Preliminary notes meiotic arrest and failure to divide [17], although during pachytene the two X chromosomes may form an XXY body [15]. The observed alterations in the sex chromosome axes (see Results) may indicate that this cell (and most likely all polyploid spermatocytes) would not complete meiotic prophase and would degenerate. The absence of trivalents in this cell is in agreement with the almost complete absence of trivalents and univalents in autotetraploid meiotic cells [8]. It may also be significant that in tetraploid spermatocytes of grasshoppers, the two X chromosomes do not synapse, although they lie close together [18]. From the ultrastructural viewpoint, Moens [9] has demonstrated the switching of partners between axes in an autotetraploid of Lilium longiflorum. Thus, it can be said that ultrastructural axes of chromosomes reflect the pairing behavior of chromosomes in polyploids as they do in structural rearrangements as seen with 3-dimensional reconstructions from serial sections [16] and with the microspreading technique [11]. The diagrammatic nature of the axial pattern of the chromosomes described here, and the rapid results obtainable with the microspreading technique may be advantageous for diagnosing ploidy alterations in meiotic cells of mammals, where observation of later stages with the light microscope is impossible because of pachytene arrest and degeneration.
We gratefully acknowledge the assistance of Mr Todd Gambling and Ms Marlene Johnson. During the period of this study, A.J.S. held a Hargitt Fellowship from the Department of Zoology, Duke University. This research was supported by NSF grant no. GB-40562 to M.J.M., and by NIH grants 5-S01-RR.05404 and CA-14236 to Duke University.
467
References 1. Counce, S J & Meyer, G F, Chromosoma 44 (1973) 231. 2. Beatty, R A, Lim, M C & Coulter, V J, Cytogenet cell genet 15 (1975) 256. 3. Darlington, C D, Cytology, 3rd edn, p. 119. Churchill, London, 1965. 4. Fawcett, D W, Ito, S & Slautterback, D, J biophys biochem cytol 5 (1959) 453. 5. Fechheimer, N S, J reprod fertil 2 (1961) 68. 6. Ford, C E & Evans, P E, Nature 230 (1971) 389. 7. Hult~n, M, Karlman, A, Londsten, J & Tiepolo, L, Hereditas 65 (1970) 197. 8. John, B & Lewis, K R, The meiotic system, p. 116. Springer-Verlag, Berlin and New York (1965). 9. Moens, PB, J cell sci7 (1970) 55. 10. Moses, M J, Chromosoma 60 (1977) 99. 11. Moses, M J, Russell, L B & Cacheiro, N, Science 196 (1977) 892. 12. Painter, T S, J exp zool 37 (1923) 291. 13. Pogosianz, H E & Brujako, E T, Cytogenetics 10 196 (1977) 892. 14. Sasaki, M & Makino, S, Chromosoma 16 (1965) 637. 15. Slizynski, B M, Genet res 5 (1964) 328. 16. Solari, A J, Chromosoma 34 (1971) 99. 17. - - Int rev cytol 38 (1974) 273. 18. White, M J D, Cytologia 5 (1933) 135. Received April 13, 1977 Accepted May 31, 1977
Contamination of detergent-purified rat liver nuclei by cytoplasmic ribosomes M. D. DABEVA, P. T. PETROV, A. S. STOYKOVA and A. A. HADJIOLOV, Institute of Bio-
chemistry, Bulgarian Academy of Sciences, Sofia, Bulgaria
Contamination of isolated nuclei by cytoplasmic ribosomes adhering to the outer nuclear membrane is a major obstacle in studies on metabolic activities of nuclei and nucleo-cytoplasmic interactions. Quantitative estimates yielded figures ranging from 5% [1] to 15% [2] of total nuclear RNA. It is generally accepted that treatment of nuclei with non-ionic detergents removes the outer nuclear membrane together with attached ribosomes [3-6]. However, the fate of contaminating cytoplasmic riboExp Cell Res 108 (1977)
468
Preliminary notes
Preliminary notes somes during detergent treatment of nuclei has not been clarified. In this work we present evidence that detergent-purified rat l i v e r n u c l e i a r e still c o n t a m i n a t e d
by cyto-
plasmic ribosomes.
Experimental Pure rat liver nuclei are isolated by the widely used procedure of Blobel & Potter [3] and by a two-step hypertonic sucrose-detergent procedure developed by us [7] in order to minimize degradation and leakage of nuclear RNA. Immediately after sacrificing the experimental rats (about 150 g body wt), the livers are dissected out, rinsed in cold saline and processed further in the cold. Ten grams of liver are homogenized with a glass-Teflon homogenizer in 20 ml 2.3 M sucrose containing 0.01 M Tris-HCI (pH 7.0) and 0.01 M MgC12. The homogenate is filtered through two layers of cheesecloth, adjusted to 30 ml with the same medium, layered over 8 ml 2.3 M sucrose (as above) and centrifuged for 40 min at 25 000 rpm and 0°C in the SW27 rotor of a Beckman L5-50 ultracentrifuge. The nuclear pellet is rinsed with 0.25 M sucrose and suspended in 20 ml 0.5 M sucrose, containing 0.01 M Tris-HCl (pH 7.0), 0.01 M MgCI~ and Triton X-100 (final conc. 0.1-1%), by four tractions of a loose homogenizer. The suspension is underlaid with 10 ml 1 M sucrose (as above, without Triton X-100) and centrifuged for 5 rain at 5000 g. The nuclear pellets are fixed in 1.6% glutaraldehyde at 4°C for 1 h. Post-fixation with 2% OsO4 is at room temperature for 1 h, this treatment being omitted when Bernhard's EDTA method is used [8]. After dehydration in ethanols and propylene oxide, the material is embedded in Epon-812. Ultrathin sections are examined in the JEM 100B electron microscope at 80 kV. Two nuclear RNA fractions, corresponding to "nucleosor' and "nucleolar" RNA are obtained by subsequent extraction of detergent-purified nuclei at 4 and 50°C with phenol, 0.1 M Tris-acetate (pH 6.0) as described earlier [9, 10]. Agar-urea gel electrophoresis of RNA is according to Dudov et al. [11] and the amount of RNA and DNA determined by a twowave length method [12].
469
T a b l e I. Contamination of detergent-puri-
fied nuclei by cytoplasmic ribosomes Rat liver RNA is labelled in vivo for 6 days with 150 txCi of [~4C]orotate (19 mCi/mmole) per animal. The cytoplasmic fraction is obtained from a 30% homogenate in 1 M sucrose by centrifugation at 17000 g for 15 min. This cytoplasmic fraction is mixed with 2 vol 2.5 M sucrose and used as homogenizing medium for the isolation of nuclei from unlabelled livers. The amount of cytoplasmic fraction is equivalent to the livers used for isolation of nuclei on a wet weight basis. Nuclei are isolated either by the method described in this work [7] or according to Blobel & Potter [3]. Purification is achieved by treatment with different concentrations of Triton X-100 as described in the text. The sucrose solutions used are buffered with 10 mM Tris-HC1 (pH 7.0) and 10 mM MgCI~ (our method) or 50 mM Tris-HC1 (pH 7.6), 25 mM KCI and 5 mM MgC12 (Blobel-Potter method). The radioactivity of nuclear RNA (cpm/mg RNA) is determined after alkaline hydrolysis of the total nuclear preparation or of "nucleosor' and "nucleolar" RNA extracted as described. Aliquots of the final liver homogenate used for isolation of nuclei are diluted 6fold with the respective Tris-HCl buffer, the nuclei sedimented at 2000 g for 10 min and the RNA from the supernatant is extracted with cold phenol. The labelling of this RNA is determined as above and its specific radioactivity taken as 100 Percentage contamination by cytoplasmic RNA in Method for isolation of nuclei
total nuclear RNA
"Nucleosol" 4°C RNA
"Nucleolar" 50°C RNA
This work +0.5% Triton +1.0% Triton Blobel-Potter +1.0% Triton
7.0 6.7 4.6 8.0 5.7
14.8 14.2 10.8 16.6 13.2
6.9 5.9 3.4 7.5 4.4
plete removal brane,
while
of the outer nuclear their
integrity
mem-
is p r e s e r v e d
Results and Discussion
(fig. I a ) . T h e r e m o v a l o f t h e o u t e r n u c l e a r
Treatment of nuclei, isolated by our method
membrane
w i t h 0 . 1 % T r i t o n X - 1 0 0 r e s u l t s in t h e c o r n -
a n d B e r n h a r d ' s t e c h n i q u e s (fig. 1 b). H i g h e r
Fig. 1. (a) Overall view of rat liver nuclei isolated
m a g n i f i c a t i o n (fig. 1 c) fails to r e v e a l r i b o somes on the surface of Triton X-100 puri-
and purified by the method described in the text showing the good preservation and purity of nuclei and the removal of the nuclear membrane. ×4000; (b) the same nuclear fraction as in (a) stained by Bernhard's EDTA method for ribonucleoprotein particles, x20000; (c) inset of (b) at higher magnification (x80000). The nuclear surface is apparently free of ribonucleoprotein particles. Arrows indicate presumed nuclear pore complex material.
is e v i d e n c e d
by both standard
fied nuclei, the only RNP structures visuali z e d in t h i s a r e a b e i n g r e p r e s e n t e d b y p u t a tive nuclear Essentially
pore
complex
material
[13].
the same results are obtained
when the concentration
o f T r i t o n X - 1 0 0 is
r a i s e d t o 0.5 o r 1 % , as w e l l as u p o n d e t e r -
Exp CellRes 108(1977)
470
Preliminary notes
gent treatment of nuclei isolated by the Blobel-Potter method. The RNA/DNA ratio of nuclei isolated by our procedure is 0.205+0.004 before and 0.195+0.006 after Triton X-100 treatment (7 experiments). These results show that Triton X-100 treatment causes a loss of about 5 % o f total nuclear RNA. The above results seem to indicate that, by both electron microscopic and chemical criteria, detergent treatment results in the removal of contaminating cytoplasmic ribosomes in parallel with the lysis of the outer nuclear membrane. That this is not the case is evidenced by experiments in which the liver homogenate is mixed with a cytoplasmic fraction with its rRNA labelled for 6 days in vivo with [14C]orotate. The nuclei, subsequently isolated from this mixture by either procedure, are purified by Triton X-100 and the radioactivity in the fractions of total nuclear, "nucleosol" and "nucleolar" RNA is determined. The results (table 1) demonstrate that a substantial amount of added labelled cytoplasmic RNA (about 3-5% of total nuclear RNA) adheres to nuclei and is not removed by Triton X-100 treatment, although this contamination could not be revealed by either electron microscopy or chemical analyses. This RNA contaminates both the "nucleosol" and the "nucleolar" RNA fractions known to contain all the nuclear rRNA [9, 10]. The electrophoretic and labelling profiles of the "nucleosor' RNA extracted from Triton X-100 purified nuclei (fig. 2) show that the contaminating radioactivity is confined to 28 S and 18 S rRNA and therefore originates from added cytoplasmic ribosomes. The above results reveal that a substantial amount of cytoplasmic ribosomes adheres to liver nuclei and resists purification by detergent treatment. Moreover, contamination of detergent purified nuclei by cytoExp Cell Res 108 (1977)
0.7 I
, ~ j 28 S
0.7
0.6
0.6
0.5 ¸
05 18S
O,t..
0.4
0.3-
03
0,2
0.2
0,1.
0.1 ~'......,,..,... 6
7
8
9
10
11
Fig. 2. Abscissa: distance from origin (cm); ordinate: (left) E~60; (right) E550. Agar-urea gel electrophoresis of " n u c l e o s o l " R N A
from detergent purified rat liver nuclei isolated in the presence of [~4C]orotate labelled cytoplasm. --, A28o; ---, radioactivity of autoradiogram recorded at 550 nm. The results demonstrate that contamination of nuclei is by cytoplasmic28 S and 18S rRNA. plasmic ribosomes may be even higher, since under our experimental conditions, the ribosomes attached in situ to the outer nuclear membrane are not labelled. Thus, when liver nuclear RNA fractions enriched in rRNA are considered, contamination by cytoplasmic ribosomes may account for a substantial part of their 28 S and 18 S rRNA. Consequently, special purification or analysis techniques are required in studies on intranuclear ribosomes in order to take into consideration the contamination of detergent-purified nuclei by cytoplasmic ribosomes.
References 1. Whittle, E D, Bushnell, D E & Potter, V R, Biochim biophys acta 161 (1968) 41. 2. Smith, S J, Adams, H R, Smetana, K & Busch, H, Exp cell res 55 (1969) 185.
Preliminary notes 3. Blobel, G & Potter, V R, Science 154 (1966) 1662. 4. Sadowski, P D & Howden, J A, J cell bio137 (1968) 163. 5. Laval, M & Bouteille, M, Exp cell res 76 (1973) 337. 6. Goidl, J A, Canaani, D, Boublik, M, Weissbach, H & Dickerman, H, J biol chem 250 (1975) 9198. 7. Dabeva, M D, Dudov, K P, Todorov, B N, Petrov, P T, Stoykova, A S & Hadjiolov, A A, Compt rend acad sci Bulgaria 29 (1976) 1841. 8. Bernhard, W, J ultrastr res 27 (1969) 250. 9. Hadjiolov, A A, Dabeva, M D & Mackedonski, V V, Biochem j 138 (1974) 321. 10. Dabeva, M D, Todorov, B N & Hadjiolov, A A, Biokhimiya, 41 (1976) 458. 11. Dudov, K P, Dabeva, M D & Hadjiolov, A A, Anal biochem 76 (1976) 250. 12. Tsanev, R G & Markov, G G, Biochim biophys acta 42 (1960) 442. 13. Franke, WW & Scheer, U, The cell nucleus (ed H Busch) vol. 1, pp. 220-347. Academic Press, New York (1974). Received May 18, 1977 Accepted May 31, 1977
High speed scintillation autoradiography of DNA fibres undergoing DNA synthesis at zygotene and pachytene in the lily S. K. SEN, S. C. KUNDU and J. P. GADDIPATI,
Botany Department, Bose Institute, Calcutta 700009, India I t h a s b e e n i n d i c a t e d e a r l i e r [3] t h a t o u r initial a t t e m p t s to p r o v i d e s t a n d a r d a u t o r a d i o graphic evidence for meiotic DNA synt h e s i s [1, 2] in m e i o c y t e s o f t h e lily (Lilium longiflorum v a t . P r a e c o x ) w e r e n o t v e r y s u c c e s s f u l . A f t e r l a b e l l i n g w i t h [3H]thym i d i n e ([3H]TdR) at z y g o t e n e , f e w y e t c l e a r d e v e l o p e d g r a i n s w e r e o b s e r v e d to b e p r e s e n t in c h r o m o s o m e s . O u r e v i d e n c e f o r z y g o t e n e D N A s y n t h e s i s ( Z - D N A ) in lily, in fact, supplemented the earlier findings of I t o & H o t t a [4]. A l t h o u g h it is k n o w n t h r o u g h b i o c h e m i c a l a n a l y s i s [2] t h a t an a d ditional small amount of DNA synthesis a l s o t a k e s p l a c e in lily at p a c h y t e n e (PD N A ) , t h e r e is n o a u t o r a d i o g r a p h i c evid e n c e f o r this. W e a l s o f a i l e d to p r o v i d e autoradiographic evidence for meiotic DNA
synthesis
471
in
microsporocytes of wheat (Triticum aestivum v a r . K68) a n d b a r l e y ( H o r d e u m vulgate v a r . K12) [5] d u r i n g a n y p a r t o f m e i o t i c n u c l e a r divi3ion. O n t h e b a s i s o f o u r initial s u c c e s s w i t h a u t o r a d i o g r a p h y in lily a n d as c l e a n m i c r o sporocytes free from surrounding somatic c e l l s o f a n t h e r t i s s u e a r e a v a i l a b l e in lily [6J--unlike wheat and barley--we att e m p t e d to c h a r a c t e r i z e z y g o t e n e a n d p a c h y t e n e D N A s y n t h e s i s in lily b y a u t o radiography of DNA fibres applying high speed scintillation autoradiography techn i q u e [7, 8]. T h i s is t h e e s s e n t i a l s u b j e c t o f the present communication.
Materials and M e t h o d s In Lilium, zygotene DNA synthesis occurs 3-5 days after completion of S phase synthesis. Zygotene and pachytene last for about 34 and 40 h in the variety of lily which was studied. Buds were collected according to their length [9]. This makes it possible to collect microsporocytes at specific meiotic stages [10, 11]. For adoption of DNA-fibre autoradiography, the meiocytes were explanted exactly in early zygotene and early pachytene. They were then incubated separately in basic culture media [12] containing FUdR (1 x 10-6 M) for 6 h. Subsequently they were transferred to incubate in a media containing radioactive thymidine (250/,~Ci/ml, spec. act. 10.5 Ci/mmole), separately, for 45 min and 2, 6 and 12 h. The meiocytes were harvested by freezing immediately after incubation with radioactive thymidine. The filaments of the meiocytes were subsequently cleaned from tapetal cells according to the method of Hotta & Stern [6]. Cytological checks were introduced to safeguard against contamination of the meiocytes from tapetal cells. The crude DNA from the cleaned filaments were extracted by the chloroform-isoamylalcohol method of Marmur [13]. Pronase, RNase and phenol treatments were avoided. DNA was precipitated with 2 vol of ethanol and the precipitate was collected on a glass rod and dissolved in 0. I x SSC. A hundred-fold dilution of the DNA was made and was spread on clean slides by stretching a fraction of a drop of the DNA-containing fluid on a clean slide in one direction. After spreading, the slides were air-dried and Kodak AR-10 stripping films were then mounted. The slides were then dried and stored for 24 h at 4°C. Following this, the prepared slides were immersed in scintillation fluid (6 g PPO and 0.1 g dimethyl POPOP in 1000 ml Toluene) in the dark for 72 h at 20*(]. In complete darkness, slides were passed through xylene, grades of ethanol and distilled water allowing 5 min for each step. Slides were finally developed in a Kodak D19 developer for 6 min.
Exp CellRes 108 (1977)
We gratefully acknowledge the assistance of Mr Todd Gambling and Ms Marlene Johnson. During the period of this study, A.J.S. held a Hargitt Fellowship from the Department of Zoology, Duke University. This research was supported by NSF grant no. GB-40562 to M.J.M., and by NIH grants 5-S01-RR.05404 and CA-14236 to Duke University.
467
References 1. Counce, S J & Meyer, G F, Chromosoma 44 (1973) 231. 2. Beatty, R A, Lim, M C & Coulter, V J, Cytogenet cell genet 15 (1975) 256. 3. Darlington, C D, Cytology, 3rd edn, p. 119. Churchill, London, 1965. 4. Fawcett, D W, Ito, S & Slautterback, D, J biophys biochem cytol 5 (1959) 453. 5. Fechheimer, N S, J reprod fertil 2 (1961) 68. 6. Ford, C E & Evans, P E, Nature 230 (1971) 389. 7. Hult~n, M, Karlman, A, Londsten, J & Tiepolo, L, Hereditas 65 (1970) 197. 8. John, B & Lewis, K R, The meiotic system, p. 116. Springer-Verlag, Berlin and New York (1965). 9. Moens, PB, J cell sci7 (1970) 55. 10. Moses, M J, Chromosoma 60 (1977) 99. 11. Moses, M J, Russell, L B & Cacheiro, N, Science 196 (1977) 892. 12. Painter, T S, J exp zool 37 (1923) 291. 13. Pogosianz, H E & Brujako, E T, Cytogenetics 10 196 (1977) 892. 14. Sasaki, M & Makino, S, Chromosoma 16 (1965) 637. 15. Slizynski, B M, Genet res 5 (1964) 328. 16. Solari, A J, Chromosoma 34 (1971) 99. 17. - - Int rev cytol 38 (1974) 273. 18. White, M J D, Cytologia 5 (1933) 135. Received April 13, 1977 Accepted May 31, 1977
Contamination of detergent-purified rat liver nuclei by cytoplasmic ribosomes M. D. DABEVA, P. T. PETROV, A. S. STOYKOVA and A. A. HADJIOLOV, Institute of Bio-
chemistry, Bulgarian Academy of Sciences, Sofia, Bulgaria
Contamination of isolated nuclei by cytoplasmic ribosomes adhering to the outer nuclear membrane is a major obstacle in studies on metabolic activities of nuclei and nucleo-cytoplasmic interactions. Quantitative estimates yielded figures ranging from 5% [1] to 15% [2] of total nuclear RNA. It is generally accepted that treatment of nuclei with non-ionic detergents removes the outer nuclear membrane together with attached ribosomes [3-6]. However, the fate of contaminating cytoplasmic riboExp Cell Res 108 (1977)
468
Preliminary notes
Preliminary notes somes during detergent treatment of nuclei has not been clarified. In this work we present evidence that detergent-purified rat l i v e r n u c l e i a r e still c o n t a m i n a t e d
by cyto-
plasmic ribosomes.
Experimental Pure rat liver nuclei are isolated by the widely used procedure of Blobel & Potter [3] and by a two-step hypertonic sucrose-detergent procedure developed by us [7] in order to minimize degradation and leakage of nuclear RNA. Immediately after sacrificing the experimental rats (about 150 g body wt), the livers are dissected out, rinsed in cold saline and processed further in the cold. Ten grams of liver are homogenized with a glass-Teflon homogenizer in 20 ml 2.3 M sucrose containing 0.01 M Tris-HCI (pH 7.0) and 0.01 M MgC12. The homogenate is filtered through two layers of cheesecloth, adjusted to 30 ml with the same medium, layered over 8 ml 2.3 M sucrose (as above) and centrifuged for 40 min at 25 000 rpm and 0°C in the SW27 rotor of a Beckman L5-50 ultracentrifuge. The nuclear pellet is rinsed with 0.25 M sucrose and suspended in 20 ml 0.5 M sucrose, containing 0.01 M Tris-HCl (pH 7.0), 0.01 M MgCI~ and Triton X-100 (final conc. 0.1-1%), by four tractions of a loose homogenizer. The suspension is underlaid with 10 ml 1 M sucrose (as above, without Triton X-100) and centrifuged for 5 rain at 5000 g. The nuclear pellets are fixed in 1.6% glutaraldehyde at 4°C for 1 h. Post-fixation with 2% OsO4 is at room temperature for 1 h, this treatment being omitted when Bernhard's EDTA method is used [8]. After dehydration in ethanols and propylene oxide, the material is embedded in Epon-812. Ultrathin sections are examined in the JEM 100B electron microscope at 80 kV. Two nuclear RNA fractions, corresponding to "nucleosor' and "nucleolar" RNA are obtained by subsequent extraction of detergent-purified nuclei at 4 and 50°C with phenol, 0.1 M Tris-acetate (pH 6.0) as described earlier [9, 10]. Agar-urea gel electrophoresis of RNA is according to Dudov et al. [11] and the amount of RNA and DNA determined by a twowave length method [12].
469
T a b l e I. Contamination of detergent-puri-
fied nuclei by cytoplasmic ribosomes Rat liver RNA is labelled in vivo for 6 days with 150 txCi of [~4C]orotate (19 mCi/mmole) per animal. The cytoplasmic fraction is obtained from a 30% homogenate in 1 M sucrose by centrifugation at 17000 g for 15 min. This cytoplasmic fraction is mixed with 2 vol 2.5 M sucrose and used as homogenizing medium for the isolation of nuclei from unlabelled livers. The amount of cytoplasmic fraction is equivalent to the livers used for isolation of nuclei on a wet weight basis. Nuclei are isolated either by the method described in this work [7] or according to Blobel & Potter [3]. Purification is achieved by treatment with different concentrations of Triton X-100 as described in the text. The sucrose solutions used are buffered with 10 mM Tris-HC1 (pH 7.0) and 10 mM MgCI~ (our method) or 50 mM Tris-HC1 (pH 7.6), 25 mM KCI and 5 mM MgC12 (Blobel-Potter method). The radioactivity of nuclear RNA (cpm/mg RNA) is determined after alkaline hydrolysis of the total nuclear preparation or of "nucleosor' and "nucleolar" RNA extracted as described. Aliquots of the final liver homogenate used for isolation of nuclei are diluted 6fold with the respective Tris-HCl buffer, the nuclei sedimented at 2000 g for 10 min and the RNA from the supernatant is extracted with cold phenol. The labelling of this RNA is determined as above and its specific radioactivity taken as 100 Percentage contamination by cytoplasmic RNA in Method for isolation of nuclei
total nuclear RNA
"Nucleosol" 4°C RNA
"Nucleolar" 50°C RNA
This work +0.5% Triton +1.0% Triton Blobel-Potter +1.0% Triton
7.0 6.7 4.6 8.0 5.7
14.8 14.2 10.8 16.6 13.2
6.9 5.9 3.4 7.5 4.4
plete removal brane,
while
of the outer nuclear their
integrity
mem-
is p r e s e r v e d
Results and Discussion
(fig. I a ) . T h e r e m o v a l o f t h e o u t e r n u c l e a r
Treatment of nuclei, isolated by our method
membrane
w i t h 0 . 1 % T r i t o n X - 1 0 0 r e s u l t s in t h e c o r n -
a n d B e r n h a r d ' s t e c h n i q u e s (fig. 1 b). H i g h e r
Fig. 1. (a) Overall view of rat liver nuclei isolated
m a g n i f i c a t i o n (fig. 1 c) fails to r e v e a l r i b o somes on the surface of Triton X-100 puri-
and purified by the method described in the text showing the good preservation and purity of nuclei and the removal of the nuclear membrane. ×4000; (b) the same nuclear fraction as in (a) stained by Bernhard's EDTA method for ribonucleoprotein particles, x20000; (c) inset of (b) at higher magnification (x80000). The nuclear surface is apparently free of ribonucleoprotein particles. Arrows indicate presumed nuclear pore complex material.
is e v i d e n c e d
by both standard
fied nuclei, the only RNP structures visuali z e d in t h i s a r e a b e i n g r e p r e s e n t e d b y p u t a tive nuclear Essentially
pore
complex
material
[13].
the same results are obtained
when the concentration
o f T r i t o n X - 1 0 0 is
r a i s e d t o 0.5 o r 1 % , as w e l l as u p o n d e t e r -
Exp CellRes 108(1977)
470
Preliminary notes
gent treatment of nuclei isolated by the Blobel-Potter method. The RNA/DNA ratio of nuclei isolated by our procedure is 0.205+0.004 before and 0.195+0.006 after Triton X-100 treatment (7 experiments). These results show that Triton X-100 treatment causes a loss of about 5 % o f total nuclear RNA. The above results seem to indicate that, by both electron microscopic and chemical criteria, detergent treatment results in the removal of contaminating cytoplasmic ribosomes in parallel with the lysis of the outer nuclear membrane. That this is not the case is evidenced by experiments in which the liver homogenate is mixed with a cytoplasmic fraction with its rRNA labelled for 6 days in vivo with [14C]orotate. The nuclei, subsequently isolated from this mixture by either procedure, are purified by Triton X-100 and the radioactivity in the fractions of total nuclear, "nucleosol" and "nucleolar" RNA is determined. The results (table 1) demonstrate that a substantial amount of added labelled cytoplasmic RNA (about 3-5% of total nuclear RNA) adheres to nuclei and is not removed by Triton X-100 treatment, although this contamination could not be revealed by either electron microscopy or chemical analyses. This RNA contaminates both the "nucleosol" and the "nucleolar" RNA fractions known to contain all the nuclear rRNA [9, 10]. The electrophoretic and labelling profiles of the "nucleosor' RNA extracted from Triton X-100 purified nuclei (fig. 2) show that the contaminating radioactivity is confined to 28 S and 18 S rRNA and therefore originates from added cytoplasmic ribosomes. The above results reveal that a substantial amount of cytoplasmic ribosomes adheres to liver nuclei and resists purification by detergent treatment. Moreover, contamination of detergent purified nuclei by cytoExp Cell Res 108 (1977)
0.7 I
, ~ j 28 S
0.7
0.6
0.6
0.5 ¸
05 18S
O,t..
0.4
0.3-
03
0,2
0.2
0,1.
0.1 ~'......,,..,... 6
7
8
9
10
11
Fig. 2. Abscissa: distance from origin (cm); ordinate: (left) E~60; (right) E550. Agar-urea gel electrophoresis of " n u c l e o s o l " R N A
from detergent purified rat liver nuclei isolated in the presence of [~4C]orotate labelled cytoplasm. --, A28o; ---, radioactivity of autoradiogram recorded at 550 nm. The results demonstrate that contamination of nuclei is by cytoplasmic28 S and 18S rRNA. plasmic ribosomes may be even higher, since under our experimental conditions, the ribosomes attached in situ to the outer nuclear membrane are not labelled. Thus, when liver nuclear RNA fractions enriched in rRNA are considered, contamination by cytoplasmic ribosomes may account for a substantial part of their 28 S and 18 S rRNA. Consequently, special purification or analysis techniques are required in studies on intranuclear ribosomes in order to take into consideration the contamination of detergent-purified nuclei by cytoplasmic ribosomes.
References 1. Whittle, E D, Bushnell, D E & Potter, V R, Biochim biophys acta 161 (1968) 41. 2. Smith, S J, Adams, H R, Smetana, K & Busch, H, Exp cell res 55 (1969) 185.
Preliminary notes 3. Blobel, G & Potter, V R, Science 154 (1966) 1662. 4. Sadowski, P D & Howden, J A, J cell bio137 (1968) 163. 5. Laval, M & Bouteille, M, Exp cell res 76 (1973) 337. 6. Goidl, J A, Canaani, D, Boublik, M, Weissbach, H & Dickerman, H, J biol chem 250 (1975) 9198. 7. Dabeva, M D, Dudov, K P, Todorov, B N, Petrov, P T, Stoykova, A S & Hadjiolov, A A, Compt rend acad sci Bulgaria 29 (1976) 1841. 8. Bernhard, W, J ultrastr res 27 (1969) 250. 9. Hadjiolov, A A, Dabeva, M D & Mackedonski, V V, Biochem j 138 (1974) 321. 10. Dabeva, M D, Todorov, B N & Hadjiolov, A A, Biokhimiya, 41 (1976) 458. 11. Dudov, K P, Dabeva, M D & Hadjiolov, A A, Anal biochem 76 (1976) 250. 12. Tsanev, R G & Markov, G G, Biochim biophys acta 42 (1960) 442. 13. Franke, WW & Scheer, U, The cell nucleus (ed H Busch) vol. 1, pp. 220-347. Academic Press, New York (1974). Received May 18, 1977 Accepted May 31, 1977
High speed scintillation autoradiography of DNA fibres undergoing DNA synthesis at zygotene and pachytene in the lily S. K. SEN, S. C. KUNDU and J. P. GADDIPATI,
Botany Department, Bose Institute, Calcutta 700009, India I t h a s b e e n i n d i c a t e d e a r l i e r [3] t h a t o u r initial a t t e m p t s to p r o v i d e s t a n d a r d a u t o r a d i o graphic evidence for meiotic DNA synt h e s i s [1, 2] in m e i o c y t e s o f t h e lily (Lilium longiflorum v a t . P r a e c o x ) w e r e n o t v e r y s u c c e s s f u l . A f t e r l a b e l l i n g w i t h [3H]thym i d i n e ([3H]TdR) at z y g o t e n e , f e w y e t c l e a r d e v e l o p e d g r a i n s w e r e o b s e r v e d to b e p r e s e n t in c h r o m o s o m e s . O u r e v i d e n c e f o r z y g o t e n e D N A s y n t h e s i s ( Z - D N A ) in lily, in fact, supplemented the earlier findings of I t o & H o t t a [4]. A l t h o u g h it is k n o w n t h r o u g h b i o c h e m i c a l a n a l y s i s [2] t h a t an a d ditional small amount of DNA synthesis a l s o t a k e s p l a c e in lily at p a c h y t e n e (PD N A ) , t h e r e is n o a u t o r a d i o g r a p h i c evid e n c e f o r this. W e a l s o f a i l e d to p r o v i d e autoradiographic evidence for meiotic DNA
synthesis
471
in
microsporocytes of wheat (Triticum aestivum v a r . K68) a n d b a r l e y ( H o r d e u m vulgate v a r . K12) [5] d u r i n g a n y p a r t o f m e i o t i c n u c l e a r divi3ion. O n t h e b a s i s o f o u r initial s u c c e s s w i t h a u t o r a d i o g r a p h y in lily a n d as c l e a n m i c r o sporocytes free from surrounding somatic c e l l s o f a n t h e r t i s s u e a r e a v a i l a b l e in lily [6J--unlike wheat and barley--we att e m p t e d to c h a r a c t e r i z e z y g o t e n e a n d p a c h y t e n e D N A s y n t h e s i s in lily b y a u t o radiography of DNA fibres applying high speed scintillation autoradiography techn i q u e [7, 8]. T h i s is t h e e s s e n t i a l s u b j e c t o f the present communication.
Materials and M e t h o d s In Lilium, zygotene DNA synthesis occurs 3-5 days after completion of S phase synthesis. Zygotene and pachytene last for about 34 and 40 h in the variety of lily which was studied. Buds were collected according to their length [9]. This makes it possible to collect microsporocytes at specific meiotic stages [10, 11]. For adoption of DNA-fibre autoradiography, the meiocytes were explanted exactly in early zygotene and early pachytene. They were then incubated separately in basic culture media [12] containing FUdR (1 x 10-6 M) for 6 h. Subsequently they were transferred to incubate in a media containing radioactive thymidine (250/,~Ci/ml, spec. act. 10.5 Ci/mmole), separately, for 45 min and 2, 6 and 12 h. The meiocytes were harvested by freezing immediately after incubation with radioactive thymidine. The filaments of the meiocytes were subsequently cleaned from tapetal cells according to the method of Hotta & Stern [6]. Cytological checks were introduced to safeguard against contamination of the meiocytes from tapetal cells. The crude DNA from the cleaned filaments were extracted by the chloroform-isoamylalcohol method of Marmur [13]. Pronase, RNase and phenol treatments were avoided. DNA was precipitated with 2 vol of ethanol and the precipitate was collected on a glass rod and dissolved in 0. I x SSC. A hundred-fold dilution of the DNA was made and was spread on clean slides by stretching a fraction of a drop of the DNA-containing fluid on a clean slide in one direction. After spreading, the slides were air-dried and Kodak AR-10 stripping films were then mounted. The slides were then dried and stored for 24 h at 4°C. Following this, the prepared slides were immersed in scintillation fluid (6 g PPO and 0.1 g dimethyl POPOP in 1000 ml Toluene) in the dark for 72 h at 20*(]. In complete darkness, slides were passed through xylene, grades of ethanol and distilled water allowing 5 min for each step. Slides were finally developed in a Kodak D19 developer for 6 min.
Exp CellRes 108 (1977)
