1941
The Nucleolus : its place in the cell.
A review of the literature and an account of some research by Joshua Lederberg
Zoology 1 Papers
December 29, 1941
to H. B. Steinbach
The work mentioned by the author was done at the American Institute Science Laboratory and in the Histology Laboratory, Sociology Dept., Columbia University, To the directors and supervisors of which grateful acknowledgement is made.
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The Nucleolus : Bibliography
1. Sharp, L.W. 1934 Introduction to Cytology  , Wiley.
2.
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The cell was first discovered as a structural unit of plants by Hooke in 1665, but it was not until the 1820's and "'30's" that Dutrochet, Schleiden and
Schwann formulated the cell theory as we recognize it today.  In 1831, Robert Brown published the first close description of the nucleus, shortly after
which this body was recognized as a universal component of the cells of higher organisms.  We can date the modern science of cytology from this time.
he remainder of the 19th Century was occupied largely with ascertaining precisely what the morphology of cells consisted of.  In the 1880's. Flemming, Altman
and Strasburger demonstrated what are now known to be mitochondria, and universally present in animal and plant cells.  In 1898, Golgi discovered the argentophil, osmophil Golgi apparatus, which has since been found generally in animal cells, and to which various homologues have been suggested in plants and protozoa.
Chromosomes were first seen in 1848 by Hofmeister, but their place in cell-division, or the conception of mitosis, appeared only in the 1880's.  Under the impetus of genetic researches and the formulation of the chromosome theory of heredity most modern cytology is the cytology of the chromosomes.  Mitochondria and the Golgi material have also been studied intensively
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lthough there can not yet be any definitiveness about the matter, the Golgi material and the chondrioine are widely felt to have an important bearing on the
secretory processes of the cell.  In spermatogenesis, they have been shown to play important and specific parts, the Golgi organizing the aciosome[?] of the sperm head and the mitochondria forming the substance of the middle piece.
There are three items of the cell that I have not yet mentioned.  These are, firstly, the cytoplasm or hydroplasm which has no visible structural differentiations in the typical cell.  There are also, of course, carious mitusions such as plastids , vacuoles of various sorts, blapharoplasts, contractile structures, febrillae for nervous coordination, cell wall, and others, but we cannot concern ourselves here.  Secondly, there is the problem of external environment of the cell, which was studied by Carrell[?] at the Rockefeller Institute, and thirdly, and the phase that this paper discusses, 
there is the nucleolus, which receives scant enough attention in the textbooks and the literature.
It is difficult to give a comprehensive definition of this structure, as it shows some diversity.  However we can call it a body, found within a "resting" nucleus, that can be differentially stained from the chromosomes, makes no direct contribution to
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the chromatic figure or the chromosomes division, and general disappears in metaphase.  In this discussion, the word "nucleolus" is substantially identical with 'plasmosome'.
The nucleolus is almost ubiquitously associated with nucleus.  Wherever these are found, some nucleolus-like structure is commonly found also.  Where there is no formal body corresponding to the nucleus, no nucleolus can be recognized.  Thus the lower plants - bacteria ; Cyanophy case, are excluded.  In Protozoa and in other lower plants, there is no true nucleolus, the nuclear  inclusion being within a karyosome, contributing to chromosomes, or an intra-nuclear division center.  In some instances, there is an amphimecliolus[?] which is a double structure, being composed of a plasmosome and a condensed  chromosome.  Such bodies are frequently found in insect spermatocytes. True
nucleoli appear consistently in almost all cells of higher organisms.
Perhaps the cleanest appearance of the nucleoli rests in the somatic cells of higher plants where they are usually by far the most prominently staining methods are applicable to the morphological study of nucleoli; of which only a few need be mentioned.  They stain, with acid feuchsin[?] preferentially in
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the Champy-IT cell method or in acid function methyl green.  A very pretty (aesthetically) picture is given by the Flemming triples stain where they take safianim [?] in contrast to chromosomal violet.  They react negatively to the Feulgen Reaction, far 'nucleic acid', but otherwise stain well in basic dyes.  This is particularly the case in Allium cepa wool-tips, on which the author has worked.
The nucleoli of oocyte germinal vesicles represent a special case which will be discussed later.
Hofmeister in 1848 was the first to report the dissolution of the nucleolus in cell division in Tradescantia[?].  Since that time this phenomenon has been notes repeatedly ; so that cases of persistent nucleoli are exceptional.  Zirkle offers some meager evidence for the inflow of the nucleolus substance into the core 
of the chromosomes, in Zea mayo root-tips, using special fixtures[?].  He also describes the extension of some nucleolar figments in metaphase.   In Pinus strobus cambium and root-tip he describes again the flow of nucleolus disappear before the orientation of the figure into an equatorial plate.  Inclusions in the living nucleolus are also reported.
Filsey question this interpretation.  In Rumex
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scutatus typicus, tetraploid, he reports fusion of nucleoli in pre-meiotic
phase.  At no time is a definite, fixed connection with the spireme seen.  As different fixtures give a different picture of nucleolus, inclusions, these are considered artifacts.  Bitumen diskenesis and Metaphase I the nucleolus enlarges and becomes more diffuses, giving the appearances of solution in the karyolymph.  It has disappeared by the time of appearance of multipolar spindle.  It is pointed out that for the nucleolus to make a direct contribution to all the chromosomes requires a continuous spireme thread which does not exist.  Citing Meyer (?) on parenchyma of Galtonia candicans, he states that the nucleolus diminished in emaciation, and gives as its function only that it is merely ergastic material.
The author would like to point out at this junction that Zirkle's material is somatic Zea and Pinco, while Filsey's is meotic Rumex.
An interesting deviation had been demonstrated by Frankel (12) in the meiotic divisions of certain species of Fritillaria.  In F. oblique, in the earliest meiotic prophages, the nucleolus is a cap-like structure on the periphery of the reticulum, just inside the nuclear membrane.  The suggestions is of strong pressure on the nucleolus 3 of the
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bivalent chromosomes may be attached, probably corresponding to 3 pairs of "SAT chromosomes" in the somatic metaphase.  At pro-metaphase, the nucleolus globularizes[?].  This is evidently a sudden process as related to the release of pressure when the nuclear membrane dissolves.  The chromosomal attachments thenceforth are lost.  The nucleolus pursuit, may fragment somewhat, and is randomly distributed in the sporogenetic[?] divisions.  The somatic divisions of this species show the typical picture.  Similar behavior seems to be exhibited by F. plurifola, but no-cap nucleoli are more frequent.  In F. meleagics cantorta, no prophase material was available, but two persistant [sic] nucleoli were seen at metaphase I.  In early Telophase I of F. citrina and other forms, globules form close to the distal ends of anaphase chromosomes as the approach the poles.  This swiftly disconnect from the chromosomes, are not visible in Prophase II, but reappear in telophase II, not showing in the tetrads.  In somatic mitosis, none of this is seen.  Afterosmic fixation, both nucleoli and telophase globules react to the Feulgen.  There is no evidence of matrix swelling as McClintock's theory would seem to demand.  
These phenomena clearly represent a divergint[SIC] and atypical case in which the nucleoli are not equivalent to the usual types and history.  Their significance will be touched upon
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below.
Another aberrant is noted in use.  Selim working on various varieties of Oryza sativa L., particularly Kochwutu [?], whatever that means, gives the following account of nucleolar changes in the meiotic divisions.
In presyniyesis[?], there is ordinarily a single nucleolus or perhaps two on opposite sides of the nucleus.  In prophase, during reticular contraction and spireme formation, the nucleolus buds of a secondary one which remains attached.  Some races vary.  The same phenomenon ensues in the megasporocyte divisions.  The following figures are given for the frequency of this phenomenon.
[TABLE]
The conclusion might be drawn that there is further budding between fusion of nucleoli between leptonema and syniyesis[?].  This is a substantiated phenomenon, but the figures hue statistically are rather insignificant.  The author calcuated sigma = 5.74%.  Inasmuch as there is a difference of only 7.7% in the di-nuceloate forms, which would be reduced to 2% if all of the doubtful cases in happened to be dinucleolate, etc..., the statistical significance of these figures 
is very small, being of the order of 10, indicating
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that there is only about 1 chance in three that this difference is not due to chance sampling alone.
Selim suggests that the primary nucleolus contributes to the spindle, and the secondary to the substance of the chromosomes.
For a fairly modern review of the nucleolus, Gates (16) can be consulted.  An exhaustive review of the early work [19th century] is given in Montgomery, also Ludford.
In Montgomery, evidence is presented for the movement, fusion and division of nucleoli; this is evident in oocytes.  Because of the rather aberrant behavior of nucleoli in oocytes, it may be that these nucleoli are not homologous to those found in plant material.  Ludford describes the extension of nucleolar oxyphilic material into the cytoplasm in Lymnae oocytes.  The nucleolus differentiates into a "basophil"and an "oxyphil" act[?], the latter being extended and possible have some role in yolk-formation.  At the end of oogensis, the basophil part fragments and distributesthrough the cytoplasm.  A correlation is noted between size of nucleolus and cell-activity.  The early (1900-1903) work on animals does not contribute much to the connection between chromosomes and nucleolus.  Connexions [sic] in plant material have long been noted.  The situation is most animal cells is [ . . .] now to follow that in plants, as elucidated in part be McClintock.  (v. infra.)
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Interaction in some form or another between nucleoli and chromosome has been suspected by many observers for many years, although others, as Filsey have questioned it.  Perhaps the most striking and obvious relationship concerns the nucleolar number, particularly in polyploid plants.  In 1927, De Mol published some observations on diploid and triploid hyacinths.  It was noted in the root tips of many varieties that a diploid nucleus was associated with a maximum of two nucleoli, a triploid three. His interpretation was that there was a relationship to the entire genome, and is in part superseded by modern developments.  Certain exceptions were noted in the matter of nucleolar number which seem temptingly susceptible to analysis on the basis of deficient or supernumerary individual chromosomes, today.
Nawaschin (cited in Sharp, originals inaccessible) observed in Galtonia candicans that the chromosome satellites lie on the surface of the nucleolus in late somatic prophase.  In 1931, Heitz showed in somatic Vicia faba the equivalence of an SAT region (sine acids thymonucleo, because it was believed to be achromatic to Feulgen) to the satellite - separation 
of a chromosome.
More recently, McClintock has shown that, in Zia, there is a definite, chromatic, nucleolar organizing region in chromosome VI.The chromosomes in Zia
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completely formed geneme, no distinct nucleoli develop, but a large number of nucleolus-like bodies develop along the superficial matrix of the chromosome, 
evidently by the coalescence of this substances the "chromosomal matrix." There are two larger bodies of the organizers of the chromosomes, however.
In other X-rated material in which the organizer is entirely lost, a similar situation prevails except that there are no larger bodies representing organizer activity.
The evidence is rather definite and indicated that the number of nucleoli that develop is dependent on the number if organizer loci and a fairly complete
chromosomal complement.  This evidence is confirmed on somatic (root-tip) material.  In normal plants there may be two nucleoli in the telophase , but as Heitz had already shown, (V. Sharp), where nucleoli develop in proximity in the telophase they may fuse.  The maximum number of nucleoli, then reflects the number of organic loci. Plants heterozygous for this interchange (6 .9, 69 x 96) show 3 nucleoli, 2 large and 1 small evidently from the regions in 6,96 and 69 respectively.  Plants homozygous for this interchange show 4 nucleoli, representing the 2"64" and the 2"96" regions.  In Zia, there is ordinarily one pair of SAT chromosomes.  Thus, the nucleolus is shown to
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have been mapped out genetically to some extent and can be distinguished
by the position of spindle fiber attachments, size, and chromatic knots.  In prophase, the chromosome is always in contact with the organizer, while the satellite may be some distance away in the surface of the nucleolus.
By means of X-Rays, McClintock was able to procure plants in which there was a reciprocal translocation with chromosome 9, the break in 6 being across the 
chromatic nucleolar organizer.  Both parts of the organizer now functioned independently.  The part that is more proximal to the achromatic region seemingly is more active as large nucleoli develop in connection with chromosome 9 than 6, although 6 has the larger part.  The findings are
 as follows, with respect to the situation in spores to which different chromosome combinations were distributed in the reduction divisions of a plant 
heterozygous for the interchnage, i.e. whose genotype was 6-69, 9-96: 6 is the normal chromosome, 69 is the translocated.
I. 6-9.  This is normal, and produces one nucleolus at 6
II. 69.96 This has a complete complement, but with a divided organizer, one at 6 one at 9 and produced nucleoli correspondingly.
III.  69.9 This is missing the translocated portion of 6, but contains only one organizer and produces one nucleolus accordingly
IV 6 96 This is missing the rather large translocated portion of 9, and has two organizers.  Presumably because of the m-
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Have a direct morphological relationship to one of chromosomal elements - a chromatic region set off by an 'SAT' region at the other end of which may be a satellite, or which may be merely a 'secondary constriction.'
The formation of nucleolar-like material from the matrix leads McClintock to suggest that the nucleolus is formed by confluence of this matrix, or that there is 
certainly a close relationship between matrix, and nucleolus.  This is particularly well-known when the organizer is inactive, and nucleolus-like bodies form as droplets on chromosomes "from matrix".  The behavior of this substance suggests that nucleolus organization is a means of distributing "chromatics" to reform the metabolic nucleus, and that "complete release of the matrix from the chromatin in necessary before the chromatin can function properly in metabolism".
This process of nucleolus formation has been confirmed more or less exactly in Alluim in Tractonus (a beetle) to cite two references and by many
other observers in both plants and animals, particularly insects.  However, not sufficient, work has been done to show how general this process is.  It  is, however, quite clear that in many instances the chromosomes do make definitive contribution and control to the nucleolus.
Nucleoli in plant and animal cells present such a diversity of form that it may not be possible to rationalize them all into a
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single unified conception of this structure.  In plant root-tips, the nucleolus is relatively "basophilic", in animal material they are more characteristically
"oxyphile" - except in the case of oocytes already cited.  Another disturbing factor is their discontinuity.  In regularly dividing cells, there may be a certain 
similarity in nucleolar positions between adjacent cells.  This, however, is a manifestation of the continuity of the chromosomes and thence of 
the nucleolar organizer.
No positive conclusion can yet be drawn as to the physiology of the nucleolus.  Its metabolism is a mystery; its chemical composition hardly less so.  Its morphological history, however, is not consistent.   In the cases already cited, there has been evidence for extension of nucleolus into ooplasm, possibly in yolk formation. It is still somewhat open to doubt whether these changes actually do take place, but certainly there is sufficient evidence to show that the oocyte nucleolus is rather anamolous [sic].  Furthermore, Nakahara (31) has described a similar extension in nucleoli of silk gland cells of a caterpillar (Pieres repae) and of caddis-fly dawae (Neuronica poetica Walkland, but here the evidence is even more obscure.  Some great chemical changes in the nucleolar substance is presumes to account for changes in stain after the "nucleoli" have extruded.  This "nucleolar" substance is regarded as part of the substance of the secreted silk.  In view of the uncertainty regarding the
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nature of these nucleoli even when mitia[?]-nuclear, this account must be held in abeyance until further evidence of the history of these "nucleoli" is presented.
That aberrations are not limited to animals is shown by the papers of Selim and Frankel.  Most of these deviations occur in the meiotic phase as where highly specialized cells are concerned.  The seratinization and pigmentation of skin are cited in  as another example.  See also Ludford, 1924, in which exctuded nucleoli and mitochondria and Golgi are seen to be somehow interrelated in the formation of "heratohyatin" and herative.
Enough has been presented to show that, although these is a fundamental ground place for the formation and nature of nucleolus in cells just after a mitotic
or mitotic phase, - the formation in relation to chromosomes - in cells that have become highly specialized, the nucleolus may assume structural and functional characters so far removed from the fundamental that me can question their homology with it.
Very little has been done on the chemical relations of the nucleoli.  The present author is now investigating the "isoelective point " of nucleolar and chromosomal substances in fixed onion root tip in an effort to find some relation.  However, as Conn points out: "a physical chemist defines the isoelective point as that where minimum dissociations takes place.
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A protein occurring in a solid form as in sections of fixed tissue can hardly be expected to dissociate and accordingly it is a question whether it can have p can properly be said to have an insoelective permit."
The chemistry and physics of the nucleus are obscure.  Evidence has accumulated that it is the heaviest component of the nucleus.  In many eggs, it is influenced by gravity to stay at the bottom of the nucleus.  In particular, the results of centrifuging show that it is probably somewhat denser than the chromosomes.  A general discussion of the results of centrifuging is given.  In tissue culture, the nucleolus can readily be moved through the nuclear "fluid" without hindrance.
In the course of an attempt to rationalize fixation images Zuikle found that certain fixatures were followed by the straining of nucleoli with dion-Hematoxylin, and others not.  Particularly, acetic acid causes non-stainability; formalin causes a large degree of staining.  Mixtures may be diffused differentially to give the effects noted.  The author has preparations of Alluim root tip fixed in formalin and acetic acid.  Some of the central cells have stained nucleoli.  In an appreciable number of cases, there are nuclei with two nucleoli of which one may be stained and the other not!
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It is too early to draw any conclusions on this sort of thing; the author is working on it now.  However, there is little question that all the nucleoli in onion root tip cells are equivalent, and "good" plasmosomes. Quantitative data on diffusion rates in guinea pig liver are given in.  Toostoochav has done some work on the chemical effects (an isoelectic point) of fixatines that maybe a starting point for similar work on the nucleolus Shinke and Shigeniga gene histo-chemical evidence for the presence of lipoids (solvents for Sudan III) in the nucleolus and chromosomes, and cytoplasm, reticulum of the resting nucleus, phiagnoplast[?], with a little question as to spindle fetus[?].  This may be more a demonstration of the non-specificty of the reaction than of the general occurrence of fats in any concentration Mellon's reagent is used as a test for proteins and turns out to be indefinite.  Feulgin is negative.  This is interpreted as + for lipoids ? for proteins and - for "thymonucleic acid".  These tests are only very vague and far from specific.  On this basis, the far-fetched speculations of Mensinkai may be dismissed. Francini in an inaccessible paper notes a positive reaction to Ruthenium Red in nucleolus of the orchid Paphiopedilum spicerianum.  This is interpreted as indicating the presence of a pectin or other polysaccharide.
In prophases these material is dissolved in the karyotymph.  The equivalences
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of nucleolus with spindle is suggested.  This may be recurrence of many fantastic ideas that appeared in the past.
By now, we should be more firmly convinced than ever of the hazy situation respecting nucleoli.  They must perform some important function within the typical cell - what it is we cannot say.  As cells specialize, they specialize also.
But time is not for speculation or baseless guessing but for further experimental elucidation in the laboratory.
Joshua Lederberg 12/23/41.