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CANCER RESEARCH

VOLUME 23 MARCH 1963 NUMBER 3

The Nucleolus of the Cancer Cell : A Reviews

HARRIS BUSCH, PAUL BYVOET,t AND KAREL SMETAN4

(Department of Pharmacology, Baylor University College of Medicine, Houaton@ Texas)

Introduction 314 Identification and Morphology of the Nucleolus 314 Isolation of Nucleoli from Cells 314 Staining of nucleoli for biochemical procedures Methods for disruption of the nucleus Purification of the nucleoli Composition and Internal Structure of the Nucleolus 316 DNA RNA Quantity of Nucleolar RNA Protein Shape, Size, and Number of Nucleoli 3@O Hypertrophy of the nucleolus in tumor cells Other nucleolar changes in tumor cells Modification of Nucleolar Size, Shape, and Number in Nontumor Cells 3@1 Enlargement of the nucleolus Diminution in size of nucleoli External influences on nucleolar RNA Origin of Nucleoli Functions of the Nucleolus 3@4 Synthesis of RNA in the nucleus Role of the nucleolus in biosynthesis of RNA Biochemical evidence for rapid RNA synthesis in the nucleolus Role of DNA in biosynthesis of cytoplasmic RNA Role of the nucleolus in the biosynthesis of ribonucleoproteins Role of the nucleolus in the biosynthesis of protein Nucleolus as a storage depot Relationship of Nucleolar RNA to RNA of the Remainder of the Cell 3@6 â€œMessengerâ€•-RNA s-RNA Ribosomes Discussion 3@8

* The original studies reported in this manuscript have been t Research Associate of the American Cancer Society. supported in part by grants from the U.S. Public Health Serv- Present address: Department of Pharmacology, University of ice, the American Cancer Society, and the Jane Coffin Childs Florida, Gainesville, Florida. @ Fund. Postdoctoral Trainee in Pharmacology, on leave from the Received for publication September 20, 1962. National Academy of Science, Prague. 313 Thia One @1@II@II@\I\\\UII\IIIIU@ POB6-BTA-D1L4

INTRODUCTION species (179, 9249).The technics employed included Several factors have contributed to the in light microscopy of fixed and stained specimens, creasing interest in the role of the nucleolus in the as well as fresh, supravitally stained material, cancer cell. Among these are included the accumu ultraviolet-absorption microscopy of fresh or fixed lating evidence that the nucleoli of mammalian material (9245â€”48), phase contrast microscopy, in celLs (a) occupy a central role in the production or terference microscopy, and electron microscopy. distribution of RNA, (b) have a significant role in Although fixation of tissues produces many arti the production of components of the cytoplasmic facts (130, 314, 4092),cytochemical strains are gen ribosomes, and (c) can now be isolated as purified erally made with fixed material. The solutions entities from both neoplastic and other mam used vary from aqueous solutions, such as formal malian cells. The development of important new dehyde, 1 per cent chromic acid, Zenker's fluid, technics for the isolation of RNA and fractionation Cajal's fluid, or formol sublimate to partly or of nuclear proteins has further increased interest largely organic solutions, such as Bouin's fluid, in both the mechanisms and kinetics of biochemi Zenker-formol, Carnoy's fluid, formaldehyde-ab cal events occurring in the nucleolus. These tech solute alcohol-propionic acid, 95 per cent ethanol, flies are added to previously existing procedures or methanol (68, 158, 196, 9213, 92924â€”928,255,9257, which have served to bring attention to the impor 307, 3928, 3929, 3492, 4092). Fixation by freeze-drying tance of the nucleolus by showing that the nude or air-drying has also been employed. Similarly, a olus had a high concentration of RNA and by large variety of stains is available for studies on permitting determination of its size in specific the nucleolus. Phase contrast microscopy, and cells as well as the effects of changes in cell func especially supravital staining of fresh or living tion on nucleolar size. Staining technics and ultra material, have proved to be the most successful violet-absorption measurements also showed that in making the nucleolus clearly visible. Stains rec the nucleoli of neoplastic cells were increased in ommended for the nucleolus include methylene size by comparison with a variety of other types blue or its homolog Azure C (Fig. 1), Giemsa's of cells. The primary objective of this review is to stain, brilliant cresyl blue and dilute toluidine blue correlate some of the extensive earlier studies with (9231â€”33,9259,9260,9288,301, 3928,374â€”76,386, 387). the biochemical information now rapidly increas Recently, Seman (3927) reported that the borate ing with regard to the nucleolar components, their salt of methylene blue was useful for visualization turnover and functional role in the cell. For more of the nucleolus. More classical examples of this extensive reviews of the morphological aspects of class of stains are hematoxylin or hematoxylin the subject, the interested reader is referred to a iron, and impregnation technics with silver or number of previous excellent reviews (392,33, 158, osmium tetroxide. Tanaka (3792) has successfully 335). used acetocarmine and hematoxylin solution so as to stain both nucleoli and chromosomes. IDENTIFICATION AND MORPHOLOGY OF THE NUCLEOLUS ISOLATION OF NUCLEOLI Nucleoli of mammalian cells are readily visual FROM CELLS ized by a variety of optical procedures, of which Until recently, satisfactory methods for the the simplest is phase microscopy. In unstained isolation of highly purified nucleolar preparations mammalian cells or cell nuclei one can readily were unavailable. The recent reports (50)1 that visualize, within the nuclei, one or more bodies nucleoli can be isolated in reasonable yields from with a different refractive index from that of the liver and neoplastic cells in a reproducible manner remainder of the nucleus. Depending upon the suggest the possibility that direct analysis of the concentration of divalent ions in the medium, the components and functional activity of the nucleo nucleoli may be stippled either lightly or heavily. lus will soon be accomplished. Fundamentally, Some questions have arisen whether the bodies there are three basic problems in the isolation of seen by phase microscopy are identical with those nucleoliâ€”namely, rapid recognition of nucleoli, which are stained by commonly employed meth release of the nucleoli from the nucleus, and puri ods (9217); but there is very little doubt that this fication of the nucleoli away from the attached is the case, since the particles isolated by proce nuclear debris and other nuclear components. dures to be discussed later not only look like those Staining of nucleolifor bÃ¼cheirticalprocedures.â€” in the nuclei, but also are stained with â€œnucleolarâ€•One of the problems in studies on the isolation of stains. nucleoli was the need for a method for rapid stain The morphology of the nucleolus has been ing of preparations. Although a variety of stains

studied in a variety of cells of tissues of different 1 R. Maggio, P. Siekevitz, and G. Palade, in manuscript.

would seem to be potentially useful for this pur which differ considerably in density. One of the pose (9217, 3927), the method which proved most simplest procedures for purification of the nucleoli satisfactory was the simple procedure of mixing a was that of Monty et al. (9250), who permitted the drop of 0.1 per cent Azure C in distilled water or nucleoli and other components of the nuclei to 0.925M sucrose with the tissue preparation (Fig. 1). settle slowly in tragacanth solutions. There are a Within a few seconds the nucleoli were stained a number of problems involved, particularly the deep purple, and the cytoplasmic ribonucleopro long time required (366). Another procedure is teins were stained to a lesser extent (Fig. 1). This differential centrifugation of the nucleoli with the procedure also permitted quantitation of the num use of sucrose gradients or several steps in centri ber of nucleoli and determination of recovery in fugation so as to provide separations of the heavier various steps of the purification procedure.2 nucleolar masses from the lighter nuclear con Methods for disruption of the nucleus.â€”Methods taminants. Recent reports have suggested that for isolation of nuclei from tumor cells and liver similar methods have been effective in the prepa cells have been improved (51, 67), but the condi ration of nucleoli from plants (928, 311). A purely tions required for isolation of nuclei are different chemical method for obtaining a â€œnucleolo-chro from those required for isolation of nucleoli. The mosomal apparatusâ€• was reported by Georgiev et nuclei of tumor cells can be freed from cytoplasmic al. (1926â€”929),whoextracted nuclei with dilute salt tags when isolated in media which do not contain solutions and then subjected the extracts and the divalent ions, but the presence of latter is abso residues to differential centrifugation. They re lutely essential for isolation of nucleoli. According ported that this method permitted the separation ly, in preparation of the nucleoli of tumor cells, it of nuclear components into a number of types of has been necessary to use a less satisfactory prepa particles such as microsomes, ultramicrosomes, a

ration of the nuclei as a starting material. â€˜â€˜nucleolochromosomal apparatus,â€• and the nuclear Sonication is one of the procedures employed sap. As indicated by data from other laboratories, for disruption of the nuclei and has proved useful, nucleoli from a number of nonmammalian species inasmuch as nucleolar morphology and staining are simpler to isolate than those of mammalian characteristics are unchanged (9250). Another pro cells (928, 311). cedure employed is grinding nuclear preparations The criteria for purity of nucleoli are not yet between a rapidly rotating plate and a stationary completely established because of the presence in one separated by a very narrow spaceâ€”i.e., about nucleolar preparations of a mass of chromatin 0.0001 inch apart. Poort (9291â€”93)has devised an which is related by some authors (9254) to the

instrument called a â€œpankerâ€•inwhich the distance â€˜â€˜nucleolus-associated chromatinâ€• (1923) . Appar between the rotating plate and the stationary one ently, the nucleolus is embedded in a substantial is controlled by a micrometer. However, this pro mass of Feulgen-positive chromatin which not cedure has not been widely successful for the de only is in close proximity, but also forms a con struction of nuclei. Another method used is that densed surface substance that may prevent the of Morin et al. (9254), who isolated nuclei with the nucleolus from destruction. In media which do not aid of polyvinylpyrrolidone and osmotic shock; the contain divalent ions the nucleoli are readily de nuclei were then suspended in a viscous dextran stroyed by sonication, but in media that contain @ solution and were agitated in a mixer. The homog substantial amounts of Ca@ (0.005 M) or enate obtained was apparently a mixture of the nucleoli resist destruction by sonication. broken nuclei and the nuclear components. In re Under these circumstances, either the chromatin cent studies on plants, use has been made of closely around the nucleolus that forms a Feulgen-posi pressed rollers to break up nuclei (928, 178, 311). tive shell, or the ribonucleoproteins of the nucleo Purification of the nucleoli.â€”Several methods lus become condensed to the point that differential have been employed to purify the nucleoli away destruction of nuclear components is possible. In from the nuclear components, and in each case the almost all experiments on isolation of nucleoli high method is based upon the assumption that the concentrations of DNA have been found to be nucleoli are more dense than the other nuclear present in the final preparations (9250). In part, components or than the nuclei themselves. While this may result from the inability of available this may be true in most instances, it is always methods to remove the contaminating chromatin found that there are some nuclei which are sedi of high density from the medium. However, it may mented in very low force fields (less than 100 Xg) also be possible that the DNA associated with the and that the nucleoli are a population of particles nucleolus may not be a random contaminant but 2M. Muramatsu, K. Smetana, and H. Busch, Cancer Re actually may be a highly specific segment of the search (in press). DNA which differs functionally from the other

chromatin of the nucleus (9254). This intriguing strands in the nucleoli which are apparently de possibility is under study. The values for DNA rived from â€œnucleolarâ€• chromosomes. Pretreat and RNA in isolated mammalian nucleoli are pre ment of their tissue-cultured cells with adenosine sented in Table 1. was required, and the method of fixation was im portant in revealing the Feulgen-positive threads COMPOSITION AND INTERNAL STRUC inside the nucleolus. O'Donnell (9271) has studied TURE OF THE NUCLEOLUS the nucleoli of Spirogyra and has shown depoly DNA.â€”From the descriptions of the relation merization of nucleic acid following treatment of ships between the chromosomes and the formation the nucleoli with either DNase or RNase. Follow of nucleoli to be presented later, it would seem prob ing treatment of the nucleoli with RNase, fibers able that DNA would be a constituent of the nu were noted in the nucleoli which were Feulgen cleoli, either as a part of the surface of the nucleoli positive and were presumably composed of double or as a part of the inner core of the nucleolus.3 On thin chromonemata from the â€œnucleolus-organiz the basis of results obtained with the Feulgen stain erâ€•chromosomes. In the Spirogyra there are ap the general opinion has been that the nucleolus parently two chromosomes which are responsible does not contain any DNA but is surrounded by a for formation of nucleoli, one short and one long; shell of the so-called â€œnucleolus-associated chro these produce a small and large nucleolus, re TABLE 1 CoMPosITIoN OF THE NUCLEOLUS Values presented are percentages of the dry weight or pg. per nucleolus.

cent TissueRef.RibonucleicComponentPer . dry weightpg/ Nucleolus.

acid7 cells 63, 9.22) 14 Rat liver (344) (246)Deoxyribonucleic 1â€”1.5 Rat liver 3.9 Rat liver* 5.80.5 2.4MammalianWalker tumor*(61,

acid12â€”18 liver 7.8 Walker tumor* Ratliver*(9.46)Protein70 10.43.2 1.5Rat

liver 74 Rat liver* 8713 38Rat Walker tumor*(344â€”46)

* M. Muramatsu, K. Smetana, and H. Busch, Cancer Research (in press).

matinâ€• or heterochromatin (334, 885, 357, 366, spectively, from which the chromonema inter 380). An increasing number of reports have mdi twined with the nucleolar mass is presumably cated the presence of Feulgen-positive structures derived. inside the nucleolus (1923, 9213â€”15,9257,9271, 366, Precise quantitative estimates of DNA associ 380). DNA-containing threads or granules can also ated with nucleoli are not yet possible because of be found inside the nucleolus. Indeed, the adrenal the difficulty of isolation of completely pure nucle cortical cells of the pigeon have been reported to oli. The high concentration of DNA in the nucleoli contain nucleoli which do stain with the Feulgen of liver cells reported by Monty et al. (9250) has technic (927) but not with stains for RNA. LettrÃ© been questioned, but part of the relatively high and Siebs (9213â€”15)have found that nucleoli of concentration of DNA may have been due to the tumor cells and fibroblasts in tissue culture contain fact that some of the RNA may have been lost by nucleoli which stain positively with methyl green; their extraction procedures, particularly those in they suggest that chromosomal material is pres which solutions of low ionic strength were used. ent. They have noted the presence of filamentous Moreover, no calcium or other divalent ions were added to their medium for isolation of the nucleo 3Some cytologists suggest that chromatin within the nucle olus should not be considered to be nucleolar substance. How li, and the lack of such ions may have resulted in ever, precise analyses of the composition of DNA within the solubilization of the ribonucleoproteins of the nu nucleolus have not been possible, in view of previous difficulties cleolus. Although they found that the DNA com in isolationofnucleoli.Withimproved technicsforisolation of nucleoli, it is possible that a â€œspecificDNAâ€•may be isolated prised approximately 192â€”18per cent of the dry from the nucleolus. weight of the nucleolus, similar experiments in this

Downloaded from cancerres.aacrjournals.org on September 25, 2021. © 1963 American Association for Cancer Research. BUSCH et al.â€”Nucleolus of the Cancer Cell: Review 317 laboratory have shown that DNA comprised less by silver-impregnation and electron-microscopic than 10 per cent of the dry weight of the nucleolar studies. In these studies a filamentous network preparations of the liver4 and only about 7.5 per called â€œnucleolonemaâ€•(Figs. 3â€”9)continuous with cent of the total dry weight of nucleolar prepara the nucleolini (105, 92926)wasfound to be surround tions of the Walker tumor. As indicated previous ed by and embedded in a structureless mass, the ly, some of this DNA appears to be condensed on â€œparsamorphaâ€• (925,926,36, 89, 141). The nucleo the surface of the nucleoli, but it is probable that lonema has been related to the nucleolini or intra some is present as chromosomal tags adherent to nucleolar vacuoles by Estable and de Sotelo (104, the nucleolar masses. It seems probable that DNA 105). Love and Suskind (92928)suggested from their directly in the nucleolus would stain only faintly studies that at least nine different types of ribo with the Feulgen stain, since it would be diluted nucleoproteins exist in animal cells, of which one with other nucleolar components such as RNA and type, the perichromosomal RNA, apparently sur protein or be confined to structures too small to be rounds and outlines the nucleolus (92927).Improve resolved with the light microscope. ments in this technic have recently been described RNA.â€”In cytochemical studies on the RNA (9292@5).Evidencehas been provided for the presence content of the nucleolus a number of dyes have of other specialized particles in the nucleoli of the been used including methyl green-pyronine, meth plant Aloe vera (364). Dense particles in the nucleo ylene blue, toluidine blue (Fig. 92),Azure B, and Ii have been noted in electron microscopic studies gallocyaninechromalum (16, 147, 158, 92926,9278, (9205, 366). 340, 358, 366). These dyes are less specific than the Among the five RNA moieties, which, according Feulgen stain, inasmuch as they react with both to Love, exist in the nucleus (92924-928),the nuclear DNA and RNA. Methyl green and also pyronine, and nucleolar parachromatin seem to be strongly for example, will react with either DNA or RNA related. The other three RNA moieties are the depending on the conditions under which the reac nucleolarâ€”â€•pars amorphaâ€•â€”RNA, the chromo tion is carried out (9288). An increased specificity somal, and the perichromosomal RNA. It was of these dyes has been achieved by pretreatment noted that in some cell species the â€œparsamorphaâ€• of the material to be stained with RNase or showed a marked tendency to persist throughout DNase. Extraction with trichloroacetic acid, per the mitosis (92928),incontrast to the nucleolar para chloric acid, KOH, or HC1 before staining has also chromatin, which was probably extruded, inas been employed (365). much as it disappeared in the early stages of Using such methods, Brachet (39, 40) found cell division. Estable and de Sotelo (104, 105) that the pyroninophilia of nucleoli disappeared have suggested that the nucleolonemata persist after RNase treatment. Caspersson and â€¢Schultz throughout mitosis. Swift (366) distinguished (63) also concluded that RNA was present in the three types of RNA-containing materials inside nucleolus when they found an ultraviolet-absorp the nucleus : chromosomal (extra-nucleolar) ribo tion spectrum typical for nucleic acids in the Feul nucleoprotein, spindle remnants, and nucleoli. gen-negative nucleolus. Although since then nu In contrast, LettrÃ©and Siebs (9213â€”15)suggest merous cytological studies indicated that the nu ed that the nucleolonema consists of parts of the cleolus contains much of the nuclear RNA, few nucleolar chromosomes and that the â€œparsamor quantitative investigations have been published. phaâ€• represents the RNA or ribonucleoprotein A differential staining technic for nucleoproteins which is being synthesized or accumulated by the of HeLa cells was recently developed by Love and nucleolar chromosomes. The finding of two types Liles and has shown the presence of two different of RNA (92926,359,360,881) inside the nucleolus is types of ribonucleoproteins inside the nucleolus in agreement with the results of several studies, (92924â€”928,359,360).Oneof these, in the â€œparsamor which indicated the presence of two metabolically phaâ€• was distributed diffusely throughout the different types of RNA inside the nucleus and the nucleolus, and the other, consisting of masses of nucleolus and which will be discussed in more de ribonucleoprotems present in or around nucleolar tail in later sections. vacuoles, is apparently identical to the â€œnucleoliniâ€• Quantity of nucleokir RNA.â€”Almost all the his referred to in older literature. These authors relat tological evidence obtained in the last 30 years has ed both types of ribonucleoproteins to the struc shown that there is a heavy concentration of RNA tural morphology of the nucleolus as revealed in the nucleoli (39â€”48,61â€”68,384,385,366). At the moment, there is still considerable uncertainty as 4Valu@ for the liver are average values for liver cells. The polyploidy of liver cells affects the size and components of the to the precise amount per nucleolus and the pre nucleolus.Thefusednucleolusofoctaploidcellsislessthan4 cise number of types of RNA present in the nude times larger. than the nucleolus of diploid liver cells. oius (Table' 1). Early studies of Mauritzen et al.

(9239) established that the RNA in the nucleus of the liver cellâ€”i.e., 92.4pg. as compared with 0.5 comprised about 4 per cent of the dry weight of pg. for the liver cell.2 In growing plant cells, the the nucleus and this value has been confirmed in nucleoli have been reported to contain 1.4 times this and other laboratories. In the nucleolus, as much RNA as DNA (928). Monty et al. (9250) reported that RNA comprised The type ofRNA in the nucleolus is not yet corn 1â€”1.5per cent of the dry weight, although ultra pletely defined. As noted previously, histochemical violet absorption studies had suggested that RNA studies have suggested the presence of two types comprised up to 7 per cent of the nucleolar dry of ribonucleoproteins in the nuclei of a number of mass (61, 63, 92926,380). In studies in which a com different types of cells (2926, 92927,9236,359, 360). bination of ultraviolet and x-ray absorption tech Osawa et al. (9276)and Logan and Davidson (929292) nics were used, it was reported RNA comprised 92 had indicated that there were two types of RNA per cent of the nucleolar dry matter (9267).Stenram fractions in nuclei which could be isolated by cx used interferometric and refractometric methods tracting cells with 0.15 Mand 92.0MNaC1 solutions. to determine that the concentration of dry matter These methods are now commonly employed to in the nucleolus was 920â€”30per cent (19, 353). Of extract first the ribosomes and then the deoxyribo this mass, approximately 14 per cent was reported nucleoproteins from cells. Later, Sibatani et al. to consist of RNA (351), and, hence, RNA would (3392,333) and Yamana et al. (411â€”13)fractionated comprise 5â€”7per cent of the total mass. In studies RNA into two fractions with the aid of the Kirby on isolated nucleoli of oocytes, Vincent (379) technic. The homogenate of the tissue was initially found that the RNA content ranged from 1.7 to 8 shaken with phenol saturated with phosphate buf per cent of the dry weight of nucleoli, with smaller fer, and the phenol layer was separated from the values being found for immature nucleoli of small aqueous layer. The phenol layer was then extract er sizeâ€”i.e., with a diameter of 92.5 z than for the ed with phosphate buffer and the extracted RNA mature nucleoli with a diameter of 10 p. The per was referred to as p-RNA whereas the residue re centage of dry weight comprised by RNA of nude maining either at the interphase or in the phenol oh isolated from media containing sucrose was was referred to as r-RNA. The p-RNA has a high approximately 5 per cent (17, 18, 380). In studies concentration of guanylic acid and comprised a in this laboratory, RNA of the nucleolus of the larger amount of the total RNA in neoplastic than liver cell was found to comprise 3.5 per cent of the in nontumor tissues. However, the turnover rate dry weight, and the RNA of the nucleolus of the of the r-RNA was considerably higher than that tumor cell comprised 5.3 per cent of the dry of the p-RNA. These results were obtained in tu weight. mors as well as normal liver (3392, 333, 411â€”13). The discrepancies in the values obtained by the Recently, Sibitani et at. (331) found that phenol histochemical methods and the direct analyses treatment of thymus nuclei produced similar re may be related to the washing out of RNA or ribo suits. In further studies, they treated the residual nucleoprotein in the course of the purification pro fraction with phenol after extraction of calf thy cedures by which the nucleoli are isolated as well mus nuclei with Tris buffer and 1 M NaCl to re as to the large mass of deoxyribonucleoprotein move ribonucleoproteins and deoxyribonucleopro which is adherent to or part of the nucleolus. Al teins. After incubation of the cells with labeled though the cytochemical technics do not suffer RNA precursors, they found that the r-RNA of from these problems, a critical problem is the pre this fraction had the highest specific activity of all cise standardization of the cytochemical methods. the RNA fractions studied, and, moreover, the At present it would appear that the RNA of the base ratio of this fraction was remarkably similar to nucleolus comprises between 5 and 14 per cent of that of DNA (Table 92).They suggested that this the dry mass; the gap between these determina fraction is equivalent to â€œm-RNAâ€•ormessenger tions may be closed as methods for isolation and RNA. Although morphological evidence was not purification of nucleoli improve as well as the cor presented that this RNA is a nucleolar component, responding cytochemical analyses, although some the authors have referred to the very highly active variation may be dependent upon cell function. RNA as â€œnucleolarRNA 92â€•onthe grounds that Interestingly, the average DNA content of the the nucleolar components would not be extracted nucleolus of the Walker tumor cells was twice that by the procedure which they used in the salt ex of the liver nucleolus, inasmuch as the former con tractions; moreover this fraction contained 192â€”14 tained 3.92 pg. per nucleolus, whereas the latter per cent RNA by weight. contained 1.5 pg. per nucleolus. However, the nu Protdn.â€”The presence of proteins inside the cleolus of the Walker tumor cell contained an nucleolus has been demonstrated by means of average of almost 5 times as much RNA as that ultraviolet absorption (61, 63), x-ray absorption

(9267),interferencemicroscopy(383),and various protein in the nucleoli of the Walker tumor and stains like bromophenol blue (92492,388), Fast the liver were 87 and 74 per cent of the lipide-free green (367), and the Millon-reaction (9290,367). dry weight, respectively. It was also found that the Although several reports have indicated a high nucleolus contains both acid-soluble and acid-in percentage of basic proteins present in the nucleo soluble proteins, of which the latter are present in lus (61, 63, 92492,3929),others have questioned this significantly greater concentration than the for (380). Pollister and Ris (9290) pointed out that, mer, which may be histones linked to the chroma after removal of all histone-material, the nucleolus tin present in the nucleolar preparations. On the showed only a slight decrease in tyrosine-content, basis of incorporation of isotope, the acid-insoluble as determined by the Millon-reaction; histones nucleolar proteins would appear to be significantly contain very little tyrosine (47, 48). Using the different from those of the remainder of the nu Fast green technic, Horn and Ward provided evi cleus. It remains to be seen whether either of these dence that some basic proteins were present in the groups of proteins has a molecular weight equiva nucleolus (171). lent to that found in the proteins of the cytoplas Among the reports indicating the presence of mic ribonucleoproteinsâ€”i.e., about 925,000 (87) lipoproteins in the nucleolus (92, 19) is that of which is similar to that of the nuclear histones Zagury (4920), who found liporibonucleoprotein (47-49). complexes containing sulfhydryl groups in the cor Such studies as have been carried out on the

TABLE 9. NUCLEOTIDE COMPOSITION OF NUCLEAR RIBONUCLEIC ACID FRACTIONS OF CALF THYMUs (881)

MOLESTOTALNUCLEOTIDEPRESENTA5

MATERIAL -, A+LG+CSPECIFIC ACTIVITY ANALYZEDMoLsn/100 . . . AdenylicRNA. Uridylic Guanyhc Cytidylic OF acidâ€œNucleolarâ€• acid. acid. acid.

RNA Total:

Fraction! 18.9 19.1 34.2 28.0 87.9 62.1 1,760 Fraction!! 9.7.5 9.9.0 23.8 19.6 56.5 43.5 19,000 DNA9.1.8 27.69.3.9.28.3 22.69.5.7 19.945.0 55.956.9. 42.56,240 (thymine)30.5 tical zone of the nucleolus. Albertini (92) studied enzymes of the nucleolus have used cytochemical the sulfhydryl groups of nucleolar proteins with methods (90, 131). By reduction of formazan dyes the aid of tetrazolium dyes and found that the evidence has been obtained for the presence of nudeolar surface showed intense staining, whereas succinic dehydrogenase in the nucleolus (90). In the concentration of sulfhydryl groups in the cen nucleoli of muscle cells, phosphatase activity has ter was much lower. On the basis of these studies been reported to be increased both by denervation (92,4920), Albertini suggested that there was a cor of the muscle and by injection of specific nucleo relation between the synthetic activity of the cell tides such as nicotinamide-adenosine-8'-mono and the number of -SH groups on the nucleolar phosphate (131). There are a variety of concepts surface. which have been presented regarding the func Very little biochemical information is available tions of ribonucleases in the biosynthesis of pro about the proteins of the nucleolus, despite the teins, in the biosynthesis of nucleic acids, and in fact that they are by far the largest component of the degradation of various types of RNA (77,9207). the nucleolus. Estimates of the protein content of Most of the concepts have relatively little founda nucleoli have ranged from 40 to 90 per cent (9267, tion other than the concept that the ribonucleases 9290,379, 381, 383). Since the maximal values for of the gastrointestinal tract function to hydrolyze RNA and DNA have been reported to be 14 and nutritionally available ribonucleic acid. Recently, 18 per cent, respectively, of the dry weight of the highly purified RNases have been obtained from a nucleolus, the proteins may account for approxi number of sources (15, 9240,344,369), and remark mately 70 per cent of the dry weight. In studies in able studies have been made on the sequences of this laboratory, the values for the percentage of amino acids in these proteins (9, 9208, 369, 388,

401). The presenceof inhibitorsof RNase in cells which aid in their recognition. One of these fea has also been demonstrated (77, 317, 318). Thus tures is the enlargement of the nucleolus which is far, however, it is difficult to find studies on the proportionally in excess of that of the nucleus (692, concentration or role of ribonucleases in the nude 892,115,183, 9214,9231â€”33,301,3920,3921,419). olus. At present, the role of RNA polymerase (177, Careful measurements carried out by Stenram 400) in the nucleolus is also unknown. (349, 850) revealed that the nucleolar volume of azo dye-induced rat hepatoma cells was approxi SHAPE, SIZE, AND NUMBER mately 8.8 cu. @L,ascompared with 1.8 cu. j@for the OF NUCLEOLI corresponding normal liver cells. However, this The shape of the nucleolus is dependent upon the enlargement does not appear to be pathognomonic procedures used to prepare either cells or nuclei for malignancy (16, 83, 9230, 3928). As Caspersson for observation. As Kopac and others have pointed and his colleagues showed (61â€”63),a large nucleo out (199), in some preparations, the nucleoli may lus accompanies active protein synthesis, as can be have rounded or spherical shapes, but in other seen in growing or secreting cellsâ€”e.g., in regen preparations the variety of shapes presented by crating rat liver or pancreas (349, 361, 3692). The nucleoli is great (179, 199, 9249). Perhaps the most nucleolar volume of rat liver cells increases or de physiological preparations of cells and cell nuclei creases depending on whether the animals are are those suspended in saline solutions or in tissue placed on a high protein diet or starved. When the cultures and in these the nucleoli are frequently rats were fed a diet low in protein, the size of the triangular, with a number of projections some of nucleoli increased even more than on a high pro which extend to the nuclear membrane (9213â€”15). tein diet (348â€”54). On a low protein diet, the aver Occasionally, the nucleoli appear to be clearly as age nucleolar volume of liver cells reached a vol sociated with the nuclear membrane (Fig. 3). ume of 3.3 cu. .t, a value close to that of the hepa The number of nucleoli in cells is variable. Earli toma cells. Stenram noted that the percentage of er studies by Woods (407) showed that in plants dry matter in the nucleoli of rat liver cells was ap there were as many as 928nucleoli in a cell. Haenel proximately 37 per cent and that of RNA was (147) has found as many as 40 nucleoli in mega approximately 14 per cent and did not change on karyocytes. In studies on the number of nucleoli a low protein diet. There must have been a con in the Walker tumor, it was found that there were siderable excess biosynthesis of nucleolar material, an average of 1.5 nucleoli per nucleus, with a range especially ribonucleoprotein. This effect apparent of one to six nucleoli per nucleus. In most of the ly was caused by a lack of certain essential amino cells at least one large nucleolus was present. In acids in the diet and was not seen in tumor cells. normal liver cells, approximately two nucleoli were The feeding of azo dye carcinogens increased the present per nucleus, and the range was one to six size of the nucleoli of the normal liver cells, which nucleoli per nucleus. In normal human lympho became even larger on a nonprotein diet (63, 351). cytes there is one nucleolus per nucleus (140, 141, Early nucleolar enlargement during carcinogenesis 340, 358), and in leukemic lymphocytes an in in the skin of mice has also been reported by creased number is found; this is also the case in Cooper (80) and Cowdry and Paletta (83) ; this lymphocytes of infants and young children. In enlargement may be related to hyperplastic, studies on HeLa cells in tissue culture, Swift (366) rather than neoplastic, changes. reported that the number of nucleoli per cell was Other nucleolar changes in tumor cells.â€”Though related to the medium in which the cells were in less common than hypertrophy, a number of other cubated. In pure salt solutions, the number of nu changes of the nucleoli of malignant cells have cleoli per cell averaged 3.7 and ranged from 1 to 8. been reported. Irregularity in shape of tumor nu However, on incubation of the cells in a nutrition cleoli (16, 115, 92929,301, 307, 3921, 419) has been ally adequate medium the number of nucleoli de noted but has also been observed in a number of creased to an average of 1.7 per cell. In addition rapidly growing nonmalignant tissues (16, 1192, to the change in number, the nucleoli increased in 3920, 361, 3692, 366). An increase in number of nu size and became circular in outline. Among the cleoli (16, 83, 301) may be related to the aneu procedures which affect the number of nucleoli as ploidy of cancer cells (892, 9269), but does not seem well as their size in cells are treatment of animals to be typical for malignancy (16, 387, 419). A with thioacetamide (196) and partial hepatecto splitting up of the nucleolar substance into one to my (197, 366). eight small spherical globules was noted when Hypertrophy of the nucleolus in tumor cell8.â€”A1- HeLa cells were cultured in salt solutions lacking though cancer cells may vary morphologically, amino acids (366). Dissociation of the nucleoli of they have some morphological characteristics cultured cells into small pyroninophilic globules

has also been observed after treatment with adeno in number (16, 301), irregularity in the shape of sine (9214). Following treatment of leukemic pa the nucleolus-associated chromatin (307), fine nu tients with folic acid analogs, the nucleoli dimin cleolar strands between nucleoli, and abnormality ished in size in white blood cells (147). of position of the nucleoli (16). In normal cells the An increase in intranucleolar bodi,.@sin tumor position of the nucleolus is also variable, as has cells was reported by Page et at. (9277), who found been shown with the aid of refined cinematograph an increase in number of both refractive and ar ic technics (9215). gentophile bodies; they doubted that these were identical structures, although previous reports had MODIFICATION OF NUCLEOLAR SIZE, suggested that the refractive bodies were the SHAPE, AND NUMBER IN â€œnegativesâ€•ofthe argentophile bodies (819). In a NONTUMOR CELlS critical study, Lewis (9216) could not find a con Enlargenient of the nucleolus.â€”Both increase in sistent difference between the number of nucleolar size and abnormality of configuration of nucleoli vacuoles of normal and malignant fibroblasts. have been reported for a large number of rapidly An interesting aspect of nucleolar structure re growing nonmalignant tissues, such as the corpus lated to the age of cells was reported by Soudek luteum (386) , megaloblasts of pernicious anemia (343), who found the presence of vacuoles in the (147), abnormal lymphocytes in infectious mono nucleolus (Fig. 4) and related their number to the nucleosis and pleural exudates (340), proliferating age of the nucleolusâ€”i.e., the more vacuoles pres endometrium, fetal tissues, granulation tissue (16, ent, the greater the age of the cell. An increase in 1192), and regenerating rat liver cells (361, 3692). the number of intranucleolar vacuoles of normal Nucleolar enlargement in nonmalignant cells has cells has been produced by irradiation (9286,3923), been produced in a number of ways in addition to cholinergic stimulation of the mouse pancreas feeding carcinogens or a nonprotein diet. A re (416), treatment of cells with acridine derivatives markable increase in size of liver nucleoli has been (9209),and growing cell cultures at low tempera observed after partial hepatectomy (361, 3692), tures (9286). The relation of the vacuoles to intra which became very striking after a pretreatment nucleolar argentophile bodies is not certain ; re with thioacetamide (146, 194, 196, 197, 304). Fol ticular structures have been observed in various lowing repeated intraperitoneal injections of pilo normal and malignant cell species with the aid of carpine, the nucleoli of the mouse pancreas were both silver-impregnation technics (104, 105, 141) found to be significantly hypertrophic (416). and electron microscopy (925, 86, 1492). The fila Giant nucleoli developed after incubation of tissue mentous structure found is probably equivalent to culture cells with 0.925 per cent colcemide (169, the nucleolonema, the nucleolar network of fibrils; 361) and in tumors after treatment with fluoroura thus far, no constant ultrastructural differences cil (9219). have been found between nucleoli of tumors and Huge nucleoli have been found in the nuclei of other tissues with the aid of the electron micro animals treated with thioacetamide for a period scope (925,1492). and then hepatectomized. In the cells of these Prominence of the metachromatic nÃ¼cleolar livers there was a marked decrease in the protein parachromatin in tumor cells was recently report and RNA content of the microsomal fraction ed by Love and co-workers (92924-928)and Stolk which contains the ribonucleoprotein fraction. The (359, 360). These authors used the differential block in formation of ribonucleoproteins of micro staining technic for nucleoproteins noted previous somes (9206) induced by thioacetamide parallels the ly to distinguish two types of ribonucleoproteins increased content of ribonucleic acid of the nucleoli inside the nucleolus. One of these was distributed of these cells and provides another kind of evi evenly throughout the nucleolus, whereas the nu dence which suggests that the nucleolus occupies cleolar parachromatin was present in the form of an important role in the biosynthesis of these par small metachromatic staining granules or vacu ticulates. Among the noteworthy effects of thio oles. The latter structures, which the authors acetamide are the remarkable increase in amount proved to be identical with the â€œnucleoliniâ€•ofthe and the suppression of biosynthesis of r-RNA in older investigators (104, 105), appeared to be espe the nucleus as well as the marked suppression of cially prominent in transplantable tumors and release of the RNA which had been synthesized in Ehrlich ascites cells (9236) as well as intestinal tu the nucleus.3 mors induced by injectiOns of deoxyribonucleic Another cause of nucleolar enlargement is irrad acid (359â€”60). iation, as has been reported by Scherer et al. (3923). Other less frequently reported features of the An increased size of the nucleolar apparatus was nucleolarapparatusof tumor cellsare:variation noted in the spleen, bone marrow, and ascites tu

mor and was said to be due to an increase in the creased RNA synthesis in Euglena exposed to RNA content of the nucleoli. Recently, Skreb and light ; they suggested that the RNA represented Bevilacqua (339) have reported that irradiation ribosomal material essential for synthesis of pro produces a remarkable disintegration of cytoplas teins of the plastids. Vogt-Kohne (384) carried out mic RNA in enucleated amebas. Enlargement of studies on nucleolar RNA content in Chironomus the nucleoli of the liver has also been found when thummi, as well as the changes resulting from cool mice and rats have been fed diets low in essential ing, hunger, and overfeeding. A number of studies amino acids (348â€”54),but the number of nucleoli have shown a relationship between the synthesis did not change. These data are somewhat different of ribonucleic acids and the external influences on from those obtained with HeLa cells, as indicated cells such as the reduction of RNA synthesis by previously (366). As was reported previously by nicotinamide (310), the increased RNA synthesis Stenram (348â€”54),the quantity of RNA per nude in the livers of starving animals or animals bearing olus did not change appreciably. This phenomenon hepatomas (57, 58), and the increased synthesis of of increased nucleolar size seems somewhat of a RNA brought about by enzyme induction as in the paradox, since one might expect a decrease in case of tryptophan pyrrolase (107â€”10,143). How RNA synthesis if there were a decreased supply of ever, the role of the nucleolus in many of these bio amino acids and other nutrients, rather than a synthetic reactions remains to be clarified, particu stimulation of RNA synthesis. It is possible, how larly since functions have not been identified for ever, that there was a decrease in the release of many of the RNA's (116). If the nucleolus is a RNA in these cells. That suppression of RNA syn major center of biosynthesis of cytoplasmic RNA, thesis may occur in the presence of inhibition of it may be influential in the reactions involved in protein synthesis is evident from studies on viral maintenance of ATP levels in cells (410), the re RNA synthesis (399). In studies on bacteriophage, sponse of cells to growth hormone which apparent fluorophenylalanine produced a suppression of ly induces an increase in the biosynthesis of ribo viral multiplication and also produced total sup somes (9200â€”9203),maintenance of â€œmemoryâ€•(81), pression of the synthesis of RNA induced by the the reproductive capacity of cells (1921), the pro virus ; under these conditions, however, incorpora duction of hemoglobin in megaloblasts (147) and tion of labeled lysine into proteins of the bacterial perhaps even the production (59, 60) or mainte cells was decreased only to the extent of about 50 nance of the non-neoplastic state (147, 9263). per cent. Diminution in size of nucleoli.â€”These are few ORIGIN OF NUCLEOLI reports of decreases in size of nucleoli as a result In mitosis, the nucleolus apparently disappears of one or more types of treatment. However, total as an organized cellular structure in mammalian disintegration or fragmentation of the nucleolus cells. Although there are a number of different has been observed following incubation of cells opinions as to its fate, such as disintegration into with adenosine or some of the nucleotides (1792, smaller bodies of subnucleolar size or total loss of 9213,9214). The fate of the components of the nucle nucleolar structure, the precise fate of the sub oli under these conditions, as well as the fate of the molecular or submicroscopic particles comprising nucleolar components in the course of cell division, the nucleolus is not known.6 Love (92924)has mdi is not clear at present. cated that in prophase most of the parachromatin External influences on nucleolar RNA.â€”The re diffuses into the cytoplasm and is not reincorpo sponsiveness of nucleoli to a variety of external rated into the nucleus in telophase. At the same influences has been discussed in terms of the effects time, there is a decrease in the parachromatin of of media on the size and number of nucleoli. the cytoplasmic granular RNA and an increase in Adaptation of the RNA content of cells to environ perichromosomal RNA. He has suggested that in mental conditions has been reported by Rasch et mitosis there is an exchange of RNA between the al. (303), who noted that the nucleolar volume and cytoplasm and the nucleus. In preprophase and RNA were about 50 per cent of normal values in prophase there is accumulation of RNA and pro the retinal ganglion cells of animals which were tein in the nucleolus of a number of types of cells reared in the dark. Similar effects to those noted (405, 406) at the same time as there is accumula in ganglion cells were also noted in the bipolar cells tion of deoxyribonucleoprotein in the nucleus. and receptor cells. In animals exposed to light for During the period of chromosomal separation short periods of time each day the values were there is a marked loss of cytoplasmic RNA, chro intermediate. Similar results for total cellular 51n nonmammalianspeciesnucleolimay persist throughout RNA have been recently reported by Brawerman mitosis,maybe extrudedintothe cytoplasmor may disperse et al. (44) and Pogo et al. (9289),who noted an in rapidlyatthebreakdown ofthe nuclearmembrane.

mosomal protein, and total nucleolar RNA and capacity to produce nucleoli. Interestingly, there protein as measured by cytochemical studies (9241). was an increase in cytoplasmic dry weight and A marked increase occurs in the synthesis of cytoplasmic RNA when there was an increase in nucleolar protein and RNA in telophase and the the number of nucleoli. Evidence for the specificity post-telophase period, along with the increase in of nucleolus-organizing chromosomes has also been the nuclear content of DNA and histone in Tetra obtained for cells of the guinea pig (9274). Although hymena and Acetabularia (9296â€”98).During mitosis there is ample evidence for the function of a â€œnu it was found that there was essentially no bio cleolus-organizer,â€• there is little evidence in mam synthesis of RNA (300), although there was con malian cells as to the precise chromosomes in tinued protein synthesis. During telophase the nu volved or the mechanism by which the nucleoli are cleolus is reformed in specific regions of specific limited in size and number. chromosomes (91, 155) ; the regions of the chromo In chromosomes of Chironomus, a genus of mos somes involved are referred to as â€œnucleolus-or quito-like flies or gnats, there are apparently a ganizing regionsâ€• and have been reported to be number of loci which are involved in the formation â€œsecondary constrictionsâ€• of the chromosomes of special RNA's ; these may be heterochromatic during telophase. At this time, the chromosomal regions and the kinetochores of the chromosomes threads are apparently pushed apart by the nude (187). Some of the characteristics of these gnats olar products (920, 61, 69, 70, 91, 9278). The phe are related to the specific regions of the chromo nomena involved in the formation of the nucleoli somes, including secretions of the salivary glands. are believed to be similar to those involved in for In a very interesting study on Balbiani rings of the mation of other RNA-containing structures such Chironomus, Beermann (921)has related the pro as the chromosomal bands, puffs, and Balbiani duction of specific secretory granules to a specific rings (184, 366). As indicated previously there is chromosomal locus which produces a Balbiani ring. evidence that DNA strands are interlocked with The glands active in the production of normal sali the RNA of the nucleoli and there are DNA vary secretions contain four cells which produce threads throughout the nucleolar mass, as well as granules called SZ granules. In a simple Mendelian DNA in the â€œnucleolus-associated chromatinâ€• recessive of a cross between two species of Chi (184). ronomus, the cells producing the SZ granules Evidence that specific chromosomes are respon either are not present or are not active, and as a sible for the formation of nucleoli has been ob result the secretion differs in type from that of the tamed in a variety of of mutants of Triticum aesti normal flies. Apparently, the region of the chromo vum, a species of wheat. This species has a great somes which controls the production of the cells variety of chromosomal aberrations including producing the SZ granules produces a specific Bal monosomes in which there is one instead of the biani ring in chromosome number 4. The absence two chromosomes of a given pair, tetrasomes in of the Balbiani ring is associated with an absence which there are four chromosomes in place of the of these cells. Thus, the BR4 (Balbiani ring on normal two of a pair,and a varietyof other chromosome 4) is a normally activated locus which types (92923).Normally, four of the chromosomes controls a specific function ; presumably other produce nucleoli, and the average of nucleoli in puffs and possibly larger RNA masses such as the normal cells is about 1.5. When the tetrasomics nucleoli are biochemically active loci by virtue of were examined, those containing twice the normal which the chromosomes express their genetic in number of chromosomes I and X had three nucle formation. oli per cell. However, if the tetrasomic contained Evidence that the chromosomes are capable of twice the normal number of chromosomes XIV forming a multiplicity of nucleoli has been present and XVIII, there were two nucleoli per cell. When ed above, and, in a sense, such findings suggest other chromosomes were tetrasomic, such as chro that the capacity to form nucleoli is not limited to mosomes XI and XXI, there was no increase in the a single chromosomal locus. As Swift (366) has number of nucleoli per cell. These studies suggest indicated, the number of nucleoli markedly in that there are â€œstrongâ€•nucleolus-forming chromo creases in HeLa cells when the cells are placed in somes and â€œweakâ€•nucleolus-forming chromo nutritionally inadequate salt solutions, but appar somes. Doubling of the strong nucleolus-forming ently in normal media a number of these are delet chromosomes produced a doubling of the number ed, since the number falls from an average of 3.7 to of nucleoli per cell, whereas doubling of the weak 1.7 in the â€œfedâ€•cells.These studies suggest that, nucleolus-forming chromosomes resulted in only a if the chromosomes normally involved in the pro 925â€”30percent increase in the number of nucleoli duction of the nucleoli are specific, there are minor per cell. Other chromosomes apparently lack the loci which can produce nucleoli and that they are

normally inactive. In other terms, it would appear amebas. The isotope moved from thelabeled nude that there is a competition between the chromo us to the cytoplasm but was not transferred to the somal masses for production of nucleoli and that, unlabeled nucleus. Considerably more evidence of under the appropriate conditions, some chromo this type was provided by studies on comparative somes predominate. Presumably some of the labeling of RNA in normal and enucleated cells phenotypic characteristics of the organs of an mdi (136, 9295â€”300).In enucleated cells there was little vidual are governed by the activity of those chro or no labeling of the ribonucleic acids in Acantha mosomes which are more successful or active in moeba and in HeLa cells (9299). Although some ex producing the nucleoli. ceptions have been reported, notably in some but Most of the evidence suggests that a single chro not all Acetabularia (492, 48, 318), and in macro mosome and possibly a single locus on a chromo phages (151, 1592),the bulk of the evidence supports some is responsible for the production of the nude the concept that biosynthesis of RNA is largely, if olus (335, 384). Yet it is not certain whether a not exclusively, a nuclear function. number of chromosomes may coordinate the ac Role of the nucleolus in biosynthesis of RNA.â€” tivity of a number of loci to produce a single nu Interest in the role of the nucleolus in the biosyn cleolus. In a coiled system such as is presented by thesis of RNA has arisen from two types of experi the chromatin in the interphase cell, this possibili mentsâ€”primarily those on labeling of nucleoli as ty is not excluded. measured by autoradiographic evidence and those One of the interesting findings of the combined on labeling of â€œnucleolarRNAâ€• as determined by cytochemical and biochemical studies carried out studies on uptake of isotope by chemically isolated in this laboratory is the evidence that suggests the fractions. Evidence for rapid early labeling of nucleolus may play a role in formation of chromo RNA in the nucleolus was obtained in studies on somes or â€œcondensationâ€•of chromosomes; the labeling of the nucleolus with tritiated cytidine chromosomes may emerge from the nucleolar sur and tritiated uridine as measured by grain count face in their discrete forms and shapes. ing of HeLa cells, pancreas, kidney, liver, Osgood leukemia, salivary cells of Drosophila, adrenal cor FUNCTIONS OF THE NUCLEOLUS tical cells, esophagus, fibroblasts, as well as plant Synthesis of RNA in the nucleus.â€”One of the cells(8,103,111,9210,9215,9235,92792,92892,9288,300, most significant recent developments in cell physi 3926, 406, 408, 409). In most of the studies, the ology has been the establishment of the role of the most concentrated labeling in the cell was found cell nucleus as the primary site of biosynthesis of in the nucleolus, although other fractions at one RNA. Experiments of various types support the time or another had a higher total amount of iso concept that the nucleus and its DNA exert their tope. Most of these studies have provided the type â€œgoverningâ€•functions by synthesis of ribonucleo of curves indicated in Chart 1. From such studies protein and/or by synthesis of â€œmessengerâ€•-RNA it can be concluded that the nucleolus contains an (47). Early studies on the size of nucleoli in cells RNA fraction which turns over very rapidly. in various tissues established a relationship be Moreover, the high specific activity of this fraction tween nucleolar size and the activity of the tissue suggests that it is a precursor of one or more types in protein synthesis (61, 68). Estimates of the of RNA either in the nucleus or in the cytoplasm RNA content of the nuclei of cells showed that tis or both. The higher total activity in the chro sues like the heart, kidney, and muscle, which are matin-linked RNA suggests the possibility that characterized by a high rate of metabolic activity this RNA or the associated DNA also may be the but a low biosynthetic function, had a low content site of biosynthesis of RNA which is transferred of RNA (89â€”48).On the other hand, tissues like to the cytoplasm (1925). It is not possible to distin the pancreas, gastric mucosa, and cells secreting guish between the relative roles of the other nu silk had a high RNA content. clear RNA and nucleolar RNA as being the more Goldstein and his colleagues (86, 1392â€”37)later important in biosynthesis of cytoplasmic RNA on established that the nucleus of the ameba was the the basis of data of this kind (8, 1925,9210,9235,9236, cell fraction labeled to the greatest extent in the 92792,300, 3926,336, 408, 409, 4921,49292). first hour following exposure of the cell to nucleic Some additional information on the relative ac acid precursors. Later, the isotope was distributed tivity of the nucleolus and chromatin in the bio throughout the cell, and after 924hours the isotope synthesis of cytoplasmic RNA has been obtained was present only in the cytoplasm. Evidence that from studies on suppression of nucleolar function the pathway from nucleus to cytoplasm was a by ultraviolet microbeam irradiation (43, 103, â€œone-waystreetâ€•was provided by studies in which 9280â€”83).In these studies it has been found that labeled nuclei were transplanted into unlabeled there was a marked suppression of biosynthesis of

cytoplasmic RNA when the nucleolus was irradi greater in the nucleolus than in the remainder of ated. Approximately 30 per cent of the labeling of the nucleus. nuclear RNA was suppressed when the nucleolus Tandler (373â€”75)found that the nucleolus may was irradiated, but 70 per cent of labeling of cyto be characterized cytochemically by the presence plasmic RNA was suppressed when the nucleolus of substances which are able to reduce ammoniacal was irradiated. This technic of â€œenucleolationâ€•by silver nitrate and other salts in the dark. These ultraviolet microbeam irradiation thus provides methods have to be distinguished from other pro evidence that a large amount of the RNA rapidly cedures in which impregnation with silver salts is synthesized in the nucleolus is transferred to the employed. On the basis of his experiments, cytoplasm. Moreover, the RNA synthesized in the Tandler (373â€”75)concluded that the reducing ma nucleolus accounts for a large proportion of the terial could not be identified as RNA, DNA, RNA formed in the nucleus which is transferred to the cytoplasm. Interestingly, for 4â€”6hours after ultraviolet irradiation of the nucleolus there NUCLEOLUS was no change in the functional activity of Elela cells (9247). SPECIFIC Biochemical evidencefor rapid RNA synthesis in ACTIVITY z the nucleolus.â€”In studies on nuclear fractions ex tracted initially to remove nuclear ribosomes, it was found that labeling of the residual â€œnucleolarâ€• U) fraction with either labeled orotic acid or adeno sine was remarkably greater than labeling of other nuclear RNA. It seemed probable from the technic employed that both RNA linked to the chromatin as well as the nucleolar RNA was studied in these experiments (6, 7). As indicated previously, Siba tani and Yamana found that the RNA fraction 0@ with the highest turnover in the nucleus was the C) r-RNA fraction, which was later associated with TOTAL COUNTS the nucleolus, although the methods utilized would not be considered completely adequate from a cy tological point of view (8892,338, 411â€”13).Similar types of experiments have recently been carried out by Georgiev et a]. (1926â€”929)whichprovide fur 1.@NUCLEOLt@@@â€”. ther biochemical support for the concept that there is a high turnover of nucleolar RNA. HOURS Recent studies in this laboratory6 have shown CHART 1.â€”Schematic representation of data obtained by that in normal liver cells there is a very rapid label auto-radiographic analysis following incubation of tissues with ing of two fractions of nucleolar RNAâ€”i.e., both RNA precursors.The kinetics of labeling are shown for both specific activity and C'@incorporated into RNA of the nucleus, the p-RNA and the r-RNA fraction. In these two nucleolus,and cytoplasm (8, 108,111,9.10,9.15,9.85,9.72,282, nucleolar components the specific activity reached 9.88, 800, 826, 406, 408, 409). values of 14,000 counts/mm/mg at periods of 15 minutes following injection of 3 pc. orotic acid-92- -NH2, or SH-groups, but seemed to be orthophos C'4 into normal rats. In rats treated with thioacet phate. The reducing substance disappeared from amide the nucleoli contained increased amounts of the nucleolus during prophase but could then be both of these types of RNA, but the greatest in - detected in the cytoplasm, and at the end of cell crease was in the r-RNA. In nucleoli of normal division it disappeared from the cytoplasm and liver cells the content of r-RNA was slightly great reappeared in the nucleolus. er than that of p-RNA, but in the nucleoli of the Role of DNA in biosynthesis of dytoplasmic livers of thioacetamide-treated rats, the content of RNA .â€”Avery substantial mass of evidence has accumulated which has indicated that, in a num r-RNA was 4 times that of the p-RNA. These data ber of bacterial systems and probably in mam suggest that r-RNA is an important component o f malian cells as well, DNA, probably double the nucleolus and, furtler, tiat tl@erates cf thin stranded, serves as the primer for the biosynthesis over of both the p-RNA and the r-RNA are much of RNA (7, 35, 9250,385, 400, 4925). The reactions H. A. Adams and H. Busch, Cancer Research (in press). involved are catalyzed by an RNA polymerase

(177, 400) which requires as substrates the triphos microscope (9215), nucleoli have been observed to phates of uridine, cytidine, adenosine, and guano discharge particles into the cytoplasm. sine. Evidence to support the concept that DNA Role ofthe nucleolus in the biosynthesis of protein. is the template for biosynthesis of RNA has arisen â€”The function of the nucleolus in protein synthe from studies on the role of chromatin in the bio sis is less remarkable than its apparent role in the synthesis of RNA (35, 38), from formation of biosynthesis of RNA (300). The evidence which â€œhybridsâ€•ofDNA and RNA (153, 385), from the shows the marked localization of labeled precur demonstration of the requirement for DNA in the sors of RNA in the nucleolar mass is very different biosynthesis of inducible enzymes (9265), the block from the results of studies which show that there induced by chloramphenicol (18, 14, 148), as well is relatively little selective localization of labeled as the net synthesis of RNA in systems described amino acids in the nucleolus. However, Ficq has by Weiss and others (400). Such evidence raises suggested that the nucleolus is also a site of syn the question of the role of the nucleolus in RNA thesis of protein, and such studies have been con biosynthesis particularly if future studies fail to firmed by other workers (113, 114, 9256,9275,389). reveal intranucleolar DNA. There seems little Although it is generally conceded that there is a doubt that considerable synthesis of RNA occurs great deal of protein synthesis on ribosomes, the in the nucleus outside of the nucleolus (9280â€”83), possibility has not been excluded that other cellu Rho and Bonner (311) have shown that the chro lar sites are active, including the chromosomes matin is the primary site of synthesis of RNA in (338) and the mitochondria (180, 181, 315). The the nuclei of pea seedlings. Studies on RNA syn intimate relationship between RNA synthesis and thesis need to answer the questions : (a) what is the protein synthesis has been shown in a number of function of the RNA from the chromatin ; (b) what experiments in bacterial systems (92192),but the is the function of the nucleolar RNA; and (c) which results in HeLa cells were considerably different of these is equivalent to the â€œmessengerâ€•-RNA (103). Irradiation of the nucleolus with a micro about which so much has recently been discussed? beam of ultraviolet light did not change amino Although the biosynthesis of RNA is apparently acid incorporation into the nucleus and produced almost entirely a nuclear function, some biosyn only a 30 per cent decrease in amino acid incorpo thesis of RNA may occur outside the nucleus (41, ration in the cytoplasm. Perhaps the half-life of 43, 151, 1592).There is suggestive evidence that the cytoplasmic RNA is sufficiently long to permit at least one or even three nucleotides can readily maintenance of normal cellular function for pro be added to the nucleotide chain in the cytoplasm tracted periods despite the failure of further RNA (54, 55, 156, 157, 177, 3924). Strauss and Goldwas biosynthesis in either enucleated cells or those in ser (363) have suggested that purely cytoplasmic which the nucleoli had been irradiated (9245, 9248, systems in which ribosomes were present could 9295, 9296). carry out net biosynthesis of RNA. Evidence has Nucleolus as a storage depot.â€”The possibility been obtained that polynucleotide phosphorylase has been considered that the nucleolus simply or at least enzymes which are capable of degrading serves as a kind of depot to which products of the RNA to ribonucleoside 5â€•-phosphates are present chromatin are moved and from which these prod in the ribosomes of E. coli (377, 378). In addition, ucts are released depending upon the need of the Yeas (414) has made the interesting suggestion cell (9215). However, recent studies in this labora that s-RNA may also function as a means for car tory on the rate of labeling of nucleolar RNA rying activated nucleotides to sites of synthesis of would seem to exclude this possibility, inasmuch nucleic acids. At least in the cytoplasm this may as the specific activities of nucleolar RNA in thio be a mechanism of activation of the nucleotides to acetamide-treated animals are far in excess of those re-form hydrolyzed s-RNA (43). of other cellular or nuclear RNA's. A number of Role of the nucleolus in the biosynthesis of ribo lines of evidence suggest that RNA synthesis occurs both in the nucleolus and in the chromo nucleoproteins.â€”The nucleolini which are visible somes, as mentioned previously (8, 9236,335). in the nucleolus with special stains have been sug gested as possible precursors of the ribosomes of RELATIONSHIP OF NUCLEOLAR RNA the nucleus (891â€”96)and of the cytoplasm (9215). TO RNA OF THE REMAINDER Electron microscopic studies (Figs. 5â€”10) have OF THE CELL shown the presence of a large number of discrete â€œMessengerâ€•-RNA.â€”â€œMessengerâ€•-RNAis a small bodies in the nucleolus (925, 926). In studies form of RNA which has been found in bacteria and of the fate of nucleoli by means of motion pictures yeast and is believed to have the characteristics of made of cells under observation through the phase extremely rapid biosynthesis, a purine and pyrimi

Downloaded from cancerres.aacrjournals.org on September 25, 2021. © 1963 American Association for Cancer Research. BUSCH et al.â€”Nucleolus of the Cancer Cell: Review 327 dine base composition similar to that of DNA, a has not been studied, and it remains to be learned sequence complementary to that of DNA, a low whether these enzymes are present in the nucleo molecular weight, and the capacity to adhere to lus. The possibility that there is a group of en ribosomes and direct their metabolic activity (192â€”zymes bound to particles that differ from those 14,119,1920,144,9234,92692,9264,306,378,377,385, found already has been suggested by recent studies 4925). Although the evidence for rapid biosynthesis, on biosynthesis of RNA in ribosomes (363). based upon labeling with radioactive precursors, is s-RNA.â€”The questions regarding the role of good and there is some evidence supporting the the nucleolus with regard to biosynthesis of concept that the base composition .resembles that m-RNA and the evidence for rapid biosynthesis of of DNA, the molecular weight has not been fully RNA within the nucleolus have suggested that the defined, and the mechanism whereby the activity nucleolus may be involved in the biosynthesis of of the ribosomes is directed is not known . The con other types of RNA . s-RNA's are RNA's of small cept of â€œmessengerâ€•-RNAisone of the most impor molecular weightâ€”i.e., about 925,000â€”to which tant of modern biology and has far-reaching influ amino acids are transferred after they are con ences in the many disciplines contributing to the verted to amino acyl adenylates (3â€”5,923,924, 41, cancer problem (47) . A number of workers have 45, 106,185,186,9244,9266,9294,316,355).These suggested that a similar fraction exists in mam RNA's are apparently a group of compounds malian cells (3925, 331). Scholtissek has suggested which have specific configurations designed to pro that the ribosomes have the ability to select and vide specificity for their linkage to individual stabilize the different types of RNA produced in amino acids and to govern their positioning on the the nucleus (3925, 415) based upon data obtained surface of the ribosomes (9292,97, 98, 163â€”68,189, from experiments involving release of RNA from 92920,9273,403,404,418,4923,4924).Inpart,this the nucleus after a period of hours in which the specificity may be related to the presence of some nuclei are incubated in a medium which had a low recently discovered purine and pyrimidine bases oncotic pressure. The fraction released had an end in these molecules which are apparently present group turnover different from that of the nuclear in extremely small amounts in other RNA's (75, RNA and RNA of cytoplasmic fractions and dif 100,101,92921,341).Theterminalendofthes-RNA fered from tissue to tissue. The data, however, that is the receptor for amino acids is CCA (cyti were not sufficiently definitive to establish the na dine, cytidine and adenine) as has been shown by ture of the RNA in terms of molecular weight, many experiments (54, 55, 1924,154, 156, 157, 170). number of types and turnover in the normal cell. The linkage of this trinucleotide to the over-all Although such data suggest that an RNA may molecule is apparently much more labile than the leave the nucleus to move to the cytoplasm, an other nucleotide linkages. Recently, Vincent and other possibility is that, in mammalian cells, the Baltus (3892) have suggested that one of the pos chief site of RNA synthesis is in the nucleolus and sible functions of the nucleolus is the addition of the activity of this organelle is governed by CCA trinucleotide to the s-RNA. The data which m-RNA derived from chromosomes (311). Siba they obtained were based upon rates of addition tani et al. (331) have found a â€œnucleolarâ€•fraction of various labeled nucleotides to the ribonucleic which has a high rate of turnover in normal liver acids in starfish nucleoli. Evidence that the entire and is equivalent to r-RNA which has been re s-RNA molecule was produced in the nucleolus ferred to previously. Moreover, this RNA has a was not obtained, but the possibility cannot be base composition which is remarkably similar to excludedby data availableatthe presenttime. the over-all base composition of the DNA, and Alltheevidencecitedpreviouslywhichhasshown hence they have suggested that this â€œnucleolarâ€• an intimate relationship between nucleolar size fraction is m-RNA (Table 92). In this laboratory,Â° and the rate of protein synthesis in cells may have it has been found that both the r-RNA and the as a common basis the synthesis of s-RNA. p-RNA of the isolated nucleoli of normal livers and Ribosom,es.â€”The extensive studies in recent the liver of thioacetamide-treated rats have a years on the ribosomes have established their spe much more rapid turnover than do most of the cific role in the biosynthesis of proteins originally other cellular RNA's studied. Interestingly, the by in vivo experiments and later in direct biosyn specific activities of both of the fractions were al thetic assays in vitro (1, 34, 592,53, 792â€”74,992â€”96, most identical at 5 and 15 minutes after injection 159â€”692,173â€”76,188,189, 9200â€”92092,92592,9253,9261, oflabeledoroticacidintorats. 305, 308, 309, 39292, 370, 871, 897, 408, 404, 417). Although evidence has been provided for the The ribosomesare the activeribonucleoprotein role of specific enzymes in biosynthesis of RNA components of the microsomes on which the amino (92692,9270, 400), their localization in the nucleus acyl-s-RNA's are positioned prior to the polymeri

zation of the amino acid residues to form the pro biosynthesizes only a special type of RNA, such tein molecules (929,30, 368). They are composed of as a-RNA, which is essential to protein synthesis high molecular weight RNA and of proteins of and hence undergoes enlargement when this re which there are one or more basic proteins (74, 84, quirement is great ; (b) the nucleolus represents a 87, 149). The structure of the ribosome is complex, storage system for accumulation of a large amount and its active form consists of two or more types of RNA which is released into the cytoplasm at a of RNA which are linked together under the influ specific timeâ€”evidence for such a concept has ence of divalent ions, particularly magnesium (192, been obtained by phase microscopy, which has 38,45,66,9204,9211,9218,9287,9251,9285,9287,330, shown that the nucleolus may approach the nu 377, 398). The evidence which has suggested a re clear membrane and discharge small bodies or lationship between nucleolar function and the bio droplets into the cytoplasm in much the same way synthesis of ribosomes has been of three types: that the contents of the zymogen granules are (a) evidencethatin the nucleolustherearea large released into the extracellular fluid ; (c) the nucleo number of small ribonucleoprotein particles, as lus may represent a depot for accumulation of well as nucleolini, which have been referred to RNA from a number of chromosomal loci from one earlier; (b) evidence that the nucleolus contains or more chromosomes which are particularly ac many ribonucleoprotein granules (Figs. 5â€”10) tive and may be the important components for which may be discharged into the cytoplasm (925, specialized cellular functions (9279)â€”evidence for 926,9218â€”15);and (c) suppression of biosynthesis of a high activity of the nucleolus-associated chro microsomal RNA and protein in livers of thioacet matin has been obtained by Sirlin et al. (334â€”38) amide-treated animals (9206,9258).It is conceivable who carried out studies with labeled cytidine on that the parts of the ribosome which are different chromosomes of salivary glands of Chironomids from one another in both size and enzymatic con and noted a high labeling in the juxta-nucleolar stitution (1092, 390) are formed in different parts chromatin; (d) the nucleolus may contain RNA of the nucleus. It is possible that part of the RNA which is genetically functional and may be neces is added in the nucleolus along with a considerable sary for completion or biosynthesis of RNA which portion of the protein moieties. is important to cell functionâ€”it is well established There have been a number of reports which from studies on viruses that RNA is the genetical have shown that, at least in calf-thymus nuclei, ly active material which is replicated intracellular there is a ribonucleoprotein particle which has the ly and is essential to definition of the characteris same capacity for incorporation of labeled amino tics of the virus (78, 79, 1892,9238, 3892, 333), and acids into protein as has been found for the cyto the suggestion has been made (331) that the RNA plasmic ribonucleoproteins (119, 1920,391â€”95).Al of the nucleolus consists of two parts, one of which though the existence of these ribonucleoprotein turns over rapidly and one of which turns over particles has not been unequivocally demonstrated much more slowly ; (e) the nucleolus may be re in the nuclei of other species or other cell types, quired for the synthesis of special types of RNA it is possible that they are present and may be which contain some of the newer purine and pyrim related to the biosynthetic activity of the nucleo idine bases Such as those described by Dunn and lus (Figs. 6â€”10).None of these considerations ex others (75, 100, 101, 92921,341) to occur predomi plain satisfactorily the cytochemical and auto nantly in the s-RNA of the liver cells ; (J) the nu radiographic data referred to previously, which cleolus may fabricate an essential component of areacceptedasshowingthatthereisa transferof ribosomes which may be fed into the cytoplasm nbonucleoproteinsfromthe nucleusto the cyto whether this is part of the RNA-specifying protein plasm. At the moment, one must agree with Vin synthesis, or the protein or lipide components of cent and Baltus (3892), who have stated that there ribosomes, cannot be evaluated at the present, but is no evidence which establishes the specific role of these components are all present in the ribosomes the nucleolus in the biosynthesis of RNA in the â€¢orparticles of the ergastoplasm, although their nucleus or transfer of RNA to the cytoplasm. functional significance remains to be assessed. One However, there are many interesting avenues to must also recognize that many cells, such as those explore, and it is indeed possible that the nucleolus treated with thioacetamide, contain a number of plays a key role in biosynthesis of each of the ribo nucleoli. Many nucleoli may develop in nuclei of nucleic acid-containing cytoplasmic and nuclear these and other cells, some of large size (Fig. 9) components. and others very small; each is surrounded by a DISCUSSION dense mass of nucleolus-associated chromatin. Among the possibilities for nucleolar function These nucleoli may subserve a number of different that have been suggested are: (a) the nucleolus functions.

Many questions remain to be answered with 8. Asw@o, M., and LnSwm, C. P. Comparison of the Spe regard to the nucleolar RNA. Among these are: cific Activity Time Curves of Ribonucleic Acid in Chro matin, Nucleolus and Cytoplasm. Exp. Cell Res., 20:250â€” (a) the types, baseratios, and sequencesofthe 53, 1960. RNA ; (b) the relative metabolic activities of the 9. Ai@irmssr@i,C. B.; H@nzn, E.; Snx@, M.; and WHrrz, RNA; (c) molecular weights of the RNA; and F. H., JR. The Kinetics of Formation of Native Ribonu (d) physical structure of the RNA. Although it has clease during Oxidation of the Reduced Polypeptide been postulated that â€œmessengerâ€•-RNAis of high Chain. Proc. Natl. Acad. Sci., 47:1309â€”14, 1961. 10. ANSEVIN, A, T., and LAUFFER, M. A. Native Tobacco and/or low molecular weight, it is certain that in Mosaic Virus Protein of Molecular Weight 18,000. Na ribosomes and also in the nucleolus there are ture,183:1601â€”2,1959. RNA's of high molecular weight similar to viruses 11. A@roM, F.: VARGA,L.; and Hmvsxii, E. J. Untersuchung (10, 71, 117, 118) in which the molecular weight der Ribonukleinslture-Heterogenitat in der Kaninchen leber. Ada PhysioL, 16: 1â€”6,1958. of RNA may be 1.6 X 10g. Inasmuch as highly 12. ARONSON, A. I., and MCCARTHY, B. J. Studies of E. cdi purified nucleolar preparations can now be ob Ribosomal RNA and Its Degradation Products. Biophys. tamed with relative ease (50), it would seem worth J.,1:215â€”26,1961. while to attempt chromatographic fractionation of 13. ARONSON, A. I., and SPIEGELMAN, S. Protein and Ribo nucleolar RNA (11, 37, 46, 85, 138, 189) as well nucleic Acid Synthesis in a Chloramphenicol-inhibited System. Biochim. Biophys. Acts, 53:70â€”84,1961. as further structural analysis of the nucleolar 14. . On the Nature of the Ribonucleic Acid Synthe RNA's to determine whether the sequences and sized in the Presence of Chloramphenicol. ibid., pp. 84â€”95. helical configurations conform more closely to 15. As@o, K., and EoAau, F. Ribonuclease T1 Digestion of cytoplasmic RNA or to DNA (81, 76, 88, 99, 19292, Yeast Soluble RNA. J. Biochem., 50:467â€”70, 1961. 3192,845â€”47,376) and further to repeat the ana 16. Avnzs, W. W. A Method of Staining Nucleoli of Cells in lytical studies on base composition of the nucleolar Fresh Benign andMalignant Tissues. Cancer Res., 8:352- 56, 1948. RNA to determine whether the differences can be 17. BALTUS, E. Observations sur le role biochimique du nu found between the nucleolar RNA of neoplastic clÃ©ole.Arch. Tnt. Physiol., 62:281â€”8%,1954. cells and that of other cells C198). Until such time 18. . Observations sur le role biochimique du nuclÃ©ole as the RNA is isolated in a highly purified form, Biochim. Biophys. Acts, 15:268-67, 1954. 19. Bsnss, D., and Dicx, D. A. T. Interferometry and Re sequential analysis would not seem to be particu fractometry of Cells in Tissue Culture. Exp. Cell Res., 13 larly profitable, despite the fact that methods for (Suppl.4):103â€”35,1957. such analyses are improving (47). 20. BAUER, H., and CASPERSSON,T. Cytochemical Observa tions on Nucleolus Formation in Chironomus. Proc. ACKNOWLEDGMENTS VIlith mt. Congr. Genetics,1948. 21. BEnm&.&srsr, W. Em Balbiana-Ring ala Locus einer The authors wish to express their deep appreciation to Dr. Hewson Swift, Department of Zoology, University of Chicago, Speicheldrllsen-Mutation.Chromosoma(Berl.), 12:1-25, for his generosity in providing electron micrograms for Figures 1961. 9 and 10, and for many helpful comments. 22. BENZEH,S.,and WEISBLUM,B.On the Species Specificity of Acceptor RNA and Attachment Enzymes. Proc. Natl. REFERENCES Acad. Sd.,47:1149â€”54,1961. 23. Bs@nG,P. Studies on the Enzymatic Utilization of Amino 1. ABDtL-Noun, B., and Waiiwrira, G. C. Biological Activi Acyl Adenylates: the formation of Adenosine Triphos ty of Reconstituted Ribosomes. Exp. Cell Res., 20:226- phate. J. Biol. Chem., 233:601â€”7, 1958. 27, 1960. 24. BERGMANN,F.H.; Bmw, P.; and DIEcxss@&r@,M.The 2. Aun@rnii, L. Etude cytophotometriquedes proteines Enzymic Synthesis of Amino Acyl Derivatives of Ribo -SH nuclÃ©olairesdela racine de Jacinthe; repartition prÃ© nucleic Acid. II. The Preparation of Leucyl-, Valyl-, Iso cise de ces composes au sein du nuclÃ©ole.Compt. Rend. leucyl-, and Methionyl Ribonucleic Acid Synthetases Acad. Sci., 248:3476â€”78, 1959. from Eacher-ichiacdi. J. Biol. Chem., 236: 1735-40, 1961. 8. Aii@mi, E. H.; GLAS8MAN,E.; Coiwzs, E.; and SQHWEET, 25. BEmia.uw, W.; BAum@,A.; GROPP,A.; HAGTJENAU,F.; R. S. Incorporation of Amino Acids into Ribonucleic and OnSRWIO, C. L'Ultrastructure du nuclÃ©olede eel Acid. II. Amino Acid Transfer Ribonucleic Acid. J. Biol. lules normales et cancÃ©reuses. Exp. Cell Res., 9:88-100, Chem., 235:1068â€”74, 1960. 1955. 4. Au@m@,E. H.; GI@tsssL&N,E.; and SCHWEET, R. S. Incor 26. Bxnini@uin, W.; HAGENAU, F.; and Oannuwo, C. L'Ul poration of Amino Acids into Ribonucleic Acid. I. The trastructure du nuclÃ©olede quelques cellules animales, Role of ActivatingEnzymes.J.Biol.Chem., 235:1061- rÃ©vÃ©lÃ©eparlemicroscopeÃ©lectronique.Experientia,8:58â€” 67, 1959. 59, 195%. 5. Aim@, E. H., and SCHWEET,R.S. Synthesis of Hemo 27. BRATTACRARYTA,T. K., and GHOSH, A. On the Occur globin in a Cell-free System. I. Properties of the Complete rence of Feulgen-positive Nucleoli in Adrenocortical Cells System. J. Biol. Chem., 237:760â€”67,1962. of the Pigeon. Naturwissenschaften, 9:389â€”90, 1961. 6. ALLFREY, V. G., and MInSKY, A. E. Some Aspects of 28. BIRNSTIEL, M. L.; CHIPCHASE, M.; and BosciEn, J. In Ribonucleic Acid Synthesis in Isolated Cell Nuclei. Proc. corporation of Leucine-H' into Subnuclear Components Natl. Acad. Sci., 43:821â€”26, 1957. of Isolated Pea Nuclei. Biochem. Biophys. Res. Comm., 7. ALLFREY, V. G.; Minaxy, A. E.; and OSAWA, S. Protein 6:161â€”66, 1961. Synthesis in Isolated Cell Nuclei. J. Gen. Fhysiol., 40: 29. Bisuop, J. 0., and SCHWEET, R. S. Role of Glutathione 451â€”90,1957. in Transfer of Amino Acids from Amino Acyl-Ribonu

cleic Acid to Ribosomes. Biochim. Biophys. Acts, 49: for Isolation of Nuclei from Cells of the Walker 256 Car 235â€”36,1961. cinosarcoma. Cancer lies., 19:684â€”87, 1959. 30. BLOEMENDAL.,H. ; LITTAUER, U. Z. ; and DANIEL, V. 52. CAMPBELL, P. N. Protein Synthesis with Special Refer Transfer of Soluble Polynucleotides to Microsomal RNA. ence to Growth Processes Both Normal and Abnormal. Biochim. Biophys. Acts, 51:66â€”7%,1961. Adv. Cancer lies., 5:97â€”155, 1958. 31. BOEDTKER, H., and SniassoNs, N. S. The Preparation and 53. CAMPBELL, P. N. ; GREENGARD, 0. ; and KERNOT, B. A. Characterization of Essentially Uniform Tobacco Mosaic Studies on the Synthesis of Serum Albumin by the Iso Virus Particles. J. Am. Chem. Soc., 80:2550â€”56, 1958. lated Microsome Fraction from Rat Liver. Biochem. J., 3%. BOLOGNARI,A. Ultrastructure of the Nucleoli. Boll. Soc. 74:107â€”17,1960. Ital. Biol. Sper., 35:479â€”83, 1959. 54. CANELLAKIS, E. S. Mechanisms Involved in the Biosyn 38. . Cellule tumorali e ovociti. Boll. Zool., 28:597â€” thesis of Ribonucleic Acids. N.Y. Acad. Sci., 81:675â€”78, 659, 1961. 1959. 34. BOMAN, H. G., and SvENSSON, I. Evidence for a New En 55. CANELLAKIS, E. S., and HERBERT, E. Studies on s-RNA zyme Connected with Protein Synthesis. Nature, 191: Synthesis. II. Assay and Method of Purification of 674â€”77,1961. s-RNA a, f3, and y. Biochim. Biophys. Acts, 45:133-38, 85. BONNER, J. ; HUANO, R. C. ; and MAHESHWARI, N. The 1960. Physical State of Newly Synthesized RNA. Proc. Natl. 56. . s-RNA Synthesis. IV. The Effect of Inorganic Aced. Sci., 47:1548-54,1961. Pyrophosphate and a Method for the Extraction of 36. BORYBKO,E., and B@o, F. B. Structure of the Nucleolus s-RNA.ibid.,47:78â€”85,1961. as Revealed by the Electron Microscope. Bull. Johns 57. CANTAROW,A. ; PASCRKIS, K. E. ; and WILLIAMS, T. L. Hopkins Hosp., 89:468â€”71, 1951. Influence of Starvation in Incorporation of Uracil-2-Câ€• 37. Boscn, L.; WENDE, G. VAN DEn; and BLOEMENDAL, H. in Liver and Hepatoma RNA in the Rat. Biochem. Bio MOdified Cellulose Ion Exchangers and Their Use in phys. Res. Comm., 1:75â€”78, 1959. Ribonucleic Acid Fractionation. Nature, 191:349-52, 58. CANTAROW, A. ; WILLIAMS, T. L. ; and PASCHKIS, K. E. 1961. Uracil Incorporation in Liver RNA of Young and Preg 38. Bowusi, T. J.; DAGLEY, S.; S@xsn, J.; and Wiw, D. G. unnt Rats and in RNA of Fetal Tissues and Certain Tu Nature of the Ribosomes Present in Bacteria and Yeast: mors. Biochem. Biophys. lies. Comm., 4: 156â€”58,1961. a Re-appraisal. Nature, 189:638-40, 1961. 59. CARYALHO, S. DE. Biologic Properties of Human Leuke 39. Bn@cnicr, J. La detection histochimique des acides pen mic and Tumoral RNA. III. The Effect of Different tosenucleiques. Compt. Rend. Soc. Biol., 1.33:88â€”89, Media on the Cytopathogenicity in Tissue Culture. J. 1940. Lab. Clin. Med., 55:694â€”705, 1960. 40. . La localisation des acides pentosenuclÃ©iques dans 60. CARVALHO,S. DE; RAND, H. J.; and MEYER, D. P. Bio les tissus ammaux et lea oeufs d'amphibiens en voie de logic Properties of Human Leukemic and Tumoral RNA. dÃ©veloppement.Arch.Biol., 53:207â€”57,194%. IV. Leukemia and Neoplasms Induced in Mice with 41. . Ribonucleic Acids and the Synthesis of Cellular Human Leukemic RNA Carried in Tissue Culture. J. Proteins. Nature, 186:194â€”99,1960. Lab. Clin. Med., 55:706â€”14, 1960. 4%. . Nucleocytoplasmic Interactions in Unicellular 61. CASPERSSON, T. 0. Cell Growth and Cell Function. A Organisms. In: J. Ba@tcnurand A. E. MInSKY(eds.), The Cytochemical Study. New York: W. W. Norton, 1950. Cell, pp. 696-745.New York: AcademicPress, 1961. 62. CASPERSSON, T., and [email protected], L. Studies on Protein 43. BRACHET, J. ; CHANTRENNE, H. ; and VANDERHAEGRE,F. Metabolism in the Cells of Epithelial Tumors. Acts Recherches sur lea interactions biochimiques entre le Radiol. (Suppl.),46: 1â€”105,194%. noyau et le cytoplasme chez lea organismes unicellulaires. 63. CASPERSSON,T. 0., and SCHULTZ,J. Ribonucleic Acids in Biochim. Biophys. Acts., 18:544â€”63, 1955. Both Nucleus and Cytoplasm and the Function of the 44. BRAWERMAN, G.; Pooo, A. 0.; and CHARGAFF, E. In Nucleolus. Proc. NatL Acad. Sri., 26:507â€”15, 1940. duced Formation of Ribonucleic Acids and Plastid Pro 64. CASTREN, H. Uber die Struktur der Zellen der Binde tein in Euglena gracilis under the Influence of Light. gewebsgeschwillste beim Menschen. Arb. Pathol. Inst. U. Biochim. Biophys. Acts, 55:326-34, 196%. Helsingfors.,4:240â€”318,1926. 45. BROWN, G. L., and ZUBAY, G. Physical Properties of the 65. CATCHPOLE,H. R., and GERSH, I. Components of Normal Soluble Ribonucleic Acid of Eacheriehia coli. J. Mol. Biol., and Abnormal Liver Cells Studied by Ultraviolet Mi 2:287â€”96, 1960. croscopy and by Differential Centrifugation. Disc. Far. 46. Bmtsrnss, A. T. H., and Vizoso, A. D. The Relation be Soc.,9:471â€”80,1950. tween Molecular Size of Ribonucleic Acid and Its Chro 66. CHAO, F. The Isolation and Characterization of Ribonu matographic Profile on Calcium Phosphate. Biochim. cleic Acid from Yeast Macromolecular Ribonucleopro Biophys. Acta, 49:225â€”28, 1961. tein. Biochim. Biophys. Acts, 53:64â€”69, 1961. 47. Buses, H. An Introduction to the Biochemistry of the 67. CHAUVEAU, J. ; Moui@, Y. ; and ROUILLER, C. Isolation Cancer Cell. New York: Academic Press, 196%. of Pure and Unaltered Liver Nuclei, Morphology and 48. BUSCH, H., and DAVIS, J. R. Nuclear Proteins of Tumors Biochemical Composition. Exp. Cell Res., 11:317â€”21, and Other Tissues: A Review. Cancer Res., 18:1241â€”56, 1956. 1958. 68. CHAYEN, J.; DAviss, H. G. ; and MILEs, U. J. Observa 49. Buscii, H.; HNILICA, L. S.; CHIEN, S. C.; DAvis, J. R.; tions on Some Plant Interphase Nuclei. Proc. Roy. Soc. and TAYLOR, C. Isolation and Purification of RP%-L, a London, s. B, 141:190â€”99, 1953. Nuclear Protein Fraction of the Walker Tumor. Cancer 69. CHEN, T. T. Observations on Mitosis in Opalinids (Proto Res., 22:637â€”45, 1962. zoa Ciliata). I. The Behaviour and Individuality of 50. Buses, H.; MURAMATSU,M.; Csiss, S. C.; and Sos&nns, Chromosomes and Their Significance. Proc. Natl. Acad. C. Isolation and Metabolic Activity of Nucleoli of the Sci., 22:594â€”60%, 1936. Walker Tumor. Proc. Am. Assoc. Cancer lies., 3:308, 70. â€”â€”. Observations on Mitosis in Opalinids (Protozoa 196%. Chats). II. The Association of Chromosomes and Nu 51. Buses, H.; STARBUCK,W. C.; and DAVIS, J. R. A Method cleoli. Ibid., pp. 602â€”7.

71. CHENG, P-Y. On the Similarity of Chain Length of Nu Particles from Rat Liver Microsomes. Exp. Cell lies., cleic Acids. Proc. Nati. Aced. Sci., 45:1557â€”60, 1959. 23:517â€”27,1961. 72. Cousi, P. Incorporation in Vitro of Amino Acids into 93. DECKEN, A. VONDER, and HULTIN, E. A Metabolic Iso Ribonucleoprotein Fractions of Microsomes. Biochim. tope Transfer from Soluble Polynucleotides to Micro Biophys. Acts, 33:284â€”85, 1959. somal Nucleoprotein in a Cell-free Rat Liver System. 73. COHN, P., and Bumsn, J. A. V. Fractionation of Micro Exp. Cell lies., 15:254â€”56, 1958. somal Proteins by a Non-ionic Detergent. Biochim. 94. . Activity of Amino Acid-activating Enzyme Sys Biophys. Acts, 25:%%%â€”%3,1957. tems during Liver Regeneration. Acts Chem. Seandinav., 74. COHN, P.; SnesoN, P.; and Bumnii, J. A. V. The Presence 12:597â€”98, 1958. of Basic Proteins in Ribonucleoproteins of Rat Liver. 95. . Activity of Rat-Liver Enzymes in the Transfer Biochem. J., 76:%3Pâ€”%4P,1960. of s-RNA-bound Amino Acids to Protein by Ribonucleo 75. CoHN, W. E. Pseudouridine, a Carbon-Carbon Linked protein Particles. Biochim. Biophys. Acts, 45:139â€”48, R.ibonucleoside in Ribonucleic Acids. J. Biol. Chem., 235: 1960. 1488â€”98,1960. 96. . The Enzymatic Composition of Rat Liver Micro 76. Coirnn, J. R. The Localization of Ribonucleic Acid in somes during Liver Regeneration. Exp. Cell lies., 19:591â€” the Egg of llyanas8a obsolete. Exp. Cell lies., 21:126â€”36, 604, 1960. 1960. 97. DocToR, B. P.; APGAR, J.; and HOLLEY, R. W. Fractions 77. COLTER, J. S.; KUHN, J.; and Eu@sas, K. A. 0. The Ribo tion of Yeast Amino Acid-Acceptor Ribonucleic Acids by nucleases of Mouse Ascites Tumors. Cancer lies., 21:48â€” Countercurrent Distribution. J. Biol. Chem., 236:1117â€” 51,1961. 20, 1961. 78. COMMONER,B.; Lxppnscorr, J. A.; and SYMINOTON,J. 98. DocroR, B. P., and CONNELLY,C. M. Separation of Yeast Replication of Tobacco Mosaic Virus. Nature, 184:1992â€” Amino Acid-Acceptor Ribonucleic Acids by Countercur 2001,1959. rent Distribution in MOdified Kirby's System. Biochem. 79. Co@mmi@s, G.; T0HA, J.; and OHLBAUM,A. Preliminary Biophys.lies.Comm., 6:201â€”4,1961. Study on the Kinetics of U@Incorporation into Protein 99. Do'ry, P. ;BOEDTKER,H. ; FRESCO,J. R. ; HASELKORN,R.; and Ribonucleic Acid of HeLa Cells Infected with Polio and Lrrrs, M. Secondary Structure in Ribonucleic Acids. virus. Biochim. Biophys. Acts, 35:268â€”69, 1959. Proc. Natl. Acad. Sci., 45:482â€”99, 1959. 80. COOPER, N. C. Early Cytological Changes Produced by 100. DUNN, D. B. Additional Components in Ribonucleic Carcinogens. Bull. N.Y. Med. Soc., 32:79â€”82, 1956. Acid of Rat-Liver Fractions. Biochim. Biophys. Acts, 81. Counm@a, W. C., and Joasi, E. 11. Effect of Ribonuclease 34:286â€”88, 1959. on Retention of Conditioned Response in Regenerated 101. DUNN, D. B., and SMITH, J. D. Occurrence of a New Base Planarians. Science, 134: 1363-65, 1961. in the Deoxyribonucleic Acid of a Strain of Bacterium 82. COWDRY, E. V. Cancer Cells, pp. 107â€”50.Philadelphia: coli. Nature, 175:336â€”38, 1955. W. B. Saunders Co., 1955. 10%. ELSON, D., and TAi@,M. Biochemical Differences in Rho 83. COWDRY,E. V., and PALETTA, F. X. Changes in Cellular, nucleo-proteins. Biochim. Biophys. Acts, 36:281â€”8%, Nuclear and Nucleolar Sizes during Methylcholanthrene 1959. Epidermal Carcinogenesis. J. Natl. Cancer Inst., 1:745â€” 103. ERRERA, M.; HELL, A.; and PERRY, R. P. The Role of 59, 1941. the Nucleolus in Ribonucleic Acid- and Protein Synthesis. 84. CRAMPTON, C. F., and PurunMANN, M. L. The Amino II. AminoAcidIncorporation into Normaland Nucleolar Acid Compositionof Proteins Isolated from the Ribonu Inactivated HeLa Cells. Biochim. Biophys. Acts, 49:58â€” cleoprotein Particles of Rat Liver. J. Biol. Chem., 234: 63,1961. %64@-â€¢44,1959. 104. EaTABLE, C., and SOTELO, J. R. Una nueva estructura 85. CRnsslca, E. H., and SPENCER, J. H. A Comparison of the celulare: el nucleolonema. Publ. Inst. Invest. Sci. Biol., Nudeic Acidsof Rat Liver and NovikoffHepatoma. Bio 1:105â€”26,1951. chem. J.,76:171â€”78,1960. 105. . Symposium on the Fine Structure of Cells. 8th 86. CROCKER, T. T., and Gowwrnin, L. Nuclear-cytoplasmic Intern. Congress of Cell Biology, pp. 170â€”90.Leyden Relationships in Human Cells in Tissue Culture. I. In and New York: Intersci., 1955. strumentation for Micro-manipulation and Cinemicrogra 106. EVERETT, G. A.; MEmin&, S. H.; and HOLLEY, R. W. phy. Mikroskopie, 12:315â€”2%,1958. Separation of Amino Acid-specific â€œSolubleâ€•Ribonucleic 87. Cuiuty, J. B., and HsamH, R. T. Molecular Weight of the Acids by Partition Chromatography. Fed. Proc., 20:388, Protein from Bovine Liver Ribosomes. Biochem. Bio 1961. phys. lies. Comm., 6:415â€”17, 196%. 107. FEIOELSON, P., and FEIGELSON, M. Alterations in Nucleic 88. DANON, D.; MARIKOVSKY,Y.; and LrrTAUER, U. Z. A AcidTurnovers in SubcellularComponentsduring Tryp Comparative Electron Microscopical Study of RNA from tophan Peroxidase Induction. Biochim. Biophys. Acts, Different Sources. J. Biophys. Biochem. Cytol., 9:253-61, 32:430â€”35,1959. 1961. 108. FEIGELSON, P. ; FEIGEisON, M. ; and FANCHER, C. Kinet 89. DAVIS, J. M. G. The Ultrastructure of the Mammalian ics of Liver Nucleic Acid Turnovers during Enzyme In Nucleolus. In: E. J. S. MITCHELL (ed.), Symposium: The duction in the Rat. Nucleic Acid Turnovers during En Cell Nucleus, 1959. zyme Induction in the Rat. Biochim.Biophys.Acts, 32: 90. Dn, P., CHArPERJEE, R.; and Mrrna, S. Nucleolar Sue 133â€”39,1959. cinic Dehydrogenease in Mouse Mammary Carcinoma 109.FEIGELSON,P.;FEIGELSON,M.;and GREENGARD,0.In Cells. Nature, 192:286â€”86,1961. Vise Relationshipsbetween Cortisone,RibonucleicAcid, 91. DEA1WW, W. H. The Material Continuity and Individu Protein Synthesis and Enzyme Induction in Rat Liver. ality of the Somatic Chromosomesof Ambyostomatigri First Intern. Congress.Endocrinol.,p. 823.SessionVIII, nui,swithSpecialReferencetothe Nucleolusasa Chro Copenhagen, 1960. mosomal Component. J. Morphol., 56:157â€”79, 1934. 110. FEIGELSON,P., and GREENGARD,0. A Microsomal Iron 9%. DECKER, A. VONDER. Influence of Preparative Media on Porphyrin Activator of Rat Liver Tryptophan Pyrrolase. Properties and Enzyme Activities of Ribonucleoprotein J. Biol. Chem.,236:158â€”57,1961.

111. FEINENDEGEN,L. E.; B0RD, V. P.; SHREEVE,W. W.; and as Shown by Transplantation Experiments in Amoeba PAINTER, R. B. RNA and DNA Metabolism in Human Proteus. Exp. Cell Res., 15:685-37, 1958. Tissue Culture Cells Studied with Tritiated Cytidine. 133. GOLDSTEIN, L. ; CAILLEAU, R. ; and Csoczza, T. T. Nu Exp. Cell Res., 19:443â€”59,1960. clear-Cytoplasmic Relationships in Human Cells in Tissue 112. FERREIRA,A. E. M. Relation of the Macronucleolus and Culture. II. The Microscopic Behavior of Enucleate Hu Nucleonucleolar Ratio of Cancer to Histologic Grading. man Cell Fragments. Exp. Cell Res., 19:332-42, 1960. J. Lab. Chin.Med., 26:161%â€”%8,1941. 134. GOLDSTEIN,L., and Mxcou, J. On the Primary Site of 113. Fic@, A. Etude autoradiographique du mÃ©tabolisme des Nuclear RNA Synthesis. J. Biophys. Biochem. Cytol., protÃ©ines et des acides nuclÃ©iqueau cours de l'oogenÃ©se 6:301â€”4,1959. dies les batraciens. Exp. Cell Res., 9:286â€”93, 1955. 135. GOLDSTEIN,L., and Micou, J. Nuclear-Cytoplasmic Re 114. FIcQ, A., and Bnscusrr, J. Distribution of RNA and In lationships in Human Cells in Tissue Culture. LU. Auto corporation of Phenylalanine-%-Câ€• into Protein. Exp. radiographic Study of Interrelation of Nuclear and Cyto Cell Res., 11:135â€”45,1956. plasmic Ribonucleic Acid. J. Biophys. Biochem. Cytol. 115. FIDLER, H. K. A. A Comparative Cytological Study of 6:1â€”6,1959. Benign and Malignant Tissues. Am. J. Cancer, 25:77%â€” 136. GOLDSTEIN,L. ; Micou, J. ; and CROCKER,T. T. Nuclear 79, 1985. Cytoplasmic Relationships in Human Cells inTissue Cul 116. FINsssoaE, F. J., and Vor@nis, E. Some Chemical Char ture. IV. A Study of Some Aspects of Nucleic Acid and acteristics of Amphibian Egg Ribonucleic Acids. J. Biol. Protein Metabolism in Enucleate Cells. Biochim. Bio Chem., 236:443â€”47,1961. phys. Acts, 45:82-86, 1960. 117. FRAENKEL-CONRAT,H.; STAEHELIIq,M.: and CRAWFORD, 137. GOLDSTEIN,L., and Pi@tu'r, W. Direct Evidence for Nu L. V. Tobacco Mosaic Virus Reconstitution Using In clear Synthesis of Cytoplasmic Ribose Nucleic Acid. activated Nucleic Acid. Proc. Soc. Exp. Biol. Med., 102: Proc. Nat!. Acad. Sci. 41 :874â€”80,1955. 118-21, 1959. 138. GOLDTHWAIT,D. A. Isolation and Characterization of 118. FnsENKEL-CONRAT, H., and Wn&@uis, R. C. Reconsti Ribonucleic Acid Fractions from Rat Liver. J. Biol. tution of ActiveTobacco MosaicVirusfrom Its Inactive Chem., 234:3245â€”50,1959. Protein and Nucleic Acid Components. Proc. Nat!. Acad. 139. . Metabolic Studies in Vitro of Two Ribonucleic Sci. 41:690â€”98, 1955. Acid Fractions from Rat Liver. Ibid., pp. 3251-56, 119. FRENaTER, J. H.; ALLYREY,V. G.; and MInSKY, A. E. 1959. Metabolism and Morphology of Ribonuc!eoprotein Par 140. GONZALEZ-GUZMAN,I.Generalities on the Nuclear Con tides from the Cell Nucleus of Lymphocytes. Proc. tent of Some Blood Cells. Blood, 3 (SuppL @2):57-65, NatI. Acad. Sci.,46(4):43%-44,1960. 1948. 120. . In Vitro Incorporation of Amino Acids into the 141. . Contribution I la reconnaissance de l'appareil nu Proteins of Isolated Nuclear Ribosomes. Biochim. Bio clÃ©olairdes cellules leucÃ©miques. In Sang, 20:225-38, phys. Acts, 47:180â€”37, 1961. 1949. 121. FITKASAWA,H. Nucleotide Composition of RNA from 142. . Etudes sur l'ultrastructure du nuclÃ©ole.I. Cel Cytoplasmic Male-sterile Wheat. Rap. Cell Res., 25:276- lules des organes hemopoiÃ©tiques normaux et patho 85,1961. logiques. Rev. Hematol., 14:333â€”43, 1959. 122. FuLLEii, W. Two-stranded Helical Configurations for 143. GREENGARD, 0., and FEIGELSON, P. The Activation and Ribonucleic Acid. J. Mol. Biol., 3:175â€”84, 1961. Induction of Rat Liver Tryptophan Pyrrolase in Vise by 123. FUElER!, P. Chromatin Association with the Nucleolus of Its Substrate. J. Biol. Chem., 236: 158-61, 1961. Various Mammals. Boll. Dell. Soc. Its!. Biol. Sper., 33: 144. Gsoss, F.; HIATT, H.; GILBERT, W. ; KURLAND, C. G.; 260â€”61,1957. RISEBROUGH, R. W. ; and WATSON, J. D. Unstable Ribo 124. FURTH, J. J. ; HURWI'rZ, J. ; KauG, R. ; and ALEXANDER, nucleic Acid Revealed by Pulse Labeling of Escherichia M. The IncorporationofAdenylicand Cytidylic Acids cdi. Nature, 190:581â€”85, 1961. into Ribonucleic Acid. J. Biol. Chem., 236:3317â€”2%,1961. 145. GRossi, L. G., and MOLDAVE,K. Transfer of Ribonucleic @ 125. J. G., and CALLAN, H. G. H' Uridine Incorporation Acid (RNA)-bound Amino Acids to Microsomal Proteins. in Lampbrush Chromosomes. Proc. Nat!. Acad. Sci. 48: Biochim.Biophys. Acts, 35:275â€”77,1959. 562â€”70,1962. 146. GUPTA, D. N. Nodular Cirrhosis and Metastasising Tu 126. GnonGlsw, G. P. Role of the Nucleus in the Synthesis of mors Produced in the Liver with Thioacetamide. J. a-RNA. Doki. Acad. Nauk, 142:211-14,1962. Pathol. Bacteriol., 72:415â€”26,1956. 127. Gzoaomv, G. P. ; ERMOLAEVA,V. P. ; and ZBARSKII,I. B. 147. HAENEL, U. VerILnderungen der Nucleolar Substanz an The Quantitative Relations between Protein and Nu Knochenmark bei pernicioser AnÃ¤mie und hire patho clear Protein Fractions of Various Tissues. Biochemistry, genetische Bedeutung. Helv. Med. Acts., 17:627â€”50,1951. 25(2):238, 1960. 148. [email protected], F. E., and Wout, A. D. Mode of Action of Chior 128. GEORGIEV, G. P., and SAMARINA,0. P. The Metabolic amphemcol. VIII. Resemblance between Labile Chlor Activity of Components of Nuclear Sap. Biochemistry, amphemcol-RNA and DNA of Bacillus cereus. Biochem. 26(3):401â€”7, 1961. Biophys. Res. Comm., 6:464-68, 1961-62. 129. GEORGIEV, G. P. ; SAMARINA,0. P. ; MANTIEvA, V. L. ; and 149. HAMILTON,M. G.; CAVALIER!,L. F.; and PETERMANN, ZBA1ISKY,I. B. Further Studies on the Nuclear Ribonu M. L. Some PhysicochemicalPropertiesof Ribonucleo cleoproteins. Biochim. Biophys. Ada, 46:399-400, 1961. protein from Rat Liver Microsomes. J. Biol. Chetn., 237: 130.Gums, I.FixationandStaining.In:J.Bnaciis'rand 1155â€”59,1962. A. E. MInSKY(eds.),The Cell,1:21-66. New York: Aca 150. HANSEN, H. J., and NADLER, S. B. Chemical Interaction @ demic Press, 1959. of S'5-6-mercaptopurine and Ribonucleic Acids. Proc. 131. GoLARz,M. N., and BouaNE, G. H. Induction and Ac Soc. Exp. Biol. Med., 107:324â€”26, 1961. centuation of Phosphatase Activity in the Nucleoli of 151. HARRIS, H. Turnover of Nuclear and Cytoplasmic Ribo Muscle Nuclei by Denervation and Injected Nucleotides. nucleic Acid in Two Types of Animal Cell, with Some Exp.Cell.Res.,25:691â€”93,1961. Further Observations on the Nucleolus. Biochem. J., 73: 182. GOLDSTEIN,L. Localization of Nucleus-specific Protein 362â€”69,1959.

152. . The Relationship between the Synthesis of Pro 171. HORN, E. C., and W@iw, C. L. The Localization of Basic tein and the Synthesis of Ribonucleic Acid in the Connec Proteins in the Nuclei of Larval Drosophila Salivary tive Tissue Cell. Biochem.J., 74:276â€”79,1960. Glands. Proc. Natl. Acad. Sci., 43:776â€”79, 1957. 158. HAYASRI, M., and SPIEGELMAN,S. The Selective Synthe 172. HUGHES, A. The Effect of Purines and Related Substances sis of Informational RNA in Bacteria. Proc. Nati. Aced. upon Cells in SChick Tissue Cultures. Exp. Cell lies., 3: Sci.,47:1564â€”80,1961. 108â€”20,1952. 154. Hzcm@, L. I.; STEPHENSON, M. L.; and [email protected]@iin,P. C. 178. HULTIN, T. Incorporation in Vise of Labeled Amino Binding of Amino Acids to the End Group of a Soluble Acids into Subfractions of Liver Cytoplasmic Fractions. Ribonucleic Acid. Proc. Nat!. Acad. Sci., 45:505â€”18, Exp. Cell Res. 3 (Suppl.):210â€”17, 1955. 1959. 174. . On the Functions of the Endoplasmic Reticulum. 155. HEITZ, E. Uber totsie mid partielle somatische Hetero Biochem. Pharmacol., 5:359â€”68, 1961. pyknose, sowie strukturelle Geschlechtschromosomen 175. . Release of Labeled Protein from Prelabeled Rat bei Drosophilafunebris. Z. Zellforsch.,19:720â€”42,1933. Liver Ribosomes. Biochim. Biophys. Acts, 51:219â€”20, 156. HERBERT,E.Reactions of Terminal Groups of Ribonu 1961. cleic Acid in Animal Systems. Ann. N.Y. Aced. Sci., 81: 176. HUNTER, G. D.; BROOKES, P.; CRATHORN, A. R.; and 679â€”89,1959. Bumun, J. A. V. Intermediate Reactions in Protein Syn 157. HERBERT, E., and CAREu@xis, E. S. s-RNA Synthesis. thesis by the Isolated Cytoplasmic-Membrane Fraction V. Reconstitution of a Biological Property of Phospho of Bacillus megaterium. Biochem. J., 73:369â€”76, 1959. diesterase-treated s-RNA. Biochim. Biophys. Acts, 47: 177. Htmwrrz, J., and BRESLER, A. E. The Incorporation of 85â€”92,1961. Ribonucleotides into Ribonucleic Acid. J. Biol. Chem., 158. Hunm, M. Zum Nucleolus Problem. Z. Zellforsch., 46: 236:542â€”48,1961. 18â€”51,1957. 178. JOHNSTON, F. B.; SurTERFIELD, G.; and S@rum, H. The 159. HOAGI.AND,M. B. On an Enzymatic Reaction between Isolation of Nucleoli from Ungerminated Pea Embryos. Amino Acids and Nucleic Acid and Its Possible Role in J. Biophys. Biochem. Cytol., 6:53â€”57,1959. Protein Synthesis. Rec. Tray. Chim. Pays-baa, 77:623- 179. JORGENSENS, M. Zellenstudien, Arch. Zellforschung, 10: 38, 1958. 1â€”202,1913. 160. HOAGLAND,M. B., and Coany, L. T. Interaction of Sol 180. KALF, G. F.; BATES, H. M.; and SIMPSON, M. V. Protein uble Ra'bonucleicAcidand Microsomes.Proc. Soc. NatI. Synthesis in Intact and Sonically Disrupted Mitochon Aced. Sci.,46:1554â€”63, 1960. dna. J. Histochem. Cytochem., 7:245â€”47, 1959. 161. HOAGLAND,M. B.; STEPHENSON,M. L.; Scorr, J. F.; 181. K@LF, G. F., and SIMPSON, M. V. The Incorporation of HECET, L. I.; and ZAMECNIK, P. C. A Soluble Ribonucleic Valine-1-C'4 into the Protein of Submitochondrial Frac Acid Intermediate in Protein Synthesis. J. Biol. Chem., tions. J. Biol. Chem., 234:2943â€”47, 1959. 231:241â€”57, 1958. 182. KAMMEN, A. v@. Infectious Ribonucleic Acid in the 162. HOAGLAND,M. B.; Z@taszcsrz@,P. C.; and STEPHENSON, Ribosomes of Tobacco Leaves Infected with Tobacco M. L. Bolesof Particulate and SolubleRibonucleicAcids Mosaic Virus. Biochixn. Biophys. Acts, 53:239-32, 1961. in Protein Synthesis. Symp. Mol. Biol., Univ. Chicago, 183. KARP, H. Die Cytodiagnostik maligner Tumoren aus pp. 105â€”14,1959. Punktaten und Sekreten. Z. Krebsforsch., 36:579â€”605, 163. Hou@y, R. W. ; APGAR, J. ; Docroa, B. P. ; FARROW, J.; 1982. MABIIfl, M. A.; and Mmuuu@, S. H. A Simplified Proce 184. KAUSIIIVA, B. S. Structural Basis of Nucleolar Function. dure forthe Preparation of Tyrosine- and Valine-Acceptor Experientia,17:455,1961. Fractions of Yeast â€œSolubleRibonucleic Acid.â€• J. Biol. 185. KELLER, E. B., and Z@uEcm@, P. C. The Effect of Gua Chem.,236:200â€”202,1961. nosine Diphosphate and Triphosphate on the Incorpora 164. Hou@y, R. W.; BRUNNGRABER, E. F.; Saw, F.; and tion of Labeled Amino Acids into Proteins. J. Biol. Chem., WILLIAMS, H. H. Partial Purification of the Threonine 221:45â€”59, 1956. and Tyrosine-activating Enzymes from Rat Liver, and 186. KELLER, E. B.; ZAMscrnz, P. C.; and LOFIFIELD, R. B. the Effect of Potassium Ions on the Activity of the Tyro The Role of Microsomes in the Incorporation of Amino sine Enzyme. J. Biol. Chem., 236: 197â€”99,1961. Acids into Proteins. J. Histochem. Cytochem., 2:878â€”86, 165. Hou@, R. W., and Doc'roit, B. P. Countercurrent Dis 1954. tribution of Amino Acid-Acceptor Ribonucleic Acids. 187. KEYL, H. G. Chromosomenevolution bei Chironomus. I. Fed. Proc., 19:348, 1960. Strukturabwandlungen an Speicheldrusenchromosomen. 166. Hoiizy, R. W.; Doc'ron, B. P.; MERRILL,S.H.; S@D, Chromosoma (Berl.), 12:26â€”47, 1961. F. M. Countercurrent Distribution of Rat-Liver â€œSo!-188. Knwtt, H. K.; HALVORSON,H.; and Bock, R. Release ubleâ€•-Fraction Ribonucleic Acids. Biochim. Biophys. of Soluble Protein from Yeast Ribosomes. Biochim. Bio Acts., 35:272â€”73,1959. phys. Acts, 49:212â€”14, 1961. 167. Hou@sy, R. W., and L&z@ts, V. A. Metal Content of 189. KUJARA, H. K.; Hu, A. S. L.; and HALVORSON, H. 0. The â€œSoluhleâ€•-Fraction Ribonucleic Acids. J. Biol. Chem., Identification of a Ribosomal-bound B-Glucosidase. 286:1446â€”47,1961. Proc. Nat!. Aced. Sd., 47:489â€”97, 1961. 168. Hou@ny, R. W., and Mnami@, S. H. Countercurrent Dis 190. KIRBY, K. S. A New Method for the Isolation of Ribo tribution of an Active Ribonucleic Acid. J. Am. Chem. nucleic Acids from Mammalian Tissues. Biochem. J., 64: Soc., 81:753, 1959. 405â€”8,1956. 169. HoawiN, W., and OTTo, H. Einige Probleme der Behand 191. KrrAzuME, P.; Yc@s, M. ; and Vn@cEr@rr,W.S. Metabolic lung b@sartiger Geschwlllste mit cytostatischen Substan Properties of a RibonucleicAcid Fraction in Yeast. Proc. zen am Beispieldes Colcemid.Z. Krebsforsch., 60:780- NaIl. Aced. Sd., 48:265â€”82, 1962. 74, 1955. 192. KJELDGAARD,N. 0. The Kinetics of Ribonucleic Acid 170. Ho@x@s, J. W.; ALLFREY, V. G.; and MIRSKY, A. E. and Protein Formation in Salmonella typhimurium dur Adenosine as the Receptor End Group in Nuclear Amino ing the Transition between Different States of Balanced Acid-Transfer RNA. Biochim. Biophys. Acta, 47:194-96, Growth. Biochim. Biophys. Acts, 49:64â€”76, 1961. 1961. 193. KLEE, W. A., and C@oNI, G. L. Studies on Soluble

Ribonucleic Acid (s-RNA) of Rabbit Liver. I. Amino 215. . Film sur des etudes du nuclÃ©olede cellules culti Acid AcceptorSpecificityand MolecularWeight. Proc. vÃ©esin vitro. Chemotherapia, 2:223â€”25, 1961. Natl. Aced. Sri., 46:322â€”23,1960. 216. LEWIS, W. H. Nucleolar Vacuoles in Living Normal and 194. KizniitLD, R. G. Early Changes in Rat Liver and Kid Malignant Fibroblasts. Cancer lies., 3:531â€”86,1948. ney Cells Induced by Thioacetamide. Cancer Res., 17: 217. Lnrn, R. D. Histopathologic Technic and Practical 954â€”62,1957. Histochemistry. New York: Blakiston, 1954. 195. KLEINTELD,R. G., and KOULISH, S. Cytological Aspects 218. LINDIGREIT, R., and COIJTELLE,R. Der Abbau ribo of Mouse and Rat Liver Containing Nuclear Inclusions. somaler Ribonukleinsaure aus E. coli in Untereinheiten. Anat. Eec., 128:433â€”45, 1957. Acts Biol. Med. Germ., 8:79â€”87,1962. 196. KLznirzu, R. G., and VON HAAM, E. The Effect of 219. LINDNER, A. Cytochemica! Effects of 5-Fluorouracil on Thioacetsmide on Rat Liver Regeneration. I. Cytological Sensitive and Resistant Ehrlich Ascites Cells. Cancer Studies. Cancer lies., 19:769â€”78, 1959. lies., 19:189â€”94, 1959. 197. . Effect of Thioacetamide on Rat Liver Regenera 220. Lxp@w@,F.;Huiaawiii, W. C.; HARTMANN,G.;BOMAN, tion. II. Nuclear RNA in Mitosis. J. Biophys. Biochem. H. G.; and Ace, G. AminoAcid Activationand Protein Cytol., 6:893â€”98, 1959. Synthesis. J. Cell Comp. Physiol., 54 (Suppl. 1) :75â€”88, 198. KtsrnscuMnvr, W. J. The Composition of Ribonucleic 1959. Acidand DeoxyribonucleicAcidof Normal and Neoplas 221. LrrrLsrmw, J. W., and DUNN, D. B. Natural Occur tic Tissue. Cancer lies., 19:966â€”69, 1959. rence of Thymine and Three Methylated Adenine Bases 199. KOPAC,M. J., and MATEYKO,G.M. Malignant Nucleoli: in Several Ribonucleic Acids. Nature, 181:254â€”55, 1958. 222. LOGAN, R.,and DAVIDSON, J. N. Heterogeneity of Nuclear Cytological Studies and Perspectives. Ann. N.Y. Acad. RNA. Biochim. Biophys. Acts, 241: 196, 1957. Sci., 73:237â€”82, 1958. 223. LONGWELL,A. C., and Svirn@, G. Specific Chromosomal 200. Kounza, A. Incorporation in Vitro of C'4 Amino Acids Control of the Nucleolusand of the Cytoplasmin Wheat. into Proteins of Rat-Liver Microsomal Particles. Bio Exp. Cell lies., 20:294â€”312,1960. chim. Biophys. Acts, 35:554â€”55, 1959. 224. LovE, R. Interchange of Ribonucleic Acid between the 201. . Studies on Incorporation of Amino Acids into Nucleus and Cytoplasm during Mitosis of Tumor Cells. Protein in Isolated Rat-Liver Ribosomes. Biochem. J., Acts Unio Internat. contra Cancrum, 16: 1150â€”52,1960. 81:168â€”78,1961. 225. . Improved Staining of the Nucleoproteins of the 202. . The Effect of Hypophysectomy and Growth Hor Nucleolus. J. Histochem. Cytochem., 10:227, 1962. mone Treatment of the Rat on the Incorporation of 226. LovE, R., and BHARADWAJ,T. P. Two Types of Ribonu Amino Acids into Isolated Liver Ribosomes. lbid., pp. cleoprotein in the Nucleolus of Mammalian Cells. Nature, 292â€”97. 183:1453â€”54,1959. 203. . Incorporation of Radioactive Amino Acids into 227. LovE, R., and Lmna, R. H. Differentiation of Nucleopro Serum Albumin by Isolated Rat-Liver Ribosomes. Ibid., teins by Inactivation of Protein-bound Amino Groups 83:69â€”74,1962. and Staining with Toluidine Blue and Ammonium 204. Kuz'r, E. L., and ZEIGEL,R. F. Cytoplasmic Ribonucleo Molybdate. J. Histochem. Cytochem., 7: 164â€”81,1959. protein Components of the Novikoff Hepatoma. J. Bio 228. LovE, R., and SUSKIND, R. G. Further Observations on phys. Biochem.Cytol., 7:465â€”78,1960. the Ribonucleoproteins of Mitotically Dividing Mam 205. LA FONTAINE,J. G. A Particulate Component Found in malian Cells. Exp. Cell lies., 22:193-207,1961. Nucleolus of Allium cepa and Vicia faba. J. Biophys. 229. Lunroiw, R. J. The Morphology and Physiology of the Biochem. Cytol., 4:229-80, 1958. Nucleolus. I. The Nucleolus in the Germ-Cell Cycle of 206. LAnm, A. K. Nuclear Changes Induced in Rat Liver the Mollusc Limnaea titagnahe. J. Roy. Micr. Soc., pp. Cells by Thioacetamide. Arch. Biochem. Biophys., 46: 113â€”50,1922. 119â€”27,1953. 230. LUDFORD, R. J. Nuclear Structure and Its Modifications 207. LAwRANDE, G. DE, and Au@iw, C. Studies on the Dis in Tumours. Brit. J. Cancer, 8:112â€”31, 1954. tribution of Intracellular Ribonucleases. Ann. N.Y. Aced. 231. MACCARTY, W. C. The Value of the Macronucleolus in Sci., 81:570â€”84, 1959. the Cancer Problem. Am. J. Cancer, 26:529â€”32,1936. 208. LANNI, F. Analysis of Sequence Patterns in Ribonuclease. 232. . Further Observations on the Macronucleolus of I. SequenceVectorsandVectorMaps.Proc.Nat!. Acad. Cancer. Ibid., 31: 104-6, 1937. Sci., 46:1563â€”76, 1960. 283. MACCARTY,W. C., and HAUMEDER,E. Has the Cancer 209. LASNITSKI,L., and Wn@nisoN, J. H. The Effect of Acri CellAny DifferentialCharacteristics?Am.J. Cancer,20: dine Derivatives on Growth and Mitosis of Cells in Vitro. 403â€”7,1984. Brit. J. Cancer, 2:369â€”75, 194& 234. McC@uerirr, B. J., and ARONSON, A. I. The Kinetics of 210. LERLOsm, C. P., and AMANO,M. Synthetic Processes in the Synthesis of RibosomalRNA in E. coli.Biophys.J., the Cell Nucleus. IV. Synthetic Activity in the Nucleus 1:227â€”45, 1961. as Compared to the Rest of the Cell. J. Histochem. Cyto 235. McM@srxa-Krn@, R. Symposium: Synthetic Processes diem.,10:162â€”74,1962. in the Cell Nucleus. III. The Metabolism of Nuclear 211. LEDERBEEG, S., and LRDumzaG, V. Hybridization be Ribonucleic Acid in Salivary Glands of Drosophila repteta. tween Bacterial Ribosomes. Exp. Cell Res., 25:198-200, J. Hist. Cytol., 10:154â€”61, 1961. 1961. 236. MCMASTER-KAYE, R., and TAYLOR, J. H. Evidence for 212. LEDERBEBG, S., and MAZIA, D. Protein Synthesis and Two Metabolically Distinct Types of RibonucleicAcid Cell Division in a Pyrimidine-starved Protozoan. Exp. in Chromatin and Nucleoli. J. Biochem. Biophys. Cytol., Cell lies., 21:590â€”95, 1960. 4:5â€”Il, 1958. 213. Licrn@, R., and Sium, W. Beobachtungen am Nucleolus 237. MAEDA,A. Some Properties of Ribonucleic Acid from in Vitro gezUchteter Zellen. Z. Krebsforsch., 60:19-80, Yeast 805 Particle; Effect of Magnesium Ions. J. Bio 1954. them., 50:377â€”85, 1961. 214. â€”@. Beobachtungen zur Struktur des Nucleolus in 288. MARTIN, E. M. The Effect of Encephalomyocarditis normalen Zellen sowie in Tumorzellen. ibid., pp. 564â€”80. Virus Infection on the Ribonucleic Acid Metabolism of

Krebs II Mouse Ascites Cells. Biochem. J., 71:P1â€”2, 259. NAEGELI,0. Blutkrankheiten und Blutdiagnostik. Zweite 1959. Auflage, pp. 38â€”41.Leipzig:Veit & Co., 1912. 239. MAURITZEN, C. M.; Roy, A. B.; and STEDMAN, E. The 260. . Blutkrankheiten und Blutdiagnostik. Vierte Ribosenucleic Acid Content of Isolated Cell Nuclei. Proc. Aufiage, pp. 23â€”26.Berlin: J. Springer, 1923. Roy. Soc. s.B, 140:18â€”31, 1952. 261. NATHANS, D., and LIPMANN, F. Amino Acid Transfer 240. MAYER, M. E. ; PETERSON, E. A. ; SOBER, H. A. ; and from Amino-acyl-ribonucleic Acids to Protein on Ribo GRECO, A. E. Purification and Characterization of Ribo somes of Eacheriehia coli. Proc. Nati. Aced. Sci., 47:497â€” nuclease of Calf Spleen. Ann. N.Y. Aced. Sci., 81:599â€” 504,1961. 610,1961. 262. NrsG, C.; STEVENS, A.; and LOPER, J. C. Stimulation of 241. M@tzu, D., and DAR, K. The Isolation and Biochemical Amino Acid Incorporation into Protein by Phage DNA Characterization of Mitotic Apparatus of Dividing Cells and an RNA Polymerase Fraction. Biochem. Biophys. Proc. Nat!. Aced. Sci. 38:826â€”38,1952. Res. Comm., 7:30â€”35, 1962. 242. MENZIES, D. W. Nucleolar Definition in Hemalum and 263. Nm, M. D.; CORDOVA,C. C.; and Nm, L. C. Ribonucleic Eosin Staining. Stain Technol., 37:41-42, 1962. Acid-induced Changes in Mammalian Cells. Proc. Nati. 243. MInSKY, A. E., and OSAWA, S. The Interphase Nucleus Aced.Sci.,47:1689â€”1700,1961. (Chemical Composition). In: J. BRACHur and A. E. Mm 264. NOMURA, M.; HALL, B. D.; and SPIEGELMAN, S. Charac SKY (ads.), The Cell, 2:677â€”770. New York: Academic terization of RNA Synthesized in Eaclierichia coli after Press,1961. Bacteriophage â€˜[â€˜2Infection. J. Mol. Biol., 2:306â€”26, 244. Mor'nsm, R. ; STEPHENSON,M. L. ; and ZAMECNIX,P. C. 1960. The Preparation and Some Properties of a Low Molecu 265. Novza.u, G. D. Mechanisms Involved in Control of En lar Weight Ribonucleic Acid from Baker's Yeast. Bio zyme Synthesis. In: Molecular Basis of Neoplasia, pp. chim. Biophys. Acts, 43: 1â€”8,1960. 435â€”60.Austin: Univ. of Texas Press, 1962. 245. MONTGOMERY,P. O'B., and BONNER, W. A. A New 266. NovELu, G. D., and DEMOSS, J. A. The Activation of Technique for Ultraviolet MicrobeamIrradiation of Liv Amino Acids and Concepts of the Mechanism of Protein ing Cells. A.M.A. Arch. Pathol., 66:418â€”21, 1958. Synthesis. J. Cell. Comp. Physiol., 50 (Suppl. 1): 173â€”97, 246. MONTGOMERY,P.O'B.; BONNER,W.A.; and ROBERTS, 1957. F. F. Ultraviolet Damage to Living HeLa Cells as Re 267. NURNBERGER, J.; ENGSTROM, A.; and LINDSTROM, B. A corded by Time-Lapse Motion Picture Studies. Proc. Soc. Study of the Ventral Horn Cells of the Adult Cat by Two Exp. Biol. Med., 95:589â€”91, 1957. Independent Cytochemicel Techniques. J. Cell Comp. 247. MONTGOMERY, P. O'B., and HUNDLEY, L. L. Effects of Physiol., 39:215â€”54, 1952. Selective Ultraviolet Irradiation of Cytoplasm of Living 268. NYGAARD, 0. F.; Gt@rrna, S.; and RuscH, H. P. Nucleic Cells. Proc. Soc. Exp. Biol. Med., 105:117â€”20, 1960. Acid Metabolism in a Slime Mold with Synchronous 248. . Ultraviolet Microbeam Irradiation of the Nucleoli Mitosis. Biochim. Biophys. Acts, 38:298â€”306, 1960. of Living Cells. Exp. Cell lies., 24:1-5, 1961. 269. OBERLING,C., and BERNHARD,W. The Morphology of the 249. MONTGOMERY,T.H. ComparativeCytologicalStudies Cancer Cells. In: J. BRACHarand A. E. MInSKY(eds.), with Special Regard to the Nucleolus. J. Morphol., 15: The Cell, 5:405â€”96,1960. 265â€”564,1898. 270. OcsoA, S., and Mu, S. Enzymatic Synthesis of Polynu 250. Mos'ry, K. J.; Lrcr, M.; KAY, E. R. M.; and DOUNCE, cleotides. IV. Purification and Properties of Polynucleo A. L. Isolationand Propertiesof LiverCell Nucleoli.J. tide Phosphorylase of Azotobacter vinelandii. J. Biol. Biophys. Biochem. Cytol., 2: 127â€”45,1956. Chem., 236:3303â€”11, 1961. 251. MORGAN, R. S. A New Form of Ribosome from Yeast. 271. O'DONNELL, E. H. J. Deoxyribonucleic Acid Structures J.Mol.Biol.,4:115â€”17,1962. in the Nucleolus. Nature, 191: 1325â€”26,1961. 252. MORGAN, W. S.; Dr@c@EN,A. VON DER; and HULTIN, T. 272. OEHLERT, W. Autoradiographische Untersuchungen zur Amino Acid Incorporation into Interior Protein Sites by RibonuldeinsÃ¤ure-Synthese in den verschiedenen Struk Purified Rat Liver Systems. Exp. Cell Res., 20:655â€”57, turen der Zelle Beitr. Pathol. Anat., pp. 811â€”50,1961. 1960. 273. OFENGAND, E. J.; DIECKMANN, M.; and BERG, P. The 258. MORGAN, W. S., and HULTIN, T. In Vise and in Vitro Enzymatic Synthesis of Amino Acyl Derivatives of Ribo Distribution of Labeled Amino Acids in Soluble Protein nucleic Acid. III. Isolation of Amino Acid-Acceptor Fractions of Rat Liver as Shown by Column Chromatog Ribonucleic Acids from Escherichia coli. J. Biol. Chem., raphy. Exp. Cell lies., 23:431â€”35, 1961. 236:1741â€”47,1961. 254. MoRiir, G. A.; ZAJDELA,R.; and COSTEROUSSE,0.Meta 274. OnNo, S.; WEILER, C.; and S'rmmjs, C. A Dormant Nu bolic Heterogeneity of Mouse Liver Desoxyribonucleic cleolus Organizer in the Guinea Pig, carla Cobaya. Exp. Acid. Exp. Cell lies., 13:204â€”7,1957. Cell Res., 25:498â€”503, 1961. 255. MORRISON, J. H.; LEAK, L. V.; and WILSoN, G. B. Corn 275. OLazEwsKA, M. J., and BRACEur, J. Incorporation de la bined Staining of Nucleolus and Chromosomes in Squash DL-mÃ©thionine5 S dana les fragments nuclÃ©eset anuc!@es Preparations of Plant and Animal Somatic Tissues. d'acetabularia mediterranea. Exp. Cell lies., 22:370â€”SO, Trans. Am. Micr. Soc., 76:355â€”6%,1959. 1961. 256. MORTREUUJ-LANGLOIS, M. Etude comparative du mets 276. OSAWA, S.; TAKATA, K. ; and HOTIA, Y. Some Aspects of bolisme des protÃ©ineset de l'acide ribonuclCique dana les the Relation between Nuclear and Cytoplasmic RNA noyaux hÃ©patiques de la sours en fonction de lear dia Synthesis. Biochim. Biophys. Ada, 25:656â€”57, 1957. metre. Exp. Cell Res., 24:46â€”51, 1961. 277. PAGE, R. C.; REGAN, J. F.; and MACCARTY, W. C. Intra 257. Mtaas@um, J. Presence d'inclusions Feulgen positives nucleolar Bodies in Normal and Neoplastic Human Tis dans lee nuclÃ©o!eslarvairesde quelques DiptÃ¨res.Arch. sue. Am. J. Cancer, 32:383â€”94, 1938. Biol., 67:485â€”98,1956. 278. PATAU, K. SAT-Chromosomen and Spiralstruktur der 258. MURAMATSU,M.,and BUSCH,H. Effects of Thioacetarn Chromosomen der extrakapsuliLrenKorper. Cytologia ide on Metabolism of Proteins of Normal and Regen Fujii Jubilee Vol., pp. 667â€”80,1937. erating Liver. Cancer Res., 22:1100â€”1104,1962. 279. PELLING, G. Chromosomal Synthesis of Ribonucleic Acid

as Shown by Incorporation of Uridine Labeled with Macronuclei of Two Ciliated Protozoa. J. list. Cyto Tritium. Nature, 184:655-56, 1959. chem.,10:145â€”53,1962. 280. Pgiusy, R. P. On the Nucleolar and Nuclear Dependence 300. Paseco@r, D. M., and BENDER, M. A. Synthesis of RNA of Cytoplasmic RNA Synthesis in HeLa Cells. Exp. Cell and Protein during Mitosisin MammalianTissueCulture lies., 20:216â€”20, 1960. Cells. Exp. Cell lies., 25:260-68, 196%. 281. Pznuiy, R. P., and ERRERA, M. The Influence of Nucleo 301. QUENSEL, U. Zur Frage der Cytodiagnostik der Ergilase lar RibonucleicAcid Metabolism on That of the Nucleus serÃ¶serHÃ¶hlen. Acts Med. Scandinav., 68:427â€”501, 1928. and Cytoplasm. In: J. S. Mrrcnsu. (ed.), The Cell Nu 30%. RABINOVITZ,M., and FISHER, J. M. Formation of a Ribo dens. New York: Academic Press, 1959. somal Lesion in Rabbit Reticulocytes by the Lysin An 282. Pznny, R. P.; Emmns, M.; HELL, A.; and DURwALD, H. tagonist, S-(B-aminoethyl) Cysteine. Biochem. Biophys. Kinetics of Nucleoside Incorporation into Nuclear and Res. Comm., 6:449â€”Si, 1961-6%. Cytoplasmic RNA. J. Biophys. Biochem. Cytol., 11:1â€”13, 303. RAscii, E.; Swwr, H.; Rinsun, A. H.; and CHOW,K. L. 1961. Altered Structure and Composition of Retinal Cells in 283. PEnny, R. P.; HEii, A.; and Eunxa@, M. TheRole of the Dark-reared Mammals. Exp. Cell lies., 25:848-63, 1961. Nucleolus in Ribonucleic Acid- and Protein Synthesis. 304. RaTHER, L. J. Experimental Alteration of Nuclear and I. Incorporation of Cytidine into Normal and Nucleolar Cytoplasmic Components of the Liver Cell with Thio Inactivated HeLa Cells. Biochim. Biophys. Acts, 49:47â€” acetamide. I. Early Onset and Reversibility of Volume 57,1961. Changes of the Nucleolus, Nucleus and Cytoplasm. Bull. 284. PrrERawr@i, M. L. Ribonucleoprotein from a Rat Tu Johns Hopkins Hosp., 88:38â€”58,1951. mor, the Jensen Sarcoma. I. The Effect of Magnesium 305. REm, E. Ribonudeic Acids in Rat-Liver Microsomes. Binding on U!tracentrifugal and Electrophoretic Proper Biochim. Biophys. Acts, 49:218â€”21,1961. ties. J. Biol. Chem., 235:1998â€”2003,1960. 306. REID, E., and STEVENS, B. M. â€œRapidlyLabelledâ€• Ribo 185. Ps@rziueANN, M. L., and PAVLOVEC,A. Ribonucleoprotein nucleic Acid in Rat-Liver â€œCalcium Supernatantâ€• Frac from a Rat Tumor, the Jensen Sarcoma. J. Biol. Chem., tions.Biochim.Biophys.Acts,49:215â€”18,1961. 236:8235â€”39,1961. 307. REITALU, R. The Appearance of Nucleoli and Hetero 9.86. P@rEns, K. Variationsstatistische Untersuchungen tiber chromatin in Mesothelial Cells and Cancer Cells of As das Auftreten von Vakuolen in den Nuldeolen von cites Tumours of the Mouse. Acts Path. Microbiol. Hlihnerherzfibroblasten in Vitro nach der Einwirkung Scandinav., 41:257-66, 1957. von B2sntgenstrahlen,Megaphen mid KItlte. Z. Zell 308. RENDI, R., and CAMPBELL, P. N. The Role of Cytoplas forsch., 44:14â€”26,1956. mic Ribonucleic Acid in the Incorporation of Amino 287. PETERs, T., Jn. Cytoplasmic Particles and Serum Albu Acids into Microsomal Proteins. Biochem. J., 72:435â€”41, mm Synthesis. J. Histochem. Cytochem., 7:224-34, 1959. 1959. 288. PnuE, N. W. The Recognition, Distribution, and Action 309. Rzisrn, R., and HULTIN,T. Preparation and Amino Acid of NucleicAcids. Conferenceon Chromosomes,pp. 105â€” Incorporating Ability of Ribonucleoprotein-Particles 29, 1956, Wageningen, The Netherlands. Zwolle, The from Different Tissues of the Rat. Exp. Cell Res., 19:253â€” Netherlands: W. E. J. Tjeenk WiIIInk. 66,1960. 289. P000, A. 0.; Bnswumwi, G.; and CHARGAYF, E. New 810. REVEL, M. ; MANDEL, L. ; WINTZERITH, M. ; Ki@sm, N.; RibonucleicAcid SpeciesAssociatedwith the Formation and MANDEL, P. Regulation de la biosynthÃ¨se des acides of the Photosynthetic Apparatus in Euglena gracilis. ribonuclÃ©iques. Action de la nicotinamide. Bull. Soc. Biochemistry, 1:128â€”Si, 196%. Chim. Biol., 43:91â€”101, 1961. 290. POLLISTER, A. W., and Ris, H. Nucleoprotein Determina 311. limo, J. H., and BONNER, J. The Site of Ribonudeic Acid tion in Cytological Preparations. Cold Spring Harbor Synthesis in the Isolated Nucleus. Proc. NatI. Aced. Sd., Symp. Quant. Biol., 12:147â€”57, 1947. 47:1611â€”19,1961. 291. POORT,C. A New Homogenizer for the Isolation of Nu 31%. RICE, R. V. An Electron Microscopical Examination of dci in Concentrated Glycerine. Biochim. Biophys. Acta, Configurations of Ribonucleic Acid and Other Polyelec 25:32â€”84,1957. trolytes. Biochim. Biophys. Acts, 53:29-43, 1961. 292. . Electrophoretic Comparison of Nuclear and Nu 313. RICHTER, G. Regeneration mid RNS-Synthese bei der deolar Proteins. I. Beef Pancreas. ibid., 46:373-80, 1961. Einwirkung eines artfremden Zeilkernes auf gealterte, kernlose Zellteile von Acetabularia Mediterranea. Natur 293. . Electrophoretic Comparison of Nuclear and Nu cleolar Proteins. II. Rat Pancreas. ibid., 51:286-40, 1961. wissenschaften, 45:629â€”30, 1958. 314. ROBERTIS, E. D. P. DE; Nowisani, W. W.; and SAEZ, 294. Paziss, J.; Dmcxawoi, M.; and BERG, P. The Enzymic F. A. General Cytology, Philadelphia: W. B. Saunders, Synthesis of Amino Acyl Derivatives of Ribonudeic Acid. IV. The Formationof the S'-HydroxylTerminal 1960. 315. ROODYN, D. B.; REm, P. J.; and Wonx, T. S. Protein Trinudeotide Sequence of Amino Acid-Acceptor Ribo Synthesis in Mitochondria. Requirements for the Incor nucleicAcid. J. Biol. Chem., 236:1748â€”57,1961. poration of Radioactive Amino Acids into Mitochondrial 295. PaEaco'r'r, D. M. The Nucleus and RNA Synthesis in Protein. Biochem. J., 80:9â€”21, 1961. Amoeba. Exp. Cell Res., 12:196â€”98, 1957. 316. ROSENBAUM, M., and BROWN, R. A. Simplified Prepara 296. . The Nuclear Dependence of RNA Synthesis in tion and Properties of Undegraded Rat Liver Transfer Acanthamoeba ISp. Ibid., 19:29-34, 1960. Ribonucleic Acid. Anal. Biochem., 2: 15â€”2%,1961. 297. - . Nuclear Function and Nuclear-Cytoplasmic In 317. RoTH, J. S. Comparative Studies on Tissue Ribonu teractions. Ann. Rev. Physiol., 22:17-44, 1960. deases. Ann. N.Y. Acad. Sci., 81:611â€”17, 1959. 298. â€” . Relation between Cell Growth and Cell Division. 318. ROTH,J. S., and Wo.nw@, R. An Impurity in RNA Prep IV. The Synthesis of DNA, RNA and Protein from Divi arations That Interferes with Assays for Ribonuclease sion to Division in Tetrahymena. Exp. Cell Res., 19:228- and Ribonuclease Inhibitor. Biochem. Biophys. Res. 38, 1960. Comm., 6:151â€”54,1961. 299. . Symposium: Synthetic Processes in the Cell Nu 319. SAGUCHI,S. Studies on the Glandular Cells of the Frog's cleus. U. Nucleic Acid and Protein Metabolism in the Pancreas. Am. J. Anat., 26:347-42%, 1920.

320. SAvINOVSXAL, A. A. Structural and Functional Signifi Methylated Guanines in Ribonucleic Acids from Several cance of the Nucleolus. Uspekhi Sovremennoi Biol. Sources. Biochem. J., 72:294â€”301, 1959. (Moskwa)., 46:156â€”73, 1958. 34%. SNOAD, B. The Behavior of Nucleoli in P/iaedrannassa 321. SAXEN,A. Pathologisch-anatomische und klinische Stu carmioli. Exp. Cell Res., 10:78â€”87,1956. dien ueber die Primaeren von der Nasenkavitilt der an 343. SounEic, D. The Correlation between the Growth of the grenzenden Nebenhoehlen ausgehenden Papillome und Cell and Nucleolar Secretion in Basidiobolus ranarurn Karzinome. Arb. Path. Inst. Helsingfors,4:1-180, 1926. eidam. Exp. Cell lies., 20:447-5%, 1960. 322. SCHACHTSCHABEL,D., and ZuuG, W. Untersuchungen 344. SPAHR, P. F., and HOLLINGWORTH,B.R. Purification and zur Biosynthese der Proteine. I. tber den Einbau â€œC Mechanism of Action of Ribonuclease from Escherichia markierter Aminosauren ins Protein zellfreier Nucleo coli Ribosomes. J. Biol. Chem., 236:823â€”31, 1961. proteid-Enzym-Systeme. Ztschr. Physiol. Chem., 314: 345. STAEHELIN,M. Studies on Nucleotide Sequences in Ribo 262â€”75,1959. nucleic Acids. I. Separation of Oligonucleotides on 828. SCHEBER, E. ; RINGLEB, D. ; and VmcrsxE, L. E. tber den DEAE-Cellulose. Biochim. Biophys. Acts, 49:11-19, Einuluss der BA$ntgenstrahlen auf den Nucleolarapparat 1961. der Zellen. Strahientherapie, 90:41â€”59, 1953. 346. . Studies on Nucleotide Sequences in Ribonu 324. SCHNEIDER,J. H. The Level of Phosphorylation of Nu cleic Acids. II. Spectroscopic Properties of Oligoribonu cleotides as Related to the Incorporation of (6-â€•C)Orotic cleotides.ibid., pp. 20-26. Acid into Ribonucleic Acid by Rat-Liver Homogenates. 347. . Studies on Nucleotide Sequences in Ribonucleic Biochim. Biophys. Acts, 51:60â€”65, 1961. Acids. III. Amounts of Oligonucleotides in Pancreatic 325. ScHourxumK, C. An Unstable Ribonucleic Acid in Rat Ribonuclease Digests. Ibid., pp. 27â€”35. Liver. Nature, 194:353â€”55, 1962. 348. SrEsaAus, U. The Nucleolar Size in the Liver Cell of 826. ScimvrzR, B.; OEHLERT,W.; and MAURER, W. tYber eine Rats Fed High and Non-protein Diets. Exp. Cell lies., ailgemeine Beziehung zwischen der Umsatzrate der Ribo 5:539â€”41,1953. nuldeinsÃ¤ure mid des Eiweisses im Organismus von 349. . Nucleolar Size in the Liver of Rats Fed Diets Mans und Ratte. Biochim. Biophys. Acts, 49:35â€”46, Deficient in Essential Amino Acids. Acts Pathol. Micro 1961. biol. Scandinav., 38:364â€”74, 1956. 827. Snaaar@, G. La misc en evidence des nuclÃ©olessur frottis 350. . Nucleolar Size in the Liver of Rats Fed on High par le bleu de mCthylÃ¨ne borate. Rev. Franc. etudes din. and Nonprotein Diets after Starvation. Acts Anat., biol., 5:196â€”98, 1960. 26:352â€”61,1956. 828. Suasssses, C. S., and Bnamium, P. N. Staining the Nu 351. . The Volume and Ribose Nucleic Acid Concentra cleolus. Stain Technol., 16:119â€”20, 1941. tion of Nucleoli in Liver and Hepatoma Cells of Rats Fed on High and Nonprotein Diets. Acts Pathol. Microbiol. 329. Snnaa, J. A., and Quzmoz, L. A. Direkter Nachweis Scandinav., 44:239â€”46, 1958. basiseber Proteine in den Chromosomen mid im Nude olus. Naturwissenschaften, 32:47-48, 1944. 35%. . Interferometric Determinations of the Ribose Nucleic Acid Concentration in Liver Nucleoli of Protein 830. SHERMAN,J. H., and Purumwiw, M. L. Some Physical fed and Protein-deprived Rats. Exp. Cell lies., 15:174â€” Properties of Calf-Liver Ribonucleoprotein. Biochim. 83, 1958. Biophys. Acts, 47:188â€”89, 1961. 353. . InterferometricandRefractometricDetermina 331. SIBATANI,A.; Kws'r, S. R. DE; ALLFREY, V. G.; and Mm tions of the Dry Matter Concentration of Nuclei and Nu SKY, A. E. Isolation of a Nuclear RNA Fraction Resem cleoli. Ibid., 22:545â€”558, 1961. bling DNA in Its Base Composition. Proc. NatI. Aced. 354. . On Protein Deficiency in Mice with Special Ref Sci., 48:471â€”77, 1962. erence to Liver Nucleolar Size. Z. Zellforsch., 56:125â€”29, 332. SIBATANI, A. ; Y@awiA, K. ; Kmwn@&,K. ; and OKAGAKI, 196%. H. Fractionationwith Phenol of RibonucleicAcid of 355. STEPHIENSON,M.L., and ZAMzcin@, P. C. Purification of Animal Cells. Biochim. Biophys. Acts, 331:590â€”91, 1959. Valine Transfer Ribonucleic Acid by Combined Chro 383. SLBATANI,A.; YAMANA,K.; Kmsuu@, K.; and TAKAHASHI, matographic and Chemical Procedures. Proc. NatL Aced. T. Fractionation ofTwoMetsbolicallyDistinct Classesof Sci.,47:1627â€”35,1961. RNA's in Animal Cells and Its Bearing on Cancer. Na 356. STITCH, H. Experimentelle karyologische und cyto ture, 186:215â€”17, 1960. chemische Untersuchungen an Acetabularia Mediter 334. Smus, J. L. The Nucleolus Problem. Nature, 186:275â€” ranea. Z. Naturforsch., 6B:319â€”26, 1951. 77, 1960. 357. . Bau mid Function der Nukleolen. Experientia, 335. . The Nucleolus. Prog. Biophys. Biophys. Chem., 12:7â€”14,1956. 12:25â€”59,319â€”26,1962. 358. STOCKINGER, L., and KnLLieun, G. Der Lymphozyten 336. Smi@m, J. L., and JACOB, J. Cell Function in the Ovary nukleolus. Wien. Ztschr. Inn. Med., 33: 135-41, 195%. of Drosophila. II. Behavior of RNA. Exp. Cell Res., 20: 359. STOLK,A. Two Types of Ribonucleoprotein in the Nude 283â€”93,1960. olus of Tumor Cells. Naturwissenschaften, 23:654, 1959. 337. Smuw, J. L.; KATO, K.; and JONES, K. W. Synthesis of 360. . Two Types of Ribonucleoprotein in the Nude Ribonucleic Acid in the Nucleolus. Biochim. Biophys. olus of Intestinal Carcinoma of the Newt Following In Acts, 48:421â€”23,1961. jection of Herring-Sperm Deoxyribonucleic Acid. Nature, 338. Smus, J. L., and KNIGHT, G. R. Chromosomal Synthesis 192:1215â€”16, 1961. of Protein. Exp. Cell Res., 19:210â€”19,1960. 361. STowELL, R. E. Nucleic Acid and Cytological Changes in 339. SKREB, Y., and BEVILACQUA,L. Acide ribonuclÃ©iqueet Regenerating Rat Liver. Arch. Pathol., 46: 164â€”78,1948. protÃ©lneschezlea fragments d'amibes irradiÃ©s.Biochim. 362. . Alterations in Nucleic Acids during Hepatoma Biophys. Acts, 55:250-52, 196%. Formation in Rats Fed p-Dimethylaminoazobenzene. 340. SMHT&NA,K. A Further Contribution to the Question of Cancer, 2:121â€”31,1949. the Incidence of Nucleoli in the Nuclei of Mature Lym 363. STRAUSS,D. B., and GOLDWASBER,E. Uridine Nucleotide phocytes of Man. Fol. BioL, 7:268â€”74, 1961. Incorporation into Pigeon-LiverMicrosome Ribonucleic 341.San@, J. D., and Dines,D. B. The Occurrenceof Acid. J. Biol. Chem., 236:849â€”56, 1961.

364. Sine, C. N. The Occurrence of Dense Granules of Un 382. VINCENT, W. S., and BALTUS, E. The Ribonucleic Acids known Function in the Nucleoli of Certain Plant Cells. of Nucleoli. In: J. S. MITCHELL (ed.), The Cell Nucleus, Exp.CellRes.,25:213â€”iS,1961. pp. 18-23. New York: Academic Press, 1960. 365. Swirr, H. Cytochemical Techniques for Nucleic Acids. 383. VINCENT, W. S., and HUXLEY, A. H. The Dry Matter in: E. CHARGAFF and J. N. DAVIDSON (eds.), The Nude Content of Starfish Oocyte Nucleoli. Biol. Bull., 107:290â€” id Acids, 2:51â€”92, 1955. 91,1952. 366. . Studies on Nucleolar Function. In: R. E. 384. VOGT-KÃ¶HNE,L. Quantitative cytochemische Unter ZIRKLE (ed.), Symp. Mol. Biol., pp. 266â€”303. Chicago: suchungen an Nukleolen aus Speicheldrtlsenkernen von Univ. of Chicago Press, 1959. Chironomus Thummi. Chromosoma, 12:382â€”97,1961. 367. Swivr, H., and RASCH, E. M. The Nucleoproteins of Nor 385. Vouur@, E. Synthesis and Function of the DNA-like mal and Irradiated Cells. J. Natl. Cancer Inst., 12:2.32, RNA. Fed. Proc.,21:112â€”19,1962. 1951. 386. VON HAAM, E., and ALEXANDER, H. G. A New Method of 368. SZAFRANSKI, P., and BAGDASARIAN,M. Nucleopeptides Vital Staining of the Nucleus and the Nucleolus in Cell and Protein Biosynthesis. Post. Biochemii, 7:49-62, 1961. Suspensions and Frozen Sections. J. Tech. Meth., 15:70â€” 369. TAKAHASKI, K. The Structure and Function of Ribonu 73, 1936. clease T1. I. Chromatographic Purification and Proper 387. . Cytological Studies of Malignant Tumours. Am. ties of RibonucleaseT1.J. Biochem.,49: 1â€”8,1961. J. Clin. Pathol., 6:394â€”414, 1936. 370. TAKANAMI, M. A Stable Ribonucleoprotein for Amino 388. VUNAKIS, H. V. ; LEIXHIM, E. ; DELANEY, R. ; LEVINE, L.; Acid Incorporation. Biochem. Biophys. Acts, 39:318â€”26, and BROWN, R. K. Studies on the Antigenic Structure of 1960. Ribonuclease. II. Partially Deaminated Ribonuclease. 371. T@uc&s@MI,M., and OKAMOTO,T. Isolation of an Enzyme J. Biol. Chem., 235:8430â€”36, 1960. Catalyzing the Transfer of Amino Acids from Soluble 389. WADDINGTON,C. H. The Role of the Nuclear Structures RNA to Microsomal Ribonucleoprotein. Biochim. Bio in the Incorporation of Amino Acids into Protein. Proc. phys. Acts, 44:379â€”Si, 1960. Inter. Genetics Symp., pp. 167â€”70,1956. 372. TANAKA, R. Aceto-basic Fuchsin as a Stain for Nucleoli 390. WADE, H. E., and L0VHTT, S. Polynucleotide Phosphoryl and Chromosomes of Plants. Stain Technol., 36:325-27, ase in Ribosomes of E. coli. Biochem. J., 81:319â€”28, 1961. 1961. 391. W@ieG, T.-Y. The Globulin of Calf Thymus Nuclei and 373. TANDLER, C. J. The Reaction of Nucleoli with Ammonia the in Vitro Incorporation of (Câ€•)ATP into Globulin cal Silver Nitrate in Darkness; AdditionalData. J. Histo RNA. Biochim. Biophys. Acts, 45:8â€”14, 1960. chem. Cytochem., 3:196-20%, 1955. 392. . Incorporation of (â€˜IC)Amino Acids into Ribo 374. -@ . The Silver Reducing Property of the Nucleolus nucleoprotein Fraction of Isolated Thymus Cell Nuclei. and the Formation of Prenuclear Material during Mitosis. Ibid., 49:108â€”1%,1961. Exp. Cell Res., 17:560-64, 1959. 393. . Saline Soluble Proteins of Isolated Thymus Nu 375. . The Localization of Intracellular Phosphate. The clei. Ibid., pp. 239â€”44. Role of the Nucleolus.Biochim.Biophys. Acta, 44:536â€” 394. . Ribonucleoprotein Particles from Isolated Calf 42, 1960. Thymus Nuclei. Ibid., 51:180â€”83, 1961. 376. TIMASHEFF, S. N.; Wrrz, J.; and Luzz.&TI, V. The Struc 395. . Stability of Nuclear Ribosomes. Ibid., 53: 158â€”OS, tare of High Molecular Weight Ribonucleic Acid in Solu 1961. tion. A Small-angle X-Ray Scattering Study. Biophys. J., 396. . The Chemical Composition of Nuclear Ribo 1:525â€”37,1961. somes of Calf Thymus. Arch Biochem. Biophys., 97:387â€” 377. TISSIERES, A. Structure of E. coli Ribosomes and Incor 92, 1962. poration of Amino Acids. In: The Molecular Basis of 397. WARREN, W. A., and GOLDTHWAIT,D. A. The Isolation Neoplasia, pp. 507â€”18.Austin: Univ. Texas Press, 1962. of Yeast Ribosomes Associated with THose Phosphate 378. TISSIERES, A., and Hopzrns, J. W. Factors Affecting Dehydrogenase. Proc. Nat!. Aced. Sci., 48:698â€”709, Amino Acid Incorporation into Proteins by Escherichia 1962. coli Ribosomes. Proc. Natl. Acad. Sci., 47:2015â€”23, 1961. 398. WEBSTER, G. C. The Biosynthesis of Amide and Peptide 379. VINCENT, W. S. Isolationand Chemical Propertiesof Bonds. Symp. Soc. Exp. Biol., 13:330â€”44, 1959. NucleoliofStarfishOocytes.Proc.Nati.Acad. Sci.,38: 399. WECKER, E., and SCHONNE, E. Inhibition of Viral RNA 139â€”45,1952. Synthesis by Parafluorophenylalanine. Proc. Natl. Aced. 380. . Structure and Chemistry of Nucleoli. Int. Rev. Sci.,47:278â€”82,1961. Cytol., 4:269â€”98,1955. 400. WEiss, S. B. Biosynthesis of Ribopolynucleotides. Fed. 381. . A Biochemical Approadh to Cell Morphology. Proc., 21:120â€”26, 1962. Anat. Rec., 133:346, 1959. 401. WHITE, F. H.,and ANFINSEN, C. B. Some Relationships

FIG. 1.â€”Nucleoli in Walker tumor cells and nuclei, stained with Azure C. X980. FiG. 2.â€”Nucleoli in Walker tumor cells, stained with to luidine blue. X980. FIG. 3.â€”A: Electron microgram of Walker tumor cells fixed in osmium tetroxide. In one nucleus, the nucleolus (NO) is closely associated with the nuclear membrane and in the other, the nucleolus (arrow) is in the center of the nucleus. X7,000. In this and in following figures the line represents l@u(micron). B: Nucleolus (NO) of Walker tumor cellin closeproximity to the nuclear membrane. X20,000.

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Downloaded from cancerres.aacrjournals.org on September 25, 2021. © 1963 American Association for Cancer Research. FIG. 4.â€”Vacuoles in nucleoli of leukemic lymphocyte. Aroundthenucleolusisnucleolus-associatedchromatin(C/i). Fixationwasinchrom-formol.)<14,000. FIG. 5.â€”Section of a nucleus of a Walker tumor cell showing nucleoluscomposedof nucleolonemata(arrow)which contain many smallparticles.X20,000. FIG. 6.â€”Nucleolus of a Walker tumor cell showing dense and lessdenseparticlescomposingthe nucleonernata(arrow). â€˜Themagnificationis 26,000.

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Downloaded from cancerres.aacrjournals.org on September 25, 2021. © 1963 American Association for Cancer Research. FiG.7.â€”Junctionofthenucleolus(NO)and chromatinin the nucleus of the Walker tumor cell. The dense granules noted in the nucleolus are seen elsewhere in the nucleus (dg) and may form part of a ribonucleoprotein reticulum in the nucleus. X26,000. FIG. 8.â€”Isolated nucleolus of a Walker tumor cell fixed in osmium tetroxide. The nucleolonemata are composed of small particles. )(60,000. In this case only, the measured line is 0.5@i.

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Downloaded from cancerres.aacrjournals.org on September 25, 2021. © 1963 American Association for Cancer Research. FIG. 9.â€”Nucleolus of a normal liver cell adjacent to the nuclear membrane. The nucleolus is composed of masses of dense granules as well as less dense granules which are present in the nucleonemata. This electron microgram was made by Dr. Ilewson Swift.

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Downloaded from cancerres.aacrjournals.org on September 25, 2021. © 1963 American Association for Cancer Research. FIG. 10.â€”Electron microgram of a nucleolus of the liver of a thioacetamide-treatedrat.The nucleolusiscomposed of a large number of dense and less dense particles. The normal structureofthe nucleolonemataisobscured.This electron microgram was made by I)rs. H. Swift and R. Kleinfeldt.

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of Structure to Function in Ribonuclease. Ann. N.Y. 413. â€”.Fractionation of Ribonucleic Acids with Phenol. Aced. Sci., 81:515â€”23,1959. II. The Relationship between RNA Fractions in Rat 40%. WOLMAN,M. Problems of Fixation in Cytology, Histology Ascites Hepatoma. Exp. Cell Res., 21:535â€”40, 1960. and Histochemistry. Intern. Rev. Cytol., 4:79â€”102, 1955. 414. YeAS, M. Aspects of Ribonucleic Acid Synthesis. In: 403. WONG, K. K.; MEISTER, A.; and MOLDAVE, K. Enzymat R. E. ZIRKLE (ed.), Symp. Mo!. Biol., pp. 115-21. id Formation of RNA-amino Acid from Synthetic Amino Chicago: Univ. of Chicago Press, 1959. acyladenylate and Ribonucleic Acid. Biochim. Biophys. 415. Yc@sa,M., and VINCENT, W. S. The Ribonucleic Acid of Acta, 36:531â€”33, 1959. Epanaphe moloneyi druce. Exp. Cell lies., 21:513â€”22, 404. WONG, K. K., and MOLDAVE, K. Enzymatic Participa 1960. tion of Enzyme-bound Amino Acyl Adenylates in Amino 416. YOKOYAMA,H. 0., and STOWELL, R. E. Nucleolar Volume Acid Incorporation into Protein. J. Biol. Chem., 235:694â€” Changes in the Mouse Pancreas after Repeated Pilocar 99, 1960. pine Injections. J. Nat!. Cancer Inst., 11:939â€”45, 19@1. 405. WOODARD,J. ; GELBER, B. ; and Swwr, H. Nucleoprotein 417. YOUNG, R. J.; KIHARA, H. K.; and HALVORSON, H. 0. Changes during the Mitotic Cycle in Paramecium aurella. Studies on the Kinetics of Protein Synthesis in Yeast. Exp. Cell lies., 23:258â€”64, 1961. Proc.Natl.Aced. Sci.,47:1415-23,1961. 406. WOODARD,J.,RASCH,E.; and Swwr, H. Nucleic Acid and 418. YU, C. T., and ZAMECNIK, P. C. A Hydrolytic Procedure Protein Metabolism during the Mitotic Cycle in Vicia for Ribonucleosides and Its Possible Application to the Sequential Degradation of RNA. Biochim. Biophys. faba. J. Biophys. Biochem. Cytol., 9:445â€”6%,1961. Acta, 45: 148â€”54,1960. 407. WOODS, M. W. The Nucleolus in Tulipa. Am. J. Bot., 419. ZADEK, J. Die zytodiagnostischen Kennzeichen der 24:528â€”86, 1937. Krebszellen. Adta Med. Scandinav., 80:78â€”9%,1933. 408. WOODS, P. S. RNA in Nuclear-cytoplasmic Interaction 420. ZAGURY, D. Existence d'un complexe liporibonuc!eopro Brookhaven Symp. Biol. No. 12, pp. 153â€”74,1959. tÃ©iquea groupement sulphhydryles au rein du nucleoli. 409. WOODS,P. 5., and TAYLOR,J. H. Studies of Ribonucleic Compt. Rend. Aced. Sd., 244: 182,5â€”27,1957. Acid Metabolism with Tritium-labeled Cytidine. Lab. 421. ZALOKAR,M. Nuclear Origin of Ribonucleic Acid. Nature, Investigation, 8:309â€”18, 1959. 183:1380,1959. 410. WmGHT, L. D., and TRAGER, R. Additional Evidence for 422. . Sites of Protein and Ribonucleic Acid Synthesis Involvement of RNA in Maintenance of ATP Levels in in the Cell. Exp. Cell lies., 19:559â€”76, 1960. Homogenates. Proc. Soc. Exp. Biol. Med., 106:21â€”24, 423. ZAMECNIK,P. C., and STEPHENSON,M. L. Partial Purifi 1961. cation of Transfer RNA. Ann N.Y. Aced. Sci., 88:708- 411. YAMANA, K. Metabolic Heterogeneity of Ribonucleic 17, 1960. Acid in Ehrlich Ascites Tumor Cells. Gann, 50:201â€”8, 424. ZAMECNIK, P. C.; STEPHENSON, M. L.; and YU, C. T. 1959. Studies on Preparation, Fractionation and Degradation 41%. YAMANA, K., and SIBATANI, A. Fractionation of Ribonu of Soluble Ribonucleic Acid. Symp. Protein Biosyn. cleic Acids with Phenol. I. Two Metabolically Distinct Wassenaar,1960. Classes of Ribonucleic Acids in Animals Cells. Biochim. 425. ZUBAY, G. A Theory on the Mechanism of Messenger Biophys. Acts, 41:295â€”308, 1960. RNA Synthesis. Proc. Natl. Acad. Sci., 48:456-61, 1962.

Harris Busch, Paul Byvoet and Karel Smetana

Cancer Res 1963;23:313-339.

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