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Circadian rhythm in size and 3H-uridine incorporation of single puffs of Drosophila salivary glands in vitro
436
Preliminary notes
9. Naha, P M, Nature 223 (1969) 1380. 10. Meiss, H K &Basilico, C, Nature new biol 239 (1972) 66. Received August 1, 1974 Revised version received September 6, 1974
Circadian rhythm in size and 3H-uridine incorporation of single puffs of Drosophila salivary glands in vitro G. NAGEL and L. RENSING, I. Zoolog. I~nstitut der Universitiit
Giittingen, D 34 Giittingen, BRD
Summary. Salivary glands of late third instar larvae of Drosophila melanogaster were kept in a chemically defined medium at 25”C, under a light-dark cycle (light time from 9-21 h). Puff size and SH-uridine incorooration of various single uuffs changed sianificantiy during a 24 h period invitro. Maxima occurred at 8 h and 21 h. minima at 24 h and 11 h. In the unpuffed region’ 3A6-3C2, only one maximum of 3H-uridine incorporation-at 17 h-was observed.
Circadian rhythms have been described in cell cultures of various tissues [l, lo], and in isolated DrosophiZa salivary glands in particular [24]. From this evidence, it can be concluded that each cell generates its own circadian rhythm-the basic mechanism of which is still unknown, however. Even if nuclear activities may not contribute to this mechanism [30, 311,it seemsto be important to test sequential or synchronous changes of various chromosome regions over 24 h. The results should then be discussedas to possible control mechanisms of gene activity that could be deduced from different models of circadian oscillators [8, 20, 321. Materials and Methods Salivary glands of late third instar larvae of Drosophila Meigen (115 h after egg laying at 25°C [4,22, 231)were prepared and then kept in an artificial medium [18, 241 under a 12:12 h light-dark cycle (light time from 9-21 h) at 25°C. Chromosome regions and puffs were localized in squash preparations with reference to the chromosome maps of Bridges & Brehme [5] and photographs of Ashburner [2-4].
melanogaster
Puff size. Measurements were made after squashing and orcein staining of cells and chromosomes [19] Exptl
Cell Res 89 (1974)
taking the width of the puffed region and of a reference band as measures to calculate the puff quotient (PQ, [26]). A certain puff size at a certain time of day reoresents the mean of 20 values from 5-8 experiments-at different days; each of the 20 values is the mean of 5 PQ measurements in a single gland. The puff size is plotted together with the threefold standard error. Autoradiography. Glands were incubated with SHuridine 1 h before squashing (10 ,&i/ml medium). For each puff at each time of day 30 autoradiographs were evaluated. The autoradiographs for each time of day were made during 5-8 series of experiments at different days. Grains were counted by use of a microscope. Because of the different background and the different overall incorporation of the chromosomes in single preparations, a correcting factor had to be introduced. The background of each slide was determined by counting the &ains of a defined area close to the chromosomes, whereas the incorporation rate of the chromosomes was estimated by measuring the grain number of the region 58A-60F. This was done five times for each slide and the mean values were calculated. For a series of experiments of a certain time of day. a mean value for background and general incorp&ation level was then caliulated from the individual means. The deviation of the background of a single slide from the mean value of an experimental series at a certain time of day and the deviation of the individual chromosome incorporation from the mean incorporation were used as correcting factors. The corrected grain number of a puff area was then divided by the diameter of a reference band to account for differences in polyteny and squashing (autoradiographic quotient, AQ). The incorporation into a certain puff at a certain time of day is plotted together with the confidence intervals (ca 95 % [33]).
Results Puff size. In different chromosomal regions
bimodal rhythmic changes in puff size are registered. These changes are significant in the caseof the largest puffs 23E, 50CD (fig. I), 74EF/75B (fig. 2), 93D. Other puffs such as 47A/BC, 58BC/CD (fig. l), 22E, 62E, 68C, 78D, 82F, 83E, and 85F show the same rhythmic pattern, the level of significance being lower in these cases.The puffed regions 50CD, 74EF, 75B and 58BC/CD were chosen as examples to represent rhythmic changes measured in the other puffed regions [18]. Maxima of puff size occur at 8 h shortly before light-on and at 21 h at the time of lightoff. The 8 h values were calculated from
Preliminary notes
437
experiments made after 24 and 48 h of incubation in the medium. Incorporation of 3H-uridine. The incorporation
of 3H-uridine was measured in the following chromosome regions: 3A6-3C2, the genetic locus for rhythm mutants [14], 58A-60F as an estimate of the overall incorporation of the chromosomes, 2B5-6/13-17; 47A, 47BC, 50CD, 58BC/DE, 63BC, 66E, 67F, 74EF/75B, 93D, 85F, and the nucleolus. Maxima of 3H-uridine incorporation in most casesappear shortly before the maxima of puff size (figs 1, 2). In the region 3A6-3C2 on the X chromosome, no puffing was observed; the 3H-uridine incorporation, however, changes
125
I
i k
--750 ,ocI- *
k---q-; T*O” ,J, :m ,’ ’ __--- d I Jr -41 ‘,r4- I
.. v /’ 9’ 1
Fig. 2. Abscissa: time of day, partly repeated in order to give a clear picture of the rhythmic pattern; ordinate: (left) AQ, autoradiographic quotient; for explanation see Methods; (right) PQ, puff quotient; for explanation, see Methods. Puff size (- ) and 3H-uridine incorporation (-) of two puffed chromosomal regions on the third chromosome.
Fig. I. Abscissa: time of day, partly repeated in order to give a clear picture of the rhythmic pattern; ordinate: (left) AQ, autoradiographic quotient; for explanation see Methods; (right) PQ, puff quotient; for explanation see Methods. Puff size (---) and SH-uridine incorporation (&-) of two puffed chromosomal regions on the second chromosome.
during 24 h. This rhythmicity might be explained by a short adsorption of heterogeneousRNA from other parts of the genome to this site or by a low synthetic activity of this region. It may be of interest that the maximum of incorporation seems to occur at 17 h whereas another unpuffed region (58A-60F) doesnot show significant rhythmic changes. The nucleolar 3H-uridine incorporation parallels the rhythmic pattern of the puffs. An observation should be mentioned concerning differences in the rhythmic pattern between males and females that had first been described by Rensing et al. [271 in the case of oxygen consumption: in the female Exptl Cell Res 89 (1974)
438
Preliminary notes
oscillator have been considered [6, 251: conformations of membrane proteins together with ion fluxes and ion pumps are elements of one model [20], energy transductions between cell compartments are essential functions of another model [32]. A metabolic Discussion system with coordinative metabolites, such Circadian rhythms of transcription have been as for example cyclic nucleotides, may also reported for rat and mouse liver [7, 9, 121, function as a cellular oscillator. Evidence of influences on transcription can be provided and have been shown to exist also in Drosophila salivary glands in vivo [21], in algae for all three possibilities [17, 25, 281. It [34], and in other cells and tissues. In rat cannot be decided at present, however, which of these models is actually realized. liver the histone and non-histone protein content, the 3H-lysine incorporation into References these proteins [29] and the phosphorylation 1. Andrews, R V & Folk, G E Jr, Comp biochem of nuclear proteins [lq seem to be correlated physiol 11 (1964) 393. with the transcriptional rhythmicity. Further2. Ashburner, M, Chromosoma 21 (1967) 398. 3. - Ibid 31 (1970) 356. more, the activity of protein kinases [15] 4. - Developmental studies on giant chromosomes and the permeability of nuclear membranes (ed W Beermann). Snrinaer-Verlag. Berlin. Heidelberg, New York (1972): -’ [13] have been shown to change during 24 h. 5. Bridges, C B & Brehme, K S, Carnegie inst A major point of interest is the question Wash pub1 552 (1944) 256. 6. Btinning, E, The physiological clock. Springerwhich control mechanism is responsible for Verlag, New York (1973). the rhythmicity of transcription. This question I. Daring, R & Rensing, L, Comp biochem physiol 45B (1973) 285. has to, be discussed from the point of view 8. Ehret, C & Trucco, E, J theor biol 15 (1967) 240. of a basic circadian oscillator within the cell. 9. Glasser, S R & Spelsberg, T C, Biochem biophys res commun 47 (1972) 951. Theoretically, transcription could be a neces10. Hardeland, R, J interdiscip cycle res 3 (1972) 109. sary part of the oscillator as has been pro11. Jacob, F & Monod, J, Cytodifferentiation and macromolecular svnthesis. Academic Press, New posed more generally by Jacob & Monod York, London (1963). [1 l] and has been specifically worked out 12. Jardetzkv. C D. Barnum, C P & Halberg, F, Am j physioii87 (1956) 608.. in the case of a circadian oscillator by Ehret 13. Kittlick, P D, Exptl path01 4 (1970) 143. & Trucco [8]. The latter model assumed a 14. Konopka, R J & Benzer, S, Proc natl acad sci US 68 (1971) 2112. sequential gene transcription, each gene being Langner, R. Unpublished results. coupled to the next by secondary gene 15. 16. Letnansky, K & Reisinger, L, Biochem biophys res commun 49 (1971) 312. products giving rise to a 24 h cycle. From 17. Lezzi, M & Robert,‘M, Developmental studies the above-described findings, this model on giant chromosomes (ed W Beermann). Springer-Verlag, Berlin, Heidelberg, New York is not likely to be realized in Drosophila, since all puffs seem to be in phase with each 18. (1972). Nagel, G. In preparation. other. Since Acetabularia shows circadian 19. Nicoletti, B, Drosophila inf serv 33 (1959) 181. Njus, D, Sulzmann, F M & Hastings, J W, rhythms even without nucleus [30, 311, it 20. Nature 248 (1974) 116. can be concluded that nuclear transcription 21. Probeck, H D & Rensing, L, Cell differentiation 2 (1974) 337. is not an element of the basic mechanism but 22. I&sing, -L, Z vergl physiol 53 (1966) 62. is rather controlled by an oscillator that is 23. - Z Zellforsch 74 (1966) 539. 24. - J insect physiol 15 (1969) 2285. localized in the cytoplasm. Biologische Rhythmen und Regulation. Fischer 25. &lag Stuttgart (1973). Various mechanisms of the cytoplasmic chromosomes, the evening maximum appears to be higher and the morning maximum lower, when compared with the respective maxima in male chromosomes.
Exptl Cell Res 89 (1974)
Preliminary notes
439
26. Rensing, L & Hardeland, R, J insect physiol 13 (1967) 1547. 21. Rensing, L, Brunken, W & Hardeland, R, Experientia 24 (1968) 509. 28. Rensing, L & Hardeland, R, Exptl cell res 73 (1972) 311. 29. Rzepka, P, J interdiscip cycle res 5 (1974) 167. 30. Vanden Driessche. T, J interdisciu_ cycle - res 1 (1970)21. 31. - Ibid 2 (1971) 133. 32. Wagner, E. Personal communication. 33. Weber, E, Grundriss der biologischen Statistik, p. 420. VEB Gustav Fischer Verlag Jena (1956).
independent and insensitive to sulfhydryl blockers [2]. DNA synthesis apparently similar to the latter type can be stimulated in non-S phase nuclei by exposing them in vitro to low concentrations of DNase I [2]. In addition to being independent of ATP and insensitive to sulfhydryl blockers, this type of DNA synthesis is not semiconservative, is either not inhibited or is markedly stimulated by monovalent cations, and is only Received September 18, 1974 partially sensitive to actinomycin D [2, 31. We have termed these two distinctive forms of in vitro DNA synthesis in hepatocyte nuclei, types A and C; type A synthesis has been shown to represent DNA replication [2, 4, 161, and we have presented evidence Effect of Ara-CTP on DNA replication that type C synthesis may represent DNA and repair in isolated hepatocyte nuclei repair 121. M. L. STENSTROM,l M. EDELSTEIN and J. W. DNA replication in whole mammalian GRISHAM,l Department of Pathology, Washington University School of Medicine, St Louis, MO 63110, cells is inhibited by the cytidine analog, l-pUSA D-arabinofuranosylcytosine (ara-C) [S-7]. The triphosphate of ara-C (ara-CTP) is believed Summary. Ara-CTP differentially inhibits two types to be the active form, since this compound of DNA synthetic activity occurring in isolated hepatocyte nuclei in vitro. Ara-CTP inhibits type A inhibits in vitro crude DNA polymerase synthesis (replication) with a Ki of 5 x 10-T M, extracted from a variety of mammalian cells whereas type C synthesis (presumed renair) is much less sensitive, the Ki being 5 x 1O-4 M. Significant [8-131. Recent studies with permeabilized inhibition of type C synthesis does not occur until bacteria indicate that ara-CTP specifically type A synthesis is suppressed by more than 50 %. inhibits polymerase II but that it has little Isolated rat liver nuclei can incorporate deoxy- or no effect on polymerase I, the presumed ribonucleoside triphosphate (dNTP) pre- repair polymerase 114, 151. Similarly, only cursors into DNA in vitro in the absence of one of two polymerase activities in isolated S phase hepatocyte nuclei was sensitive to both exogenous primer-template and DNA polymerase [l]. At least two synthetic activi- ara-CTP, and the sensitive activity was ties are involved [2]. DNA synthesis in thought to represent replicative polymerase [16]. In addition, a differential sensitivity undamaged S phase nuclei is predominantly was noted for nuclear and cytoplasmic DNA ATP-dependent, is semiconservative, and is inhibited by sulfhydryl blockers, actinomycin polymerases purified from cultured human D, and monovalent and divalent cations other lymphocytes [17, 181. The cytoplasmic pothan Mg2+ and Mn2+ [l, 21.A small fraction lymerase, presumably responsible for DNA to total synthesis in S phase nuclei is ATP- replication, was more sensitive to ara-CTP [17, 1X]. 1 Present address: Department of Pathology, University of North Carolina School of Medicine, Chapel Hill, N.C. 27514, USA, to which reprint requests should be addressed. 29
- 741802
We have examined further the effect of ara-CTP on replicative and presumed reparative DNA syntheses in isolated hepatocyte Exptl Cell Res 89 (1974)
Preliminary notes
9. Naha, P M, Nature 223 (1969) 1380. 10. Meiss, H K &Basilico, C, Nature new biol 239 (1972) 66. Received August 1, 1974 Revised version received September 6, 1974
Circadian rhythm in size and 3H-uridine incorporation of single puffs of Drosophila salivary glands in vitro G. NAGEL and L. RENSING, I. Zoolog. I~nstitut der Universitiit
Giittingen, D 34 Giittingen, BRD
Summary. Salivary glands of late third instar larvae of Drosophila melanogaster were kept in a chemically defined medium at 25”C, under a light-dark cycle (light time from 9-21 h). Puff size and SH-uridine incorooration of various single uuffs changed sianificantiy during a 24 h period invitro. Maxima occurred at 8 h and 21 h. minima at 24 h and 11 h. In the unpuffed region’ 3A6-3C2, only one maximum of 3H-uridine incorporation-at 17 h-was observed.
Circadian rhythms have been described in cell cultures of various tissues [l, lo], and in isolated DrosophiZa salivary glands in particular [24]. From this evidence, it can be concluded that each cell generates its own circadian rhythm-the basic mechanism of which is still unknown, however. Even if nuclear activities may not contribute to this mechanism [30, 311,it seemsto be important to test sequential or synchronous changes of various chromosome regions over 24 h. The results should then be discussedas to possible control mechanisms of gene activity that could be deduced from different models of circadian oscillators [8, 20, 321. Materials and Methods Salivary glands of late third instar larvae of Drosophila Meigen (115 h after egg laying at 25°C [4,22, 231)were prepared and then kept in an artificial medium [18, 241 under a 12:12 h light-dark cycle (light time from 9-21 h) at 25°C. Chromosome regions and puffs were localized in squash preparations with reference to the chromosome maps of Bridges & Brehme [5] and photographs of Ashburner [2-4].
melanogaster
Puff size. Measurements were made after squashing and orcein staining of cells and chromosomes [19] Exptl
Cell Res 89 (1974)
taking the width of the puffed region and of a reference band as measures to calculate the puff quotient (PQ, [26]). A certain puff size at a certain time of day reoresents the mean of 20 values from 5-8 experiments-at different days; each of the 20 values is the mean of 5 PQ measurements in a single gland. The puff size is plotted together with the threefold standard error. Autoradiography. Glands were incubated with SHuridine 1 h before squashing (10 ,&i/ml medium). For each puff at each time of day 30 autoradiographs were evaluated. The autoradiographs for each time of day were made during 5-8 series of experiments at different days. Grains were counted by use of a microscope. Because of the different background and the different overall incorporation of the chromosomes in single preparations, a correcting factor had to be introduced. The background of each slide was determined by counting the &ains of a defined area close to the chromosomes, whereas the incorporation rate of the chromosomes was estimated by measuring the grain number of the region 58A-60F. This was done five times for each slide and the mean values were calculated. For a series of experiments of a certain time of day. a mean value for background and general incorp&ation level was then caliulated from the individual means. The deviation of the background of a single slide from the mean value of an experimental series at a certain time of day and the deviation of the individual chromosome incorporation from the mean incorporation were used as correcting factors. The corrected grain number of a puff area was then divided by the diameter of a reference band to account for differences in polyteny and squashing (autoradiographic quotient, AQ). The incorporation into a certain puff at a certain time of day is plotted together with the confidence intervals (ca 95 % [33]).
Results Puff size. In different chromosomal regions
bimodal rhythmic changes in puff size are registered. These changes are significant in the caseof the largest puffs 23E, 50CD (fig. I), 74EF/75B (fig. 2), 93D. Other puffs such as 47A/BC, 58BC/CD (fig. l), 22E, 62E, 68C, 78D, 82F, 83E, and 85F show the same rhythmic pattern, the level of significance being lower in these cases.The puffed regions 50CD, 74EF, 75B and 58BC/CD were chosen as examples to represent rhythmic changes measured in the other puffed regions [18]. Maxima of puff size occur at 8 h shortly before light-on and at 21 h at the time of lightoff. The 8 h values were calculated from
Preliminary notes
437
experiments made after 24 and 48 h of incubation in the medium. Incorporation of 3H-uridine. The incorporation
of 3H-uridine was measured in the following chromosome regions: 3A6-3C2, the genetic locus for rhythm mutants [14], 58A-60F as an estimate of the overall incorporation of the chromosomes, 2B5-6/13-17; 47A, 47BC, 50CD, 58BC/DE, 63BC, 66E, 67F, 74EF/75B, 93D, 85F, and the nucleolus. Maxima of 3H-uridine incorporation in most casesappear shortly before the maxima of puff size (figs 1, 2). In the region 3A6-3C2 on the X chromosome, no puffing was observed; the 3H-uridine incorporation, however, changes
125
I
i k
--750 ,ocI- *
k---q-; T*O” ,J, :m ,’ ’ __--- d I Jr -41 ‘,r4- I
.. v /’ 9’ 1
Fig. 2. Abscissa: time of day, partly repeated in order to give a clear picture of the rhythmic pattern; ordinate: (left) AQ, autoradiographic quotient; for explanation see Methods; (right) PQ, puff quotient; for explanation, see Methods. Puff size (- ) and 3H-uridine incorporation (-) of two puffed chromosomal regions on the third chromosome.
Fig. I. Abscissa: time of day, partly repeated in order to give a clear picture of the rhythmic pattern; ordinate: (left) AQ, autoradiographic quotient; for explanation see Methods; (right) PQ, puff quotient; for explanation see Methods. Puff size (---) and SH-uridine incorporation (&-) of two puffed chromosomal regions on the second chromosome.
during 24 h. This rhythmicity might be explained by a short adsorption of heterogeneousRNA from other parts of the genome to this site or by a low synthetic activity of this region. It may be of interest that the maximum of incorporation seems to occur at 17 h whereas another unpuffed region (58A-60F) doesnot show significant rhythmic changes. The nucleolar 3H-uridine incorporation parallels the rhythmic pattern of the puffs. An observation should be mentioned concerning differences in the rhythmic pattern between males and females that had first been described by Rensing et al. [271 in the case of oxygen consumption: in the female Exptl Cell Res 89 (1974)
438
Preliminary notes
oscillator have been considered [6, 251: conformations of membrane proteins together with ion fluxes and ion pumps are elements of one model [20], energy transductions between cell compartments are essential functions of another model [32]. A metabolic Discussion system with coordinative metabolites, such Circadian rhythms of transcription have been as for example cyclic nucleotides, may also reported for rat and mouse liver [7, 9, 121, function as a cellular oscillator. Evidence of influences on transcription can be provided and have been shown to exist also in Drosophila salivary glands in vivo [21], in algae for all three possibilities [17, 25, 281. It [34], and in other cells and tissues. In rat cannot be decided at present, however, which of these models is actually realized. liver the histone and non-histone protein content, the 3H-lysine incorporation into References these proteins [29] and the phosphorylation 1. Andrews, R V & Folk, G E Jr, Comp biochem of nuclear proteins [lq seem to be correlated physiol 11 (1964) 393. with the transcriptional rhythmicity. Further2. Ashburner, M, Chromosoma 21 (1967) 398. 3. - Ibid 31 (1970) 356. more, the activity of protein kinases [15] 4. - Developmental studies on giant chromosomes and the permeability of nuclear membranes (ed W Beermann). Snrinaer-Verlag. Berlin. Heidelberg, New York (1972): -’ [13] have been shown to change during 24 h. 5. Bridges, C B & Brehme, K S, Carnegie inst A major point of interest is the question Wash pub1 552 (1944) 256. 6. Btinning, E, The physiological clock. Springerwhich control mechanism is responsible for Verlag, New York (1973). the rhythmicity of transcription. This question I. Daring, R & Rensing, L, Comp biochem physiol 45B (1973) 285. has to, be discussed from the point of view 8. Ehret, C & Trucco, E, J theor biol 15 (1967) 240. of a basic circadian oscillator within the cell. 9. Glasser, S R & Spelsberg, T C, Biochem biophys res commun 47 (1972) 951. Theoretically, transcription could be a neces10. Hardeland, R, J interdiscip cycle res 3 (1972) 109. sary part of the oscillator as has been pro11. Jacob, F & Monod, J, Cytodifferentiation and macromolecular svnthesis. Academic Press, New posed more generally by Jacob & Monod York, London (1963). [1 l] and has been specifically worked out 12. Jardetzkv. C D. Barnum, C P & Halberg, F, Am j physioii87 (1956) 608.. in the case of a circadian oscillator by Ehret 13. Kittlick, P D, Exptl path01 4 (1970) 143. & Trucco [8]. The latter model assumed a 14. Konopka, R J & Benzer, S, Proc natl acad sci US 68 (1971) 2112. sequential gene transcription, each gene being Langner, R. Unpublished results. coupled to the next by secondary gene 15. 16. Letnansky, K & Reisinger, L, Biochem biophys res commun 49 (1971) 312. products giving rise to a 24 h cycle. From 17. Lezzi, M & Robert,‘M, Developmental studies the above-described findings, this model on giant chromosomes (ed W Beermann). Springer-Verlag, Berlin, Heidelberg, New York is not likely to be realized in Drosophila, since all puffs seem to be in phase with each 18. (1972). Nagel, G. In preparation. other. Since Acetabularia shows circadian 19. Nicoletti, B, Drosophila inf serv 33 (1959) 181. Njus, D, Sulzmann, F M & Hastings, J W, rhythms even without nucleus [30, 311, it 20. Nature 248 (1974) 116. can be concluded that nuclear transcription 21. Probeck, H D & Rensing, L, Cell differentiation 2 (1974) 337. is not an element of the basic mechanism but 22. I&sing, -L, Z vergl physiol 53 (1966) 62. is rather controlled by an oscillator that is 23. - Z Zellforsch 74 (1966) 539. 24. - J insect physiol 15 (1969) 2285. localized in the cytoplasm. Biologische Rhythmen und Regulation. Fischer 25. &lag Stuttgart (1973). Various mechanisms of the cytoplasmic chromosomes, the evening maximum appears to be higher and the morning maximum lower, when compared with the respective maxima in male chromosomes.
Exptl Cell Res 89 (1974)
Preliminary notes
439
26. Rensing, L & Hardeland, R, J insect physiol 13 (1967) 1547. 21. Rensing, L, Brunken, W & Hardeland, R, Experientia 24 (1968) 509. 28. Rensing, L & Hardeland, R, Exptl cell res 73 (1972) 311. 29. Rzepka, P, J interdiscip cycle res 5 (1974) 167. 30. Vanden Driessche. T, J interdisciu_ cycle - res 1 (1970)21. 31. - Ibid 2 (1971) 133. 32. Wagner, E. Personal communication. 33. Weber, E, Grundriss der biologischen Statistik, p. 420. VEB Gustav Fischer Verlag Jena (1956).
independent and insensitive to sulfhydryl blockers [2]. DNA synthesis apparently similar to the latter type can be stimulated in non-S phase nuclei by exposing them in vitro to low concentrations of DNase I [2]. In addition to being independent of ATP and insensitive to sulfhydryl blockers, this type of DNA synthesis is not semiconservative, is either not inhibited or is markedly stimulated by monovalent cations, and is only Received September 18, 1974 partially sensitive to actinomycin D [2, 31. We have termed these two distinctive forms of in vitro DNA synthesis in hepatocyte nuclei, types A and C; type A synthesis has been shown to represent DNA replication [2, 4, 161, and we have presented evidence Effect of Ara-CTP on DNA replication that type C synthesis may represent DNA and repair in isolated hepatocyte nuclei repair 121. M. L. STENSTROM,l M. EDELSTEIN and J. W. DNA replication in whole mammalian GRISHAM,l Department of Pathology, Washington University School of Medicine, St Louis, MO 63110, cells is inhibited by the cytidine analog, l-pUSA D-arabinofuranosylcytosine (ara-C) [S-7]. The triphosphate of ara-C (ara-CTP) is believed Summary. Ara-CTP differentially inhibits two types to be the active form, since this compound of DNA synthetic activity occurring in isolated hepatocyte nuclei in vitro. Ara-CTP inhibits type A inhibits in vitro crude DNA polymerase synthesis (replication) with a Ki of 5 x 10-T M, extracted from a variety of mammalian cells whereas type C synthesis (presumed renair) is much less sensitive, the Ki being 5 x 1O-4 M. Significant [8-131. Recent studies with permeabilized inhibition of type C synthesis does not occur until bacteria indicate that ara-CTP specifically type A synthesis is suppressed by more than 50 %. inhibits polymerase II but that it has little Isolated rat liver nuclei can incorporate deoxy- or no effect on polymerase I, the presumed ribonucleoside triphosphate (dNTP) pre- repair polymerase 114, 151. Similarly, only cursors into DNA in vitro in the absence of one of two polymerase activities in isolated S phase hepatocyte nuclei was sensitive to both exogenous primer-template and DNA polymerase [l]. At least two synthetic activi- ara-CTP, and the sensitive activity was ties are involved [2]. DNA synthesis in thought to represent replicative polymerase [16]. In addition, a differential sensitivity undamaged S phase nuclei is predominantly was noted for nuclear and cytoplasmic DNA ATP-dependent, is semiconservative, and is inhibited by sulfhydryl blockers, actinomycin polymerases purified from cultured human D, and monovalent and divalent cations other lymphocytes [17, 181. The cytoplasmic pothan Mg2+ and Mn2+ [l, 21.A small fraction lymerase, presumably responsible for DNA to total synthesis in S phase nuclei is ATP- replication, was more sensitive to ara-CTP [17, 1X]. 1 Present address: Department of Pathology, University of North Carolina School of Medicine, Chapel Hill, N.C. 27514, USA, to which reprint requests should be addressed. 29
- 741802
We have examined further the effect of ara-CTP on replicative and presumed reparative DNA syntheses in isolated hepatocyte Exptl Cell Res 89 (1974)