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Effects of cordycepin on morphology and RNA synthesis of amphibian lampbrush chromosomes
Copyright All rights
8
1972
by Academic Press, Inc. in any form reseroed
of reproduction
Experimental
EFFECTS SYNTHESIS
Cell Research 75 (1972) 11-14
OF CORDYCEPIN
ON MORPHOLOGY
OF AMPHIBIAN L. FIUME,’
LAMPBRUSH
I. NARDI,2
AND
RNA
CHROMOSOMES
S. BUCCP and G. MANCINO’
IInstitute of General Pathology, University of Bologna, Bologna, and Thair of Histology and Embryology, Institute of Zoology and Comparative Anatomy, University of Pisa, Pisa, Italy
SUMMARY The action of certain inhibitors of RNA and protein syntheses on the morphology of the loops of lampbrush chromosomes in amphibian oocytes was studied. Cordycepin, which affects the synthesis of nuclear heterogeneous RNA, induces a complete retraction of the loops into the chromomeres. On the other side puromycin and cycloheximide do not induce any changes in the morhology of lambrush chromosomes.
Lampbrush chromosome loops of amphibian oocytes and polytene chromosome puffs of dipteran salivary glands are uncoiled parts of chromosomes active in RNA synthesis. They disappear after treatment with actinomycin D [l-5] or cr-amanitin [6, 71 which inhibit RNA synthesis by binding the former to DNA [cf 81, the latter to the form II of nuclear RNA-polymerases [cf 9, lo]. This disappearance has been interpreted as evidence that loops and puffs cannot exist without the continuous synthesis of a type of RNA formed by RNA-polymerase which is sensitive to cc-amanitin [6, 71. In the present experiments we studied the behaviour of loops after treatment with cordycepin (3-deoxyadenosine). This substance suppressesthe labelling of messenger RNA (mRNA) associatedwith polyribosomes [l 1, 121and affects the synthesis of nuclear heterogeneous RNA (HnRNA) [12]. We
found that loops disappear after treatment with cordycepin. Since this drug suppresses the labelling of mRNA in polyribosomes it would have been possible to suppose that what loops require for their existence is a continuous supply of some proteins whose synthesis is depending upon mRNA: therefore we studied also the action on the loops of two inhibitors of protein synthesis: puromycin [cf 131and cycloheximide [cf 141. MATERIAL
AND TECHNIQUE
The newt specimens used in the present work belong to the species Triturus cristatus carnifex (Laurenti). They have been collected at the beginning of winter, when ovaries are fully developed. Manipulation of oocyte germinal vesicles and isolation of lampbrush chromosomes and nucleoli have been made according to a technique previously described [15]. Five experiments were performed at room temperature, using pieces of ovaries of 50-60 growing oocytes of varying sizes (the volumes of the samples were of about 0.3 ml): (1) The samples were incubated in about 200 pug Exptl
Cell Res 75 (1972)
12
L. Fiume et al.
of cordycepin (Sigma Chemical Co.) for incubation periods ranging from a few min to 5 h: oocytes were kept moist by addition of a tiny amount of coelomic fluid. Single oocytes were removed at regular intervals starting from 15 min of incubation, for testing the mornhological state of lampbrush chromosomes. (2) The samples were incubated in 200 fig of cordvceuin for 2: h and 28 h and then rinsed in 5 : 10.1 M K/NaCl and transferred in 250 ,&i of 3H-uridine (spec. act. 5.4 Ci/mM; The Radiochemical Centre, Amersham, UK), for incubation times of 4 to 6 h. The preparations of isolated chromosomes and nucleoli were mounted autoradiographically, following both the stripping film technique (Kodak Plates AR-IO; exposure of about 25 days; Kodak Developer D19 and Kodak Unifix) and the emulsion method (Ilford Emulsion K2; exposure of 7 to 15 days). (3) The samples were incubated in 30 pg of puromycin (Serva Entwicklungslabor, Heidelberg) or in 30 ,rhgof cycloheximide (Sigma) for incubation periods ranging from a few minutes to 10 h in the case of puromycin, or to 17 h in the case of cycloheximide. Single oocytes were removed at regular intervals starting from 15 min of incubation, for testing possible morphological variations of lampbrush chromosomes. (4) The samnles were first treated with 30 ~.a of purdmycin for-34 h; soon after they were incubated in 500 uCi of SH-ohenvlalanine (snec. act. 1 CiimM: Radiochemical Centrej for 3 to -69 h. Finally the preparations of isolated chromosomes and nucleoli were mounted autoradiographically. (5) The samples were treated with 30 ,ug of puromycin for 3 h; then they were incubated in 300 ,uCi of SH-uridine (spec. act. 4.6 Ci/mM, Radiochemical Centre) for 44, 7, 22 and 24 h. Preparations were also made using oocytes excised from untreated pieces of ovaries as a control for expts (1) and (3); from pieces of ovaries incubated only in SH-uridine as a control for expts (2) and (5); from pieces of ovaries only incubated in 3H-phenylalanine for expt (4).
RESULTS Effects of cordycepin on lampbrush loops. The loops of lampbrush chromosomes gradually retract into chromomeres, beginning from 45 min of incubation in cordycepin; the retraction is noticeable after 3 h of incubation for medium and large size oocytes (fig. 1); it is complete at about 6 h. Smaller oocytes require longer incubation. Among the lateral structures, fibrillar loops (Fl), giant fusing loops (GFL) and lumpy objects [cf 161 remain well extended when condensation of normal loops is nearly complete (figs 1, 2). Exptl
Cell Res 75 (1972)
Effects of cordycepin on incorporation of 3Huridine into lampbrush loops and nucleoli. Labelling is evident along lampbrush chromosomesuntil loops are still present, even if partly retracted (fig. 3): this incorporation probably corresponds to synthesis of heterogeneous RNA which is only partly affected by cordycepin [12]. Radioactivity cannot be detected when loops are completely condensed. Giant fusing loops are poorly labelled or appear less labelled in comparison with the controls. Giant fibrillar loops and lumpy objects do not show any radioactivity; however, these kinds of loops are unlabelled or covered with a few silver grains also in control preparations. Both nucleoli free in the nucleoplasm and those still inserted on the nucleolus organizing regions, recently identified in the lampbrush set of the species [17], are radioactive, although incorporation of 3H-uridine seems to be lower than in controls. Effects of puromycin and cycloheximide on lampbrush loops. These inhibitors of protein synthesis do not affect the morphology of lampbrush chromosomes: the loops and the other lateral structures remain well developed even after a very prolonged incubation period. A certain diffuse toxic effect on germinal vesicles can be noticed after several hours of incubation in puromycin. Effects of puromycin on incorporation of 3Hphenylalanine into lampbrush loops and nucleoli. Puromycin blocks the incorporation of phenylalanine into lampbrush chromosomes and nucleoli which appeared completely unlabelled. Effects of puromycin on incorporation of 3Huridine into lampbrush loops and nucleoli. Lampbrush chromosomes and nucleoli are heavily labelled even after several hours of
Effects of cordycepin on lampbrush chromosomes
13
Fig. 1. Lampbrush chromosomes from a medium-size oocyte incubated in cordycepin for 3 h . Giant fusing loops (arrows) are still well developed. x 170. Fig. 2. Lampbrush chromosome from a medium-size oocyte incubated in cordycepin for 2$ h: normal loops are remarkably retracted, while fibrillar loops (FI) are still well extended. x 380. Fig. 3. Lampbrush chromosome from a medium-size oocyte treated with cordycepin for 3 h and i.hen incubated in SH-uridine for 5t h: labelling is evident along the chromosome axis since normal loops are rlot completely retracted. x 1 360.
Exptl
Cell Res 75 (1972)
14 L. Fiume et al. incubation of the ovarian samples inhibitor of the protein synthesis.
in the
DISCUSSION The finding that inhibitors of protein synthesis, such as puromycin or cycloheximide, do not affect the morphology of loops suggests that loops do not require for their existence a continuous supply of some proteins. Assuming that the only function of mRNA is that of allowing protein synthesis to be accomplished, such a finding would indicate that mRNA is not the kind of RNA necessaryfor the existence of loops. Cordycepin which causes loops to collapse lowers the synthesis of HnRNA [12]. Probably different kinds of RNA with different functions belong to HnRNA which is formed in part by RNApolymerase II (sensitive to a-amanitin) and in part by RNA-polymerase III [18]. It is possible that the kind of RNA necessary for the existence of loops belongs to, or derives from, a fraction of HnRNA whose synthesis is inhibited by cordycepin and which is formed by RNA-polymerase sensitive to a-amanitin. This RNA might be chromosomal RNA (cRNA). cRNA has a large fraction of its sequences in common with HnRNA [cf 191and there is evidence that it is involved in the process of gene activation [20, 21, 221. Since the uncoiled parts of chromosomes (loops, puffs, euchromatin) are those active in RNA synthesis, it is likely that the RNA required for chromosome uncoiling is the one which is involved in the process of gene activation. This suggestion is supported by the finding that a-amanitin which causesloops and puffs to collapse and euchromatin to condense [23] brings about
Exptl
Cell Res 75 (1972)
an early and strong inhibition of the synthesis of cRNA in rat liver [24]. This work was supported by grants from the Italian National Council (CNR), Rome.
REFERENCES 1. Izawa. M. Allfrev. V G & Mirskv. A E. Proc natl acad sci US 49 (1963) 544. -’ ’ 2. Ebstein, B S, J cell biol 35 (1967) 709. 3. Mancino, G Barsacchi, G & Nardi, I, Atti accad naz Lincei 45 (1968) 591. 4. Snow, M H L & Callan, H G, J cell sci 5 (1969) 1. 5. Beermann. W. J exptl zoo1 157 (1964) 49. 6. Mancino, ‘G, Nardi, I, Corvaja; N, Fiume, L & Marinozzi, V, Exptl cell res 64 (1971) 237. 7. Beermann, W, Chromosoma 34 (1971) 152. 8. Reich, E & Goldberg, I H, Progr nucleic acid res mol biol 3 (1964) 183. 9. Fiume, L & Wieland, Th, FEBS letters 8 (1970) 1. 10. Lindell, T J, Weinberg, F, Morris, P W, Roeder, R G & Rutter, W J, Science 170 (1970) 447. 11. Penmann, S, Fan, H, Perlman, S, Rosbash, M, Weinberg, R & Zylber, E, Cold Spring Harbor symp quant biol 35 (1970) 561. 12. Darnell, J E, Philipson, L, Wall, R & Adesnik, M, Science 174 (1971) 507. 13. Hartmann, G, Behr, W, Beissner, K A, Honikel, K & Sippel, A, Angew Chem (int ed) 7 (1968) 693. 14. Traub, P, Inhibitor tools in cell research (ed Th Biicher & H Sies) p. 79. Springer, Berlin (1969). 15. Gall, J G, Methods in cell physiology (ed D Prescott) p. 37. Academic Press, New York (1965). 16. Callan, H G & Lloyd, L, Phil trans roy sot (London) B 243 (1960) 135. 17. Mancino, G, Nardi, I & Ragghianti, M, Experientia. In press. 18. Zylber, E A & Penman, S, Proc natl acad sci US 68 (1971) 2861. 19. Mayfield, J E & Bonner, J, Proc natl acad sci US 69 (1972) 7. 20. Bonner, J, Dahmus, M E, Fambrough, D, Huang, R C, Marushige, K & Tuan, D, Science 159 (1968) 47. 71 Bekhor, I, Kung, G & Bonner, J, J mol biol 39 (1969) 351. 22. Mayfield, J E & Bonner, J, Proc natl acad sci US 68 (1971) 2652. 23. Marinozzi, V & Fiume, L, Exptl cell res 67 (1971) 311. 24. Montecuccoli, G, Novello, F & Stirpe, F. Personal communication. IA.
Received March 20, 1972
8
1972
by Academic Press, Inc. in any form reseroed
of reproduction
Experimental
EFFECTS SYNTHESIS
Cell Research 75 (1972) 11-14
OF CORDYCEPIN
ON MORPHOLOGY
OF AMPHIBIAN L. FIUME,’
LAMPBRUSH
I. NARDI,2
AND
RNA
CHROMOSOMES
S. BUCCP and G. MANCINO’
IInstitute of General Pathology, University of Bologna, Bologna, and Thair of Histology and Embryology, Institute of Zoology and Comparative Anatomy, University of Pisa, Pisa, Italy
SUMMARY The action of certain inhibitors of RNA and protein syntheses on the morphology of the loops of lampbrush chromosomes in amphibian oocytes was studied. Cordycepin, which affects the synthesis of nuclear heterogeneous RNA, induces a complete retraction of the loops into the chromomeres. On the other side puromycin and cycloheximide do not induce any changes in the morhology of lambrush chromosomes.
Lampbrush chromosome loops of amphibian oocytes and polytene chromosome puffs of dipteran salivary glands are uncoiled parts of chromosomes active in RNA synthesis. They disappear after treatment with actinomycin D [l-5] or cr-amanitin [6, 71 which inhibit RNA synthesis by binding the former to DNA [cf 81, the latter to the form II of nuclear RNA-polymerases [cf 9, lo]. This disappearance has been interpreted as evidence that loops and puffs cannot exist without the continuous synthesis of a type of RNA formed by RNA-polymerase which is sensitive to cc-amanitin [6, 71. In the present experiments we studied the behaviour of loops after treatment with cordycepin (3-deoxyadenosine). This substance suppressesthe labelling of messenger RNA (mRNA) associatedwith polyribosomes [l 1, 121and affects the synthesis of nuclear heterogeneous RNA (HnRNA) [12]. We
found that loops disappear after treatment with cordycepin. Since this drug suppresses the labelling of mRNA in polyribosomes it would have been possible to suppose that what loops require for their existence is a continuous supply of some proteins whose synthesis is depending upon mRNA: therefore we studied also the action on the loops of two inhibitors of protein synthesis: puromycin [cf 131and cycloheximide [cf 141. MATERIAL
AND TECHNIQUE
The newt specimens used in the present work belong to the species Triturus cristatus carnifex (Laurenti). They have been collected at the beginning of winter, when ovaries are fully developed. Manipulation of oocyte germinal vesicles and isolation of lampbrush chromosomes and nucleoli have been made according to a technique previously described [15]. Five experiments were performed at room temperature, using pieces of ovaries of 50-60 growing oocytes of varying sizes (the volumes of the samples were of about 0.3 ml): (1) The samples were incubated in about 200 pug Exptl
Cell Res 75 (1972)
12
L. Fiume et al.
of cordycepin (Sigma Chemical Co.) for incubation periods ranging from a few min to 5 h: oocytes were kept moist by addition of a tiny amount of coelomic fluid. Single oocytes were removed at regular intervals starting from 15 min of incubation, for testing the mornhological state of lampbrush chromosomes. (2) The samples were incubated in 200 fig of cordvceuin for 2: h and 28 h and then rinsed in 5 : 10.1 M K/NaCl and transferred in 250 ,&i of 3H-uridine (spec. act. 5.4 Ci/mM; The Radiochemical Centre, Amersham, UK), for incubation times of 4 to 6 h. The preparations of isolated chromosomes and nucleoli were mounted autoradiographically, following both the stripping film technique (Kodak Plates AR-IO; exposure of about 25 days; Kodak Developer D19 and Kodak Unifix) and the emulsion method (Ilford Emulsion K2; exposure of 7 to 15 days). (3) The samples were incubated in 30 pg of puromycin (Serva Entwicklungslabor, Heidelberg) or in 30 ,rhgof cycloheximide (Sigma) for incubation periods ranging from a few minutes to 10 h in the case of puromycin, or to 17 h in the case of cycloheximide. Single oocytes were removed at regular intervals starting from 15 min of incubation, for testing possible morphological variations of lampbrush chromosomes. (4) The samnles were first treated with 30 ~.a of purdmycin for-34 h; soon after they were incubated in 500 uCi of SH-ohenvlalanine (snec. act. 1 CiimM: Radiochemical Centrej for 3 to -69 h. Finally the preparations of isolated chromosomes and nucleoli were mounted autoradiographically. (5) The samples were treated with 30 ,ug of puromycin for 3 h; then they were incubated in 300 ,uCi of SH-uridine (spec. act. 4.6 Ci/mM, Radiochemical Centre) for 44, 7, 22 and 24 h. Preparations were also made using oocytes excised from untreated pieces of ovaries as a control for expts (1) and (3); from pieces of ovaries incubated only in SH-uridine as a control for expts (2) and (5); from pieces of ovaries only incubated in 3H-phenylalanine for expt (4).
RESULTS Effects of cordycepin on lampbrush loops. The loops of lampbrush chromosomes gradually retract into chromomeres, beginning from 45 min of incubation in cordycepin; the retraction is noticeable after 3 h of incubation for medium and large size oocytes (fig. 1); it is complete at about 6 h. Smaller oocytes require longer incubation. Among the lateral structures, fibrillar loops (Fl), giant fusing loops (GFL) and lumpy objects [cf 161 remain well extended when condensation of normal loops is nearly complete (figs 1, 2). Exptl
Cell Res 75 (1972)
Effects of cordycepin on incorporation of 3Huridine into lampbrush loops and nucleoli. Labelling is evident along lampbrush chromosomesuntil loops are still present, even if partly retracted (fig. 3): this incorporation probably corresponds to synthesis of heterogeneous RNA which is only partly affected by cordycepin [12]. Radioactivity cannot be detected when loops are completely condensed. Giant fusing loops are poorly labelled or appear less labelled in comparison with the controls. Giant fibrillar loops and lumpy objects do not show any radioactivity; however, these kinds of loops are unlabelled or covered with a few silver grains also in control preparations. Both nucleoli free in the nucleoplasm and those still inserted on the nucleolus organizing regions, recently identified in the lampbrush set of the species [17], are radioactive, although incorporation of 3H-uridine seems to be lower than in controls. Effects of puromycin and cycloheximide on lampbrush loops. These inhibitors of protein synthesis do not affect the morphology of lampbrush chromosomes: the loops and the other lateral structures remain well developed even after a very prolonged incubation period. A certain diffuse toxic effect on germinal vesicles can be noticed after several hours of incubation in puromycin. Effects of puromycin on incorporation of 3Hphenylalanine into lampbrush loops and nucleoli. Puromycin blocks the incorporation of phenylalanine into lampbrush chromosomes and nucleoli which appeared completely unlabelled. Effects of puromycin on incorporation of 3Huridine into lampbrush loops and nucleoli. Lampbrush chromosomes and nucleoli are heavily labelled even after several hours of
Effects of cordycepin on lampbrush chromosomes
13
Fig. 1. Lampbrush chromosomes from a medium-size oocyte incubated in cordycepin for 3 h . Giant fusing loops (arrows) are still well developed. x 170. Fig. 2. Lampbrush chromosome from a medium-size oocyte incubated in cordycepin for 2$ h: normal loops are remarkably retracted, while fibrillar loops (FI) are still well extended. x 380. Fig. 3. Lampbrush chromosome from a medium-size oocyte treated with cordycepin for 3 h and i.hen incubated in SH-uridine for 5t h: labelling is evident along the chromosome axis since normal loops are rlot completely retracted. x 1 360.
Exptl
Cell Res 75 (1972)
14 L. Fiume et al. incubation of the ovarian samples inhibitor of the protein synthesis.
in the
DISCUSSION The finding that inhibitors of protein synthesis, such as puromycin or cycloheximide, do not affect the morphology of loops suggests that loops do not require for their existence a continuous supply of some proteins. Assuming that the only function of mRNA is that of allowing protein synthesis to be accomplished, such a finding would indicate that mRNA is not the kind of RNA necessaryfor the existence of loops. Cordycepin which causes loops to collapse lowers the synthesis of HnRNA [12]. Probably different kinds of RNA with different functions belong to HnRNA which is formed in part by RNApolymerase II (sensitive to a-amanitin) and in part by RNA-polymerase III [18]. It is possible that the kind of RNA necessary for the existence of loops belongs to, or derives from, a fraction of HnRNA whose synthesis is inhibited by cordycepin and which is formed by RNA-polymerase sensitive to a-amanitin. This RNA might be chromosomal RNA (cRNA). cRNA has a large fraction of its sequences in common with HnRNA [cf 191and there is evidence that it is involved in the process of gene activation [20, 21, 221. Since the uncoiled parts of chromosomes (loops, puffs, euchromatin) are those active in RNA synthesis, it is likely that the RNA required for chromosome uncoiling is the one which is involved in the process of gene activation. This suggestion is supported by the finding that a-amanitin which causesloops and puffs to collapse and euchromatin to condense [23] brings about
Exptl
Cell Res 75 (1972)
an early and strong inhibition of the synthesis of cRNA in rat liver [24]. This work was supported by grants from the Italian National Council (CNR), Rome.
REFERENCES 1. Izawa. M. Allfrev. V G & Mirskv. A E. Proc natl acad sci US 49 (1963) 544. -’ ’ 2. Ebstein, B S, J cell biol 35 (1967) 709. 3. Mancino, G Barsacchi, G & Nardi, I, Atti accad naz Lincei 45 (1968) 591. 4. Snow, M H L & Callan, H G, J cell sci 5 (1969) 1. 5. Beermann. W. J exptl zoo1 157 (1964) 49. 6. Mancino, ‘G, Nardi, I, Corvaja; N, Fiume, L & Marinozzi, V, Exptl cell res 64 (1971) 237. 7. Beermann, W, Chromosoma 34 (1971) 152. 8. Reich, E & Goldberg, I H, Progr nucleic acid res mol biol 3 (1964) 183. 9. Fiume, L & Wieland, Th, FEBS letters 8 (1970) 1. 10. Lindell, T J, Weinberg, F, Morris, P W, Roeder, R G & Rutter, W J, Science 170 (1970) 447. 11. Penmann, S, Fan, H, Perlman, S, Rosbash, M, Weinberg, R & Zylber, E, Cold Spring Harbor symp quant biol 35 (1970) 561. 12. Darnell, J E, Philipson, L, Wall, R & Adesnik, M, Science 174 (1971) 507. 13. Hartmann, G, Behr, W, Beissner, K A, Honikel, K & Sippel, A, Angew Chem (int ed) 7 (1968) 693. 14. Traub, P, Inhibitor tools in cell research (ed Th Biicher & H Sies) p. 79. Springer, Berlin (1969). 15. Gall, J G, Methods in cell physiology (ed D Prescott) p. 37. Academic Press, New York (1965). 16. Callan, H G & Lloyd, L, Phil trans roy sot (London) B 243 (1960) 135. 17. Mancino, G, Nardi, I & Ragghianti, M, Experientia. In press. 18. Zylber, E A & Penman, S, Proc natl acad sci US 68 (1971) 2861. 19. Mayfield, J E & Bonner, J, Proc natl acad sci US 69 (1972) 7. 20. Bonner, J, Dahmus, M E, Fambrough, D, Huang, R C, Marushige, K & Tuan, D, Science 159 (1968) 47. 71 Bekhor, I, Kung, G & Bonner, J, J mol biol 39 (1969) 351. 22. Mayfield, J E & Bonner, J, Proc natl acad sci US 68 (1971) 2652. 23. Marinozzi, V & Fiume, L, Exptl cell res 67 (1971) 311. 24. Montecuccoli, G, Novello, F & Stirpe, F. Personal communication. IA.
Received March 20, 1972