its effect over a gene in the other, just as one allele over another in a diploid or polyploid nucleus. Heterosis also is exhibited in heterokaryonts. If we look for functional significance of the multinucleate condition it seems to be found not in anything connected with differentiation but in the occurrence of nuclear competition, whereby nuclei containing disadvantageous genes are apparently subject to adverse selection, as found in Neurospora. The reported stability of the polykaryotic Mucor species after an initial adaptive period when first placed on artificial media (Hesseltine, unpublished thesis, University of Wisconsin) could also have this explanation. Fankhauser (1948) has shown that while a polynucleate condition causes abnormal cleavage in frogs and toads, it does not do so in most Urodeles. I n these, fertilization is normally polyspermic but at the critical stage in development the principal sperm nucleus unites with the egg nucleus, and the accessory nuclei begin to degenerate. The latter may go through prophase, and hence doubtless through chromosome reproduction, but not through mitosis. Barber (1942) found that orchid pollen grains with sub-haploid nuclei could divide normally if separated by only a thin cell wall from grains with a full haploid chromosome complement. As shown by Clark (1942) even fragmentation of the nucleus has no necessary effect on development and germination of corn pollen so long as all the fragments remain in the cell. To conclude this section of our discussion on the limited significance of nuclear or cell division for differentiation and development, brief reference may be made to a few data on the time and scope of gene action relative to mitosis and cytokinesis. Berrill and Huskins (1936) stimulated discussion of this issue by proposing that “energic” replace the term “resting” nucleus. C. Stern (1938) showed that in a number of decisive cases specific genes interact with the cytoplasm during the energic state of the nucleus. He pointed out that in some cases it is possible, as in some examples of pollen dimorphism, that gene-controlled substances exert visible effects only after the breakdown of the nuclear membrane, but decisive evidence of this was, at that time lacking. I am not aware of any more definitive evidence having been advanced since then. H. Stern
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C. LEONARD HUSKINS
(1946) showed that there is an increased permeability to sucrose of the plasma membrane during meiosis and postmeiotic mitosis in Trillium pollen and that it begins to rise before breakdown of the nuclear membrane. Data on permeability during endomitosis are lacking and should be sought, but sugar intake need, of course, bear little or no relation to diffusion of nuclear products, Jones (1947) shows that changes in the nucleus may have visible effects in the cytoplasm and that gene-determined pigments in corn may be either cell-limited or diffusible over a considerable area of tissue. Commoner (1949), from further analyses of somatic mutations at the A locus, suggests that genetic determination of the anthocyanin content occurs before cell enlargement and that the specific action of the A gene is based on initiation of production of a precursor. The radial pattern suggests to him distribution of the gene product during formation of constituent cells ; evidence on the issue of influence during mitosis might be found here. 111. CHROMOSOME REPRODUCTION Let us turn to the problem of chromosome reproduction without nuclear division. Almost without exception, biology textbooks teach that the chromosome number is constant in all somatic cells of a multicellular organism. A brief sketch of the development of this concept may be of interest and not without value for future work and concept formulation. Weismann (1893) wrote: “With certain exceptions . . , the number of chromosomes is constant for each species.” Wilson (1900) went further : “The remarkable fact has now been established with high probability that every species of plant or animal has a fixed and characteristic number of chromosomes which regularly recurs in the division of all its cells; and in all forms arising by sexual reproduction, the number is even.” I t remained for 0. Hertwig (1918) to formulate in detail the “law of constancy of chromosome numbers”: “This law tells us that the number of chromosomes in all cdls of a plant or animal species, with the occurrence of nuclear division, is always exactly the same whether we are dealing with epidermis, cartilage, muscle, or glandular cells, etc. However, . . the egg and sperm contain one half the number of chromosomes of the somatic cells. This also is a lawful phenomenon.” One embryologist later extrapolated the law to the extent of writing: “All cells whether they continue to divide or not ultimately contain the same genetic proteins in equal quantities.” There were many factors involved in the gradual consolidation of the law of constancy of chromosome numbers. Nemec in 1904 and 1910 had
.
NUCLEAR REPRODUCTION
13
discovered tetraploid nuclei in chloralized roots, but Strasburger ( 1907) was convinced that they could not persist and therefore were of no significance. H e even explained away as obviously due to some “disturbing influences, such as wounding by small animals, etc.,” his own discovery of rows of tetraploid cells in control roots. Of this Winkler (1916) wrote : [trans.] “This last remark of Strasburger is extraordinarily characteristic. I t shows that the conviction that in normal somatic tissue only diploid cells could occur has become a dogma under the influence of which the best plant cytologist comes at once to the opinion that the occurrence of tetraploid cells found therein must be pathological, without even considering any other possibilities.” Winkler himself, after establishing with certainty the occurrence of polyploid cells and tissue in Solanum species and of the production of polyploid plants from the callus of grafts, concludes : “since the germ cells always arise directly from embryonic tissues they will always have the typical chromosome number and hand it down to the next generation. The constancy of chromosome number is safeguarded even when there is vegetative reproduction, since plants grow with their growing points which, by definition, are always embryonal. . . . W e therefore come to the view that the regular occurrence of polyploid cells in the somatic tissue of higher plants by no means refutes the laws of constancy of chromosome number but must be expected in view of the importance of the chromosome number for cell size.” Except possibly for the last clause, with its teleological flavor, this is as clear and acceptable a statement on polysomaty as could be made today. Why has it so very generally been ignored, not only by textbook writers but also by most research workers ? It must be remembered that from the time the correlation of chromosome behavior in meiosis and of Mendelian factors in segregation and recombination was first clearly enunciated it took about a quarter of a century to establish the “chromosome theory of Keredity” to the satisfaction of the overwhelming majority of biologists. As late as the 1920’s there were some who still considered the chromosome complement as a variable characteristic which was no more and no less a part of the plant phenotype than, say, the number and shapes of the leaves. The important general concepts involved in the chromosome theory of heredity were most conclusively established by showing that in exceptional organisms with chromosome numbers or arrangements of their parts that deviated from the norm of the species, such as haplo-IV Drosophila, trisomic Oenothera, tetraploid Datura and translocation stocks of Drosophila, maize, etc., the genetic behavior was altered correlatively.
14
C. LEONARD HUSKINS
By 1937 it was realized that colchicine produces polyploid cells by inhibiting spindle action, and in view) of the importance of polyploidy in plant breeding there was immediately a wide search for other chemical agencies that would induce it. I t is not surprising, therefore, that when polyploid divisions were found in mature plant tissues that had been stimulated by “growth substances” it was assumed that the polyploidy was caused by the treatment. It seemed to me, however, that the effect of “auxins” was more probably stimulation of divisions in already polyploid nuclei. Geitler (1948) apparently reached this conclusion at about the same time and during the war he effectively established that “polysomaty” is common in the leaves and stems of a large series of plants, as he had earlier found it is in many insects. He had stimulated divisions by wounding. Unaware of his work, we had initiated complementary studies on roots with indole acetic acid in dosages that gave results quickly enough for us to determine that at least the higher polyploid nuclei had not been produced after initiation of the treatment (Huskins and Steinitz,
1948). Since polysomaty has not been considered to have any effect on genetic behavior, it was of no significance in that stage of the rapidly developing science of genetics where establishment of rules of transmission was the major goal. The question today is whether or not it is significant in developmental or physiological genetics. Let us first consider from this point of view some of the recent work, including that of members of our own group, on polysomaty. Incidentally, there is much confusion due to unsatisfactory terminology in many discussions of nuclear reproduction. I have been accused of confusing endomitosis, endodivision, polysomaty, and polyteny because I have not always in all contexts differentiated sharply between them. I risk this charge again and for the same reason as previously, namely, that I think the distinctions unimportant at the present time in discussions of possible functions. They are not unimportant in descriptive cytology, and I do not ignore them in that context. Further, since I have been so widely misunderstood on another point, despite two separate and specific warnings in my original speculative discussion on the possible significance of polysomaty ( Huskins, 1947), let me here emphasize that polysomaty as suck cannot possibly be of any great general significance in differentiation. It may be in special cases; in all cases it docs prove that chromosome, and therefore gene, reproduction continues after nuclear or cell division ceases and thereby opens the way to coilsideration of the possibility that gene action i s correlated zcuWa gene reproduction. The cyto-
NUCLEAR REPRODUCTION
15
logical evidence, now generally accepted, that the chromatid is not a transversely unitary structure, shows that Mendelian segregation involves units at a higher level of integration than those resolvable by even such a relatively crude analyzer as the light microscope. We may therefore justifiably consider the possibility that the ultimate units which may be effective in differentiation could be at a very much lower level. There is considerable evidence that, at the microscopic level, reproduction of the component strands of a chromosome is not uniform throughout its length. The possibility that reproduction of the materials making up a Mendelian gene may be differential for different genes in different tissues therefore becomes almost an a priori probability. The problem is to devise methods for testing i t ; such are, of course, appearing as soon as the problem is envisaged. IV. VARIATION IN NLTCLEAR SIZE To return to simpler levels of discussion: Huskins and Steinitz (1948) attempted to analyze the great variation in nuclear size in differentiated regions of Rhoeo roots. Evidence obtained by counting the number of heterochromatic bodies (which was the method devised by Geitler for insect tissues) and by treatment with indole acetic acid, coincided in showing the variation to be correlated with degree of polysomaty. Similar results were obtained with barley (Leonard-Bennett, unpublished). Duncan and Ross (1950) have shown that in nuclei of niaize endosperm undergoing mitosis the normal triploid number of chromosomes is usually present. However, in giant energic nuclei a high degree of polyteny is observed in regions of chromosomes marked, for observational purposes, by heterochroinatic “knobs.” They have, further, shown a different range in nuclear size in different areas of the endosperm. An apparent reduction in the size of endosperm nuclei adjacent to the embroyo as its development proceeds is of special interest and is currently being studied further. Nuclear and cell volume, chromosome and chromatid number in pith cells of Nicotium towentosu are being studied by Dr. Muriel Bradley. Her data show, iiiter ulia that in this material nuclear volume is related directly to chromatid and not to chromosome number. I t seems to make little or no difference whether 8n chromatids are present as the 4n number of ordinary two-chromatid chromosomes or as 2n chromosomes each having four chromatids. The data show also that following the halving of the total cell volume after telophase there is within each of the polyploid classes a relatively slight increase in nuclear volume which is followed by something like a doubling before the next prophase. These morphological data accord with cytochemical studies. They support indirectly the as-
16
C, LEONARD HUSKINS
sumption made from microchemical analysis by Boivin, Vendrely and Vendrely (1948), Mirsky and Ris (1949), and others that with certain exceptions the DNA content is the same in all somatic cells. Certain variations in DNA content have been related, obviously correctly, to reproduction of the chromosomes and to polyploidy. Relating the constancy, apart from polysomaty, to the concept that the genotype is the same in all cells, it is usually concluded that DNA is an essential, and probably constant, component of the gene. The most recent published work along these lines is that of Swift (1950) who has shown, by absorption spectrophotometry following Feulgen staining, that the nuclei of ten different somatic tissues of young and adult mice all show approximately the same amount of DNA except for some of those in the liver, pancreas, thymus, blood lymphocytes, and Sertoli cells which contained two or four times the common amount. Occasional rare intermediate values were found and presumed to be associated with mitosis. Mouse spermatid nuclei had half the DNA of the common somatic nuclei. Primary spermatocytes had four times and secondary spermatocytes twice the spermatid value. Some premeiotic sex cells had the somatic amount and some twice as much. The former predominate in testes of 1- to 10day old mice and the latter are commoner at maturity. Nuclei of six tissues of adult frogs all had approximately the same amount of D N A (excepting for a few liver nuclei which had twice as much), and this was slightly less than twice that characteristic of the mouse. From studies of embryonic mouse liver and Amblystoma larvae, it was shown that DNA content “builds up in the interphase nucleus before the visible onset of prophase” and that “during the visible stages of mitosis no DNA is synthesized.” I n the Malpighian tuhule nuclei of a grasshopper four classes of DNA content with the ratio 1 :2:4:8 were found. In our laboratory (Bloch and Patau, unpublished) the relative DNA content of mouse liver nuclei has been determined, following Feulgen staining, with both an electro photometric and a visual microphotometer, the latter instrument having been designed by Dr. K. Patau. The results with liver of adult mice of pure lines and their hybrids agree with those of Swift and confirm that the step from one class to the next higher is a very accurate doubling of the DNA content accompanied by a doubling of nuclear size. In embryonic liver the DNA content ranges from 1 :2. It doubtless is reflecting the synthesis of DNA in nuclei preparing to undergo mitosis. Intermediate values between higher classes may be reflecting chromosome reproduction preparatory to either mitosis or endomitosis. Schrader and Leuchtenberger ( 1949) have stressed the
N U CLEAR REPRODUCTION
17
variation “which may be due to different degrees of polyteny” in different tissues of Tradescantia. Since oral presentation of this article, Swift (1950) has reported findings in maize and Tradescantia. In our laboratory, measurements are being made on Allium and Tradescantia tissues (Nelson, unpublished). For the roots the results are similar to those of Swift, excepting that he reports none of the lowest class in the elongation region of maize roots but several higher multiples, whereas in the same region of Allium roots, Nelson finds large numbers of the lowest and second classes, very few of the third, and none higher. Interesting additional findings by Nelson are : (1) that the doubling which precedes mitosis occurs early in the interphase period (unpublished data of Dr. Alma Howard, Radiotherapeutic Research Unit, Hammersmith, on uptake of radioactive phosphorus, are in accord) ; ( 2 ) that the guard cells of stomata have constantly the lowest amount of DNA normal for diploid cells, while other cells of the epidermis have double this amount ; (3) immediately after the first division of the microspore nucleus the resulting “vegetative” and “generative” nuclei have the same (haploid) amount of DNA but as the pollen grain “ripens” the generative nucleus doubles its content - in readiness for its division - while the content of the vegetative remains constant, contrary to the opinion of the many descriptive cytologists who have noted its fainter staining. In all of the foregoing, the findings confirm the ideas of Jacobj (1925) and many subsequent workers that increase of nuclear size may be caused by geometric increase of chromosome number, i.e., by polysomaty. It has, however, also long been clear that nuclear size can increase greatly without change in chromosome number. In some cases it is now evident that this may be correlated with increase in degree of polyteny, as shown by Duncan and Ross in maize endosperm. In yet others it may have little or no relation to the “chromatin” content. Schrader and Leuchtenberger ( 1950) have shown cytochemically that in the very different-sized nuclei which characterize different lobes of the testes of Armelitis albopunctatus, a hemipteran insect, the DNA content is approximately the same; it is the total protein and the RNA content that are correlated with nuclear, nucleolar and cytoplasmic volume. Apart from internally regulated “permanent” changes in nuclear volume, there are, of course, changes which appear to be correlated with developmental or physiological factors. Metz and his students (Buck and Roche, 1938) have shown that osmotic and mechanical pressure changes may cause reversable increase or decrease in nuclear and chromosome size as great as 25 per cent. The diverse effects of various fixatives on nuclear size are also, of course, well recognized.
18
C. LEONARD HUSKINS
To summarize this discussion of polysomaty we may safely say: (1) The concept of genic and chromosome identity of all cells obviously needs both amplification and circumscription; (2) Many more data on its occurrence are needed before we can even decide whether or not it, as stich, is at all likely to be found of any general significance in differentiation and development. (Regular and characteristic differences in number of chromosomes in certain tissues such as Geitler and others have found in various insects and in DNA content such as Nelson is finding in plant epidermis seem to point affirmatively, whereas the difference in these regards between morphologically similar regions of maize and onion roots and many other data point negatively.) ; (3) The newer techniques if used on specially chosen materials, particularly if these permit a concomitant genetic analysis, are capable of giving us answers to problems that were unassailable by the methods of cytological study available until very recently; (4) Whatever the relationship of D N A to ultimate gene structure, it seems certain that it is precisely related to the reproduction of the chromatid, which is the subdivision of the chromosome that is significant for Mendelian heredity-so far there is no evidence of a chromosome’s being able to divide without first doubling its DNA content, but, of course, very few studies of this question have yet been made: ( 5 ) Though chromosome reproduction must “normally” precede nuclear division, either of the two processes can proceed independently of the other -the relationship is parallel to that of cytokinesis to karyokinesis.
v.
REDUCTION IN
CHROMOSOME
NUMBER
We may next consider the process of reduction of chromosome number. To the long-established account of the process in germ cells the only important item to be added from current studies with the newer techniques seems to be the discovery (Swift, Nelson, and probably many others unpublished) that the D N A content reaches the same heightened level by pachytene of the first meiotic division as that typical for early mitotic prophase. Therefore, two divisions without an intervening increase during their interkinesis are required to restore the normal relationship between number of chromosomes and nuclear D N A content since the former has been halved. W e have moved far from the concept held by many cytologists not so long ago that the first division was the reduction division, and the second an unexplained concomitant ; genetics has long shown that both divisions are essential to genetic reduction, the occurrence at first prophase of four chromatids in each bivalent indicated the same from the descriptive cytological aspect, and now we see that they are needed to restore cytochemical balance also.
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Somatic reduction and segregation have long been known but little investigated until recently. Many textbooks list Winkler’s ( 1910) Solununz danoriniaiticm as having originated by somatic reduction, but none that I know of list his 1916 cases. I believe (see Huskins, 1948) the former to be an erroneous interpretation while the latter are clearly valid. Winkler’s 1916 remarks warrant quotation as a basis for discussion of current studies. [translation] To obtain such reversions to the diploid normal form from a tetraploid gigas type, the cells which gave rise to the atavistic tissue complex must have undergone a reduction-division. We may therefore not doubt the possibility that reductiondivisions occur in somatic cells. What stimulates them has to be left undecided. That the tetraploid condition of the nucleus by itself should have caused a tendency for the reduction of the increased chromosome number cannot be assumed, since tetraploid types exist and in general persist. It will also have to remain undecided for the time being whether the halving of the chromosome number occurs by typical reduction-divisions or otherwise. As a matter of fact it will be very difficult to find such a reduction-division since the reversions, at least so far, have occurred rarely and quite irregularly, that is in places which could not be predicted.
Two cases are reported in which somatic reduction appears to be a regular process: (1) prior to meiosis in the hermaphrodite gonad of a coccid (Hughes-Schrader, 1927) and (2) in the ileum of mosquito larvae (Berger, 1941 ; Grell, 1946). Somatic pairing and segregation are, of course, well known in the Diptera, but adequate data are lacking on reduction, though it was early reported by Bridges. Bateson (1926) insisted that there was much genetic evidence for somatic segregation in various tissues, but most such cases investigated in the past quarter century have been interpreted as somatic mutation, which he warned against as likely to obscure the issue. Recent evidence indicates that in any specific case both must be considered as possible explanations until the one is ruled out. The sporadic occurrence of chromosome pairing and/or reduction in somatic tissues has frequently been recorded. For example, Gates (1912) observed it in the nucellus of Oenotheru Zutu; Ludford (1935) and others have recorded it in tumors and tissue cultures of tumors; Metz (1942) found in a Sciara hybrid a salivary gland nucleus containing chromosomes from only one of the parents; Love (1936) found pollen mother cells that had undergone reduction prior to meiosis. East ( 1934), Nishiyama (1933), Kiellander (1941), Sparrow (1941), and Vaarama (1949) obtained plants with reduced, ancestral, chromosome numbers among the progeny of polyploid strawberries, oats, Poa, wheat, and Ribes, respectively. Brown (1947) found a reduced sector in an unbalanced poly-
20
C. LEONARD HUSKINS
ploid cotton plant. Upcott ( 1939) observed irregularly reduced chroniosome numbers in tetraploid Primnula kewensis; it is possible that by attributing these to “split spindles” she may have diverted attention from the problem as Strasburger, in Winkler’s opinion, quoted earlier lierein (1916), diverted attention from polysoniaty by assuming that it must be pathological. Following sporadic discovery of haploid cells in various plant roots, consideration of Caspersson’s ( 1939) suggestion that nucleic acid plays a role in synapsis and chromosome division led to a search for somatic reduction in preparations of Allium root tips treated by Dr. M. Kodani (1948) with sodium nucleate. Many cells with two reduced groups of chromosomes were found (Huskins, 1948). They were later found also in roots grown in solutions rich in phosphates (Galinsky, 1949). More recently it has been found (Huskins and Cheng, 1950, and unpublished) that prolonged low temperature treatment also increases the frequency with which “reductional grouping” occurs. There is also evidence that genetic factors affect the frequency with which reduced tissues or organs occur. In one strain of tetraploid Rhoeo we have obtained diploid and triploid roots and shoots with and without treatment. In another strain, treatments increase the frequency of reductional groupings, but no reduced tissues have yet been obtained. Allen, Wilson, and Powell (1950) have recently compared the sodium nucleate results with the chromosome groupings that occur after colchicine treatment. An extensive study has been made by Patau and Steinitz (1951) on the origin of reductional groupings and of reduced cells (see also, Patau, 1950). It is clear that somatic reduction is of not infrequent spontaneous occurrence, that its frequency can be increased by various treatments and “natural” conditions such as low temperatures, and therefore that polyploidy is a reversible evolutionary process. Battaglia ( 1948) has recently reported that somatic reduction occurs regularly in the basal portion of the style of Sambucus, and Christoff and Christoff (1948) report it in the integumental cells of Hieracium. If these and the cases in insects mentioned earlier are confirmed and extended, the “law of constancy of chromosome numbers” and our concepts of nuclear reproduction will have to be extended in this direction also. Besides polysomaty, polyteny, and reduction, which affect the total chromosome complement, there is also aneusomaty, i.e., the occurrence of cells with variable numbers of individual chromosonles, to be taken into account. It appears probable that aneusomaty (not to be confused with aneuploidy which refers to deviations between, not within, organisms)
NUCLEAR REPRODUCTION
21
most commonly involves chromosomes that are wholly or in large part heterochromatic. Too little carefully controlled work has yet been done on aneusomaty to warrant any conclusions on either the mechanism of its occurrence or its significance. Duncan (1945) concluded that in the root tips of an orchid the occurrence of variable numbers of chromosomes was due to a differential rate of reproduction of euchromatic and heterochromatic chromosomes. Darlington and Thomas ( 1941) attributed similar variable numbers in Sorghum to selective elimination in the roots but not in the shoots. Randolph (1941) presents numerous cytogenetic data which show that in maize the problem of B chromosome function and behavior is very complex. In Cimex, Darlington (1939) found from 0 to 12 extra X chromosomes in the males, with the average number higher in natural populations (9.0) than in mass cultures (4.3). H e correlates cycles of chromosome and centromere division with the “differential precocity” of autosomes, M chromosomes, and sex chromosomes and functionally relates the various changes observed to “adaptive balance” in sex-determining mechanisms. The mechanism of variability is related to “the state or precocity” of the centromere and the relative size of the chromosomes. These issues would carry us far beyond the scope of the present review, but they lead up (as Darlington points out in his Appendix 11) to the problem of preferential segregation, which is also beyond present scope except that it must be pointed out that special spindle mechanisms exist which provide for elimination of whole sets of chromosomes. The best analyzed of such cases is probably Sciara, for which both genetic and cytological data are available. It seems probable that such elimination is functionally related to chromatin diminution that involves only parts of chromosomes and that some form of differential reproduction may be basic to all such. Investigation with the newer techniques and with wider concepts in mind than those which guided earlier descriptive studies may lead to the establishment of some of the generalizations which at present are almost entirely speculative.
VI. CONCLUSIONS To conclude we may summarize a few of the data which, though none alone may be conclusive, together suggest a need for revision and extension of some of our more orthodox concepts on various aspects of the reproduction of the nucleus and its components and of the role of the nucleus in differentiation and development. First, against the simplest unitary concept of the gene there is, to repeat, the cytological evidence that there are, frequently at least, more microscopically separable strands in both the
22
C. LEONARD HUSKINS
mitotic and meiotic chromosomes than there are chromatids, which are the unitary gene strings of Mendelian segregation. The concept of the Mendelian gene as made up of identical “lamellae” would fit this. (The term “lamella” must not be taken to connote an undue simplicity in either the concept or the writer ; it may at our present stage of concept-forming serve as the equivalent of the beads-on-a-string model which was useful when the linear order of the genes was the issue.) Against it are the onehit radiation data, together with the occurrence of reverse mutations. It would be easy to imagine a change in one “lamella” being transmitted to all the others if the process went only one way, but not if it goes either way with anything like the same frequency, as some few gene mutations do. However, the one-hit hypothesis is not unassailable. For recent discussions of it see Muller (1950) and Opatowski (1950). Secondly, against the concept of gene identity of all cells (leaving aside whole chromosome changes for the moment) there are observations which are taken to indicate : (1) that heterochromatic and euchromatic parts of chromosomes may reproduce at different rates (Schultz, 1941) ; (2) that the banding pattern and length and breadth is visibly different in the same giant chromosomes from different tissues (see Kosswig, 1948) ; (3) that “specific chromosome loci [produce] lateral loops” (Duryee, 1950) ; and (4) that different parts of the salivary gland nuclei give strikingly unequal phosphatase reaction (see Brachet and Jeener, 1948), which suggests the possibility of variation from one gene to another in speed of renewal of phosphorus in the DNA. This would leave open the alternatives of phosphorus renewal in DNA playing a role in synthesis of proteins concerned with growth or of differential reproduction of chromosome regions. Differential reproduction of gene lamellae would provide a mechanism for differentiation not envisaged by Goldschmidt and probably not compatible with his present ideas on the gene, but it can be related conceptually to his early theory of timed, sequential physiological activity of the genes. Thirdly, it now seems fairly certain that the DNA content of nuclei from different tissues is constant, or, more strictly speaking, that it is constant relative to the total number of chromatids per nucleus, whatever the tissue. However, the R N A and protein content are both variable in different tissues. Chargaff (1950) reports that the D N A from different species differs in chemical composition and puts out the interesting suggestion: “It would be gratifying if one could say-but this is for the moment no more than an unfounded speculation-that just as the desoxy-pentose nucleic acids of the nucleus are species-specific and con-
NUCLEAR REPRODUCTION
23
cerned with the maintenance of the species, the pentose nucleic acids of the cytoplasm are organ-specific and involved in the important task of differentiation.” Daly, Allfrey, and Mirsky (1950), however, dispute his findings. As for the possible role of the nucleus in differentiation, it is pointed out by Dunn ( 1949), Gluecksohn-Schoenheimer (1949), and others that the developmental reactions controlled by mutated genes are in some cases very like those occurring in normal differentiation, and that they are possibly “more fundamental and perhaps much closer to gene action than we suspect now.” Weiss (1950) emphasizes that the term differentiation has been very loosely used and that morphological criteria have played too large a part in the classification of cellular changes. H e classes as “modulations” those changes which are reversible and stresses the fact that many specialized cells cannot “dedifferentiate.” It is, of course, generally agreed that though permanent changes take place in many cells, the early crude concepts of differentiation always being determined by segregation of particles during early segmentation of animal eggs, or of its determination by any simple type of regulated gene segregation or mutation, are quite untenable. Further, the concept of genic identity of all cells was an essential step in the development of our understanding of Mendelian heredity and of the essential differences between asexual and sexual reproduction for which the basic mechanisms are mitosis and meiosis. Have we now reached the stage when we can profitably consider the possibility that our concept of the gene of hereditary transmission subsumes the “gene of differentiation and development” ( 1947) and that the two must now be distinguished? So long as we assume the chromosomal genes to be the same in all cells we are forced either to consider the cytoplasm as the seat of the primary differentiating materials on which the genes act or as (see Schultz, 1950) the variable member of the reciprocally interacting units-these are analytically the same, though the latter is conceptually more satisfying. The assumption that there must be units in the cytoplasm that determine differentiation has led to the very fruitful discoveries of the entities that currently are most frequently referred to as plasmagenes though the concepts this term implies cause its rejection by many (see Schultz, 1950). Plasmagenes that are found to be dependent on chromosome genes in their function, even though autonomous in reproduction (Ephrussi, 1950) do not of course conflict with the orthodox concept of gene identity of all cells. If, however, we should be forced to the conclusion maintained by Darlington (1949) that the only difference between nuclear genes and plasmagenes is that the latter “have been denied . . . the gift . . . of coordinated seg-
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C. LEONARD HUSKINS
regation at meiosis,” we run into logical, or at least semantic difficulties. Though nucleus and cytoplasm are probably always interacting, the Acetabularia and other evidence seem to me to indicate that the nucleus primarily controls differentiation. But if cellular differentiation should in other cases be determined primarily by fully autonomous cytogenes which arise during ontogeny, as Sonneborn (1949) and others have suggested, then such cells are by definition genetically different even though we call their new “genes” plasmagenes. These cells should show different hereditary capacities not only in vegetative reproduction but also in the sexual reproduction of plants, if such differentiated cells can ever give rise, however remotely and indirectly, to female ,gametes. This latter would demonstrate definitely that the plasmagenes really are genes in the accepted meaning of the term, but it is the concept of chromosome identity, not of genic identity, of all cells that can be saved by the concept of two sorts of genes differing only in their location. This with the data on constant DNA content appeals to those of us who picture the chromosomes, but not necessarily the genes, as characterized by DNA at least during their period of reproduction and also of “division.” W e have, however, seen the limitations of “the law of constancy of chromosome numbers” and the constancy of chromosome parts seems very likely to prove even more limited. Which brings us back to nuclear reproduction: it is a very complex process which normally comprises many subsidiary processes which for the present we can safely classify into only two, viz., chromosome reproduction and chromosome separation. Various types of separation are accomplished by mitosis, endomitosis, and meiosis, each with many variants, but in each there is an essential basic uniformity. To these processes we must add the very incompletely known mechanisms of somatic reduction and of aneusomaty and differential elimination of parts of chromosomes, whole chromosomes of special types, and of genomes or sets of chromosomes. W e do not have to consider amitosis, for most of the descriptive work on it has long ago been shown to include errors of interpretation and the evidence of genetics shows clearly that it cannot be a normal process of nuclear reproduction, if by normal we mean the production of nuclei continuing to have potentially unlimited capacity for further reproduction. VII. REFERENCES Allen, N. S., Wilson, G. B., and Powell, S. (1950) J. Hered. 41, 159. Barber, H. N. (1942) J. Getwt., 48, 97. Bateson, W. (1926) 1. Genet., 16, 201: Battaglia, Emilio (1948) Nuovo G. bot. it&., 65, I.
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Nemec, B. (1904) Jb. z k s . Bot., 89, 645. Nemec, B. (1910) Das Problem der Befruchtungsfrage. Berlin. Nishiyama, I. (1933) Jap. 1. Genet., 8, 107. Opatowski, I. (1950) Genetics, I, 56. Patau, Klaus (1950) Genetics, MI 128. Patau, Klaus, and Steinitz, L. M. (1951) (In press.) Randolph, L. F. (1941) Genetics M, 608. Schrader, F., and Leuchtenberger, Cecilie (1949) Proc. nat. Acad. Sci., Wash, 86, 464. Schrader, F., and Leuchtenberger, Cecilie (1950) Ezp. Cell Res., 1, 421. Schultz, Jack (1941) Cold Spr. Harb. Symp. qzcant. Biol., 9, 55. Schultz, Jack (1950) Science, 111, 403. Schulze, K. L. (1939) Arch. Protistertk., 92, 167. Sonneborn, T. M. (1949) AMY. Scient., 87, 33. Stern, C. (1938) Amer. Nat., 7!2, 350. Stern, Herbert (1946) Philos. Tram., 40, 141. Strasburger, E. (1907) Jb. w'ss. Bot., 44, 482. Sparrow, A. H., Huskins, C. L., and Wilson, G. B. (1941) Canud. J. Res., 19, 323. Swift, H. H, (1950a) Proc. nut. Acad. Sci., Wash., SS, 643. Swift, H. H. (1950b) Physiol. ZoGI., 2S, 169. Upcott, M. (1939) J. Genet., 89, 79. Vaarama, A. (1949) Hereditas I,136. Weismann, August (1893) The Germ Plasm. W. Scott Ltd. London. Weiss, P. A. (1950) Quart. Rev. Biol., I,177. Weisz, P. B. (1947) J . Morph., 81, 45. Wilson, E. B. (1900) The Cell. Macmillan Co., N. Y. Winkler, Hans (1910) Ber. dfsch. bot. Ges., 28, 116. Winkler, Hans (1916) 2. Bot., 8, 417.