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Localization of ribosomal cistrons in metaphase chromosomes of Vicia faba (L.)
Preliminary
rRNA at 19°C demonstrates that there is not tight coordination of the syntheses of mitochondrial rRNA and cytosol rRNA. This could be concluded from the earlier observation of specific syntheses using antibiotics [I], but the present demonstration is closer to a physiological separation. The specificity of labelling at 19°C should permit studies of the metabolism of mitochondrial rRNA in the absence of antibiotics. The rate of synthesis and of processing of mitochondrial rRNA was slow at 19°C and even by 4 h about half of the mitochondrial RNA sedimented at greater than I8 S. The turnover of the latter RNA is much faster at 37°C [8-IO]. By labelling at 19°C the processing of mitochondrial RNA might be followed more readily because of its slower turnover. As reported previously [ll-141 a 21s peak is seen at short times of labelling. In addition, there is a peak at about 33 S as previously reported by Storrie & Attardi [I 11. The relationship of these species to the mitochondrial rRNA is not known at present. This work was supported by the MRC of Canada, grant MT-1940. One of us (R. B. W.) is the holder of an MRC Studentship.
References 1. Dubin. D T. Biochem bionhvs . _ res commun 29 (1967) ‘655. ’ 2. Sawicki. S & Godman. , G. J cell biol 50 (1971) ~ , 746. ’ 3. Soeiro, R & Amos, H, Biochim biophys acta 129 (I 966) 406. 4. Honig, G R, Smulson, M E & Rabinovitz, M, Biochim biophys acta 129 (1966) 576. 5. Goldstein, F S & Penman, S, J mol biol 80 (1973) 243. 6. Stevens, R H & Amos, H, J cell biol 50 (1971) 818. 7. Bartoov. B. Mitra. R S & Freeman. , K B. Biothem j 120’ (1970)‘455. 8. Attardi. B & Attardi. , G., J mol biol 55 (1971) . 231. ’ 9. Aloni, Y & Attardi, G, J mol biol 70 (1972) 375. 10. Dubin, D T, J biol them 247 (1972) 2662. 11. Storrie, B & Attardi, G, J mol biol 71 (1972) 177. 12. Dubin, D T & Czaplicki, S M, Biochim biophys acta 224 (1970) 663.
notes
463
13. - Ibid 238 (1971) 491. 14. Fukamachi, S, Bartoov, B & Freeman, K B, Biochem j 128 (1972) 299. Received September 13, 1974
Localization of ribosomal cistrons in metaphase chromosomes of Vieie fuba (L.) W. SCHEUERMANN’ and MARITA KNALMANN, Znstitut fir Strahlenbotanik der GeselIschaft fiir Strahlen und Utnweltforschung m.b.H., Miinchen, Hannover, and Znstitut fCr Biophysik der Technischen UniversitGt Hannover, BRD Summary. Tritiated ribosomal RNA (rRNA) was prepared from the roots of Vicia faba after incubation in 3H-uridine. Separation of the nucleic acids by MAK chromatography yielded fractions of specific activity of 4-5 x lo5 dpm/pg. 4+ 5S, 18s and 25s RNA fractions were used for cytological hybridization on squash preparations of Vicia faba root tip meristems. Autoradiographs of the 18s and 25s RNA preparations exhibited a clear labelling in the secondary constriction of the satellite (SAT) chromosomes after exposition times of 28 weeks.
RNA-DNA hybridization at the cytological level can be a sensitive tool for the localization of specific genes. This has been shown for ribosomal cistrons of several species [14]. Localization of specific genes is in principle possible by autoradiographic techniques if the multiplicity of the genes concerned and the specific activity of the RNA fraction used for the hybridization are sufficiently high. Cytological rRNA-DNA hybridizations were first detected with 18 S and 25s rRNA in oocytes of amphibia [2] and later also in giant chromosomes of several Diptera (e.g. Drosophila hydei, Sciara coprophila, Rhynchosciara hollaenderi [9]; Drosophila melanogaster [ 131)as well as Phaseoluscoccineus[ 11.
The only cytological rRNA-DNA hybridization in diploid nuclei of which we are presently aware was reported in human chromosomes (spec. act. of rRNA: 3.5-5 x lo6 dpm/ 1 Address: Arbeitsgruppe Molekulare Pflanzenzytologie, Ruhr-Universitlt, D-463 Bochum, Postfach 2148, Geblude ND 05/593. Exptl Cell Res 90 (1975)
464
Preliminary
notes Results and Discussion
Table 1. RNA fraction 4t5S 18s 25s
Concentration bg/ml]a
1 Specific activity
tdpm/~d
0.7
5x106
::‘:
4x 105
4x 105
a 1 OD unit/ml -2 30 pg RNA/ml.
pug [4]), in Macaca mulatta (spec. act. of rRNA: 5 x lo6 dpm/,ug [5] and in Xenopus laeuis (spec. act. of the 5s rRNA: 2.8 x IO6
dpm/pg P 11). We know of no corresponding detection in diploid nuclei of somatic plant tissue. Brady & Clutter [l] in fact did not find hybridization in Phaseolusdiploid nuclei (only in the giant chromosomes) using a specific activity of 3.6 x lo6 dpm/pg. A localization of ribosoma1 cistrons using cytological hybridization should be possible, since Cullis & Davies [3] reported that in plant material studied to date, the number of ribosomal cistrons varies between 1 580 and 27 000 copies. Similar values were found in 4 species of the genus Vicia (V. faba: 9 500 copies [7]). Material and Methods Seeds of Vicia faba (L.) var. minor were germinated according to the method described by Scheuermann & Tlgder [12]. Seedlings with roots of a mean length of about 30 mm were incubated in a well aerated 3Huridine solution (200 ,&i/ml tap water; 51 Ci/mM uridine-5.6-3H. 24 h). The uH was adiusted to 5.5 with a 7 % SGrensen ‘phosphate buffer. The extraction of the nucleic acids was carried out using a modified phenol cresol method [6]. The nucleic acids were senarated in a methvlated albumin kieselguhr (MAK) column according-to Mandell & Hershey [8]. From the collected fractions, which were identified by UV-absorption measurements, those containing 4 + 5 S, 18 S and 25 S RNA were selected. and their- nucleic acid concentrations and activities were determined. The cytological rRNA-DNA hybridization was performed by following closely the technique described by Pardue & Gall [lo]. The squash preparations were coated with a K 5-film emulsion (Ilford Ltd). Giemsa staining was performed after development of the emulsion. The exposure time of the microautoradiographic preparations was at least 28 weeks. Exprl
Cell Res 90 (1975)
The concentration and specific activity of the RNA fractions used for the hybridization are summarized in table 1. The cytological evaluation yielded the following results: (1) Hybridization with 18s and 25s fractions results in clear labelling of the secondary constriction in the SAT-chromosome (fig. 1) and in the formation of certain ‘hot spots’ over the interphase nuclei. (2) The 4+5S fraction does not yield a correspondingly clear labelling. However, a weak labelling of the secondary constriction cannot be excluded. As expected, the results show an obvious cytological hybridization of the 18 S and 25 S fractions in the secondary constriction of the SAT-chromosomes. It is known that the nucleolus organizer region is situated in this area. The weak labelling of the secondary constriction of the SAT-chromosome when using the 4+ 5 fraction has to be interpreted with extreme caution, since the investigations of Wimber & Steffensen [13], among others, show that MAK-separated 5 S RNA fractions
Fig. 1. Microautoradiograph of cytological rRNADNA (25s) hybridization showing labelling in the secondary constriction of the Giemsa-stained SATchromosomes.
Preliminary
contain impurities of other nucleic acids. These may lead to hybridization and labelling of gene sites of high molecular weight RNA. To our knowledge, data concerning the multiplicity of 5s ribosomal cistrons in Vicia faba do not exist at present. In order to obtain a clear response to hybridization of the RNA fractions on the chromosomes and to ensure an unequivocal interpretation, further investigations using other fractionating methods (e.g. polyacrylamide gel electrophoresis), higher specific activities and the addition of cold RNA in the case of the 4 + 5S RNA fractions are being undertaken. The skilful technical assistance of Mrs U. Lindemann (GSF) and Mrs I. Orozco (GSF) is gratefully acknowledged. We are indebted Mr C. Bunnenberg for translating the manuscript. The investigations were partly supported by grants of the Deutsche Forschungsgemeinschaft (Sche 95/5). This work is dedicated to Professor A. Schraub on the occasion of his 65th birthday (December 12, 1974).
References 1.
2. 3. 4. 5. 6. 7. 8. 9. IO.
11. 12. I3 14
Brady, T & Clutter, M E, J cell biol 53 (1972) 827. Brown, D D & Dawid, I B, Science 160 (1968) 272. Cullis, C & Davies, D R, Chromosoma (Eerl.) 46 (1974) 23. Henderson, A S, Warburton, D & Atwood, K C, Proc natl acad sci US 69 (1972) 3394. - Chromosoma (Berl.) 4‘4 (1954) 367. Kirby, K S, Biochem j 96 (1964) 266. Maher, B A & Fox, D P, Nature new biol 245 (1973) 170. Mandell, D J & Hershey, A D, Anal biochem 1 (1960) 66. Pardue, M L, Gerbi, S A, Eckhardt, R A & Gall, J G, Chromosoma (Berl.) 29 (1970) 268. Pardue, M L & Gall, J G, Methods in enzymology (ed E L Grossman & K Moldave) vol. XXI; Part D, p. 470. Academic Press, New York (1971). Pardue, M L, Brown, D D & Birnstiel, M L, Chromosoma (Berl.) 42 (I 973) 191. Scheuermann, W & Tlgder, K, Rad res 36 (1968) 483. Wimber, D E & Steffensen, D M, Science 170 (1970) 639. Tschermak-Woess, E, Fortschritte der Botanik (ed H EII-nberg, K Esser, H Merxmiiller, E Schnepf & H Ziegler) vol. 35, p. 11. Springer Verlag, Berlin-Heidelberg-New York, 1973.
Received September 16, 1974 30- 741804
notes
465
Concanavalin A inhibits the dispersionof the cortical granule contents of sand dollar eggs V. D. VACQUIER and D. S. O’DELL, Department of Zoology, University of California, Davis, Calif. 9S616, USA, and Department of Zoology, University College, London WClE GBT, UK Summary. Dendraster excentricus eggs fertilized in Con A (10 Pg/ml) elevate vitelline layers and expel cortical granule contents into the perivitelline space, The granule material does not disperse but remains composed as discrete spheres. The elevated vitelline layer remains thin and weak. It is not a true fertilization membrane because it lacks the structural material supplied by the granules.
The cortical granules of echinoderm eggs have been the subject of many investigations [I]. Recently, attention has focused on the contents of the granules and the functions they serve in the fertilization process [2-51. While studying the effect of Concanavalin A (ConA) on fertilization, we found that it inhibits the dispersion of the dense structural material released from the cortical granules of sand dollar eggs. Materials and Methods Gametes of the California sand dollar Dendraster excentricus were spawned into sea water by the injection of 0.5 M KC]. Concanavalin A (MilesYeda) was dissolved in sea water to a final concentration of 10 /Lg/ml. Eggs were exposed to Con A sea water 10 min prior to insemination. Twenty minutes after fertilization (23°C) the eggs were fixed for 2 h in 2 % glutaraldehyde in 80 ?,-sea water. They were washed in sea water 1 h and then post-fixed 1 h in 1 % 0~0, in sea water. Gradual hehydration was accomplished in a graded series of ethanols, followed by propylene oxide and embedding in epon. This sections were stained with lead citrate and examined in the electron microscope.
Results and Discussion
1. Normal fertilization. Cortical granules of unfertilized eggs are shown in fig. 1. The granules are composed of a dense inner mass surrounded by a less dense fibrous material. Upon fertilization the granules fuse with the egg membrane and extrude their contents into the perivitelline space which forms Exptl
Ccl1 Rcs 90 (1975)
rRNA at 19°C demonstrates that there is not tight coordination of the syntheses of mitochondrial rRNA and cytosol rRNA. This could be concluded from the earlier observation of specific syntheses using antibiotics [I], but the present demonstration is closer to a physiological separation. The specificity of labelling at 19°C should permit studies of the metabolism of mitochondrial rRNA in the absence of antibiotics. The rate of synthesis and of processing of mitochondrial rRNA was slow at 19°C and even by 4 h about half of the mitochondrial RNA sedimented at greater than I8 S. The turnover of the latter RNA is much faster at 37°C [8-IO]. By labelling at 19°C the processing of mitochondrial RNA might be followed more readily because of its slower turnover. As reported previously [ll-141 a 21s peak is seen at short times of labelling. In addition, there is a peak at about 33 S as previously reported by Storrie & Attardi [I 11. The relationship of these species to the mitochondrial rRNA is not known at present. This work was supported by the MRC of Canada, grant MT-1940. One of us (R. B. W.) is the holder of an MRC Studentship.
References 1. Dubin. D T. Biochem bionhvs . _ res commun 29 (1967) ‘655. ’ 2. Sawicki. S & Godman. , G. J cell biol 50 (1971) ~ , 746. ’ 3. Soeiro, R & Amos, H, Biochim biophys acta 129 (I 966) 406. 4. Honig, G R, Smulson, M E & Rabinovitz, M, Biochim biophys acta 129 (1966) 576. 5. Goldstein, F S & Penman, S, J mol biol 80 (1973) 243. 6. Stevens, R H & Amos, H, J cell biol 50 (1971) 818. 7. Bartoov. B. Mitra. R S & Freeman. , K B. Biothem j 120’ (1970)‘455. 8. Attardi. B & Attardi. , G., J mol biol 55 (1971) . 231. ’ 9. Aloni, Y & Attardi, G, J mol biol 70 (1972) 375. 10. Dubin, D T, J biol them 247 (1972) 2662. 11. Storrie, B & Attardi, G, J mol biol 71 (1972) 177. 12. Dubin, D T & Czaplicki, S M, Biochim biophys acta 224 (1970) 663.
notes
463
13. - Ibid 238 (1971) 491. 14. Fukamachi, S, Bartoov, B & Freeman, K B, Biochem j 128 (1972) 299. Received September 13, 1974
Localization of ribosomal cistrons in metaphase chromosomes of Vieie fuba (L.) W. SCHEUERMANN’ and MARITA KNALMANN, Znstitut fir Strahlenbotanik der GeselIschaft fiir Strahlen und Utnweltforschung m.b.H., Miinchen, Hannover, and Znstitut fCr Biophysik der Technischen UniversitGt Hannover, BRD Summary. Tritiated ribosomal RNA (rRNA) was prepared from the roots of Vicia faba after incubation in 3H-uridine. Separation of the nucleic acids by MAK chromatography yielded fractions of specific activity of 4-5 x lo5 dpm/pg. 4+ 5S, 18s and 25s RNA fractions were used for cytological hybridization on squash preparations of Vicia faba root tip meristems. Autoradiographs of the 18s and 25s RNA preparations exhibited a clear labelling in the secondary constriction of the satellite (SAT) chromosomes after exposition times of 28 weeks.
RNA-DNA hybridization at the cytological level can be a sensitive tool for the localization of specific genes. This has been shown for ribosomal cistrons of several species [14]. Localization of specific genes is in principle possible by autoradiographic techniques if the multiplicity of the genes concerned and the specific activity of the RNA fraction used for the hybridization are sufficiently high. Cytological rRNA-DNA hybridizations were first detected with 18 S and 25s rRNA in oocytes of amphibia [2] and later also in giant chromosomes of several Diptera (e.g. Drosophila hydei, Sciara coprophila, Rhynchosciara hollaenderi [9]; Drosophila melanogaster [ 131)as well as Phaseoluscoccineus[ 11.
The only cytological rRNA-DNA hybridization in diploid nuclei of which we are presently aware was reported in human chromosomes (spec. act. of rRNA: 3.5-5 x lo6 dpm/ 1 Address: Arbeitsgruppe Molekulare Pflanzenzytologie, Ruhr-Universitlt, D-463 Bochum, Postfach 2148, Geblude ND 05/593. Exptl Cell Res 90 (1975)
464
Preliminary
notes Results and Discussion
Table 1. RNA fraction 4t5S 18s 25s
Concentration bg/ml]a
1 Specific activity
tdpm/~d
0.7
5x106
::‘:
4x 105
4x 105
a 1 OD unit/ml -2 30 pg RNA/ml.
pug [4]), in Macaca mulatta (spec. act. of rRNA: 5 x lo6 dpm/,ug [5] and in Xenopus laeuis (spec. act. of the 5s rRNA: 2.8 x IO6
dpm/pg P 11). We know of no corresponding detection in diploid nuclei of somatic plant tissue. Brady & Clutter [l] in fact did not find hybridization in Phaseolusdiploid nuclei (only in the giant chromosomes) using a specific activity of 3.6 x lo6 dpm/pg. A localization of ribosoma1 cistrons using cytological hybridization should be possible, since Cullis & Davies [3] reported that in plant material studied to date, the number of ribosomal cistrons varies between 1 580 and 27 000 copies. Similar values were found in 4 species of the genus Vicia (V. faba: 9 500 copies [7]). Material and Methods Seeds of Vicia faba (L.) var. minor were germinated according to the method described by Scheuermann & Tlgder [12]. Seedlings with roots of a mean length of about 30 mm were incubated in a well aerated 3Huridine solution (200 ,&i/ml tap water; 51 Ci/mM uridine-5.6-3H. 24 h). The uH was adiusted to 5.5 with a 7 % SGrensen ‘phosphate buffer. The extraction of the nucleic acids was carried out using a modified phenol cresol method [6]. The nucleic acids were senarated in a methvlated albumin kieselguhr (MAK) column according-to Mandell & Hershey [8]. From the collected fractions, which were identified by UV-absorption measurements, those containing 4 + 5 S, 18 S and 25 S RNA were selected. and their- nucleic acid concentrations and activities were determined. The cytological rRNA-DNA hybridization was performed by following closely the technique described by Pardue & Gall [lo]. The squash preparations were coated with a K 5-film emulsion (Ilford Ltd). Giemsa staining was performed after development of the emulsion. The exposure time of the microautoradiographic preparations was at least 28 weeks. Exprl
Cell Res 90 (1975)
The concentration and specific activity of the RNA fractions used for the hybridization are summarized in table 1. The cytological evaluation yielded the following results: (1) Hybridization with 18s and 25s fractions results in clear labelling of the secondary constriction in the SAT-chromosome (fig. 1) and in the formation of certain ‘hot spots’ over the interphase nuclei. (2) The 4+5S fraction does not yield a correspondingly clear labelling. However, a weak labelling of the secondary constriction cannot be excluded. As expected, the results show an obvious cytological hybridization of the 18 S and 25 S fractions in the secondary constriction of the SAT-chromosomes. It is known that the nucleolus organizer region is situated in this area. The weak labelling of the secondary constriction of the SAT-chromosome when using the 4+ 5 fraction has to be interpreted with extreme caution, since the investigations of Wimber & Steffensen [13], among others, show that MAK-separated 5 S RNA fractions
Fig. 1. Microautoradiograph of cytological rRNADNA (25s) hybridization showing labelling in the secondary constriction of the Giemsa-stained SATchromosomes.
Preliminary
contain impurities of other nucleic acids. These may lead to hybridization and labelling of gene sites of high molecular weight RNA. To our knowledge, data concerning the multiplicity of 5s ribosomal cistrons in Vicia faba do not exist at present. In order to obtain a clear response to hybridization of the RNA fractions on the chromosomes and to ensure an unequivocal interpretation, further investigations using other fractionating methods (e.g. polyacrylamide gel electrophoresis), higher specific activities and the addition of cold RNA in the case of the 4 + 5S RNA fractions are being undertaken. The skilful technical assistance of Mrs U. Lindemann (GSF) and Mrs I. Orozco (GSF) is gratefully acknowledged. We are indebted Mr C. Bunnenberg for translating the manuscript. The investigations were partly supported by grants of the Deutsche Forschungsgemeinschaft (Sche 95/5). This work is dedicated to Professor A. Schraub on the occasion of his 65th birthday (December 12, 1974).
References 1.
2. 3. 4. 5. 6. 7. 8. 9. IO.
11. 12. I3 14
Brady, T & Clutter, M E, J cell biol 53 (1972) 827. Brown, D D & Dawid, I B, Science 160 (1968) 272. Cullis, C & Davies, D R, Chromosoma (Eerl.) 46 (1974) 23. Henderson, A S, Warburton, D & Atwood, K C, Proc natl acad sci US 69 (1972) 3394. - Chromosoma (Berl.) 4‘4 (1954) 367. Kirby, K S, Biochem j 96 (1964) 266. Maher, B A & Fox, D P, Nature new biol 245 (1973) 170. Mandell, D J & Hershey, A D, Anal biochem 1 (1960) 66. Pardue, M L, Gerbi, S A, Eckhardt, R A & Gall, J G, Chromosoma (Berl.) 29 (1970) 268. Pardue, M L & Gall, J G, Methods in enzymology (ed E L Grossman & K Moldave) vol. XXI; Part D, p. 470. Academic Press, New York (1971). Pardue, M L, Brown, D D & Birnstiel, M L, Chromosoma (Berl.) 42 (I 973) 191. Scheuermann, W & Tlgder, K, Rad res 36 (1968) 483. Wimber, D E & Steffensen, D M, Science 170 (1970) 639. Tschermak-Woess, E, Fortschritte der Botanik (ed H EII-nberg, K Esser, H Merxmiiller, E Schnepf & H Ziegler) vol. 35, p. 11. Springer Verlag, Berlin-Heidelberg-New York, 1973.
Received September 16, 1974 30- 741804
notes
465
Concanavalin A inhibits the dispersionof the cortical granule contents of sand dollar eggs V. D. VACQUIER and D. S. O’DELL, Department of Zoology, University of California, Davis, Calif. 9S616, USA, and Department of Zoology, University College, London WClE GBT, UK Summary. Dendraster excentricus eggs fertilized in Con A (10 Pg/ml) elevate vitelline layers and expel cortical granule contents into the perivitelline space, The granule material does not disperse but remains composed as discrete spheres. The elevated vitelline layer remains thin and weak. It is not a true fertilization membrane because it lacks the structural material supplied by the granules.
The cortical granules of echinoderm eggs have been the subject of many investigations [I]. Recently, attention has focused on the contents of the granules and the functions they serve in the fertilization process [2-51. While studying the effect of Concanavalin A (ConA) on fertilization, we found that it inhibits the dispersion of the dense structural material released from the cortical granules of sand dollar eggs. Materials and Methods Gametes of the California sand dollar Dendraster excentricus were spawned into sea water by the injection of 0.5 M KC]. Concanavalin A (MilesYeda) was dissolved in sea water to a final concentration of 10 /Lg/ml. Eggs were exposed to Con A sea water 10 min prior to insemination. Twenty minutes after fertilization (23°C) the eggs were fixed for 2 h in 2 % glutaraldehyde in 80 ?,-sea water. They were washed in sea water 1 h and then post-fixed 1 h in 1 % 0~0, in sea water. Gradual hehydration was accomplished in a graded series of ethanols, followed by propylene oxide and embedding in epon. This sections were stained with lead citrate and examined in the electron microscope.
Results and Discussion
1. Normal fertilization. Cortical granules of unfertilized eggs are shown in fig. 1. The granules are composed of a dense inner mass surrounded by a less dense fibrous material. Upon fertilization the granules fuse with the egg membrane and extrude their contents into the perivitelline space which forms Exptl
Ccl1 Rcs 90 (1975)