Preliminary
in both cycling and in noncycling G2 blocked mouse ear epidermal cells. Labeled mitoses again represent cycling ear epidermal cells which had passed through S in the presence of 3H-TdR were moving through G2 at the time of temperature stimulation, are released into M, and now appear as labeled mitotic figures arrested in metaphase by colcemid. The appearance of unlabeled mitoses in heat stimulated ear epidermis after 2, 4, and even 8 days of continuous labeling with 3H-TdR proves the existence of a category of epidermal cells which remain blocked in G2 for at least 8 days (or even months [6]), and which still retain their capacity to proliferate in response to an appropriate stimulus -in this case high temperature. Once again, a relatively large proportion (15 to 20 %) of heat induced epidermal cells are drawn from the noncycling G2 blocked component of the basal layer of epidermis (a component estimated to be only about 5% of the total epidermal cell population [5, 61). Although this report does not study the reasons for high temperature induced proliferation of mouse ear epidermal cells, the observation that rat plantar epidermal cells can be stimulated to enter mitosis by raising the temperature of the cage floor [7] (plantar skin is also bare of an insulating coat of hair), and the suggestion that mouse ear epidermal cells live at a temperature lower than most other cells of the body [S], indicate that induction of ear epidermal cell mitosis is a result of raising intracellular temperature. In addition to looking into such explanations, high temperature can be used as a convenient and simple method for studying cell proliferation in relation to the cell cycle, in relation to the G2 period of cycling and noncycling cells, and it can be used as a tool to further investigate the concept of inherent G 1 and G2 cell cycle blocks [l, 4, 51. Finally, the observation that high tempera-
notes
46I
ture specifically releases cycling cells in G2 and noncycling G2 blocked cells may be useful in cancer therapy-as a tool for synchronizing tumor cells, and as an agent for releasing noncycling G2 blocked tumor cells. This work was supported in part by NIH research grants AM-16060 and HD-07745.
References 1.
2. 3. 4. 5. 6. 7. 8.
Gelfant, S, Methods in cell physiology (ed D M Prescott) vol. 2, p. 359. Academic Press, New York (1966). Pilgrim, C H, Lang, W & Maurer, W, Exptl cell res 44 (1966) 129. Gelfant, S, Symp int sot cell biol 2 (1963) 229. Gelfant, S & Smith, J G, Jr, Science 178 (1972) 357. Gelfant, S & Candelas, G C, J invest derm 59 (1972) 7. Pederson, T & Gelfant, S, Exptl cell res 59 (1970) 32. Storey, W F & Leblond, C P, Ann NY acad sci (1951) 537. Sherman, F G, Quastler, H & Wimber, D R, Exptl cell res 25 (1961) 114.
Received August 19, 1974 Revised version received October 8, 1974
Synthesis of mammalian mitochondrial rRNA at low temperature B. K. WATERS, R. B. WALLACE MAN, Department of Biochemistry, versity,
Hamilton,
Ont.,
Canada
and K. B. FREEL8S
McMaster 4J9
Uni-
Summary. L cells incubated at 19°C synthesize mitochondrial rRNA but not cytosol rRNA. In addition to 16 and 13s mitochondrial rRNA. mitochondrial RNA sedimenting at higher S values was also synthesized. The processing of mitochondrial rRNA is slower at 19°C than at 37°C.
Studies on the synthesis of mitochondrial RNA were greatly facilitated when Dubin [l] found that mitochondrial RNA but not cytosol rRNA of mammalian cells in tissue culture was labelled in the presence of a low concentration of actinomycin D. Mitochondrial RNA was then not masked by cytosol rRNA. Since actinomycin D has other known effects on cellular processes Exptl
Cell Res 90 (1975)
462
Preliminary
notes
aside from inhibiting cytosol rRNA synthesis [2-51, it would be advantageous to find other conditions in which mitochondrial RNA but not cytosol rRNA could be labelled. Stevens & Amos [6] found that cytosol rRNA was not labelled in HeLa cells at 19°C. In this paper we show that under these conditions mitochondrial rRNA is labelled. This demonstrates the independence of the synthesis of mitochondrial RNA from that of cytosol rRNA. Further, labelling at 19°C might be of value in following the processing of mammalian mitochondrial RNA. Methods L cells were grown in suspension culture as described previously [7]. RNA of 3 x lo* cells in 500 ml was labelled at 19°C with [5PH] uridineat 1 ,&i/ml. Mitochondria and mitochondrial RNA were isolated as described previously [7]. The RNA was treated with 10 KZ DNase/ml at 0°C for 30 min and characterized by centrifugation in a convex sucrose-density gradient. In control cultures, cells were labelled in the presence of 1.O pugethidium bromide/ml. RNA whose synthesis was inhibited by ethidium bromide was taken as that transcribed from mitochondrial DNA.
Results In fig. lA, B and C, the results of labelling L cell RNA at 19°C for 2, 4 and 18 h respectively are shown, and in fig. 1D the result of labelling at 37°C in the presence of 0.05 pg actinomycin D/ml is also shown. From fig. lC, it can be seen that the labelling of cytosol rRNA is completely inhibited at 19°C as was also found in HeLa cells [6], but mitochondrial rRNA was labelled. The pattern of the mitochondrial rRNA was the sameas that of mitochondrial rRNA labelled at 37°C (fig. 10). The total amount of labelling of mitochondrial rRNA at 19°C was about 10 y0 of that at 37°C in the presence of 0.05 pg actinomycin D/ml. Mitochondrial RNA continued to be synthesized at 14°C at a very slow rate. Synthesis at lower temperatures was not examined. Cells returned to 37°C after 18 h at 19°C grew at a normal rate without a lag. Exptl Cell Res 90 (1975)
Fig. 1. Abscissa: fraction; ordinate: (left) mitochondrial RNA (cpm) (O-O); (right) cytoplasmic RNA 4x0 (0-O). Sucrose-density-gradient centrifugation of mitochondrial RNA. Cells were labelled as described in the Methods section, either at 19°C with no actinomycin D (A-C) or 37°C with 0.05 ,ug actinomycin D/ml. The length of time labelling was (A) 2 h, (B) 4 h, (C) 18 h and (D) 18 h. Mitochondrial RNA was isolated as described previously and centrifuged at 195 000 g,, for 12 h at 3°C on a 15-33 % (w/v) sucrose gradient in 0.1 M sodium acetate pH 6.0 using the SW 41 rotor in a Beckman L2-65B ultracentrifuge. The ethidium bromide sensitive counts, usually 80% of the total, have been plotted in each case.
The major components labelled in 18 h at 19°C were the mitochondrial 16 and 13S rRNAs. At shorter times, the 16 and 13s peaks were less pronounced in comparison with RNA of higher sedimentation value. A peak at 21s and perhaps at 25 and greater than 30s are apparent at both 2 and 4 h (fig. lA, B). Discussion Mitochondrial RNA transcription continues at 19°C as well as whatever processing of the initial transcript is necessary to yield mitochondrial RNA. It is not yet known whether the mitochondrial rRNA is incorporated into mitochondrial ribosomes. The specific synthesis of mitochondrial
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)
in both cycling and in noncycling G2 blocked mouse ear epidermal cells. Labeled mitoses again represent cycling ear epidermal cells which had passed through S in the presence of 3H-TdR were moving through G2 at the time of temperature stimulation, are released into M, and now appear as labeled mitotic figures arrested in metaphase by colcemid. The appearance of unlabeled mitoses in heat stimulated ear epidermis after 2, 4, and even 8 days of continuous labeling with 3H-TdR proves the existence of a category of epidermal cells which remain blocked in G2 for at least 8 days (or even months [6]), and which still retain their capacity to proliferate in response to an appropriate stimulus -in this case high temperature. Once again, a relatively large proportion (15 to 20 %) of heat induced epidermal cells are drawn from the noncycling G2 blocked component of the basal layer of epidermis (a component estimated to be only about 5% of the total epidermal cell population [5, 61). Although this report does not study the reasons for high temperature induced proliferation of mouse ear epidermal cells, the observation that rat plantar epidermal cells can be stimulated to enter mitosis by raising the temperature of the cage floor [7] (plantar skin is also bare of an insulating coat of hair), and the suggestion that mouse ear epidermal cells live at a temperature lower than most other cells of the body [S], indicate that induction of ear epidermal cell mitosis is a result of raising intracellular temperature. In addition to looking into such explanations, high temperature can be used as a convenient and simple method for studying cell proliferation in relation to the cell cycle, in relation to the G2 period of cycling and noncycling cells, and it can be used as a tool to further investigate the concept of inherent G 1 and G2 cell cycle blocks [l, 4, 51. Finally, the observation that high tempera-
notes
46I
ture specifically releases cycling cells in G2 and noncycling G2 blocked cells may be useful in cancer therapy-as a tool for synchronizing tumor cells, and as an agent for releasing noncycling G2 blocked tumor cells. This work was supported in part by NIH research grants AM-16060 and HD-07745.
References 1.
2. 3. 4. 5. 6. 7. 8.
Gelfant, S, Methods in cell physiology (ed D M Prescott) vol. 2, p. 359. Academic Press, New York (1966). Pilgrim, C H, Lang, W & Maurer, W, Exptl cell res 44 (1966) 129. Gelfant, S, Symp int sot cell biol 2 (1963) 229. Gelfant, S & Smith, J G, Jr, Science 178 (1972) 357. Gelfant, S & Candelas, G C, J invest derm 59 (1972) 7. Pederson, T & Gelfant, S, Exptl cell res 59 (1970) 32. Storey, W F & Leblond, C P, Ann NY acad sci (1951) 537. Sherman, F G, Quastler, H & Wimber, D R, Exptl cell res 25 (1961) 114.
Received August 19, 1974 Revised version received October 8, 1974
Synthesis of mammalian mitochondrial rRNA at low temperature B. K. WATERS, R. B. WALLACE MAN, Department of Biochemistry, versity,
Hamilton,
Ont.,
Canada
and K. B. FREEL8S
McMaster 4J9
Uni-
Summary. L cells incubated at 19°C synthesize mitochondrial rRNA but not cytosol rRNA. In addition to 16 and 13s mitochondrial rRNA. mitochondrial RNA sedimenting at higher S values was also synthesized. The processing of mitochondrial rRNA is slower at 19°C than at 37°C.
Studies on the synthesis of mitochondrial RNA were greatly facilitated when Dubin [l] found that mitochondrial RNA but not cytosol rRNA of mammalian cells in tissue culture was labelled in the presence of a low concentration of actinomycin D. Mitochondrial RNA was then not masked by cytosol rRNA. Since actinomycin D has other known effects on cellular processes Exptl
Cell Res 90 (1975)
462
Preliminary
notes
aside from inhibiting cytosol rRNA synthesis [2-51, it would be advantageous to find other conditions in which mitochondrial RNA but not cytosol rRNA could be labelled. Stevens & Amos [6] found that cytosol rRNA was not labelled in HeLa cells at 19°C. In this paper we show that under these conditions mitochondrial rRNA is labelled. This demonstrates the independence of the synthesis of mitochondrial RNA from that of cytosol rRNA. Further, labelling at 19°C might be of value in following the processing of mammalian mitochondrial RNA. Methods L cells were grown in suspension culture as described previously [7]. RNA of 3 x lo* cells in 500 ml was labelled at 19°C with [5PH] uridineat 1 ,&i/ml. Mitochondria and mitochondrial RNA were isolated as described previously [7]. The RNA was treated with 10 KZ DNase/ml at 0°C for 30 min and characterized by centrifugation in a convex sucrose-density gradient. In control cultures, cells were labelled in the presence of 1.O pugethidium bromide/ml. RNA whose synthesis was inhibited by ethidium bromide was taken as that transcribed from mitochondrial DNA.
Results In fig. lA, B and C, the results of labelling L cell RNA at 19°C for 2, 4 and 18 h respectively are shown, and in fig. 1D the result of labelling at 37°C in the presence of 0.05 pg actinomycin D/ml is also shown. From fig. lC, it can be seen that the labelling of cytosol rRNA is completely inhibited at 19°C as was also found in HeLa cells [6], but mitochondrial rRNA was labelled. The pattern of the mitochondrial rRNA was the sameas that of mitochondrial rRNA labelled at 37°C (fig. 10). The total amount of labelling of mitochondrial rRNA at 19°C was about 10 y0 of that at 37°C in the presence of 0.05 pg actinomycin D/ml. Mitochondrial RNA continued to be synthesized at 14°C at a very slow rate. Synthesis at lower temperatures was not examined. Cells returned to 37°C after 18 h at 19°C grew at a normal rate without a lag. Exptl Cell Res 90 (1975)
Fig. 1. Abscissa: fraction; ordinate: (left) mitochondrial RNA (cpm) (O-O); (right) cytoplasmic RNA 4x0 (0-O). Sucrose-density-gradient centrifugation of mitochondrial RNA. Cells were labelled as described in the Methods section, either at 19°C with no actinomycin D (A-C) or 37°C with 0.05 ,ug actinomycin D/ml. The length of time labelling was (A) 2 h, (B) 4 h, (C) 18 h and (D) 18 h. Mitochondrial RNA was isolated as described previously and centrifuged at 195 000 g,, for 12 h at 3°C on a 15-33 % (w/v) sucrose gradient in 0.1 M sodium acetate pH 6.0 using the SW 41 rotor in a Beckman L2-65B ultracentrifuge. The ethidium bromide sensitive counts, usually 80% of the total, have been plotted in each case.
The major components labelled in 18 h at 19°C were the mitochondrial 16 and 13S rRNAs. At shorter times, the 16 and 13s peaks were less pronounced in comparison with RNA of higher sedimentation value. A peak at 21s and perhaps at 25 and greater than 30s are apparent at both 2 and 4 h (fig. lA, B). Discussion Mitochondrial RNA transcription continues at 19°C as well as whatever processing of the initial transcript is necessary to yield mitochondrial RNA. It is not yet known whether the mitochondrial rRNA is incorporated into mitochondrial ribosomes. The specific synthesis of mitochondrial
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)
