- Email: [email protected]
Synthesis of ribosomal RNA during growth and division in lilium
0
1966
by Academic
Experimenfal
Press
Cell Research
SYNTHESIS
Inc.
44, 1-12
1
(1966)
OF RIBOSOMAL DIVISION
RNA DURING IN LILIUM
GROWTH
AND
D. MI. STEFFENSEN Department
of Botany, University Urbana, Ill., U.S.A.
of Illinois,
Received December 27, 1965l
are made to examine the synthesis of stable RNA in developing microspores and pollen and in growing pollen tubes of Lilium longiflorum, taking advantage of its well ordered and synchronous nuclear divisions. Various stages in the nuclear cycle have been examined to find the most active periods of RNA synthesis. The experiments to follow have been directed toward understanding the relationship between the nucleolus and the synthesis of ribosomes.
ATTEMPTS
MATERIALS
AND
METHODS
organism used in this study is the Easter lily, Lilium The commercial varieties, Ace and Nellie White, were grown from bulbs in greenhouses.The microspores and pollen were labeled two ways: (a) by growing plants in hydroponics and (b) by sterile anther culture previously described [3, 151. The azP concentration in whole plant Iabeling was 0.5 ,ue/ml and in anther culture was 50 PC/ml. In order to follow stable RNA, the radioactive pollen was grown in petri dishesfor 10 to 12 hr with 5.0 ml of media consisting of: 0.5 ml M KH,PO,, 2.5 ml M Ca (NO,),, 1.0 ml M MgSO,, 50 mg boric acid, 20 g sucroseand 4 g Dextran (Sigma) diluted to 1 1, at pH 4.9. The resulting pollen tubes were washed in media, centrifuged and the pellet was frozen in ethanol. Total RNA and Dh’A analysis.-The labeled pollen or pollen tubes were washed and centrifuged twice in unlabeled media at 1°C and suspendedin 0.2 N perchloric acid (PCA). After centrifugation the cells were washed twice with 0.2 N PCA and the three supernatants combined to determine the radioactivity in the “acid soluble fraction”. The precipitate was then resuspended in 95 per cent ethanol saturated with magnesium acetate, centrifuged and washed twice with 95 per cent ethanol. The precipitate was suspendedin ethanol-ether (2: 1, v: v), heated to 60°C and centrifuged. The precipitate was washed twice with ether. The precipitate was then hydrolyzed in 1.0 ml 0.5 N KOH at 37°C for approximately 20 hr. This solution was then re-precipitated with 0.06 ml of 11 N PCA at 1°C. The hydrolysate and two subsequent washeswith 0.2 N PCA were combined, This nucleotide sample was examined for ultraviolet adsorption at 260 and 280 m/l with a Beckman DU spectrophotometer, Growth
and culture.-The
Zongiflorum.
1 Revised version received June 13, 1966. I-
661809
Experimental
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Research
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D. M. Steffensen
adsorbed on a charcoal-Celite column and eluted with 0.36 N and 0.036 N PCA until the radioactivity from inorganic phosphates was undetectable. The nucleotides, representing “total RNA”, were then removed from the column with 0.77 N ammonium hydroxide. After evaporation of most of the liquid the samples were dissolved in concentrated ammonium hydroxide, spotted onto planchets and the radioactivity determined by gas flow or scintillation counting. The remaining precipitate after alkaline hydrolysis was suspended in 1.0 N PCA and heated for 30 min at 75°C. The supernatant and two 1.0 N PCA washes were combined and used to estimate the specific activity of DNA. The ultraviolet adsorption was determined at 260 and 280 mp. The inorganic phosphate was removed as mentioned previously on a charcoal-Celite column before a sample was counted. RNA extraction and separation by column chromatogruphy.-The 32P-labeled pollen tubes were ground by mortar and pestle in a minimum of ethanol with sand at l”C, followed by the sodium lauryl sulfate procedure [9] and extraction of RNA by the phenol procedure [S]. The methylated-albumin Kieselguhr (MAK) column [5] was then employed to separate the RNA in a buffered sodium chloride gradient (0.1 to 1.0 IM NaCl). Radioactive RNA was co-precipitated with carrier DNA by trichloroacetic acid and passed through membrane filters as described by 1191. The ribosomal RNA peaks from the MAK column were hydrolyzed with carrier yeast RNA in 0.5 1cf KOH at 37°C for 20 hr and the resulting nucleotides separated on a Dowex-I x 8 (2OOG400 mesh) column using formic acid and mixtures of formic acid and ammonium formate. Extraction of ribosomes.-All of the extractions were done at 1°C or in an ice bath. Between 5 to 10 g of lily pollen was suspended in 20 ml of 0.88 M sucrose containing 0.001 M MgCl, and disrupted in a French pressure cell at 4500 p.s.i. In isotope experiments, the 3ZP labeled pollen tubes were ground separately by mortar and pestle and added to the disrupted pollen mixture to provide sufficient material for sedimentation and optical determination. Four centrifugation steps were then done: 15 min at 570 g, 60 min at 37,000 g and 60 min each at 23,000 and 36,000 rpm in a Spinco 40 rotor. The supernantant was spun again at 36,000 rpm for at least 2 hr to sediment a ribosome pellet. The ribosome pellet was suspended in 5 per cent sucrose, layered on a linear sucrose gradient (5 to 20 per cent) and spun for 5 hr at 23,000 rpm in the Spinco swinging bucket rotor, SW 25.1. Drops were collected from the bottom into tubes and the optical density determined at 260 rnp with a Beckman DU spectrophotometer. In labeling studies the RNA in the tubes at the ribosome peak was dialyzed against water to remove the sucrose, carrier yeast was added, and the solutions was precipitated in saline ethanol. Base ratios were obtained by alkaline hydrolysis, Dowex-I column chromatography and radioactive counting as mentioned previously. In studies to determine S values, the ribosome peak from the sucrose gradient was suspended in 0.05 M Tris buffer at pH 7.6 with or without 0.01 JZ MgCl,, depending on the experiment and dialyzed overnight against the same buffer to remove the sucrose, as indicated by a return to the refractive index of the buffer. The purity of the ribosome samples was verified on the Cary recording spectrophotometer over the wavelengths 220 to 320 m,u. The S values were obtained by using the Spinco Model E, analytical centrifuge, using Schlieren and ultraviolet optics, and their two photographic systems. Cytology and fir&ion.-Stages of the microspores and pollen were determined Experimental
Cell Research
44
Synthesis
of ribosomal
RNA during
growth
3
and division
either by squashes using propino-carmine or by the Feulgen method. Anthers used for sectioning were fixed in a mixture of gluteraldehyde, glacial acetic acid and 70 per cent ethanol mixture (1: 1:9, v:v:v) dehydrated, imbedded in paraffin, sectioned and stained with Azure B at pH 3.4.
RESULTS
AND
DISCUSSION
The post-meiotic stages of microspores up to mature pollen have been utilized to locate the synthesis times of RNA and to examine whether or not ribosomal or messenger RNA persist as stable molecules during growth. Some of the temporal aspects of DNA and RNA synthesis had already been done [7, 211 with Lilium and Tradescantia by cytochemical methods. Since then, the three RNA types have been established, Stern and Hotta [17] have examined the appearance of messenger RNA during meiosis, along with other problems of protein and nucleic acid synthesis. Lily microspores in mitosis go through at least three steps in buds from 50 to 65 mm in length: (a) building up enzymes and precursor pools, (b) duplication of chromosomes and (c) division [2, 10, 221. This period, as it will be shown, is of critical interest for understanding RNA synthesis, especially ribosomal RNA. RNA synthesis during microspore division Microspores were analyzed for total RNA and DNA and these data are presented in Fig. 1 as a ratio of optical densities at 260 mp (RNA/DNA) in order to locate their respective synthesis periods. The relatively wide distribution of individual values is probably due to three factors: (a) the inherent spread of microspore stages from the distal to proximal end of the anther, (b) the occasional variant bud with a length not directly correlated with mitotic stage, and (c) experimental error in analyzing small amounts of tissue. It is difficult to overcome these causes of variability and at the same time assay real differences at a precise stage. If one pools a number of samples, then real differences are hidden in a mean value, leaving no choice but to use small samples and accept the variability. The curve in Fig. 1 is reliable enough to establish the major changes in RNA and DNA synthesis and breakdown. It is well established that the DNA doubles previous to the microspore division [22]. The synthesis of DNA in microspores is completed in buds of approximately 60 mm in length. The S period is clearly indicated in Fig. 1 by the steep slope in the curve between 57 and 60 mm buds and is demonstrated directly by the recovery of labeled DNA (Figs. 2 and 3). With a DNA doubling, Experimental
Cell Research
44
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D. M. Steffensen
one would expect the RNA to remain at the previous levels, giving a ratio of 2.0 to 2.5 as estimated from Gl microspores. However, the data from Fig. 1 shows the ratio to be about 1.5, indicating that some RNA may have undergone turnover during the S and G2 stages. Further study is necessary to establish the meaning of this observation and types of RNA involved. CALCULATED BUD AT END OF LABELING
54 64 I’~~~I~‘~~l~~~~IIIIIIIIIII
LENGTH PERIOD
74 -
3 F2 c1 &Lo
----I
RNA/DNA
-
Specifvz
Actiwty
s x
Rot,o RNA
I
00
-
Division
si a “, 5.0 isI2 2 ‘u 2% cc 0: FU an Gc d u c s
4.0 3.0d zo0
LO-
40
50 BUD
60 LENGTH
Fig. Fig. i.-Ratios length in Lilium
0
70
mm
III,,IIIIIIIIIl,,,, 50
60
BUD LENGTH
1.
of RNA to DNA at different stages of microspore (var. Ace). The two sets of points represent different
70 AT START
Fig.
mm
OF CULTURE
2.
division, as related experiments.
to bud
Fig. 2.-Microspores labeled with SsP by anther culture for 46 hr. These data indicate a high rate of RNA synthesis at the G2 period and after division (division at 63-65 mm bud lengths). The first DNA synthesis as detected in the analysis precedes the microspore division, the second synthesis involves the generative nucleus and not the tube nucleus [211.
Microspores were labeled in bud culture for 46 hr and cells removed immediately from the anthers for analysis. The first 24 hr in culture was required before significant amounts of 32P were incorporated. These data regarding the synthesis of RNA are given in Fig. 2 and are expressed as specific activity. Radioactive DNA was detected at the two expected periods of synthesis [2] at bud lengths between 57 to 62 mm and 68 to 72 mm, respectively. As Fig. 2 shows these bud length values indicate the growth periods that the microspores were exposed to the isotope. The times of DNA synthesis aid in determining the stages before and after division. The specific activity of the Experimenlal
Cell Research
44
Synthesis
of ribosomal
RNA during
growth
and division
5
RNA (Fig. 2) begins to increase after DNA synthesis (G2); RNA synthesis drops at division and goes up again after division. Finally the RNA to DNA ratio climbs to between 4 to 5, accompanied by an expected decline in RNA synthesis in maturing pollen following the second DNA4 synthesis. The data in Fig. 3 exhibits a similar pattern of RNA synthesis, although the CALCULATED AT REMOVAL
40
50 60 BUD LENGTH
BUD LENGTH OF P= MEDIA
70 80 90 100 mm AT START OF LABELING
Fig. 3.-Anthers were cultured in SzP for 3 days and removed to non-radioactive media. The resulting mature labeled pollen was grown again in non-radioactive media and the resulting pollen tubes collected for analysis after 10 to 12 hr of growth. The data relate to the stable RNA synthesized in the microspore, before and after division, during 3 days in isotope. The two bracket regions indicate the detection of labeled DNA.
time of labeling was longer. The latter results were obtained by analyzing pollen tube RNA after the 32P-pollen had been germinated. The procedure was to label buds for 3 days, remove them from 32P, and allow the buds to grow in non-radioactive media until the pollen matured. The resulting labeled-pollen grains were germinated in unlabeled media, a culture regime amounting to a “double chase”. As before, the RNA with the highest specific activity was synthesized after DNA synthesis in the microspore at G2 and following division. Actually the isotope is not really “chased” at all in these lily cells but rather diluted and outgrown, since once absorbed, there is little if any exchange with the outside media. The radioactivity of the acid soluble fraction, extracted with 0.2 N perchloric acid, is reduced by only about Experimental
Cell Research
44
D. M. Steffensen half, even after growth in unlabeled media in bud culture and further growth of pollen grown in unlabeled media giving pollen tubes, as compared to microspores analyzed immediately on removal from the isotope. Fortunately the pollen tube system allows one to by-pass this pool problem due to its growth characteristics. In 12 hr or so, a pollen grain can germinate and the tube grow as much as 1 cm. Blocks of cytoplasm are left behind by the formation of callose plugs, as the nuclei and particulate cytoplasm migrate toward the growing tip. The adsorption of the carrier compound in question during growth then causes a considerable dilution of the isotopic pool. These special features have been utilized in other studies, where 45Ca and 35S labeled nuclei were shown to outgrow the labeled cytoplasm in pollen tubes [15, 161. Cytology
of nucleoli
Anthers were sectioned to examine differences in nucleoli during development. In microspores the average diameter of the nucleolus was about 5.0 ,u. After division the two nuclei in pollen differentiate at the stage where buds are about 75 mm in length. The nucleoli of each nucleus are different in size: the tube nucleus being about 6 to 7 ,D in diameter and the generative nucleus about 2 to 4 ,u. Using gluteraldehyde or formalin the fixation of microspores and pollen show the nucleolus to contain dense bodies in the center appearing white under bright field or phase, and Azure B negative. These may correspond to the “light zone” particles described by Lafontaine [4] but probably not the “spherules” that stain for RNA. Separation of RNA by column chromatography The experiments considered so far, have not attempted to separate the various types of RNA, which is necessary to interpret such evidence in light of current concepts. In order to obtain high specific RNA for MAK column chromatography, whole plants were grown in culture solution containing several mc of 32P. This regime was necessary, since it has not been possible to grow microspores in vitro with bud lengths shorter than 45 mm. It was essential to label microspores at much earlier stages (bud lengths from 25 to 35 mm) to assure the recovery of high specific RNA-a maximum amount of 32P during the active periods of svnthesis indicated previously. At maturity Y this labeled pollen was grown in vitro without isotope and the resulting pollen tubes extracted and the RNA prepared by the phenol procedure. This RNA was separated on a MAK column by elution with sodium chloride. The profile of optical densities in Fig. 4 is similar to those of E. coli [19] and the RNA of other organisms. The first few tubes (15 to 30) usually contain polyExperimental
Cell Research
44
Synthesis
of ribosomcll
RNA during
growth
7
and division
The S-RNA comes off the column between 0.15 to saccharide impurities. 0.45 M NaCl. This location for S-RNA has been confirmed (unpublished results) by examining the location of acyl-S-RNA (T-RNA) in the MAK column, as 35S-cysteine-T-RNA and 35S- or 3H-methionine-T-RNA. The presumed messenger RNA separates into discrete polydispersed peaks between 0.5 to 1.0 M NaCl as shown by pollen-tube labeling with 3H-uridine (Fig. 5). The major RNA peak is ribosomal RNA at 0.80 M NaCl in both Figs. 4 and 5. As determined from Fig. 4, the total amount of ribosomal RNA within the peak amounts to about 72 per cent. This calculation infers that nearly three quarters of the total labeled RNA is ribosomal. Therefore, the analyses of total RNA synthesis (Figs. 1, 2 and 3) reflect primarily the patterns of ribosome production rather than S-RNA or m-RNA. The ribosomal RNA peak from a MAK column was analyzed from three separate experiments similar to Fig. 4 for their base ratios. These data are given in Table I. The base composition is similar to the 70s ribosomes of a number of bacteria [13] and is comparable to several analyses of other higher plants [ll, 14, 241. 20 9-
9-
I8
6-
a-
16
7-
14
biz z -6-
G =06-
12
v, 6 5-=50 E
8 IO!? x
2d 4-g4- 4
89 E
!i 03-
3-
6 RADIOACTIVITY
2-
2-
4 2 PTICAL
IO
20
30
40
DENSITY
50 60 70 TUBE NUMBER
80
90
100
110
120
Fig. 4.-Fractionation of pollen tube RNA on a MAK column prepared Microspore were labeled with *rP in the plant and “chased” twice with main peak at 0.75 M NaCl is ribosomal RNA. The peak between 0.20 and Messenger RNA occurs at numerous small polydispersed peaks from 0.5
130
by the carrier 0.45 M to 1.0 M
Experimental
I40
phenol method. phosphate. The NaCl is S-RNA. NaCI. Cell
Research
44
8 Analytical
D. M. Steflensen centrifugation
The pollen of Lilium was burst in the French pressure cell in 0.88 M sucrose and the ribosomes separated by centrifugation. One set of ribosomes were dissociated by deoxycholate, sedimented, further purified on a sucrose gradient and finally suspended in 0.05 Tris buffer without magnesium. Two peaks were obtained with values of 20s and 39s by analytical centrifugation. Another set of ribosomes was isolated as before but with 0.01 Mg ions during all the steps, before a final dialysis against 0.01 M Tris buffer and 0.001 magnesium acetate. The latter mixture gave three Schlieren peaks in the analytical centrifuge. The ultraviolet photographic plates of these ribosomes exhibited three sharp discontinuities. The analysis of the three slopes from microdensitometer tracings gave the following S values: 76S, 60s and 37s at 20°C. No corrections were made for the viscosity of the buffer. These sedimentations coefficients correspond closely to the undissociated ribosome and The results are essentially identical with the two dissociated components. those obtained from pea ribosomes [23]. The dissociation characteristics
TUBE
NUMBER
Fig. B.-The results of MAK column chromatography of SH-uridine labeled RNA. Pollen tubes had been grown for 8 hr in unlabeled media, labeled for 1 hr, and given a 5 hr “chase” 0.001 M uridine and cytidine before RNA extraction. Note the three depressions in specific activity over the ribosomal peak (tubes 95 to 110). Experimental
Cell Research
44
Synthesis
of ribosomal
with variation of magnesium ribosomes [6]. RNA synthesis
in pollen
RNA during
growth
ion concentration
and division
are comparable
9 to E. coli
tubes
The initial interest in investigating the ribosomal RNA of Lilium arose from the observation that no nucleolus was evident in the nuclei of growing pollen tubes. None of the conventional cytochemical procedures gave any indication of the presence of a nucleolus, such as staining with Azure B at pH 4.0, or methyl green-pyronin. Autoradiographic studies using 3H-uridine or 3H-cytidine exhibited homogeneous labeling over the nucleus without “hot spots” in the conventional nucleolar site of RN.4 accumulation. There is no unequivocal cytochemical or biochemical method yet devised that enables one to distinguish between the types of RNA being synthesized in situ, thus it was necessary to resort to biochemical methods to ascertain what, if any, synthesis was missing. It is intended to present data concerning ribosomal RNA and essentially to ignore any consideration of soluble RNA and messenger RNA, which will be dealt with in further papers in preparation. Briefly then, isotope is added to growing pollen tubes at the peak of RNA synthesis, occurring from S to 10 hr after germination at 22°C. Significant amounts of newly synthesized RNA do not appear until nearly 3 or 4 hr after labeling with messenger-like RNA peaks being just detectable in 20 to 30 min. It is known from our autoradiographic evidence that the nuclei are labeled in a few minutes. The slight delay in detecting RNA chemically must be due, at least in part, that no attempt has been made to isolate the DNA-RNA complex at the interfacial layer during the phenol extraction. TABLE
I. Base ratios of 32P-ribosomal RNA.
Each sample selected as the ribosome peak after separation of pollen tube column, alkaline hydrolysis and Dowex-1 column chromatography. The initial during microspore and pollen development. Experiment 2’, 3’, nucleotides CMP AMP UMP GMP
by MAK was done
number
1
2 (Per
23.9 23.6 23.5 29.0
of RNA labeling
3 cent)
24.4 23.6 21.0 31.1
24.3 24.9 10.8 31 .o
Experimental
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44
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D. M. Steffensen
In one experiment 3H-uridine was used to label growing pollen tubes in vitro for 1 hr, non-radioactive uridine was added and growth proceeded for 5 hr. The RNA was extracted and fractionated on a MAK column by elution in a sodium chloride gradient. The results are illustrated in Fig. 5. Discrete high specific activity peaks occur between 0.5 to 1.0 M NaCl. Most of these are assumed to be messenger RNA. The specific activity of the ribosome peak at 0.8 M NaCl is low and at least 5 to 10 times less than the radioactive peaks on either side. In fact the specific activity drops at the highest optical densities in tubes 100, 101, and 103, the peaks of ribosomal RNA. All of the MAK column profiles of lily ribosome RNA done to date have shown only a slight tendency of forming bimodal peaks and never shown two distinct peaks as with RNA from E. coli [19]. The differences in RNA labeling patterns between microspores and pollen tubes (Figs. 4 and 5) should be re-emphasized. In Fig. 4, the RNA was labeled in the microspore and the radioactivity follows the optical density quite closely, especially the ribosome peak. The reverse is true in Fig. 5, in’pollen tubes where neither radioactivity and optical density coincide and the peaks and shoulders of ribosomal RNA and the ribosomes manifest the lowest specific activity. Density
gradient
centrifugation
of ribosomes
It would appear that most, if not all, of the labeled peaks (Fig. 5) might not be ribosome synthesis at all but overlapping peaks of messenger RNA. In order to examine this possibility, pollen tubes were labeled for 5 hr with 32P ( 8 to 13 hr after germination). The homogenized pollen tubes and disrupted non-radioactive pollen grains were combined. After the appropriate centrifugation steps the ribosomes were sedimented, suspended again, and put on a linear sucrose gradient. Following centrifugation, drops were collected from the bottom of the tube. The RNA found in the ribosome peak was analyzed for its base ratios with the following results in Table II. These values in Table II are quite different from the ribosome analysis in Table I, especially for guanlylic and adenlylic. Apparently most plant ribosomal RNA contains between 30-32 per cent guanlylic [ll, 14, 241. It would appear that the radioactive RNA above is not ribosome RNA but probably overlapping or attached messenger RNA. The RNA made in growing tobacco pollen tubes was analyzed on a MAK column by Tano and Takahashi [20] and a broad messenger-like peak gave base ratios with 29.3 per cent adelylic and 22.9 per cent guanlylic. These authors have assumed, as has been the case here, that synthesis in pollen tubes involves primarily messenger RNA. All in all the Experimental
Cell
Research
44
Synthesis
of ribosomal
RNA during
growth
11
and division
evidence indicates that little if any ribosomal RNA is being made in growing pollen tubes. In recapitulation it has been found that no nucleolus is detectable in pollen tube nuclei. The nucleolar organizer region is apparently not active. These and other data would indicate that when the nucleolar organizer is “turned TABLE
II. Base ratios from the ribosome peak from labeled pollen sucrose density gradient centrifugation.
The labeling of RNA was done with 32P in growing pollen tubes from These base ratios are interpreted not to be ribosome RNA labeling messenger RNA (see ribosome base ratios in Table I). 2’, 3’nucleotides
CMP AMP UMP GMP
tubes after a
8 to 13 hr after germination. but probably contaminating
Per cent
27.7 30.0 20.3 21.9
no ribosomes are being syntheoff”; no nucleolus is visible and apparently sized. A number of studies have focused their attention on localizing the site of ribosome synthesis in the nucleolus. There are now many compelling reasons to believe that ribosomes are made within the nucleolus at a segment of the chromosome called the nucleolus organizer. For example, ribosome synthesis was absent in the anucleolate mutant of Xenopus [l]. Molecular hybrids were made between ribosomal RNA and Drosophila DNA using varying doses of the nucleolar organizer, indicating the nucleolar organizer DNA is responsible for ribosome synthesis amounts to 0.27 per cent of the wild type genome [la]. Ultrastructural studies have shown that ribososomes are within the nucleolus and that they undergo various alterations during the mitotic cycle in Vicia faba [4] and in grasshopper neuroblasts [18]. These latter experiments show the appearance of nucleolar ribosomes to be especially abundant in late interphase and early prophase (G2) and again after telophase. All of the evidence considered seems consistant with the data from lily ribosomes presented here regarding the timing and site of ribosome synthesis.
Experimental
Cell Research
44
12
D. M. Steffensen SUMMARY
The synthesis of RNA has been examined in the microspore of lily buds during division and later development into mature pollen grains. RNA synthesis is most active just before division (G2) and after the microspore nuclear division. Column chromatography using a methylated-albumin Kieselguhr column has shown that ribosomal RNA makes up the major portion of RNA being synthesized during this period, when nucleoli are large and active. All of the conventional cytological criteria have failed to demonstrate a nucleolus in growing pollen tubes. RNA is synthesized in pollen tubes but the abrupt drop in specific activity at the ribosomal peaks and the base ratios of this RNA do not correspond to ribosomal RNA. The conclusion seems apparent that little, if any, ribosomal RNA is being synthesized in the pollen tube nuclei in the absence of nucleolar activity. The excellent technical assistance of Mrs Lalla Tanner and Mrs Elaine Miller are appreciated, as is the financial support from the National Science Foundation (NSF GB 1327). REFERENCES 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13.
14. 15. 16. 17. 18. 19. 20. 21. 22. 23. 24.
and GURDON, J. B., Proc. Nail Acad. Sci. U.S. 51, 139 (1964). STERN, H. J., Biophss. Biochem. C&Z. 11, 311 (1961). -Proc. Nat1 Acad. Sci. U.S. i9,-648 (1963). I LAFONTAINE, J. G. and CHOUINARD, L. A., J. Cell Biol. 17. 167 (1963). MANDELL, J: D. and HERSHEY, A. b., Aial. Biochem. 1, 86 (1960). ’ MESELSON, M., NOMURA, M., BRENNER, S., DAVERN, C. and SCHLESSINGER, D., J. MoZ. BioZ. 9, 696 (1964). MOSES, M. J. and TAYLOR, J. H., Exptl Cell Res. 9, 474 (1955). KIRBY, K. S., Biochem. J. 64, 405 (1956). ISHIHAMA, A., MIZURNO, N., TAKAE, M., OTAKA, E. and OSAWA, S., J. Mol. Biol. 5, 251 (1962). OGUH, M., ERICKSON, R. O., ROSEN, G. U., SAX, D. B. and HOLDEN, C., Exptl Cell Res. 2, 73 (1951). PETERWANN, M. L., The physical and chemical properties of ribosomes. Elsevier Publishing Co., Amsterdam, 1964. RITOSSA, F. M. and SPIEGELMAN, S., Proc. Nat1 Acad. Sci. U.S. 53, 737 (1965). ROBERTS, R. B., BRITTEN, R. J. and MCCARTHY, B. J., in J. H. TAYLER (ed.), Molecular Genetics, Part 1, p. 291. Academic Press, New York, 1963. SRIVASTAVA, B. I. S., Biochem. J. 96, 665 (1965). STEFFENSEN, D. M., Genetics 52, 631 (1965). STEFFENSEN, D. M..and BERGE~ON, J. A.,‘J. Biophys. Biochem. Cytol. 6, 339 (1959). STERN, H. and HOTTA, Y., Brookhaven Symp. Biol. 16, 59 (1964). STEVENS, B. J., J. Cell Biol. 24, 349 (1965). SUEOKA, N. and YAMANE, T., Proc. Nat1 Acad. Sci. U.S. 48, 1454 (1962). TANO, S. and TAKAHASHI, H., J. Biochem. 56, 578 (1964). TAYLOR, J. H., Exptl Cell Res. 4, 164 (1953). TAYLOR, J. H. and MCMASTER, R. D., Chromosoma 6, 489 (1954). Ts’o, P. 0. P., BONNER, J. and VINOGRAD, J., Biochim. Biophys. Acta 30, 570 (1958). WATERS, L. and DURE, L., Science 149, 188 (1965). BROWS, HOTTA,
Experimental
D. D. Y. and
Cell Research
44
1966
by Academic
Experimenfal
Press
Cell Research
SYNTHESIS
Inc.
44, 1-12
1
(1966)
OF RIBOSOMAL DIVISION
RNA DURING IN LILIUM
GROWTH
AND
D. MI. STEFFENSEN Department
of Botany, University Urbana, Ill., U.S.A.
of Illinois,
Received December 27, 1965l
are made to examine the synthesis of stable RNA in developing microspores and pollen and in growing pollen tubes of Lilium longiflorum, taking advantage of its well ordered and synchronous nuclear divisions. Various stages in the nuclear cycle have been examined to find the most active periods of RNA synthesis. The experiments to follow have been directed toward understanding the relationship between the nucleolus and the synthesis of ribosomes.
ATTEMPTS
MATERIALS
AND
METHODS
organism used in this study is the Easter lily, Lilium The commercial varieties, Ace and Nellie White, were grown from bulbs in greenhouses.The microspores and pollen were labeled two ways: (a) by growing plants in hydroponics and (b) by sterile anther culture previously described [3, 151. The azP concentration in whole plant Iabeling was 0.5 ,ue/ml and in anther culture was 50 PC/ml. In order to follow stable RNA, the radioactive pollen was grown in petri dishesfor 10 to 12 hr with 5.0 ml of media consisting of: 0.5 ml M KH,PO,, 2.5 ml M Ca (NO,),, 1.0 ml M MgSO,, 50 mg boric acid, 20 g sucroseand 4 g Dextran (Sigma) diluted to 1 1, at pH 4.9. The resulting pollen tubes were washed in media, centrifuged and the pellet was frozen in ethanol. Total RNA and Dh’A analysis.-The labeled pollen or pollen tubes were washed and centrifuged twice in unlabeled media at 1°C and suspendedin 0.2 N perchloric acid (PCA). After centrifugation the cells were washed twice with 0.2 N PCA and the three supernatants combined to determine the radioactivity in the “acid soluble fraction”. The precipitate was then resuspended in 95 per cent ethanol saturated with magnesium acetate, centrifuged and washed twice with 95 per cent ethanol. The precipitate was suspendedin ethanol-ether (2: 1, v: v), heated to 60°C and centrifuged. The precipitate was washed twice with ether. The precipitate was then hydrolyzed in 1.0 ml 0.5 N KOH at 37°C for approximately 20 hr. This solution was then re-precipitated with 0.06 ml of 11 N PCA at 1°C. The hydrolysate and two subsequent washeswith 0.2 N PCA were combined, This nucleotide sample was examined for ultraviolet adsorption at 260 and 280 m/l with a Beckman DU spectrophotometer, Growth
and culture.-The
Zongiflorum.
1 Revised version received June 13, 1966. I-
661809
Experimental
Cell
Research
44
2
D. M. Steffensen
adsorbed on a charcoal-Celite column and eluted with 0.36 N and 0.036 N PCA until the radioactivity from inorganic phosphates was undetectable. The nucleotides, representing “total RNA”, were then removed from the column with 0.77 N ammonium hydroxide. After evaporation of most of the liquid the samples were dissolved in concentrated ammonium hydroxide, spotted onto planchets and the radioactivity determined by gas flow or scintillation counting. The remaining precipitate after alkaline hydrolysis was suspended in 1.0 N PCA and heated for 30 min at 75°C. The supernatant and two 1.0 N PCA washes were combined and used to estimate the specific activity of DNA. The ultraviolet adsorption was determined at 260 and 280 mp. The inorganic phosphate was removed as mentioned previously on a charcoal-Celite column before a sample was counted. RNA extraction and separation by column chromatogruphy.-The 32P-labeled pollen tubes were ground by mortar and pestle in a minimum of ethanol with sand at l”C, followed by the sodium lauryl sulfate procedure [9] and extraction of RNA by the phenol procedure [S]. The methylated-albumin Kieselguhr (MAK) column [5] was then employed to separate the RNA in a buffered sodium chloride gradient (0.1 to 1.0 IM NaCl). Radioactive RNA was co-precipitated with carrier DNA by trichloroacetic acid and passed through membrane filters as described by 1191. The ribosomal RNA peaks from the MAK column were hydrolyzed with carrier yeast RNA in 0.5 1cf KOH at 37°C for 20 hr and the resulting nucleotides separated on a Dowex-I x 8 (2OOG400 mesh) column using formic acid and mixtures of formic acid and ammonium formate. Extraction of ribosomes.-All of the extractions were done at 1°C or in an ice bath. Between 5 to 10 g of lily pollen was suspended in 20 ml of 0.88 M sucrose containing 0.001 M MgCl, and disrupted in a French pressure cell at 4500 p.s.i. In isotope experiments, the 3ZP labeled pollen tubes were ground separately by mortar and pestle and added to the disrupted pollen mixture to provide sufficient material for sedimentation and optical determination. Four centrifugation steps were then done: 15 min at 570 g, 60 min at 37,000 g and 60 min each at 23,000 and 36,000 rpm in a Spinco 40 rotor. The supernantant was spun again at 36,000 rpm for at least 2 hr to sediment a ribosome pellet. The ribosome pellet was suspended in 5 per cent sucrose, layered on a linear sucrose gradient (5 to 20 per cent) and spun for 5 hr at 23,000 rpm in the Spinco swinging bucket rotor, SW 25.1. Drops were collected from the bottom into tubes and the optical density determined at 260 rnp with a Beckman DU spectrophotometer. In labeling studies the RNA in the tubes at the ribosome peak was dialyzed against water to remove the sucrose, carrier yeast was added, and the solutions was precipitated in saline ethanol. Base ratios were obtained by alkaline hydrolysis, Dowex-I column chromatography and radioactive counting as mentioned previously. In studies to determine S values, the ribosome peak from the sucrose gradient was suspended in 0.05 M Tris buffer at pH 7.6 with or without 0.01 JZ MgCl,, depending on the experiment and dialyzed overnight against the same buffer to remove the sucrose, as indicated by a return to the refractive index of the buffer. The purity of the ribosome samples was verified on the Cary recording spectrophotometer over the wavelengths 220 to 320 m,u. The S values were obtained by using the Spinco Model E, analytical centrifuge, using Schlieren and ultraviolet optics, and their two photographic systems. Cytology and fir&ion.-Stages of the microspores and pollen were determined Experimental
Cell Research
44
Synthesis
of ribosomal
RNA during
growth
3
and division
either by squashes using propino-carmine or by the Feulgen method. Anthers used for sectioning were fixed in a mixture of gluteraldehyde, glacial acetic acid and 70 per cent ethanol mixture (1: 1:9, v:v:v) dehydrated, imbedded in paraffin, sectioned and stained with Azure B at pH 3.4.
RESULTS
AND
DISCUSSION
The post-meiotic stages of microspores up to mature pollen have been utilized to locate the synthesis times of RNA and to examine whether or not ribosomal or messenger RNA persist as stable molecules during growth. Some of the temporal aspects of DNA and RNA synthesis had already been done [7, 211 with Lilium and Tradescantia by cytochemical methods. Since then, the three RNA types have been established, Stern and Hotta [17] have examined the appearance of messenger RNA during meiosis, along with other problems of protein and nucleic acid synthesis. Lily microspores in mitosis go through at least three steps in buds from 50 to 65 mm in length: (a) building up enzymes and precursor pools, (b) duplication of chromosomes and (c) division [2, 10, 221. This period, as it will be shown, is of critical interest for understanding RNA synthesis, especially ribosomal RNA. RNA synthesis during microspore division Microspores were analyzed for total RNA and DNA and these data are presented in Fig. 1 as a ratio of optical densities at 260 mp (RNA/DNA) in order to locate their respective synthesis periods. The relatively wide distribution of individual values is probably due to three factors: (a) the inherent spread of microspore stages from the distal to proximal end of the anther, (b) the occasional variant bud with a length not directly correlated with mitotic stage, and (c) experimental error in analyzing small amounts of tissue. It is difficult to overcome these causes of variability and at the same time assay real differences at a precise stage. If one pools a number of samples, then real differences are hidden in a mean value, leaving no choice but to use small samples and accept the variability. The curve in Fig. 1 is reliable enough to establish the major changes in RNA and DNA synthesis and breakdown. It is well established that the DNA doubles previous to the microspore division [22]. The synthesis of DNA in microspores is completed in buds of approximately 60 mm in length. The S period is clearly indicated in Fig. 1 by the steep slope in the curve between 57 and 60 mm buds and is demonstrated directly by the recovery of labeled DNA (Figs. 2 and 3). With a DNA doubling, Experimental
Cell Research
44
4
D. M. Steffensen
one would expect the RNA to remain at the previous levels, giving a ratio of 2.0 to 2.5 as estimated from Gl microspores. However, the data from Fig. 1 shows the ratio to be about 1.5, indicating that some RNA may have undergone turnover during the S and G2 stages. Further study is necessary to establish the meaning of this observation and types of RNA involved. CALCULATED BUD AT END OF LABELING
54 64 I’~~~I~‘~~l~~~~IIIIIIIIIII
LENGTH PERIOD
74 -
3 F2 c1 &Lo
----I
RNA/DNA
-
Specifvz
Actiwty
s x
Rot,o RNA
I
00
-
Division
si a “, 5.0 isI2 2 ‘u 2% cc 0: FU an Gc d u c s
4.0 3.0d zo0
LO-
40
50 BUD
60 LENGTH
Fig. Fig. i.-Ratios length in Lilium
0
70
mm
III,,IIIIIIIIIl,,,, 50
60
BUD LENGTH
1.
of RNA to DNA at different stages of microspore (var. Ace). The two sets of points represent different
70 AT START
Fig.
mm
OF CULTURE
2.
division, as related experiments.
to bud
Fig. 2.-Microspores labeled with SsP by anther culture for 46 hr. These data indicate a high rate of RNA synthesis at the G2 period and after division (division at 63-65 mm bud lengths). The first DNA synthesis as detected in the analysis precedes the microspore division, the second synthesis involves the generative nucleus and not the tube nucleus [211.
Microspores were labeled in bud culture for 46 hr and cells removed immediately from the anthers for analysis. The first 24 hr in culture was required before significant amounts of 32P were incorporated. These data regarding the synthesis of RNA are given in Fig. 2 and are expressed as specific activity. Radioactive DNA was detected at the two expected periods of synthesis [2] at bud lengths between 57 to 62 mm and 68 to 72 mm, respectively. As Fig. 2 shows these bud length values indicate the growth periods that the microspores were exposed to the isotope. The times of DNA synthesis aid in determining the stages before and after division. The specific activity of the Experimenlal
Cell Research
44
Synthesis
of ribosomal
RNA during
growth
and division
5
RNA (Fig. 2) begins to increase after DNA synthesis (G2); RNA synthesis drops at division and goes up again after division. Finally the RNA to DNA ratio climbs to between 4 to 5, accompanied by an expected decline in RNA synthesis in maturing pollen following the second DNA4 synthesis. The data in Fig. 3 exhibits a similar pattern of RNA synthesis, although the CALCULATED AT REMOVAL
40
50 60 BUD LENGTH
BUD LENGTH OF P= MEDIA
70 80 90 100 mm AT START OF LABELING
Fig. 3.-Anthers were cultured in SzP for 3 days and removed to non-radioactive media. The resulting mature labeled pollen was grown again in non-radioactive media and the resulting pollen tubes collected for analysis after 10 to 12 hr of growth. The data relate to the stable RNA synthesized in the microspore, before and after division, during 3 days in isotope. The two bracket regions indicate the detection of labeled DNA.
time of labeling was longer. The latter results were obtained by analyzing pollen tube RNA after the 32P-pollen had been germinated. The procedure was to label buds for 3 days, remove them from 32P, and allow the buds to grow in non-radioactive media until the pollen matured. The resulting labeled-pollen grains were germinated in unlabeled media, a culture regime amounting to a “double chase”. As before, the RNA with the highest specific activity was synthesized after DNA synthesis in the microspore at G2 and following division. Actually the isotope is not really “chased” at all in these lily cells but rather diluted and outgrown, since once absorbed, there is little if any exchange with the outside media. The radioactivity of the acid soluble fraction, extracted with 0.2 N perchloric acid, is reduced by only about Experimental
Cell Research
44
D. M. Steffensen half, even after growth in unlabeled media in bud culture and further growth of pollen grown in unlabeled media giving pollen tubes, as compared to microspores analyzed immediately on removal from the isotope. Fortunately the pollen tube system allows one to by-pass this pool problem due to its growth characteristics. In 12 hr or so, a pollen grain can germinate and the tube grow as much as 1 cm. Blocks of cytoplasm are left behind by the formation of callose plugs, as the nuclei and particulate cytoplasm migrate toward the growing tip. The adsorption of the carrier compound in question during growth then causes a considerable dilution of the isotopic pool. These special features have been utilized in other studies, where 45Ca and 35S labeled nuclei were shown to outgrow the labeled cytoplasm in pollen tubes [15, 161. Cytology
of nucleoli
Anthers were sectioned to examine differences in nucleoli during development. In microspores the average diameter of the nucleolus was about 5.0 ,u. After division the two nuclei in pollen differentiate at the stage where buds are about 75 mm in length. The nucleoli of each nucleus are different in size: the tube nucleus being about 6 to 7 ,D in diameter and the generative nucleus about 2 to 4 ,u. Using gluteraldehyde or formalin the fixation of microspores and pollen show the nucleolus to contain dense bodies in the center appearing white under bright field or phase, and Azure B negative. These may correspond to the “light zone” particles described by Lafontaine [4] but probably not the “spherules” that stain for RNA. Separation of RNA by column chromatography The experiments considered so far, have not attempted to separate the various types of RNA, which is necessary to interpret such evidence in light of current concepts. In order to obtain high specific RNA for MAK column chromatography, whole plants were grown in culture solution containing several mc of 32P. This regime was necessary, since it has not been possible to grow microspores in vitro with bud lengths shorter than 45 mm. It was essential to label microspores at much earlier stages (bud lengths from 25 to 35 mm) to assure the recovery of high specific RNA-a maximum amount of 32P during the active periods of svnthesis indicated previously. At maturity Y this labeled pollen was grown in vitro without isotope and the resulting pollen tubes extracted and the RNA prepared by the phenol procedure. This RNA was separated on a MAK column by elution with sodium chloride. The profile of optical densities in Fig. 4 is similar to those of E. coli [19] and the RNA of other organisms. The first few tubes (15 to 30) usually contain polyExperimental
Cell Research
44
Synthesis
of ribosomcll
RNA during
growth
7
and division
The S-RNA comes off the column between 0.15 to saccharide impurities. 0.45 M NaCl. This location for S-RNA has been confirmed (unpublished results) by examining the location of acyl-S-RNA (T-RNA) in the MAK column, as 35S-cysteine-T-RNA and 35S- or 3H-methionine-T-RNA. The presumed messenger RNA separates into discrete polydispersed peaks between 0.5 to 1.0 M NaCl as shown by pollen-tube labeling with 3H-uridine (Fig. 5). The major RNA peak is ribosomal RNA at 0.80 M NaCl in both Figs. 4 and 5. As determined from Fig. 4, the total amount of ribosomal RNA within the peak amounts to about 72 per cent. This calculation infers that nearly three quarters of the total labeled RNA is ribosomal. Therefore, the analyses of total RNA synthesis (Figs. 1, 2 and 3) reflect primarily the patterns of ribosome production rather than S-RNA or m-RNA. The ribosomal RNA peak from a MAK column was analyzed from three separate experiments similar to Fig. 4 for their base ratios. These data are given in Table I. The base composition is similar to the 70s ribosomes of a number of bacteria [13] and is comparable to several analyses of other higher plants [ll, 14, 241. 20 9-
9-
I8
6-
a-
16
7-
14
biz z -6-
G =06-
12
v, 6 5-=50 E
8 IO!? x
2d 4-g4- 4
89 E
!i 03-
3-
6 RADIOACTIVITY
2-
2-
4 2 PTICAL
IO
20
30
40
DENSITY
50 60 70 TUBE NUMBER
80
90
100
110
120
Fig. 4.-Fractionation of pollen tube RNA on a MAK column prepared Microspore were labeled with *rP in the plant and “chased” twice with main peak at 0.75 M NaCl is ribosomal RNA. The peak between 0.20 and Messenger RNA occurs at numerous small polydispersed peaks from 0.5
130
by the carrier 0.45 M to 1.0 M
Experimental
I40
phenol method. phosphate. The NaCl is S-RNA. NaCI. Cell
Research
44
8 Analytical
D. M. Steflensen centrifugation
The pollen of Lilium was burst in the French pressure cell in 0.88 M sucrose and the ribosomes separated by centrifugation. One set of ribosomes were dissociated by deoxycholate, sedimented, further purified on a sucrose gradient and finally suspended in 0.05 Tris buffer without magnesium. Two peaks were obtained with values of 20s and 39s by analytical centrifugation. Another set of ribosomes was isolated as before but with 0.01 Mg ions during all the steps, before a final dialysis against 0.01 M Tris buffer and 0.001 magnesium acetate. The latter mixture gave three Schlieren peaks in the analytical centrifuge. The ultraviolet photographic plates of these ribosomes exhibited three sharp discontinuities. The analysis of the three slopes from microdensitometer tracings gave the following S values: 76S, 60s and 37s at 20°C. No corrections were made for the viscosity of the buffer. These sedimentations coefficients correspond closely to the undissociated ribosome and The results are essentially identical with the two dissociated components. those obtained from pea ribosomes [23]. The dissociation characteristics
TUBE
NUMBER
Fig. B.-The results of MAK column chromatography of SH-uridine labeled RNA. Pollen tubes had been grown for 8 hr in unlabeled media, labeled for 1 hr, and given a 5 hr “chase” 0.001 M uridine and cytidine before RNA extraction. Note the three depressions in specific activity over the ribosomal peak (tubes 95 to 110). Experimental
Cell Research
44
Synthesis
of ribosomal
with variation of magnesium ribosomes [6]. RNA synthesis
in pollen
RNA during
growth
ion concentration
and division
are comparable
9 to E. coli
tubes
The initial interest in investigating the ribosomal RNA of Lilium arose from the observation that no nucleolus was evident in the nuclei of growing pollen tubes. None of the conventional cytochemical procedures gave any indication of the presence of a nucleolus, such as staining with Azure B at pH 4.0, or methyl green-pyronin. Autoradiographic studies using 3H-uridine or 3H-cytidine exhibited homogeneous labeling over the nucleus without “hot spots” in the conventional nucleolar site of RN.4 accumulation. There is no unequivocal cytochemical or biochemical method yet devised that enables one to distinguish between the types of RNA being synthesized in situ, thus it was necessary to resort to biochemical methods to ascertain what, if any, synthesis was missing. It is intended to present data concerning ribosomal RNA and essentially to ignore any consideration of soluble RNA and messenger RNA, which will be dealt with in further papers in preparation. Briefly then, isotope is added to growing pollen tubes at the peak of RNA synthesis, occurring from S to 10 hr after germination at 22°C. Significant amounts of newly synthesized RNA do not appear until nearly 3 or 4 hr after labeling with messenger-like RNA peaks being just detectable in 20 to 30 min. It is known from our autoradiographic evidence that the nuclei are labeled in a few minutes. The slight delay in detecting RNA chemically must be due, at least in part, that no attempt has been made to isolate the DNA-RNA complex at the interfacial layer during the phenol extraction. TABLE
I. Base ratios of 32P-ribosomal RNA.
Each sample selected as the ribosome peak after separation of pollen tube column, alkaline hydrolysis and Dowex-1 column chromatography. The initial during microspore and pollen development. Experiment 2’, 3’, nucleotides CMP AMP UMP GMP
by MAK was done
number
1
2 (Per
23.9 23.6 23.5 29.0
of RNA labeling
3 cent)
24.4 23.6 21.0 31.1
24.3 24.9 10.8 31 .o
Experimental
Cell Research
44
10
D. M. Steffensen
In one experiment 3H-uridine was used to label growing pollen tubes in vitro for 1 hr, non-radioactive uridine was added and growth proceeded for 5 hr. The RNA was extracted and fractionated on a MAK column by elution in a sodium chloride gradient. The results are illustrated in Fig. 5. Discrete high specific activity peaks occur between 0.5 to 1.0 M NaCl. Most of these are assumed to be messenger RNA. The specific activity of the ribosome peak at 0.8 M NaCl is low and at least 5 to 10 times less than the radioactive peaks on either side. In fact the specific activity drops at the highest optical densities in tubes 100, 101, and 103, the peaks of ribosomal RNA. All of the MAK column profiles of lily ribosome RNA done to date have shown only a slight tendency of forming bimodal peaks and never shown two distinct peaks as with RNA from E. coli [19]. The differences in RNA labeling patterns between microspores and pollen tubes (Figs. 4 and 5) should be re-emphasized. In Fig. 4, the RNA was labeled in the microspore and the radioactivity follows the optical density quite closely, especially the ribosome peak. The reverse is true in Fig. 5, in’pollen tubes where neither radioactivity and optical density coincide and the peaks and shoulders of ribosomal RNA and the ribosomes manifest the lowest specific activity. Density
gradient
centrifugation
of ribosomes
It would appear that most, if not all, of the labeled peaks (Fig. 5) might not be ribosome synthesis at all but overlapping peaks of messenger RNA. In order to examine this possibility, pollen tubes were labeled for 5 hr with 32P ( 8 to 13 hr after germination). The homogenized pollen tubes and disrupted non-radioactive pollen grains were combined. After the appropriate centrifugation steps the ribosomes were sedimented, suspended again, and put on a linear sucrose gradient. Following centrifugation, drops were collected from the bottom of the tube. The RNA found in the ribosome peak was analyzed for its base ratios with the following results in Table II. These values in Table II are quite different from the ribosome analysis in Table I, especially for guanlylic and adenlylic. Apparently most plant ribosomal RNA contains between 30-32 per cent guanlylic [ll, 14, 241. It would appear that the radioactive RNA above is not ribosome RNA but probably overlapping or attached messenger RNA. The RNA made in growing tobacco pollen tubes was analyzed on a MAK column by Tano and Takahashi [20] and a broad messenger-like peak gave base ratios with 29.3 per cent adelylic and 22.9 per cent guanlylic. These authors have assumed, as has been the case here, that synthesis in pollen tubes involves primarily messenger RNA. All in all the Experimental
Cell
Research
44
Synthesis
of ribosomal
RNA during
growth
11
and division
evidence indicates that little if any ribosomal RNA is being made in growing pollen tubes. In recapitulation it has been found that no nucleolus is detectable in pollen tube nuclei. The nucleolar organizer region is apparently not active. These and other data would indicate that when the nucleolar organizer is “turned TABLE
II. Base ratios from the ribosome peak from labeled pollen sucrose density gradient centrifugation.
The labeling of RNA was done with 32P in growing pollen tubes from These base ratios are interpreted not to be ribosome RNA labeling messenger RNA (see ribosome base ratios in Table I). 2’, 3’nucleotides
CMP AMP UMP GMP
tubes after a
8 to 13 hr after germination. but probably contaminating
Per cent
27.7 30.0 20.3 21.9
no ribosomes are being syntheoff”; no nucleolus is visible and apparently sized. A number of studies have focused their attention on localizing the site of ribosome synthesis in the nucleolus. There are now many compelling reasons to believe that ribosomes are made within the nucleolus at a segment of the chromosome called the nucleolus organizer. For example, ribosome synthesis was absent in the anucleolate mutant of Xenopus [l]. Molecular hybrids were made between ribosomal RNA and Drosophila DNA using varying doses of the nucleolar organizer, indicating the nucleolar organizer DNA is responsible for ribosome synthesis amounts to 0.27 per cent of the wild type genome [la]. Ultrastructural studies have shown that ribososomes are within the nucleolus and that they undergo various alterations during the mitotic cycle in Vicia faba [4] and in grasshopper neuroblasts [18]. These latter experiments show the appearance of nucleolar ribosomes to be especially abundant in late interphase and early prophase (G2) and again after telophase. All of the evidence considered seems consistant with the data from lily ribosomes presented here regarding the timing and site of ribosome synthesis.
Experimental
Cell Research
44
12
D. M. Steffensen SUMMARY
The synthesis of RNA has been examined in the microspore of lily buds during division and later development into mature pollen grains. RNA synthesis is most active just before division (G2) and after the microspore nuclear division. Column chromatography using a methylated-albumin Kieselguhr column has shown that ribosomal RNA makes up the major portion of RNA being synthesized during this period, when nucleoli are large and active. All of the conventional cytological criteria have failed to demonstrate a nucleolus in growing pollen tubes. RNA is synthesized in pollen tubes but the abrupt drop in specific activity at the ribosomal peaks and the base ratios of this RNA do not correspond to ribosomal RNA. The conclusion seems apparent that little, if any, ribosomal RNA is being synthesized in the pollen tube nuclei in the absence of nucleolar activity. The excellent technical assistance of Mrs Lalla Tanner and Mrs Elaine Miller are appreciated, as is the financial support from the National Science Foundation (NSF GB 1327). REFERENCES 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13.
14. 15. 16. 17. 18. 19. 20. 21. 22. 23. 24.
and GURDON, J. B., Proc. Nail Acad. Sci. U.S. 51, 139 (1964). STERN, H. J., Biophss. Biochem. C&Z. 11, 311 (1961). -Proc. Nat1 Acad. Sci. U.S. i9,-648 (1963). I LAFONTAINE, J. G. and CHOUINARD, L. A., J. Cell Biol. 17. 167 (1963). MANDELL, J: D. and HERSHEY, A. b., Aial. Biochem. 1, 86 (1960). ’ MESELSON, M., NOMURA, M., BRENNER, S., DAVERN, C. and SCHLESSINGER, D., J. MoZ. BioZ. 9, 696 (1964). MOSES, M. J. and TAYLOR, J. H., Exptl Cell Res. 9, 474 (1955). KIRBY, K. S., Biochem. J. 64, 405 (1956). ISHIHAMA, A., MIZURNO, N., TAKAE, M., OTAKA, E. and OSAWA, S., J. Mol. Biol. 5, 251 (1962). OGUH, M., ERICKSON, R. O., ROSEN, G. U., SAX, D. B. and HOLDEN, C., Exptl Cell Res. 2, 73 (1951). PETERWANN, M. L., The physical and chemical properties of ribosomes. Elsevier Publishing Co., Amsterdam, 1964. RITOSSA, F. M. and SPIEGELMAN, S., Proc. Nat1 Acad. Sci. U.S. 53, 737 (1965). ROBERTS, R. B., BRITTEN, R. J. and MCCARTHY, B. J., in J. H. TAYLER (ed.), Molecular Genetics, Part 1, p. 291. Academic Press, New York, 1963. SRIVASTAVA, B. I. S., Biochem. J. 96, 665 (1965). STEFFENSEN, D. M., Genetics 52, 631 (1965). STEFFENSEN, D. M..and BERGE~ON, J. A.,‘J. Biophys. Biochem. Cytol. 6, 339 (1959). STERN, H. and HOTTA, Y., Brookhaven Symp. Biol. 16, 59 (1964). STEVENS, B. J., J. Cell Biol. 24, 349 (1965). SUEOKA, N. and YAMANE, T., Proc. Nat1 Acad. Sci. U.S. 48, 1454 (1962). TANO, S. and TAKAHASHI, H., J. Biochem. 56, 578 (1964). TAYLOR, J. H., Exptl Cell Res. 4, 164 (1953). TAYLOR, J. H. and MCMASTER, R. D., Chromosoma 6, 489 (1954). Ts’o, P. 0. P., BONNER, J. and VINOGRAD, J., Biochim. Biophys. Acta 30, 570 (1958). WATERS, L. and DURE, L., Science 149, 188 (1965). BROWS, HOTTA,
Experimental
D. D. Y. and
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