Differential effects of antimitotic agents on the stability and behavior of cytoplasmic and ciliary microtubules

Summary When sea urchin gastrulae are treated with colchicine or hydrostatic pressure the cytoplasmic microtubules disappear, but the ciliary microtubules which make up the ciliary axoneme (9+2) remain. With calcium-free sea water the cytoplasmic microtubules are reduced in number yet the 9+2 complex in the cilia is unaffected. Furthermore during the administration of any of these agents the cilia continue to beat so that functionally as well as morphologically the ciliary microtubules are normal even though the cytoplasmic microtubules are broken down and their presumed function in development is interrupted.Available evidence indicates that these two types of microtubules appear to be made up of similar subunits. Since there are morphological connections between the microtubules of the ciliary axoneme, and since the ciliary microtubules appear to stain more intensely than the cytoplasmic microtubules, we conclude that the ciliary microtubules are stabilized either by the addition of material or through interactions between adjacent tubules or both.Supported by Grant #5T 1-GM-707 from the National Institutes of Health to ProfessorKeith R.Porter.     

Ultrastructure of transitional epithelium of man

Summary Blocks of human normal renal pelvis and ureter obtained at the time of surgery were fixed in glutaraldehyde and osmium with or without ruthenium red, for electron microscopic observations. The transitional epithelium is arranged in three cell layers: basal, intermediate and superficial. All epithelial cells show numerous microvilli and contain the characteristic vesicles of transitional epithelium, bundles of cytoplasmic filaments, microtubules and numerous free ribosomes. The epithelial extracellular compartment is notably large and appears as an intricate, tridimensional network of canaliculi and cisternae which are wider in the intermediate and superficial layers and in which microvilli and cytoplasmic folds of vicinal cells are often attached or interdigitated. At these sites there are desmosomes.The surface of all transitional epithelial cells is covered by a fibrillar mucous coat which is more developed at the plasmalemma of the free border of luminal cells in which microvilli are also seen. Ruthenium red stains selectively the plasmalemma and the mucous coat of the free surface of the epithelium, indicating the presence of an acid polysaccharide. With this technic (Luft, 1965), it is observed, radiating from the plasmalemma, branching filaments which measure 100 Å in diameter forming a zone of varying density which is about 400 m wide and which corresponds, at the light microscopic level, to the luminal border of the transitional epithelial cells in which a sialomucin has been identified. The slender filaments have a beaded appearance. At the free border, superficial cells are attached by functional complexes in which tight junctions seal the epithelial intercellular space, which is opened at the level of the basement membrane where only desmosomes are observed.The ultrastructure of human transitional epithelium of urinary tract resembles the duct cells of the salt gland of certain marine birds (Fawcett, 1962) and the amphibian epidermis (Farquhar and Palade, 1965) in which there are active processes of transport. The mucous surface coat, selectively stained by the ruthenium red, contains a sialomucin (Monis and Dorfman, 1965, 1967).The ultrastructure and histochemistry of the mucous fluffy coat of man transitional epithelium and the observations of Porter and Tamm (1955), on the ultrastructure of preparations of the Tamm and Horsfall mucoprotein (1952) are bases for suggesting that transitional epithelium of urinary tract of man is the site of biosynthesis of certain urinary mucoids. Present investigations are directed to obtain evidence to substantiate this hypothesis.General Abbreviations B basal cell - E exfoliating cell - I intermediate cell - L lumen - S superficial cell - SC surface coat - bm basement membrane - ci cell infolding - d desmosome (macula adhaerens) - f fibroblast - fi cytoplasmic filaments - is intercellular space - jc junctional complex - ly lysosome - lym lymphocyte - mt microtubules - m mitochondria - mv microvilli - n nucleus - r ribosomes - rv round vesicle - zo zonula occludens - za zonula adherens Dr. Monis wishes to thank Dr. E. De Robertis for the use of the electron microscope facilities of the Instituto de Anatomía General y Embriologia, Facultad de Medicina, Universidad de Buenos Aires. — Prof. E. Trabucco and Dr. R. J. Borzone (Cátedra de Clinica Genitourinaria de la Facultad de Medicina, Universidad de Buenos Aires) generously supplied the specimens which were the bases of this study. — Thanks are due to Mrs. A. M. Novara and Mrs. Defilippi-Novoa for efficient technical help and to Miss Rosa Gentile for secretarial assistance. Photomicrography by Mr. M. A. Saenz.Dr. Zambrano is investigator (CNICT).     

Ribosomes ofDunaliella

Summary The high molecular weight ribonucleic acids from the green algaDunaliella were isolated from wholeDunaliella cells and fromDunaliella ribosomes and analysed by the technique of sucrose density centrifugation. Ribonucleic acids from whole cells and fromDunaliella ribosomes showed the same sedimentation profile only when ribosomes were prepared in the presence of the RNase inhibitor polyvinyl sulfate. Otherwise ribonucleic acids fromDunaliella ribosomes were degraded to some extent, as compared with those from whole cells, although the ribosomes were still physically intact. The ribonucleic acids fromDunaliella were resolved by sucrose density centrifugation into three high molecular weight components sedimenting with 26, 23 and 17.5s. The 80s ribosomal fraction contained mainly a 26 and 17.5 s RNA, whereas the 50 s ribosomal fraction contained a 23 s RNA. The 26 s RNA and the 23 s RNA may represent the heavy ribonucleic acids from the cytosol and the chloroplast of the cell respectively, whereas the 17.5 s RNA may be a mixture of the two light RNA's from the two cell compartments.The experiments described in this paper were submitted by H. J.Rahmsdorf to the Fachbereich Biologie der Freien UniversitÄt Berlin in partial fulfillment of the requirements for a doctor's degree.     

Transitional epithelium of urinary tract in normal and dehydrated rats

Summary This report is a light microscopic histochemical and fine structural study of transitional epithelium of the urinary tract of normal and dehydrated rats. Four types of cells were recognized: basal, intermediate, squamous or luminal and bundle cells. The transitional epithelium of normal rat ureter and bladder shows distinct cytoplasmic staining of the squamous cells layer by PAS. The luminal free border stains more intensely with PAS. With the electron microscope, abundant cytoplasmic tonofilaments, free ribosomes and the characteristic thick-walled fusiform and round vesicles are observed, which were in greater number in the squamous cells. Lysosomes are identified with PAS, and Toluidine Blue 0, by their content of acid phosphatase and non-specific carboxylic esterase, and by their ultrastructural appearance. The bundle cell (Hicks, 1965) is characterized by histochemical technics. These cells form about 2.5% of the total cell population of normal transitional epithelium. The bundle cell contains basophilic metachromatic granules, which indicates the presence of a weakly acid mucosubstance. It is suggested that bundle cell granules are released in the intercellular spaces of transitional epithelium and that the mucosubstance may regulate flow of ions and metabolites in the epithelial intercellular channels.Several ultrastructural changes occur in the transitional epithelium of dehydrated rats: marked increase in number of thick-walled vesicles, development of polysomes, relative increase of cytoplasmic filaments and greater number of enlarged lysosomes. Bundle cells decrease in number. These ultrastructural changes promptly regressed by allowing the animal to drink water.It is suggested that the rate of formation of the characteristic vesicles of transitional epithelium, a function of membrane synthesis, may be under the control of the antidiuretic hormone.This investigation was supported in part by the Consejo Nacional de Investigaciones Científicas y Técnicas, Argentina, through a travel grant to Dr. Monis, who would like to thank Dr. E. de Robertis for the use of the electron microscope facilities of the Instituto de Anatomía General y Embriología, Facultad de Medicina, Universidad de Buenos Aires.     

Relation Between Basophilia and Fine Structure of Cytoplasm in the Fungus Allomyces macrogynus Em

In a fungus, Allomyces macrogynus Em., staining tests have revealed changes in the location of cytoplasmic basophilia following different phases of the developmental cycle. These variations in location were used to observe which fine structures correspond to basophile and non-basophile areas of the cytoplasm. Hyphae, gametangia, zygotes, and plants were fixed at various developmental stages in OsO₄, pH 6.1, and embedded in vestopal. Sections were examined in the electron microscope. Comparison of basophile and non-basophile cytoplasms leads to the conclusion that cytoplasmic particles of 150 to 200 A in diameter are responsible for basophilia. The possibility of these particles being ribosomes is discussed and confirmed. The present paper also describes some observations on the fine structure of other cellular components of this fungus, such as nuclei, mitochondria, various granules, and flagella.     

Hypothalamic neurosecretory activity in relation to the reproductive cycle in the common striped skunk (Mephitis mephitis nigra; order carnivora)

Summary The histological appearance of certain cell groups in the anterior hypothalamus was studied during various phases of the reproductive cycle of the common striped skunk (Mephitis mephitis nigra), a carnivore having a very restricted period of sexual activity. Pronounced changes occur in the amount of stainable neurosecretory material in cells of the supraoptic, paraventricular and anterior hypothalamic nuclei. Material staining with aldehyde fuchsin is stored (or inhibited from being released) in these cells during the sexually quiescent period in both the female and male skunk. The time of approaching sexual activity is characterized by the first signs of release of neurosecretory material from these hypothalamic cells and the peak of estrus and rut coincides with a minimal content of the material.Dedicated to Professor Berta V. Scharrer in honor of her 60th birthday.This study was supported by U.S.P.H.S. Training Grant 5 T 1-GM 102 and Research Grant BN-00840, from the National Institutes of Health. This represents a portion of a dissertation, written under the guidance of the late Professor Ernst Scharrer, and submitted in partial fulfillment of the requirements for the degree of Doctor of Philosophy at Yeshiva University, June, 1965. Preliminary reports of this work were presented at the 78th annual session of the American Association of Anatomists, Miami, Florida, and at the VIIIth International Congress of Anatomists, Wiesbaden, Germany, (Hagedoorn, 1965a, b). I thank Dr. H. W. Deane and Dr. J. Osinchak for their critical reading of this manuscipt.     

The liver of the slender salamander Batrachoseps attenuatus

Summary The structure of the crystalline inclusions found in Batrachoseps liver cells is described and it is shown that the most symmetric unit cell upon which the crystal lattice is built is a face-centered cube. Taking into consideration the physical properties of a face-centered cubic structure, an attempt is made to determine the nature of the macromolecules that comprise the crystal. It is concluded on the basis of available evidence that the macromolecules probably represent serum lipoproteins. The intracellular synthesis of the crystals and the possible functions they may subserve in the animal are discussed. A comparison is made between the crystals and granules in rat hepatocytes discussed by Bruni and Porter (1965).Research supported by grant RG-6729 from the Institute of General Medical Sciences, National Institutes of Health, United States Public Health Service.     

Pre-fertilization ovule development inCapsella: ultrastructure and ultracytochemical localization of acid phosphatase in the meiocyte

Summary Pre-meiotic and prophase I ovules ofCapsella bursa-pastoris (L.) Medic.(monosporic,Polygonum type of gametophyte development) were fixed routinely or incubated in a modified Gomori medium containing -glycerophosphate as a substrate. Prior to the beginning of meiosis the potential meiocyte is ultrastructurally similar to the other cells of the nucellus and is distinguished only by its size and position. At the initiation of prophase I dramatic ultrastructural and ultracytochemical changes take place in the female meiocyte. These include the sudden appearance of cytoplasmic structures composed of single and multiple concentric cisternae, distinctive changes in plastids and mitochondria, and the blebbing of 0.3 m double-membraned vesicles from the nuclear envelope. The concentric cisternae encapsulate portions of cytoplasm containing ribosomes, plastids, mitochondria, ER fragments and vesicles. Both single and multiple concentric cisternae localize high levels of acid phosphatase and function as autophagic vesicles (AVs) that sequester ribosomes and organelles for destruction during meiosis. Plastids stop dividing and become more spherical during prophase I. Some plastids localize acid phosphatase and many show continuities between the outer membrane and the plastid envelope and acid phosphatase-rich RER cisternae. Mitochondria appear as dense, contracted spheres or rods. Some mitochondria localize acid phosphatase but they do not show membrane confluencies with the ER. Some of the plastids and mitochondria that are segregated into the functional megaspore at meiosis II are destroyed but others apparantly survive meiosis and give rise to the plastid and mitochondrial populations of the young gametophyte (Schulz andJensen, unpublished). The lateral and end walls of the meiocyte show patches of intense aniline blue fluorescence and the chalazal end wall of the cell is perforated with large numbers of plasmodesmata.Research supported by NSF Grant PCM-79-11018. The authors gratefully acknowledge the valuable assistance of David Lee Ivans in this project.     

Morphometric analysis of parietal cell membrane transformations in isolated gastric glands

Summary Changes in parietal cell membranous structures that accompany the onset of acid secretion were studied with electron microscopy using isolated gastric glands from rabbit. A stereological analysis was performed to quantitate the morphological changes occurring within 5 min following histamine stimulation. These changes were compared to the changes resulting from osmotic expansion of parietal cell components following addition of 1mm aminopyrine (AP) to glands incubated in medium containing 108mm K⁺ (high-K⁺). Morphometric analyses, together with measurements of glandular water content, indicated that parietal cells swell in high-K⁺ medium. Addition of 1mm AP to glands incubated in high-K⁺ medium resulted in massive distention of the secretory canaliculus but no difference was observed in the amount of tubulovesicular membrane or the relative size of these cytoplasmic structures. In the histamine-treated glands the parietal cells displayed a rapid loss of tubulovesicular membrane and a reciprocal increase in canalicular membrane. These morphological changes were complete long before a maximum level of acid formation was achieved. Taken together, these results indicate that; (i) the morphological change accompanying stimulation does not require acid formationper se; (ii) the site of acid secretion is the intracellular canaliculus and not the tubulovesicles; (iii) there is no preexisting actual or potential continuity between the tubulovesicular space and the canalicular space; and (iv) the AP-induced expansion of the canaliculus in high-K⁺ medium, while yielding some valuable information, is not an appropriate model for studying the normal stimulus-induced morphological transition, despite a superficial similarity of appearance.     

A cytochemical study on the effects of energy deprivation on autophagocytosis in Ehrlich ascites tumor cells

Summary The effect of energy deprivation on autophagocytosis in Ehrlich ascites tumor cells was studied using cytochemical techniques. Autophagocytosis was induced with vinblastine incubation (0.1 mM) and the cellular ATP-level was lowered with 2-deoxy-d-glucose (0.35 mM). Acid phosphatase was used as a marker for lysosomal enzymes and imidazole-buffered osmium tetroxide impregnation in order to study the effects of energy deprivation on the maturation of autophagic vacuole (AV) membranes.Control and vinblastine treated cells maintained their ATP-levels throughout the incubation period tested (120 min). 2-Deoxy-d-glucose alone and with vinblastine decreased the intracellular ATP-level significantly after only 3 min incubation. Most of the AV's in control and vinblastine treated cells contained degraded material and acid phosphatase activity. Their membranes were stained only slightly or not at all with imidazole-buffered osmium tetroxide. 2-Deoxy-d-glucose alone as well as with vinblastine induced in particular an accumulation of early stages of AV's. These vacuoles contained undegraded cytoplasmic material and no acid phosphatase activity and their membranes were stained usually partly with imidazole-buffered osmium tetroxide. The membranes of some early AV's resembled endoplasmic reticulum and still had attached ribosomes.It was concluded that the inhibition of cellular energy production used in the present study did not inhibit autophagic sequestration but retarded the maturation of AV membranes and impaired the functioning of lysosomal hydrolases.     

The distribution of microtubules in differentiating cells of Micrasterias denticulata bréb.

Summary As an extension of earlier cytophysiological and morphological studies on differentiating cells of Micrasterias denticulata, a fine structural investigation of glutaraldehyde-osmium tetroxide fixed material has been made. Special emphasis has been placed on the distribution of cytoplasmic microtubules and on their possible role in the processes of growth and differentiation. Four distinct systems of microtubules were found: (a) a band in the cortical protoplasm of the isthmus region which surrounds the nucleus; (b) several bands in the cortical protoplasm of the old half cells, with rod-like cross bridges between individual microtubules and between the microtubules and the plasmalemma; (c) clusters of microtubules near the posttelophase nucleus, some separated by intertubular structures possibly fibrils; and (d) microtubules in the internal and cortical protoplasm of differentiating half cells.This work was supported by a National Science Foundation Senior Foreign Scientist Fellowship to Dr. Oswald Kiermayer,and by funds of Training Grant 5-T1-GM-707-06 to Dr. Keith R. Porter.     

The inhibition of kohlrabi chloroplast degeneration by kinetin

Summary Detached kohlrabi leaves of late autumn material yellowed completely after 6 days in weak light. This process was accompanied by a decrease in chlorophyll, protein, and ribonucleic acid levels. In the chloroplasts, degeneration symptoms such as reduction in chloroplast volume, the decay of grana, development of the long thylakoid system, disappearance of chloroplast ribosomes, increase in the volume of plastoglobuli, and finally a complete breakdown of plastids in the digesting vacuoles, were observed. The ultrastructural changes in degenerating kohlrabi chloroplasts resembled those described earlier for brussels sprouts (Dennis et al. 1967), which suggests that the plastid degeneration model may be specific for speciesBrassica oleracea L.Kinetin inhibited the fall in the level of chlorophyll, proteins, and RNA in relation to the control material, and even stimulated chlorophyll and protein synthesis to a level higher than that of the initial material. Treatment with kinetin also markedly delayed the loss of chloroplast ribosomes. The most evident effect of the kinetin influence on the plastid ultrastructure was the stimulation of the formation and maintenance of grana. A possible mechanism for these processes in the light of the recent studies on the chloroplast membranes is discussed.     

Hybrid 80S monomers formed from subunits of ribosomes from protozoa,fungi, plants,and mammals

Free 80S ribosomes of eukaryotic organisms are dissociated by KCl (0.8–1.0 m) in the presence of 2-mercaptoethanol and magnesium ions (10–15mm); the large and small subunits so formed can be recombined to yield 80S monomers. We have now studied the ability of ribosomal subunits from protozoa (Tetrahymena pyriformis), fungi (Allomyces arbuscula, Saccharomyces cerevisiae), plants (pea, wheat), and mammals (rat, mouse, rabbit) to combine to form hybrid ribosomes. In general, both subunits of the species studied participate in the formation of hybrid particles, with the exception of the 60S subunit of Tetrahymena, which does not combine with the small subunit of fungal, plant, or mammalian ribosomes. The interaction of subunits from rat and Tetrahymena ribosomes has been visualized by an electron microscope study of negatively stained preparations. The base sequences of the ribosomal RNAs of these organisms have been compared to those of Saccharomyces by nucleic acid hybridization-competition.This work was supported by a fellowship #PF-529 from the American Cancer Society and by United States Public Health Service, National Institutes of Health grant GM 12449.     

Ultrastructural studies of normal skin and epidermal papillomas of the flathead sole,Hippoglossoides elassodon

Summary Epidermal cells of normal fin web and of the epidermal papilloma of the flathead sole, Hippoglossoides elassodon, were compared by light and electron microscopy. The predominant cells of normal epidermis are squamous and are characterized by complex microvillous interdigitations between adjacent cells, numerous desmosome-like structures, terminal bars, prominent cytoplasmic filaments, and blunt, broad-based microvilli on the outer (free) surfaces of the superficial cells. The less numerous mucous cells of normal epidermis possess abundant ergastoplasm and mucus-filled vacuoles, but lack desmosome-like structures and cytoplasmic filaments.In contrast, in the epidermal papilloma, mucous cells are absent; and the tumor cells are without microvilli, desmosome-like structures, cytoplasmic filaments, and conspicuous ergastoplasm. The papilloma cells are in addition characterized by certain features not seen in normal epidermis, including prominent nuclear pores, very large nucleoli, nuclear dense bodies, vesicular mitochondria with few cristae and dense internal granules, and three types of distinctive cytoplasmic particles of possible viral nature.This investigation was supported by Public Health Service research grant CA 08158 from the National Cancer Institute, and American Cancer Society Institutional Grant No. IN-53 E.We are indebted to Miss Mary Bens for her indispensable technical assistance.     

Intracellular Distribution of 3H-Dihydrostreptomycin in a Streptomycin-dependent Strain of Bacillus megaterium

The cells of a streptomycin-dependent strain of Bacillus megaterium took up only 2 to 5% of the dihydrostreptomycin present in the medium when grown in the minimum concentration of streptomycin required for growth. During growth in the presence of (3)H-dihydrostreptomycin, radioactivity was accumulated intracellularly in three forms, namely, unbound, loosely bound to the ribosomes (removable by dialysis), and tightly bound to the ribosomes (retained after prolonged dialysis). More radioactivity for a given amount of ribonucleic acid was bound by ribosomes attached to the cell membrane than by supernatant ribosomes. Of the nondialyzable radioactivity associated with isolated ribonucleic acid, 40 to 60% was solubilized by treatment with ribonuclease or by dilute alkaline hydrolysis.     

The physical organization of the cytoplasm in Myxococcus xanthus and the fine structure of its components

Summary The cytoplasmic components of Myxococcus xanthus were found to be helical strands of considerable length when examined in thin sections of cells. Similar structures were obtained in a population of isolated particles from fractionated cells. The width of the strands was estimated to be approximately 250 A, a single thread was about 50 A in width. It was suggested that the helices were fibrillar. The width of single fibrils was close to the resolving power of the instrument, about 10 A. No single ribosomes were found in thin sections of cells but most of the isolated particles were round, 100–250 A in diameter. The cytoplasmic strands were built of subunits of the size known for ribosomes which could be identified as such upon fragmentation of the strands. Crystal-like structures were found in this Gram-negative organism which, in some cases, comprised a large portion of the cell. The question was raised whether this type of fabric represents the true physical organization of the cytoplasm.Dedicated to Prof. Dr. W. Schwartz on his 70th birthday.     

The synthesis of ribosomes by a mutant of Escherichia coli

1. When the methionine-requiring mutant 58–161 of Escherichia coli was starved of methionine, ribonucleic acid was made in the absence of protein synthesis. 2. Most of this ribonucleic acid was similar to that found in ribosomes but was contained in particles differing from ribosomes both in sedimentation coefficient and in chromatographic behaviour on diethylaminoethylcellulose. 3. When methionine was added to a starved culture, the ribonucleic acid synthesized during starvation was almost completely undegraded as growth resumed. A transient loss of 5–10% could be largely attributed to breakdown of messenger ribonucleic acid accumulated during starvation. 4. After the addition of methionine, ribosomes were formed from the particles, and during this period preferential synthesis of ribosomal protein took place. 5. It is suggested that under these conditions the direct synthesis of ribosomes from the particles may occur.     

The ultrastructure of the liver in thyroid-fed rats

Summary Rats were fed on a 25% casein diet or the same diet supplemented with desiccated thyroid. The rats were killed after 16 days. Histological sections of the livers of the control rats show coarse, basophilic inclusions and abundance of glycogen in the cytoplasm. In the thyroid-fed rats there is a diffuse, cytoplasmic basophilia with basophilic rods and no or almost no glycogen. Under the electron microscope large areas of glycogen are to be seen in the cytoplasm of the control animals. Mitochondria and rough-surfaced endoplasmic membranes, often in large stacks, are found together. The liver cells of the thyroid-fed rats have little or no glycogen in their cytoplasm. Mitochondria, endoplasmic reticulum, and free ribosomes and polysomes are evenly distributed all over the cytoplasm. There seems to be an increase in the ratio of free to membrane-bound ribosomes and polysomes in the thyroid-fed rats. The possible significance of this observation in relation to RNA synthesis is discussed.This work was supported by grants from the Swedish Medical Research Council (Project No. K-69-12X-623-05) and the Swedish Cancer Society (Project No. 95-K69-03X).     

Studies on the larval salivary gland of Drosophila: I. The nucleic acids

The localization and identification of the two nucleic acids, ribonucleic and desoxyribonucleic, have been determined for the larval salivary gland of Drosophila robusta. The determinations were made by the use of basic staining, Brachet's ribonuclease method, and the Feulgen reaction.The cytological observations suggest that the gland is functional as a secretory organ up to the mid-point of the third instar. It is during this period that the cytoplasmic basophilia (RNA) is most intense. When secretion ceases there is a decided decrease in RNA concentration. These findings suggest that the RNA system is concerned primarily with secretion.     

Ribosomal ribonucleic acid maturation in synchronous strain l cells

Summary The incorporation of [³H]-5-uridine into cytoplasmic 18S and 28S ribosomal ribonucleic acid (rRNA) was examined in Colcemid-synchronized strain L cells during G1 and S phases of the cell cycle in the presence of 5×10⁻⁵ m uridine, which was determined to be the saturating concentration for this system. The data show that in S phase a significant increase occurs in the level of [³H]-5-uridine incorporation into each rRNA species. During a 90-min exposure period, S phase cells incorporate 3.4 times as much [³H]-5-uridine into 18S rRNA and 1.9 times as much into 28S rRNA as do G1 cells. The time required for maturation of the ribosomal RNA species during G1 and during S phase is the same, with 18S rRNA appearing in the cytoplasm in 20 min and 28S rRNA in 40 min.