While, in recent years, cytologists have been able to ascribe some probable function and unity of form to most of the components
of cells in different species, the nucleolus of most cells remains one of the least explicable problems of cytology today.
This body is a conspicuous, typical part of the nucleus of most cells of higher animals. In different forms and situations,
however, it reveals a diversity that is not equaled by other cell components.
Inasmuch as the nucleolus undergoes a typical cycle in mitosis, disappearing at the metaphase and reappearing at the very
early anaphase, a link between the chromosomes and the nucleolus has long been suspected. The following papers are indicative
of the early approach to the problem:
The work of Zirkle, Filsey and de Mol, is out-dated and superseded by more modern developments ; Montgomery's is an exhaustive
review, largely historical, of the nucleolus up to 1898. The author does not regard it as essential to discuss the historical
aspect of nucleolus research. On that point, Sharp, 1934., Hofmeister 1848, might be consulted. Gates (1937) reviews the
more modern work.
Modern work in a number of species of animals and plants indicates a close connection between the nucleolus and a secondary
construction of a chromosome. Heitz (1931 a and b) has shown that
[END PAGE TWO]
[BEGIN PAGE THREE]
the position and number of the nucleoli in various varieties of Vicia follow the position and number of the secondary constructions
chromosomes in the reconstructing nucleus in telophase. More recently, McClintock (1934) has described the formation of the
nucleolus at a chromatic region setting off a satellite - construction in Zea mayo PMC's. Furthermore, with the assistance
of X-Rays, a reciprocal translocation was secured between the 6th and 9th chromosomes, such that the break in chromosome 6
occurred through the chromatic region : The number and position of the nucleoli in the products of subsequent crosses depended
on the number and position of such regions, wherefore it is called "the nucleolus organizer." When no organizer is
present, no single nucleolus is formed, but a number of "nucleolus-like" bodies appear to have formed by the coalescence
of matrix along the length of the chromosomes. Also, when a nucleolus is present, the extent of the chromosomal matrix is
inversely proportional to the extent of the nucleolus. McClintock suggests, therefore, that the nucleolus has some close
relationship to the chromosomal matrix. Mensinkai (1939) who confirms this process
in many species of Allium, attempts to give a chemical basis for this relationship. At present stage of our knowledge of
the chemistry of nucleolus and matrix, any conclusions are very tentative indeed.
[END PAGE THREE]
[BEGIN PAGE FOUR]
The isolation between "constriction" and nucleolus is confirmed for many other organisms (see, for example, Grasse
and Lespiron[?] " '38".) In prophase,
presumably, nucleolus contributes to matrix. (?)
A few instances have been described where the mitotic history of the nucleolus apparently does not conform with the account
given for Zia. It must be
emphasized first of all that no satisfactory description of the prophase history of the nucleolus is available. It appears
to fragment, dissolve, become extended into the cytoplasm, and disappear. See, for instance, Francini ("1938", a,b.)
and Zirkle, 1928.
Franksel (1937) describes an 'aberrant' case in the meiosis in a few strains of certain species of Fritillaria. In
F. obliqua the nucleolus, instead of a spheroid more or less has centrally located, is a cap on the periphery of the interphase
reticulum, just inside the nuclear membrane. 3 chromosomes may be attached to it, presumably the 3 which show secondary constridions[?]
in somatic metaphase. Before metaphase I, the nucleolus becomes globular and separates from the chromosomes. The appearance
suggests the sudden removal of the pressure excited by the nuclear membrane. This nucleolus persists, may fragment, and is
disturbed at random to the tetrad of spores
F. phrifola[?] exhibits somewhat similar behavior, but a smaller preposition of "cap nucleoli" are seen.
[END PAGE FOUR]
[BEGIN PAGE FIVE]
In other species, particularly F. citrina, the telophase is peculiar. Globules appear at the ends of chromosomes, looking
like nucleoli, and separates as
the figure polarizes. These disappear for Prophase II, reappear at Telophase II. After "cosmic" fixation, these
globules, as well as the regular nucleoli, are positive to Feulgen - Nuclealfarbung[?] indicating chemical similarities to
chromosomes. There is no evidence of matrix coalescence as McClintock's theory demands.
In another genus, Oryza Sativium[?] (rice) Selim[?]('30) also find peculiar behavior in the meiosis of some stains -.
In early prophase, the nucleolus buds
off a secondary nucleolus which remains attached. Different races vary in the details. Similar phenomena occur in megasporocyte[?]
divisions. Selim regards (without great foundation) that the primary nucleolus contributes to the achromatic figure and secondary
to the substance of the chromosomes. In plants, the nucleoli appear to remain quiescent in the inter-mitotic phase, and do
not show much specialization.
In animal material, there are also many peculiar specializations, but these occur in "resting" stages. There are
three well-known instances where the nucleolus
may contribute directly to the growth or secretion cells. These are the developing
[END PAGE FIVE]
[BEGIN PAGE SIX]
oocytes in many gastropods - e.g. Leinnaea or Patella ; in the spinning silk glands of many larval insects, and in the keratinizing
layers of the dermal epithelium. However, these instances are by no means thoroughly established or generally accepted (Wilson,
'25, p. 346, et. al).
Ludford has worked on the egg development in Lymmea. Here the "typical" nucleolus of the early organism differentiates
into a basophil and an oxyphil set. The latter is extended, and in conjunction with Golgi and chondriosomes play some role
in yolk-sphere formation. (Ludford, '22) At the "end" of oogenesis the basophil pact fragments and distributes
throughout the cytoplasm. He considers there is a correlation between the size of the nucleolus and degree of cell activity.
Similar phenomena are noted in many other sorts[?] of oocyte material. Wilson ('25) p. 271 cites Jorginsson on various
tracheates[?] where the chromosomes of the germinal vesicle enlarge, become less basophilic and assume a "lampbrush"
form. (although this is not to be imagined as the vertibrate[?]case.The nucleolus then becomes basophilic, seeming to ask
as resevoir [sic] of chromatin, although there is no direct chemical evidence for this. There are scattered references of
uncertain significance, in the literature, reporting the reaction of similar nucleoli to Feulgen. This question is still
open. In certain fish, the basichromatic[?] nucleoli spin out into chromosome-like segments, and at one time were mistaken
[END PAGE SIX]
[BEGIN PAGE SEVEN]
for such. Also, in some cases, the nucleolus persists after the breakdown of germinal vesicle, and thus must contribute to
the egg cytoplasm. This breakdown is the initiation of egg maturity ; it is necessary before fertilization can ensue. The
significance of the nucleoli here is speculative.
Nakahara (1917) is one of many authors who have examined nucleoli in silk glands. He concludes that silk (fibroin) is composed
of extended nucleoli. In these cells, the nucleus grows to a large size and branches so that a large surface is presented.
There is a figure in Wilson, p. 86. At the same time, the nucleolus divides many times so that there are fragments throughout
the nucleus. These particles seem to pass out of the nuclear wall into the cytoplasm, where the staining reaction changes.
The material worked on was Pieris rapae (a caterpillar) and Neuronica posteia[?] Walku[?] (the caddis-fly larva.) The nature
of this extension seems to demand further confirmation. Observations on live material would be most instructive.
Ludford (1924) has described the role of extended nucleoli, Golgi and mitochondria in the formation of 'keratohyatin[?]"
and keratin in the mouse epidermis. See also Cowdry (1928.)
This sort of work should demonstrate that we have not yet reached a unified conception of nucleolar function. In some instances
[END PAGE SEVEN]
[BEGIN PAGE EIGHT]
the nucleolus appears to have some role in secretions. It has been proposed that the chromosomes exert their genetic effects
on cytoplasm through extended nucleoli. But these, of course, are all speculations. Throughout any discussion of nucleolar
fragmentation, one must keep in mind that the chondriosomes in material inadequately prepared, may simulate nuclear products.
In any case, extreme caution must be used in any nucleolar interpretation.
In most animals and plants, of course, the nucleolus has not been so closely studied. It is probable that their usual history
is that outlined by McClintock in relation to the Zea chromosomes.
Something is known of the physical situation of nucleoli. Macklui[?] cited in Ludsford '22, reported that they are actine[?]
bodies, continually joining and fusing, in tissue-culture material. Chambers, 1924, on grasshopper spermotypes (Dessosteria
Casolina), using - the micromanipulator, has found that the nucleolus can be readily moved into the cell, so that it is not
firmly bound to any nuclear structure here. Gatenby (1938) gives a general amount of ultra-centrifuge results, but see particularly
Bearns[?], et al., ' 37.
After ultracentrifugation of Helix aspersa, and other species, spermatocytes, the nucleolus was found the heaviest objects
in the nucleus with the chromosomes follow. As in Allium cepa,
[END PAGE EIGHT]
[BEGIN PAGE NINE]
the reference for which I cannot recall, the nucleolus may break through the nuclear membrane. Superficially this is not
in accordance with the supposed lipoid nature of the nucleolus see infea[?] as lipids are ordinarilyless dense than inatu[?]
or "protoplasm" . Incidentally, the mitochondria are the densest, the Golgi and fat globules, the highest objects
in cells. This density, implying high concentration of its components is in accordance with the high stainability of nucleolus
even when a differential stain is not used
In the chemistry of the nucleolus, the most obscure mystery of nuclearbiochemistry reside Zirkle ('28, '29 '33
a,b,c,d) reports variations in staining of nucleolus by ion hematoxiglin[?] after different fixatives [?]. Underhill's
data (1932) maybe useful in interpreting the antagonistic effects of different fixatives [?] in a mixture.These data concern
the penetration of fixatives [?] into pieces of a guinea pig liver. This author's experience would confirm the list of
relative pinctiability[?] given in Underhill's paper, except that he figure for formalin is far too low when applied to
onion root tip.
The author, at another laboratory, has investigated, to some extent, the effects of fixatives, separately and in mixtures,
on onion root tips. (Lederberg, unpub.) This properly would form the subject of another report. It has been found that
[END PAGE NINE]
[BEGIN PAGE TEN]
fixes nucleoli so that they do not stain with Fe-Him; formalin so that they do. In mixtures of the two acetic fixation (simultaneously)is
evident, but some nucleoli in some nuclei stain. In some instances one nucleolus of two in a nucleus may stain, the other
not which proves nothing - yet except that fixation produces erratic results.The subject is now being re-investigated. In
connection with This, I am also studying the effects of various reagents on the apparent iso-electric points of the nucleolar
proteins. Inasmuch as Bouin's[?] fixatures confers an apparent isoelectric point near 2.5 on the onion root nucleolus,
there probably is some ampholyte present un-doubtedly a protein or proteins. This protein is not nucleoprotein, as it is
negative to Feulgin[?] (anthor's unpub.) as seems generally to be the case with typical nucleoli. The author believes
that it is this IEP which determines the stainability of the nucleolus. The lower it is, the greater should be the stainability.
This investigation is now proceeding :
The method essentially is Tolstoouhov's [?].
Other[?] authors - Minsinkai, 1939; Shinke and Shigermaga[?], 1933 ; Yasui, 1939 ; - claim the presence of lipoid substances
in nucleoli. Inasmuch as lipoids are universally present in protoplasm, we cannot yet claim an especial iopaction[?] in nucleolus;
none of the investigators claim this. Indeed, Shinke and Shienaiga find fats in "chromosomes, cytoplasm, nucleolus nuclear
reticulum, and probably spindle fibers." There certainly is no basis, as Mensinkai[?] proposed for
[END PAGE TEN]
[BEGIN PAGE ELEVEN]
the formulation of the chemical cycle whereby chromosomal matrix, nucleoprotein is converted into nucleolus, lipoid with a
protein surface so it can be electrically charged. Francini, 1939, in some orchids, finds a red coloration with Ruthenium
chloride, interpreting this as a positive test for pectin or other ploysaccharide. The author is contributing such histochemical
test in the Columbia histology laboratory. If Allium cepa, there is a protein in the nucleolus as would seem he hopes to
find from specific amino acid tests, IEP changes after formalin and HNO2 treatment, and similar analytical methods, something
of its nature and structure. It is inaccessible to analysis in the living cell because of its location and size. In all,
this subject has remained one of the most obscure in cytology and biochemistry.
Abbreviations of Journals listed :
Ann Bot Annals of Botany
B.A. Biological Abstracts
JRMS Journal of the Royal Microscopical Society
QJMS Quarterly Journal of the Microscopial Society
J.M. Journal of Morphology
St. Tech. Stain Technology
Zeits. Zellf Miki Anat Zeitschrift fur Zellforschung und Mikroskopische Anatomie
[END PAGE ELEVEN]
[BEGIN PAGE TWELVE]
Brams, et al. 1936. QJMS 78: 387-395
Chambers, R. 1924 p 268 in "General Cytology", Cowdry ed.
Fikey, M.A., 1930 JRMS 50:387-419
Frankel, O.H. 1937 Cytologia 8: 37-47
Francini "1938" cited in B.H. 12:9070 and 12639.
Gatereby 1938 pg 204-220 in "Evolution", de Beer ed.
Gates 1937 Cytologia 8: ->
Grasse and Lesperon "1938" cited in B.A. 12:14130
Heitz 1931 Plauta 12:775-884; 15:495-505
Hofmeister 1848 Botanische Zeitung 6:425-34, 671-74; V. Sharp.
Ludford 1922 JRMS III : 121-133, et sig, in JRMS.
1934 QJMS 69: 27-58
McClintock, B. 1934 Zeits. Zellf. Miki Anat ; 21:294-328
Macklin 1902 Carnegie Inst. of Wash; Cont. to Embryology #13
Meusinkai 1939 Ann Bot NS 3:763-95 cited in Ludford '22
de Mol 1927 La Cellute 38:1-64
Montgomery 1898 J.M. 15: 265-582
Nakahara 1917 J.M. 29:55-74
Selim, A.G. 1930 Cytologia 2:1-26
Shinke and Shiginaga 1933 Cytologia 5:184-221
Sharp 1934 Introduction to Cytology McGraw-Hill
[END PAGE TWELVE]
[BEGIN PAGE THIRTEEN]
Tolstoouhov 1928 St. Tech 3:49-56
Wilson, E. B. 1925 The Cell in Development and Heredity : McMillan