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Circular nucleoids isolated from chloroplasts in a brown alga Ectocarpus siliculosus
PRELIMINARY
NOTES
Copyright @ 1981 by Academic Press, Inc. All rights of reproduction in any form reserved 0014-4827/81/080457-05$002.00/0
Circular nucleoids isolated from chloroplasts in a brown alga Bctocarpus siliculosus T. KUROIWA and T. SUZUKI, Depnrtment of Cell Biology, NationalInstitute 444 Japan
for Basic Biology,
Okazaki,
Circular nucleoids have been isolated from the chloroplasts of a brown alga, Ectocarpus indicus. by Nonidet P-40 treatment. Enzymatic treatments of the isolated nucleoids reveal that the nucleoid is a circle composed of bead-like particles interconnected by DNA strands. The beads contain predominantly DNA and proteins.
Summary.
Extranuclear DNA has been shown to occur in chloroplasts [l]. The chloroplast DNA (ctDNA) is concentrated in a specific portion of the chloroplast called the chloroplast nucleoid (ct-nucleoid). In Sphacelaria, Ectocarpus, Pylaiella, Dictyota, and Sorocarpus of the Phaeophyta, Ophiocytium, Botrydium and Tribonema of the Xanthopyta [2-5, 71 and Licomophora, Melosira, Rixosolenia of the Chrysophyta [6, 71, the chloroplast possesses a circular nucleoid inside the girdle lamella. The brown alga, Ectocarpus indicus, is well suited for the study of the isolation of chloroplast nucleoid and the organization of ctDNA at various phases of the chloroplast division cycle, as (a) ctDNA molecules are located at the periphery of the chloroplast to form a distinct circular nucleoid; (b) the contour length of the circle changes as chloroplast division progresses [7]; (c) the chloroplasts can be easily isolated from the cells. We report here the isolation of circular chloroplast nucleoids in the brown algae, 30-KllX16
indicus, in a form which could be digested with deoxyribonuclease but swelled with trypsin treatment.
Ectocarpus
Material
and Methods
A brown alga, Ectocarpus indicus, was collected by Dr Nakamura from a part of Mikawa Bay, Aichi prefecture. Examples were grown under a regimen of glternating 12 h liiht, 12 h d&k. The temperature was 15°C when the lights were on and 1PC in the dark. Chloroplasts were isolated from about 10 g of fresh algae after washing the algae thoroughly. The algae were suspended in 20 ml of buffer S (0.25% sucrose, 1 mM EDTA, 0.6 mM soermidine. 0.05% mercadoethanol, 10 mM Tris-HdI at pH 7.6) containing-O.4 mM ohenvlmethvlsulfonvlfluoride (PMSF) and homogenized in a gla& mortar. 20 ml df the mixture was further homogenized for 30 set at 3000 rpm in a 40 ml Blender cup, and diluted with an equal volume of buffer S. The final suspensions were filtered by gravity through two layers of nylon mesh with pores 31 pm in diameter (NBC Inc., Japan). The filtrates were centrifuged at 100 g for 5 min to remove the debris and unbroken cells. The chloroplasts were then sedimented for 5 min at 800 g. The sedimented chloroplasts were washed twice by resuspending in 5 ml of buffer S containing 0.4 mM PMSF followed bv recentrifugation, andldiluted with equal volume of duffer C (0.25 M sucrose. 0.4 mM PMSF. 10 mM Tris-HCI at PH 7.6). The isolated chloropl&ts were lysed in buffer S containing 0.5 % Nonidet P-40 with agitation at 20°C for 10 min followed by centrifugation at 4500 g for 5 min to remove the debris. The ct-nucleoids were sedimented from the supernatant by centrifugation at 20000 g for 5 min. DNA and RNA were extracted from isolated chloroplasts or isolated ct-nucleoids by the method of Daniel & Baldwin [S]. The extract was then assayed for DNA by the method of Burton [9]. RNA was analysed by the orcinol reaction [lo], and protein was determined by the Lowry method [I 11. Calf thymus DNA, yeast RNA and serum were used as standards. Dilute solutions of chloroplasts were counted by phase-contrast microscopy in Petroff-Hauser bacterial counter. The cells, isolated chloroplasts, and isolated ctnucleoids were fixed in buffer S containing 0.4% glutaraldehyde. Enzymatic digestions were performed on the fixed samples according to the method described previously [12, 131.The isolated ct-nucleoids, which had been incubated with ribonuclease on a glass microscope slide, were treated further with deoxyribonuclease, Sl nuclease or trypsin. As controls, duplicate slides similarly prepared were incubated with a portion of the buffer used as solvent for the deoxyribonuclease. A small drop of the fixed samples was placed on a slide and stained with DNA-specific fluorochrome, 4’6-diamidino-2-phenylindole (DAPI) Exp Cd Res 134 /198/j
458
Preliminury notes
Fig. I. Phase contrast (d, f). fluorescent (a. C, g-i), and phase-contrast fluorescent micrographs (b, e) demonstrating in situ chloroplasts ((I), isolated chloroplasts (h-4) and isolated chloroplast nucleoids (r-g) of Ectocurpu.s indicus. (c.. d) Phase-contrast and fluorescent micrographs of higher magnifications of a portion of isolated chloroplasts in (b). In the isolated chloroplasts a circular nucleoid is visible. cf, g) Phasecontrast and fluorescent micrographs of higher magnification of a portion of isaated chloroplast nucleoids in (0). Isolated chloroplast nucleoids appear to be free from contaminating materials and com-
posed of small, spherical particles. Prior to DAPI staining, isolated ct-nucleoids were treated with ribonuclease. In (h), this treatment was followed by treatina with the same buffer containina trvosin. and in (ir by the buffer containing deoxyr?bonuclease. The chloroplast nucleoid in (h) swells remarkably. By contrast, the circles of chloroplast nucleoid in (i) are broken into small, spherical particles and are finally digested completely by deoxyribonuclease. Bar, 5 pm. (u. h) x1400; (c) xl 100; (d, e) x3600: (f; R, i) x3000; (I?) x2800.
Preliminary
Table 1. Content of DNA, RNA and protein chloroplast
in isolated
Ectocarpus
notes
459
chloroplasts
and
nucleoid
Fraction
DNA (mid
Protein
RNA
Protein/DNA
RNA/DNA
Chloroplast
0.58f0.12” (5.8x 10-‘2)b
24Ok25 (24x 10-9
64413 (6.4x IO-“)
413.8
110.3
Chloroplast nucleoid
0.19f0.02
2.6kO.04
0.51~0.05
13.7
2.7
n Mean value with S.D. * Mean value per single chloroplast.
fluorescent and phase-contrast images demonstrating isolated chloroplasts containing a circular ct-nucleoid. Fig. 1c, d is higher magnification fluorescence (fig. lc) and phase-contrast (fig. Id) micrographs of a portion of fig. lb. The isolated chloroplasts are free of conResults and Discussion taminating materials and contain the circuFluorescence is restricted to the ct-nu- lar ct-nucleoid. Fig. le shows a combinacleoids and nuclei of the Ectocarpus. The tion micrograph of epifluorescent and nucleus appears as large blue fluorescent phase-contrast images of the ct-nucleoids spherule roughly in the center of the cell, isolated from the chloroplasts by Nonidet whereas the ct-nucleoid appears as a blue P-40 treatment. Large and small circular ctfluorescent circle (fig. 1a). This agrees with nucleoids can be seen; they retain the origielectron microscopic studies [2] demon- nal structure as seen in fig. 1a. The constrating the ctDNA located at the periphery tour length of the large isolated circle is of the disk-shaped chloroplasts. The circle about 25 pm which is about twice as long changes its shape according to the division as the small one. Therefore, the large nucycle of chloroplast [7]: as the chloroplast cleoid appears to be isolated from the elongates, becomes dumbbell-shaped, and chloroplast just before chloroplast division; divides into two daughter chloroplasts, the the small nucleoid just after chloroplast peripheral nucleoidal circle also transforms division. These results suggest that the ct-nucleoid accordingly. The contour length of the ct-nucleoid, can be isolated as a circle from the chlorowhich is about 26 pm just prior to chloro- plasts at various stages during the chloroplast division, becomes 13 pm as the result plast division cycle. The isolated ct-nuof chloroplast division [7]. Several dumb- cleoids appear to be entirely free of conbell-shaped nucleoids among circular nu- taminating materials, since fragments of cleoids are observed in cells, suggesting broken cellular nuclei did not show a circle that the chloroplasts divide semi-synchro- but showed relatively large spherical masnously in a single cell (fig. la). Fig. 1b ses, each 2-4 pm in diameter. The protein/DNA ratio indicates a high shows a combination micrograph of both dissolved in the buffer S at the concentration of 50 pg/ml [6]. Stained samples were examined by epifluorescent illumination at 350 nm on an Olympus BH epifluorescent microscope equipped with a phase objective lens. A combination microscopy of both epifluorescence and transmission phase-contrast microscopies was used to detect simultaneously the outline of the isolated chloroplasts and the circular ct-nucleoids.
Em Cd Res 134 (1981)
460
Preliminary
notes
degree of purity of the chloroplast nucleoid (table 1). The protein/DNA ratio of the chloroplast nucleoidal fraction is about 0.03 times that of the chloroplast fraction. The difference appears to be simply a reflection of the relative amount of chloroplast and chloroplast nucleoidal substance characteristic of Ectocarpus cells. DNA/single chloroplast is similar to that observed in Euglena chloroplasts [14], Antirrbinum chloroplasts [15] and Nicotiana chloroplasts [ 161. Fig. lf, g is higher magnification fluorescence (fig. lg) and phase-contrast (fig. lf) micrographs of a portion of fig. 1e. The isolated ct-nucleoids exhibit a circular structure which is composed of small beadlike particles, each 0.2 pm in diameter. The small particles are interconnected by a fine strand (fig. lg). The strand contains DNA, since the digestion with DNase for a short period (15 min) has led to progressive fragmentation of the circular ct-nucleoids (fig. 1i). The fragmentation, however, was not observed with Sl nuclease, RNase and trypsin treatments. Trypsin treatment causes remarkable swelling of the small particles (fig. 1h), but treatment with DNase for a long period (60 min) completely digested the small particles. These experiments suggest that the double-strand ct-DNA is the primary skeletal structure of the circular nucleoid, both in the string and the small particles, and proteins play an important role in organization of small particles. Since the chloroplast contains approx. 5.8~ lo-l5 g of DNA (table 1) and the chloroplast nucleoid is composed of about 30 bead-like particles [7], it is likely that each particle contains approx. 0.19~ IO-” pg of DNA which corresponds to a mol. wt of 114x10” D. This value per particle is consistent with the fact that the fluorescence intensity of the particles is Erp Cd Rrs 134 (19X/)
similar to that of T4 phages [7]. Therefore, the beadlike particles differ in their size and DNA content from nucleosomes [17]. Bisulpusra & Burton [18] proposed, on the basis of electron microscopic observations of a brown alga, Sphacelaria sp., a model that the individual DNA molecules in the circular nucleoid are associated with photosynthetic lamellae. If DNA molecules are dependently associated with lamellae, the circular structure of the ct-nucleoids must be destroyed by treatment of lamellae with Nonidet P-40. The existence of isolated circular nucleoids after the treatment suggests that, in addition to the association of DNA molecules with lamellae, there must be a mechanism for organization of ctDNA molecules. Using this isolation method, it should be possible to further analyse the fine structure of ct-nucleoids, to characterize the trypsinsensitive protein species in the ct-nucleoids, and finally, to elucidate the mechanisms of organization of ctDNA. References Ris, H & Plaut, W, J cell biol 13 (1962) 383.
:: Bisulputra, T & Bisalputra, A A, J ultrastruct res 29 (1969) 151. - Ibid 32 (1970) 417. Gibbs, S P, J cell sci 3 (1968) 327. Coleman, A W, J cell bio182 (1979) 299. Kuroiwa, T, Suzuki, T & Kawano, S, Cell struct func 4 (1979) 399. 7. Kuroiwa, T, Suzuki, T, Ogawa, K & Kawano, S, Plant cell physiol 22 (1981) 322. 8. Daniel, J W & Baldwin. H H. Methods in cell physiology (ed D M Prescott) vol. I, pp. 9-40. Academic Press, New York (1964). 9. Burton, K, Biochem j 62 (1956) 315. IO. Ceriotti, G, J biol them 214 (1955) 59. 11. Lowry, 0 H, Rosebrough, N J, Fax, A L & Randall, R J, J biol them 193(1951) 265. 12. Kuroiwa, T, J cell biol63 (1976) 299. 13. Shenk, T E, Phodges, C, Rigby, P W J & Berg, P, Proc natl acad sci US 72 (1975) 989. 14. Chun, E H L, Vaughan, N H & Rich, J A, J mol biol 7 (1963) 130. 15. Ruppel, H G & Wyki, U D, Z Pflanzenphysiol 53 (1965) 32. 16. Tewari, K K & Wildman, S G, Science 153 (1966) 1269. 17. Olins, A L & Olins, DE. Science 183(1964) 330. 3. 4. 5. 6.
Preliminary
notes
461
18. Bisulputra, T & Burton. H, J ultrastruct res 29 (1969) 224. Received December 29, 1980 Revised version received March 30, 1981 Accepted April 2, 1981
Copyright 0 1981 by Academic Press, Inc. All rights of reproduction in any form reserved 0014.4827/81/080461-05%02.00/0
Length of human prematurely condensed chromosomes during GO and Gl phase HARD1 SCHMIADY’
a
and KARL SPERLING, In-
stitut far Humangenetik. Freie Universit6t Herrhnml~eg 6, D-1000 Berlin 19, Gerrntrn>
Berlin.
Summary. Length measurements on C-banded prematurely condensed no. 1 human chromosomes of GO and Gl lymphocytes, as well as of synchronized Gl HEp cells revealed that (i) no length difference exists between mitotic chromosomes and GO chromosomes; (ii) 1 h after PHA stimulation a clear increase in length is detectable; (iii) in isolated cases an increase by the factor 5 can be observed during Gl; (iv) the increase is significantly less for constitutive heterochromatin than for euchromatin. The possibility is discussed that these conformational changes of chromatin reflect physiological differences, i.e. the rate of RNA synthesis during interphase.
Ceil cycle-related changes/inrhe confor a-;’ tional patterns of chromatin have been CT ‘emonstrated by biochemical and histochemical techniques, but more directly by application of the phenomenon of premature chromosome condensation (PCC) [l-7]. After fusion between mitotic and interphase cells the chromatin of the latter enters into a mitosis-like condensation *r process. The morphology of prematurely condensed chromosomes clearly reflects their position in the cell cycle at the time of fusion, Gl, S or G2 phase [S, 91. However, a wide range of variability in chromosome length is observed within individual Fig. 1. (a) Human lymphocyte metaphase chromoGl and G2 cells. These length differences somes: (b) prematurely condensed chromosomes of lymphocytes; and (c) G1 lymphocytes after 20 h are influenced to only a minor degree by GO PHA stimulation. Arrows indicate the no. 1 C-banded I
1 Present address: Fritz-Haber-Institut f. Elektronenmikroskopie, Faradayweg 3-4; D-1000 Berlin 33, Germany.
chromosomes.
Exp Cd Res 134 (1981)
NOTES
Copyright @ 1981 by Academic Press, Inc. All rights of reproduction in any form reserved 0014-4827/81/080457-05$002.00/0
Circular nucleoids isolated from chloroplasts in a brown alga Bctocarpus siliculosus T. KUROIWA and T. SUZUKI, Depnrtment of Cell Biology, NationalInstitute 444 Japan
for Basic Biology,
Okazaki,
Circular nucleoids have been isolated from the chloroplasts of a brown alga, Ectocarpus indicus. by Nonidet P-40 treatment. Enzymatic treatments of the isolated nucleoids reveal that the nucleoid is a circle composed of bead-like particles interconnected by DNA strands. The beads contain predominantly DNA and proteins.
Summary.
Extranuclear DNA has been shown to occur in chloroplasts [l]. The chloroplast DNA (ctDNA) is concentrated in a specific portion of the chloroplast called the chloroplast nucleoid (ct-nucleoid). In Sphacelaria, Ectocarpus, Pylaiella, Dictyota, and Sorocarpus of the Phaeophyta, Ophiocytium, Botrydium and Tribonema of the Xanthopyta [2-5, 71 and Licomophora, Melosira, Rixosolenia of the Chrysophyta [6, 71, the chloroplast possesses a circular nucleoid inside the girdle lamella. The brown alga, Ectocarpus indicus, is well suited for the study of the isolation of chloroplast nucleoid and the organization of ctDNA at various phases of the chloroplast division cycle, as (a) ctDNA molecules are located at the periphery of the chloroplast to form a distinct circular nucleoid; (b) the contour length of the circle changes as chloroplast division progresses [7]; (c) the chloroplasts can be easily isolated from the cells. We report here the isolation of circular chloroplast nucleoids in the brown algae, 30-KllX16
indicus, in a form which could be digested with deoxyribonuclease but swelled with trypsin treatment.
Ectocarpus
Material
and Methods
A brown alga, Ectocarpus indicus, was collected by Dr Nakamura from a part of Mikawa Bay, Aichi prefecture. Examples were grown under a regimen of glternating 12 h liiht, 12 h d&k. The temperature was 15°C when the lights were on and 1PC in the dark. Chloroplasts were isolated from about 10 g of fresh algae after washing the algae thoroughly. The algae were suspended in 20 ml of buffer S (0.25% sucrose, 1 mM EDTA, 0.6 mM soermidine. 0.05% mercadoethanol, 10 mM Tris-HdI at pH 7.6) containing-O.4 mM ohenvlmethvlsulfonvlfluoride (PMSF) and homogenized in a gla& mortar. 20 ml df the mixture was further homogenized for 30 set at 3000 rpm in a 40 ml Blender cup, and diluted with an equal volume of buffer S. The final suspensions were filtered by gravity through two layers of nylon mesh with pores 31 pm in diameter (NBC Inc., Japan). The filtrates were centrifuged at 100 g for 5 min to remove the debris and unbroken cells. The chloroplasts were then sedimented for 5 min at 800 g. The sedimented chloroplasts were washed twice by resuspending in 5 ml of buffer S containing 0.4 mM PMSF followed bv recentrifugation, andldiluted with equal volume of duffer C (0.25 M sucrose. 0.4 mM PMSF. 10 mM Tris-HCI at PH 7.6). The isolated chloropl&ts were lysed in buffer S containing 0.5 % Nonidet P-40 with agitation at 20°C for 10 min followed by centrifugation at 4500 g for 5 min to remove the debris. The ct-nucleoids were sedimented from the supernatant by centrifugation at 20000 g for 5 min. DNA and RNA were extracted from isolated chloroplasts or isolated ct-nucleoids by the method of Daniel & Baldwin [S]. The extract was then assayed for DNA by the method of Burton [9]. RNA was analysed by the orcinol reaction [lo], and protein was determined by the Lowry method [I 11. Calf thymus DNA, yeast RNA and serum were used as standards. Dilute solutions of chloroplasts were counted by phase-contrast microscopy in Petroff-Hauser bacterial counter. The cells, isolated chloroplasts, and isolated ctnucleoids were fixed in buffer S containing 0.4% glutaraldehyde. Enzymatic digestions were performed on the fixed samples according to the method described previously [12, 131.The isolated ct-nucleoids, which had been incubated with ribonuclease on a glass microscope slide, were treated further with deoxyribonuclease, Sl nuclease or trypsin. As controls, duplicate slides similarly prepared were incubated with a portion of the buffer used as solvent for the deoxyribonuclease. A small drop of the fixed samples was placed on a slide and stained with DNA-specific fluorochrome, 4’6-diamidino-2-phenylindole (DAPI) Exp Cd Res 134 /198/j
458
Preliminury notes
Fig. I. Phase contrast (d, f). fluorescent (a. C, g-i), and phase-contrast fluorescent micrographs (b, e) demonstrating in situ chloroplasts ((I), isolated chloroplasts (h-4) and isolated chloroplast nucleoids (r-g) of Ectocurpu.s indicus. (c.. d) Phase-contrast and fluorescent micrographs of higher magnifications of a portion of isolated chloroplasts in (b). In the isolated chloroplasts a circular nucleoid is visible. cf, g) Phasecontrast and fluorescent micrographs of higher magnification of a portion of isaated chloroplast nucleoids in (0). Isolated chloroplast nucleoids appear to be free from contaminating materials and com-
posed of small, spherical particles. Prior to DAPI staining, isolated ct-nucleoids were treated with ribonuclease. In (h), this treatment was followed by treatina with the same buffer containina trvosin. and in (ir by the buffer containing deoxyr?bonuclease. The chloroplast nucleoid in (h) swells remarkably. By contrast, the circles of chloroplast nucleoid in (i) are broken into small, spherical particles and are finally digested completely by deoxyribonuclease. Bar, 5 pm. (u. h) x1400; (c) xl 100; (d, e) x3600: (f; R, i) x3000; (I?) x2800.
Preliminary
Table 1. Content of DNA, RNA and protein chloroplast
in isolated
Ectocarpus
notes
459
chloroplasts
and
nucleoid
Fraction
DNA (mid
Protein
RNA
Protein/DNA
RNA/DNA
Chloroplast
0.58f0.12” (5.8x 10-‘2)b
24Ok25 (24x 10-9
64413 (6.4x IO-“)
413.8
110.3
Chloroplast nucleoid
0.19f0.02
2.6kO.04
0.51~0.05
13.7
2.7
n Mean value with S.D. * Mean value per single chloroplast.
fluorescent and phase-contrast images demonstrating isolated chloroplasts containing a circular ct-nucleoid. Fig. 1c, d is higher magnification fluorescence (fig. lc) and phase-contrast (fig. Id) micrographs of a portion of fig. lb. The isolated chloroplasts are free of conResults and Discussion taminating materials and contain the circuFluorescence is restricted to the ct-nu- lar ct-nucleoid. Fig. le shows a combinacleoids and nuclei of the Ectocarpus. The tion micrograph of epifluorescent and nucleus appears as large blue fluorescent phase-contrast images of the ct-nucleoids spherule roughly in the center of the cell, isolated from the chloroplasts by Nonidet whereas the ct-nucleoid appears as a blue P-40 treatment. Large and small circular ctfluorescent circle (fig. 1a). This agrees with nucleoids can be seen; they retain the origielectron microscopic studies [2] demon- nal structure as seen in fig. 1a. The constrating the ctDNA located at the periphery tour length of the large isolated circle is of the disk-shaped chloroplasts. The circle about 25 pm which is about twice as long changes its shape according to the division as the small one. Therefore, the large nucycle of chloroplast [7]: as the chloroplast cleoid appears to be isolated from the elongates, becomes dumbbell-shaped, and chloroplast just before chloroplast division; divides into two daughter chloroplasts, the the small nucleoid just after chloroplast peripheral nucleoidal circle also transforms division. These results suggest that the ct-nucleoid accordingly. The contour length of the ct-nucleoid, can be isolated as a circle from the chlorowhich is about 26 pm just prior to chloro- plasts at various stages during the chloroplast division, becomes 13 pm as the result plast division cycle. The isolated ct-nuof chloroplast division [7]. Several dumb- cleoids appear to be entirely free of conbell-shaped nucleoids among circular nu- taminating materials, since fragments of cleoids are observed in cells, suggesting broken cellular nuclei did not show a circle that the chloroplasts divide semi-synchro- but showed relatively large spherical masnously in a single cell (fig. la). Fig. 1b ses, each 2-4 pm in diameter. The protein/DNA ratio indicates a high shows a combination micrograph of both dissolved in the buffer S at the concentration of 50 pg/ml [6]. Stained samples were examined by epifluorescent illumination at 350 nm on an Olympus BH epifluorescent microscope equipped with a phase objective lens. A combination microscopy of both epifluorescence and transmission phase-contrast microscopies was used to detect simultaneously the outline of the isolated chloroplasts and the circular ct-nucleoids.
Em Cd Res 134 (1981)
460
Preliminary
notes
degree of purity of the chloroplast nucleoid (table 1). The protein/DNA ratio of the chloroplast nucleoidal fraction is about 0.03 times that of the chloroplast fraction. The difference appears to be simply a reflection of the relative amount of chloroplast and chloroplast nucleoidal substance characteristic of Ectocarpus cells. DNA/single chloroplast is similar to that observed in Euglena chloroplasts [14], Antirrbinum chloroplasts [15] and Nicotiana chloroplasts [ 161. Fig. lf, g is higher magnification fluorescence (fig. lg) and phase-contrast (fig. lf) micrographs of a portion of fig. 1e. The isolated ct-nucleoids exhibit a circular structure which is composed of small beadlike particles, each 0.2 pm in diameter. The small particles are interconnected by a fine strand (fig. lg). The strand contains DNA, since the digestion with DNase for a short period (15 min) has led to progressive fragmentation of the circular ct-nucleoids (fig. 1i). The fragmentation, however, was not observed with Sl nuclease, RNase and trypsin treatments. Trypsin treatment causes remarkable swelling of the small particles (fig. 1h), but treatment with DNase for a long period (60 min) completely digested the small particles. These experiments suggest that the double-strand ct-DNA is the primary skeletal structure of the circular nucleoid, both in the string and the small particles, and proteins play an important role in organization of small particles. Since the chloroplast contains approx. 5.8~ lo-l5 g of DNA (table 1) and the chloroplast nucleoid is composed of about 30 bead-like particles [7], it is likely that each particle contains approx. 0.19~ IO-” pg of DNA which corresponds to a mol. wt of 114x10” D. This value per particle is consistent with the fact that the fluorescence intensity of the particles is Erp Cd Rrs 134 (19X/)
similar to that of T4 phages [7]. Therefore, the beadlike particles differ in their size and DNA content from nucleosomes [17]. Bisulpusra & Burton [18] proposed, on the basis of electron microscopic observations of a brown alga, Sphacelaria sp., a model that the individual DNA molecules in the circular nucleoid are associated with photosynthetic lamellae. If DNA molecules are dependently associated with lamellae, the circular structure of the ct-nucleoids must be destroyed by treatment of lamellae with Nonidet P-40. The existence of isolated circular nucleoids after the treatment suggests that, in addition to the association of DNA molecules with lamellae, there must be a mechanism for organization of ctDNA molecules. Using this isolation method, it should be possible to further analyse the fine structure of ct-nucleoids, to characterize the trypsinsensitive protein species in the ct-nucleoids, and finally, to elucidate the mechanisms of organization of ctDNA. References Ris, H & Plaut, W, J cell biol 13 (1962) 383.
:: Bisulputra, T & Bisalputra, A A, J ultrastruct res 29 (1969) 151. - Ibid 32 (1970) 417. Gibbs, S P, J cell sci 3 (1968) 327. Coleman, A W, J cell bio182 (1979) 299. Kuroiwa, T, Suzuki, T & Kawano, S, Cell struct func 4 (1979) 399. 7. Kuroiwa, T, Suzuki, T, Ogawa, K & Kawano, S, Plant cell physiol 22 (1981) 322. 8. Daniel, J W & Baldwin. H H. Methods in cell physiology (ed D M Prescott) vol. I, pp. 9-40. Academic Press, New York (1964). 9. Burton, K, Biochem j 62 (1956) 315. IO. Ceriotti, G, J biol them 214 (1955) 59. 11. Lowry, 0 H, Rosebrough, N J, Fax, A L & Randall, R J, J biol them 193(1951) 265. 12. Kuroiwa, T, J cell biol63 (1976) 299. 13. Shenk, T E, Phodges, C, Rigby, P W J & Berg, P, Proc natl acad sci US 72 (1975) 989. 14. Chun, E H L, Vaughan, N H & Rich, J A, J mol biol 7 (1963) 130. 15. Ruppel, H G & Wyki, U D, Z Pflanzenphysiol 53 (1965) 32. 16. Tewari, K K & Wildman, S G, Science 153 (1966) 1269. 17. Olins, A L & Olins, DE. Science 183(1964) 330. 3. 4. 5. 6.
Preliminary
notes
461
18. Bisulputra, T & Burton. H, J ultrastruct res 29 (1969) 224. Received December 29, 1980 Revised version received March 30, 1981 Accepted April 2, 1981
Copyright 0 1981 by Academic Press, Inc. All rights of reproduction in any form reserved 0014.4827/81/080461-05%02.00/0
Length of human prematurely condensed chromosomes during GO and Gl phase HARD1 SCHMIADY’
a
and KARL SPERLING, In-
stitut far Humangenetik. Freie Universit6t Herrhnml~eg 6, D-1000 Berlin 19, Gerrntrn>
Berlin.
Summary. Length measurements on C-banded prematurely condensed no. 1 human chromosomes of GO and Gl lymphocytes, as well as of synchronized Gl HEp cells revealed that (i) no length difference exists between mitotic chromosomes and GO chromosomes; (ii) 1 h after PHA stimulation a clear increase in length is detectable; (iii) in isolated cases an increase by the factor 5 can be observed during Gl; (iv) the increase is significantly less for constitutive heterochromatin than for euchromatin. The possibility is discussed that these conformational changes of chromatin reflect physiological differences, i.e. the rate of RNA synthesis during interphase.
Ceil cycle-related changes/inrhe confor a-;’ tional patterns of chromatin have been CT ‘emonstrated by biochemical and histochemical techniques, but more directly by application of the phenomenon of premature chromosome condensation (PCC) [l-7]. After fusion between mitotic and interphase cells the chromatin of the latter enters into a mitosis-like condensation *r process. The morphology of prematurely condensed chromosomes clearly reflects their position in the cell cycle at the time of fusion, Gl, S or G2 phase [S, 91. However, a wide range of variability in chromosome length is observed within individual Fig. 1. (a) Human lymphocyte metaphase chromoGl and G2 cells. These length differences somes: (b) prematurely condensed chromosomes of lymphocytes; and (c) G1 lymphocytes after 20 h are influenced to only a minor degree by GO PHA stimulation. Arrows indicate the no. 1 C-banded I
1 Present address: Fritz-Haber-Institut f. Elektronenmikroskopie, Faradayweg 3-4; D-1000 Berlin 33, Germany.
chromosomes.
Exp Cd Res 134 (1981)