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Gamma irradiation effects on two species of the green alga Sirogonium with different chromosome types
Enviromnental and Expermu.ntal Botany, Vol. '20, pp. 39 to 45 Pergamon Pre~ Ltd. 1980. Printed in Great Britain
G A M M A I R R A D I A T I O N EFFECTS O N T W O SPECIES OF THE G R E E N ALGA SIROGOWIUM W I T H D I F F E R E N T C H R O M O S O M E TYPES CHARLES V . W~.IJ.S* a n d R O B E R T
W. H O S H A W
Department of Ecology and Evolutionary Biology, University of Arizona, Tucson, Arizona 85721, U.S.A.
(Received 28 November 1978; accepted in revisedform 7 July 1979) WELLS C. V. and HOSHAW R. W. Gamma irradiation effects on two species of the green alga Sirogonium. ENWROmaErCrAL AND EXPERiraENTAL BOTANY 20, 39--45, 1980.--Irradiation effects from 6°Co at 3.0 kR and 15.0 kR on Sirogonium melanosporum (Randhawa) Transeau and Sirogonium sticticum (,J. E. Smith) Kfitzing were compared. Sirogonium melanosporum cells exposed to 3.0kR showed a reduction of cell division from 13.0% to 5.0% with chromosomal fragments occurring in 23.0% of the cells. Chromosomal fragments were not detected in S. sticticum cells exposed to 3.0kR. In S. rnelanosporum with 15.0kR, no cell division was visible on the first day after treatment, but by the third day 1.0% of the cells were undergoing mitosis and chromosomal fragments were present in 52.0% of the dividing nuclei. In S. sticticura exposed to 15.0kR, cytokinesis but no mitosis was observed on the first day after treatment. However, filaments contained giant cells ( > 500/zrn long) and micro-cells ( < 30/zm long). By days 4 and 13 there were 2.0% and 26.0% giant cells, respectively, when 15.0kR was applied to actively dividing cells. When cell division was minimal during irradiation treatment, only 5.0o/o giant cells were produced by day 13. Giant cells contained nuclei over double the length and almost double the width of untreated cells, and observation of 3500 of these cells showed no nuclear divisions. By contrast, micro-cells lacked nuclei and did not survive. Cells other than giant cells or micro-cells divided actively to produce vigorous cultures of S. sticticum during a 6-month observational period. Filaments of S. melanosporum receiving 15.0 kR survived for only 3 months. INTRODUCTION ARE chloroplast-containing organisms which are ideal for observing i r r a d i a t i o n i n d u c e d cytoplasmic and nuclear alterations because direct e x a m i n a t i o n can be m a d e microscopically on i n d i v i d u a l cells. Alterations m a y occur in mitosis, cytokinesis and cell size as well as in i n d i v i d u a l cell structures such as nuclei, chromosomes and chloroplasts. In the present investigation g a m m a irradiation was used from ALGAE
a 6°Co source to induce alterations in two species of Sirogonium, a genus closely allied to the filamentous, green alga Spirogyra. Sirogonium melanosporum a n d S. sticticum are a u n i q u e pair in which to e x a m i n e cytological alterations, including centromere structure and behavior. Sirogonium melanosporum possesses 6 relatively large (5-6 # m long) chromosomes 'with parallel separation o f chromatids occurring at a n a p h a s e ; S. sticticura has a p p r o x i m a t e l y 50 m i n u t e (1 # m d i a m e t e r ) dotlike chromosomes. This is the first
*Present address: Biology Department, Lenoir Rhyne College, Hickory, North Carolina 28601, U.S.A. 39
~)
CHARLES V. WEI,I,S and ROBERT W. HOSHAW
known investigation with g a m m a irradiation in which two closely related species have been used to compare cytoplasmic and nuclear alterations and to describe centromeric condition.
MATERIALS AND METHODS
Sirogonium rnelanosporum and S. sticticum (Figs. 1 and 2) are maintained continuously in the Algal Research Laboratory at The University of Arizona. Cultures were grown in ½ pint milk bottles of soil-water medium uT~ under 20-W cool-white fluorescent lamps of ca. 3000 lx on 12:12 L : D at 20__+2°C (hereafter referred to as standard conditions). Masses of filaments of S. sticticum were irradiated with 6°Co at 3.0kR and 15.0 kR levels during times of minimal and maximal mitotic activity. Doses of 3.0 kR and 15.0kR were administered at 320r/min and 500r/min, respectively. Irradiation was monitored by an ionization chamber detector (Radocon Model No. 575, Victoreen Instrument Co.). Irradiation of both species was identical except filaments of S. melanosporum were exposed during maximum mitotic activity only. The times of minimum and maximum mitotic activity were determined by sampling cultures at ½-hr intervals during a 24-hr period. After irradiation, filaments were subcultured in six bottles of soil-water medium and grown under standard conditions. Daily observations were made during a 28-day period to detect major cytological and nuclear alterations. Filaments were fixed in Freytag's fixative ~5) before staining with a modification of the Feulgen technique or Sudan black B~22). One hundred cells from each subculture were examined daily for 28 days. RESULTS
Effects on S. melanosporum Filaments exposed to 3.0kR and 15.0kR showed reduced cell division as well as nuclear and chromosomal damage (Figs. 3 and 4). Cells irradiated with 3.0 kR during maximum mitotic activity and examined on day 1 after treatment shmved cell division reduced from 13.0°o to 5.0"~, with chromosome damage in 23.0°o of
the cells. This damage resulted in 42.0°o of the cells with a single chromosomal fragment, 17.0°0 with two fragments and 42.0°o with three or more fragments. When cells showing maximimum mitotic activity were irradiated with 15.0kR, no mitotic figures were observed on the first day after treatment. However, a few cells ( < 1.00o) exhibited cytokinesis without mitosis, resulting in a nucleated and a nonnucleated cell. Bv day 3 after irradiation, 1.0",, of tile cells were undergoing mitosis, with chromosolnal li'agments [)resent ill 52.0",, o[" the dividing nuch'i II"iv,. 3 alld 1). All clu~ml~> somal fragments appeared to move toward the poles at anaphase. Large chromosomal fragments moved with the undamaged chromosomes and demonstrated normal parallel separation (Fig. 3). Filaments receiving 3.0kR survived for 6 months, whereas those receiving 15.0kR survived for only 3. Effects on S. sticticum Filaments exposed to 3.0kR and 15.0kR showed reduced cell division, but chromosomal fragments were not detected among the approximately 50 dotlike chromosomes. Temporary reduction in cell division was the only effect noted with irradiation at 3.0 kR and cultures were growing vigorously at the end of 6 months. On dav 1 following irradiation with 15.0 kR cytokinesis occurred in a few cells without evidence of cell division. However, many filaments contained giant cells (>500/am long) and micro-cells ( < 3 0 # m long) as compared to a range of 70-245/~m for untreated cells (Fig. 2). When actively dividing cells were exposed to 15.0kR, 2.0°0 were giant cells by day 4 and 26.0°'o by day 13. If cell division were minimal during irradiation, onlv 5.0°o giant cells were produced by day 13. Most giant cells increased in width in the nuclear region. Nuclei and nucleoli were approximately double the length and width of these organelles in untreated cells (Table 1). Chromosomes of the large nuclei were not observed because an examination of 3500 giant cells failed to reveal any cell undergoing mitosis. In abnormally wide giant cells, the typical straight ribbon-like chloroplasts became S- and U-shaped. Chloroplasts nearest the
Fins. 1-7, Microscopic views of Sirogonium melanosporum and ,Sirogomum ~ticucum, Figs. 1 and 2: Untreated living vegetative cells of S. melanosporum and S. sticticum, respectively: nucleus lnu): cell wall lc~): chloroplast (ch): pyrenoid (py): x300 and x350, respectively. Fig. 3: Camera lucida drawing of chromosomes of S. melanosporum in mitotic anaphase with chromosomal fragments (arrows) produced by exposure to 15,0kR. x6500. Fig. 4: Prophase nucleus of S. melimosporum stained with Sudan black B to show chromt~so,nal fragments {arrows) produced by exposure to 15.0kR: x 3500. Fig. 5: Portion of a giant cell of 5. Jticlicum produced by exposure to 15.0kR showing chloroplasts surrounding nucleus (arro~ I: x 160. Fig. 6: Two micro-cells and portions of two giant cells of S. sticticum produced b~ exposure to 15.(I kR showing chloroplast {arrows} continuous through cell walls: x 865. Fig. 7: Portion of giant cell ofS. aticticum produced by cxposmc
GAMMA IRRADIATION EFFECTS ON TWO SPECIES OF GREEN ALGA SIROGOA'IUM lateral walls and adjacent to the nucleus became pinched or constricted towards the center of the cell (Fig. 7), thus enclosing the nucleus (Fig. 5). Although chloroplasts often reached 700~m in length, they contained the same number of pyrenoids as untreated cells. Giant cells reached a mean length of 1129_+ 11.1 #m and died without dividing (Tabh' 1 I.
Table 1. Effects of gamma irradiation of 15.0kR on cell length and nuclear and nucleolar dimensions in Sirogonium sticticum Mean measurements (#m)* Irradiated Untreated
Structures Cell lengthl Nuclear length+ Nuclear width+* Nucleolar diameter~
1129.04- 11.1 22.24- 0.3 15.54- 0.5 8.44- 0.1
170.04- 1.9 9.84-0.3 9.1 4- 1.0 4.24-0.0
*Based on measurements, for 25 cells. 1"Measurements on living filaments. +*Measurements on nuclei and nucleoli stained with propi(wm'milw.
Micro-cells (7.5-30.0~m long) were produced by an unequal division of cells following irradiation with 15.0kR (Fig. 6). These cells lacked nuclei and did not survive. Figure 6 shows that chloroplasts may pass through the cell walls of micro-cells. While giant cells and micro-cells did not divide, other cells of filaments in irradiated cultures divided actively to produce vigorous cultures with normal growth during a 6 month observational period. DISCUSSION
Although irradiation with 3.0 kR slowed and reduced mitosis in both species of Sirogonium, the filaments resumed growth quickly and were growing vigorously after 6 months of subculturing. When irradiation was increased to 15.0 kR mitosis was considerably slowed in both species, apparently because of greater nuclear damage. Sirogonium melanosporum cultures survived for only 3 months following irradiation, whereas cultures of S. sticticum were still growing vigorously after 6 months wh('n ohs('r~ations were
43
terminated. This differential sensitivity between species may be due to chromosome sizes. The large chromosomes of S. melanosporum may provide a larger target area than the small dot chromosomes of S. sticticum. Greater irradiation sensitivity of large chromosomes has been reported earlier by SPARROW and CHRISTENSEN (19) for the flowering plants Tradescantia, Lilium and Vicia and by SAR,MAand SINGH (18) for the algae Chara and )v'itella. SPARROW eta/. (2°) postulated that species with low chromosome numbers are more sensitive to irradiation than species with high numbers. Our data agree with these observations: S. melanosporum with 6 large chromosomes received greater damage than S. sticticum with 50 small dodike chromosomes. X-irradiation and gamma-irradiation have been used to demonstrate the centromeric condition of chromosomes with parallel separation of chromatids. (3'6' ta,21,23) In these investigations chromosomes were fragmented and chromatid movement was examined at anaphase. Cells of S. melanosporum irradiated with 3.0 kR and 15.0kR contained chromosome fragments which moved to the poles during anaphase. Large fragments always continued to separate in parallel fashion. Since no fragments were left behind, they apparently have diffuse centromeres or are polycentric. Such centromeric distribution has already been reported for several species. MUGHAL and GODWARD(12) demonstrated a polycentric condition for the green alga Spirogyra majuscuta. Certain hemipteran insects, Rhodnium prolixus and Oncopeltus fasciatus, have been reported by BucK {2) and by COMINGS and OKODA (4) tO possess a diffuse type centromere over the entire chromosomal surface. BRASELTON(1} found several blocks of centromerle material at intervals along chromosomes for the flowering plants Luzula purpurea and Cyperus alternifolius. Even though our evidence and that of others suggest that chromosomes of diverse species of plants and animals have diffuse centromeres or are polycentric, observations at the ultrastructural level might clarify' this interpretation in S. melanosporum. Sirogonium melanosporum appears to be highly resistant to chromosomal fragmentation compared to the closely related alga Spirog~ra crassa which has 12 chromosomes. GODWARD(?) ir-
44
CHARLES V. WELLS and ROBERT W. HOSHAW
radiated filaments of S. crassa with 15.0 kR and found 90 chromosome fragments per damaged nucleus. By contrast, Sirogonium melanosporum with its six chromosomes had less than 10 fragments. While this alga may possess greater resistance to chromosomal fragmentation, it is possible that numerous fragments were not observed because badly damaged cells failed to survive irradiation. O u r inability to observe chromosomal fragments in S. sticticum subjected to either 3.0 or 15.0 kR may result from the difficulty in identifying fragments a m o n g dotlike chromosomes. However, no acentric fragments were seen during the movement of chromosomes at anaphase. This suggests that the entire chromosome has centromeric activity. Also, fragments m a y become surrounded by the nucleolar substance which is known to assist chromosome movement during mitosis. ~1°' 22) A conspicuous irradiation effect in S. sticticum was the formation of giant cells. U p to 26.0% of the cells in S. sticticum were giant cells following irradiation during periods of active cell division. Although the mechanism of origin is unknown, giant cell formation from ionizing radiation has also been reported for three algae: Mougeotia, ~16) Chlorella ~8) and ZygnemaJ TM Additionally, in the filamentous green alga Oedogonium cardiacum, HORSLEY et al. 0~ reported that the G 2 period of the cell cycle was most sensitive to irradiation damage, based on giantcell occurrence. The unicellular green alga Chlorella is most sensitive to irradiation prior to D N A synthesisJ s~ PAINTER et al. 113~ report the probability following X-irradiation of H e L a S 3 cells surviving to produce giant cells, independent of a cell's position in the D N A metabolic cycle. Although giant cells did not undergo mitosis, they evidently were able to continue the synthesis of R N A and the enzymes required for extensive cell wall elongation. The large nuclei of giant cells may represent polyploids as suggested by GODWARD (7) for ~VIougeotia. Since giant cells do not form cross walls that cut chloroplasts in two and distribute them to the two division products, they contain chloroplasts which constrict and accumulate around the nucleus. This behavior is difficult to interpret
because the constriction is not associated with any organellar movement. However, PICKETTHEAPS~xs) has reported in Spirogyra that constricdon of chloroplasts is mediated by the plasma membrane. Algal structure and behavior are subject to major alteration by gamma-irradiation. In addition, by selecting species of Sirogonium with rodlike and dotlike chromosome types, markedly altered behavior and structural aberrations may be demonstrated in different species of a single genus.
Acknowledgements--A portion of this research was supported by NSF grants GB-25130 and DEB76-82624 to R.W.H. The authors thank Dr. HERBERT M. HULL for his critical review of the manuscript.
RE]FEII.]KNI21~-~ 1. BRASELTONJ. P. (1971) The ultrastructure of the non-localized kinetochores in Luzula and Cyperus. Chromosoma ~ 89-99.. 2. Buck R. C. (1967) Mitosis and meiosis in Rhodnius prolixus: the fine structure of the spindle and diffuse kinetochore. 07. Ultrastruct. l~s. 18, 489-501. 3. CASTRO D. DE, CAMARA A. and MALHEIROSN. (1949) X-rays in the centromere problem of Luzula purpurea Link. Genet. Iber. 1, 49-54. 4. COMINOS D. E. and OKODA T. A. (1972) Holocentric chromosomes in Oncopeltus: kinetochore plates are present in mitosis but absent in meiosis. Chroraosoma 37, 177-i 92. 5. FREY'rAGA. H. (1964) Use of commercial bleach to improve separation of orchid chromosomes. Stain Technol. 39~ 167-169. 6. GODWARD M. B. E. (1954) Irradiation of Spirogyra chromosomes. H~;~dity ~ 293 (Abstr.). 7. GOt)WARDM. B. E. (1962) Invisible irradiation. Pages 551-566 in R. A. LEWlN, ed. Physiology and biochemistry of algae. Academic, New York. 8. HAMPL W., ALTMANN H. and BIEBL R. (1971) Unterschiede und Beeinflussung der Erholungskapazit/it von Chlorellazellen in verschiedenen Stadien des Zellzyklus. Radiat. Bot. 11, 201-207. 9. HOgSLEYR. J., BANERJEES. N. and BANERJEEN. (1967) Analysis of lethal responses in Oedogonium cardiacum irradiated at different cell stages. Radiat. Bot. 7, 465-476.
GAMMA IRRADIATION EFFECTS ON TWO SPECIES OF GREEN ALGA SIROGO~TUM 10. HosnAw R. W. and WAER R. D. (1967) Polycentric chromosomes in Sirogonium melanosporum. Can. J. Bot. 45~ 1169-1171. 11. HUOHEs-ScHR~a)ER S. and RIs H. (1941) The diffuse spindle attachment of coccids, verified by the mitotic behavior of induced chromosome fragments. J. Exp. Zool. M, 429-456. 12. MUOnAL S. and GODWARD M. B. E. (1973) K_inetochore and microtubules in two members of Chlorophyceae, Cladophorafracta and Spirogyra majuscula. Chromosoma14~ 213-229. 13. PAINTER R. B., McA~PXNE V. W. R. and GERMAmS M. (1961) The relationship of the metabolic state of deoxyribonucleic acid during X-irradiation to HeLa S3 giant cell formation. Radiat. Res. 14, 653-661. 14. PF.TROVAJ. (1962) The 'direct' and 'indirect' effects of ionizing radiation on the alga Zygnema. Folia Biol. 9~ 51-58. 15. PICKETT-HEAPSJ. D. (1975) Green algae: structure, reproduction and evolution in selected genera. Sinauer, Sunderland, Massachusetts. 606 p. 16. PRASAD B. N. and GODWARD M. B. E. (1968) Cytology of irradiated Mougeotia sp. .Nucleus (Calcutta) 11, 43-49. 17. PRINCSHmM E. G. (1946) The biphasic or soil-
18.
19.
20.
21.
22.
23.
45
water culture method for growing algae and flagellata, j . Ecol. 33, 193-204. SARMA Y. S. R. K. and SINGH S. B. (1977) Irradiation studies on the karyology of Charophyta--I. X-rays and gamma-rays. Cytologia 42, 279-290. SPARROW A. H. and CHRISTENSEN E. (1954) Tolerance of certain higher plants to chronic exposure to gamma radiation from cobalt-60. Science 118, 697-698. SPARROW A. H., UNDERBRINK A. G. and SP^RROW R. C. (1967) Chromosomes and cellular radiosensitivity--I. The relationship of D o to chromosome volume and complexity in seventy-nine different organisms. Radiat. Res. 32, 915-945. TANAKAN. and TANAKAN. (1977) Chromosome studies in Chionographis (Liliaceae) I. On the holokinetic nature of chromosomes in Chionographis japonica Maxim. Cytologia 42, 753763. WELLS C. V. and HOSnAW R. W. (1971) The nuclear cytology of Sirogonium. J. Phycol. 7, 279284. WroTE M. J. D. (1936) Chromosome cycle of Ascaris megalocephala. Nature 137, 783.
G A M M A I R R A D I A T I O N EFFECTS O N T W O SPECIES OF THE G R E E N ALGA SIROGOWIUM W I T H D I F F E R E N T C H R O M O S O M E TYPES CHARLES V . W~.IJ.S* a n d R O B E R T
W. H O S H A W
Department of Ecology and Evolutionary Biology, University of Arizona, Tucson, Arizona 85721, U.S.A.
(Received 28 November 1978; accepted in revisedform 7 July 1979) WELLS C. V. and HOSHAW R. W. Gamma irradiation effects on two species of the green alga Sirogonium. ENWROmaErCrAL AND EXPERiraENTAL BOTANY 20, 39--45, 1980.--Irradiation effects from 6°Co at 3.0 kR and 15.0 kR on Sirogonium melanosporum (Randhawa) Transeau and Sirogonium sticticum (,J. E. Smith) Kfitzing were compared. Sirogonium melanosporum cells exposed to 3.0kR showed a reduction of cell division from 13.0% to 5.0% with chromosomal fragments occurring in 23.0% of the cells. Chromosomal fragments were not detected in S. sticticum cells exposed to 3.0kR. In S. rnelanosporum with 15.0kR, no cell division was visible on the first day after treatment, but by the third day 1.0% of the cells were undergoing mitosis and chromosomal fragments were present in 52.0% of the dividing nuclei. In S. sticticura exposed to 15.0kR, cytokinesis but no mitosis was observed on the first day after treatment. However, filaments contained giant cells ( > 500/zrn long) and micro-cells ( < 30/zm long). By days 4 and 13 there were 2.0% and 26.0% giant cells, respectively, when 15.0kR was applied to actively dividing cells. When cell division was minimal during irradiation treatment, only 5.0o/o giant cells were produced by day 13. Giant cells contained nuclei over double the length and almost double the width of untreated cells, and observation of 3500 of these cells showed no nuclear divisions. By contrast, micro-cells lacked nuclei and did not survive. Cells other than giant cells or micro-cells divided actively to produce vigorous cultures of S. sticticum during a 6-month observational period. Filaments of S. melanosporum receiving 15.0 kR survived for only 3 months. INTRODUCTION ARE chloroplast-containing organisms which are ideal for observing i r r a d i a t i o n i n d u c e d cytoplasmic and nuclear alterations because direct e x a m i n a t i o n can be m a d e microscopically on i n d i v i d u a l cells. Alterations m a y occur in mitosis, cytokinesis and cell size as well as in i n d i v i d u a l cell structures such as nuclei, chromosomes and chloroplasts. In the present investigation g a m m a irradiation was used from ALGAE
a 6°Co source to induce alterations in two species of Sirogonium, a genus closely allied to the filamentous, green alga Spirogyra. Sirogonium melanosporum a n d S. sticticum are a u n i q u e pair in which to e x a m i n e cytological alterations, including centromere structure and behavior. Sirogonium melanosporum possesses 6 relatively large (5-6 # m long) chromosomes 'with parallel separation o f chromatids occurring at a n a p h a s e ; S. sticticura has a p p r o x i m a t e l y 50 m i n u t e (1 # m d i a m e t e r ) dotlike chromosomes. This is the first
*Present address: Biology Department, Lenoir Rhyne College, Hickory, North Carolina 28601, U.S.A. 39
~)
CHARLES V. WEI,I,S and ROBERT W. HOSHAW
known investigation with g a m m a irradiation in which two closely related species have been used to compare cytoplasmic and nuclear alterations and to describe centromeric condition.
MATERIALS AND METHODS
Sirogonium rnelanosporum and S. sticticum (Figs. 1 and 2) are maintained continuously in the Algal Research Laboratory at The University of Arizona. Cultures were grown in ½ pint milk bottles of soil-water medium uT~ under 20-W cool-white fluorescent lamps of ca. 3000 lx on 12:12 L : D at 20__+2°C (hereafter referred to as standard conditions). Masses of filaments of S. sticticum were irradiated with 6°Co at 3.0kR and 15.0 kR levels during times of minimal and maximal mitotic activity. Doses of 3.0 kR and 15.0kR were administered at 320r/min and 500r/min, respectively. Irradiation was monitored by an ionization chamber detector (Radocon Model No. 575, Victoreen Instrument Co.). Irradiation of both species was identical except filaments of S. melanosporum were exposed during maximum mitotic activity only. The times of minimum and maximum mitotic activity were determined by sampling cultures at ½-hr intervals during a 24-hr period. After irradiation, filaments were subcultured in six bottles of soil-water medium and grown under standard conditions. Daily observations were made during a 28-day period to detect major cytological and nuclear alterations. Filaments were fixed in Freytag's fixative ~5) before staining with a modification of the Feulgen technique or Sudan black B~22). One hundred cells from each subculture were examined daily for 28 days. RESULTS
Effects on S. melanosporum Filaments exposed to 3.0kR and 15.0kR showed reduced cell division as well as nuclear and chromosomal damage (Figs. 3 and 4). Cells irradiated with 3.0 kR during maximum mitotic activity and examined on day 1 after treatment shmved cell division reduced from 13.0°o to 5.0"~, with chromosome damage in 23.0°o of
the cells. This damage resulted in 42.0°o of the cells with a single chromosomal fragment, 17.0°0 with two fragments and 42.0°o with three or more fragments. When cells showing maximimum mitotic activity were irradiated with 15.0kR, no mitotic figures were observed on the first day after treatment. However, a few cells ( < 1.00o) exhibited cytokinesis without mitosis, resulting in a nucleated and a nonnucleated cell. Bv day 3 after irradiation, 1.0",, of tile cells were undergoing mitosis, with chromosolnal li'agments [)resent ill 52.0",, o[" the dividing nuch'i II"iv,. 3 alld 1). All clu~ml~> somal fragments appeared to move toward the poles at anaphase. Large chromosomal fragments moved with the undamaged chromosomes and demonstrated normal parallel separation (Fig. 3). Filaments receiving 3.0kR survived for 6 months, whereas those receiving 15.0kR survived for only 3. Effects on S. sticticum Filaments exposed to 3.0kR and 15.0kR showed reduced cell division, but chromosomal fragments were not detected among the approximately 50 dotlike chromosomes. Temporary reduction in cell division was the only effect noted with irradiation at 3.0 kR and cultures were growing vigorously at the end of 6 months. On dav 1 following irradiation with 15.0 kR cytokinesis occurred in a few cells without evidence of cell division. However, many filaments contained giant cells (>500/am long) and micro-cells ( < 3 0 # m long) as compared to a range of 70-245/~m for untreated cells (Fig. 2). When actively dividing cells were exposed to 15.0kR, 2.0°0 were giant cells by day 4 and 26.0°'o by day 13. If cell division were minimal during irradiation, onlv 5.0°o giant cells were produced by day 13. Most giant cells increased in width in the nuclear region. Nuclei and nucleoli were approximately double the length and width of these organelles in untreated cells (Table 1). Chromosomes of the large nuclei were not observed because an examination of 3500 giant cells failed to reveal any cell undergoing mitosis. In abnormally wide giant cells, the typical straight ribbon-like chloroplasts became S- and U-shaped. Chloroplasts nearest the
Fins. 1-7, Microscopic views of Sirogonium melanosporum and ,Sirogomum ~ticucum, Figs. 1 and 2: Untreated living vegetative cells of S. melanosporum and S. sticticum, respectively: nucleus lnu): cell wall lc~): chloroplast (ch): pyrenoid (py): x300 and x350, respectively. Fig. 3: Camera lucida drawing of chromosomes of S. melanosporum in mitotic anaphase with chromosomal fragments (arrows) produced by exposure to 15,0kR. x6500. Fig. 4: Prophase nucleus of S. melimosporum stained with Sudan black B to show chromt~so,nal fragments {arrows) produced by exposure to 15.0kR: x 3500. Fig. 5: Portion of a giant cell of 5. Jticlicum produced by exposure to 15.0kR showing chloroplasts surrounding nucleus (arro~ I: x 160. Fig. 6: Two micro-cells and portions of two giant cells of S. sticticum produced b~ exposure to 15.(I kR showing chloroplast {arrows} continuous through cell walls: x 865. Fig. 7: Portion of giant cell ofS. aticticum produced by cxposmc
GAMMA IRRADIATION EFFECTS ON TWO SPECIES OF GREEN ALGA SIROGOA'IUM lateral walls and adjacent to the nucleus became pinched or constricted towards the center of the cell (Fig. 7), thus enclosing the nucleus (Fig. 5). Although chloroplasts often reached 700~m in length, they contained the same number of pyrenoids as untreated cells. Giant cells reached a mean length of 1129_+ 11.1 #m and died without dividing (Tabh' 1 I.
Table 1. Effects of gamma irradiation of 15.0kR on cell length and nuclear and nucleolar dimensions in Sirogonium sticticum Mean measurements (#m)* Irradiated Untreated
Structures Cell lengthl Nuclear length+ Nuclear width+* Nucleolar diameter~
1129.04- 11.1 22.24- 0.3 15.54- 0.5 8.44- 0.1
170.04- 1.9 9.84-0.3 9.1 4- 1.0 4.24-0.0
*Based on measurements, for 25 cells. 1"Measurements on living filaments. +*Measurements on nuclei and nucleoli stained with propi(wm'milw.
Micro-cells (7.5-30.0~m long) were produced by an unequal division of cells following irradiation with 15.0kR (Fig. 6). These cells lacked nuclei and did not survive. Figure 6 shows that chloroplasts may pass through the cell walls of micro-cells. While giant cells and micro-cells did not divide, other cells of filaments in irradiated cultures divided actively to produce vigorous cultures with normal growth during a 6 month observational period. DISCUSSION
Although irradiation with 3.0 kR slowed and reduced mitosis in both species of Sirogonium, the filaments resumed growth quickly and were growing vigorously after 6 months of subculturing. When irradiation was increased to 15.0 kR mitosis was considerably slowed in both species, apparently because of greater nuclear damage. Sirogonium melanosporum cultures survived for only 3 months following irradiation, whereas cultures of S. sticticum were still growing vigorously after 6 months wh('n ohs('r~ations were
43
terminated. This differential sensitivity between species may be due to chromosome sizes. The large chromosomes of S. melanosporum may provide a larger target area than the small dot chromosomes of S. sticticum. Greater irradiation sensitivity of large chromosomes has been reported earlier by SPARROW and CHRISTENSEN (19) for the flowering plants Tradescantia, Lilium and Vicia and by SAR,MAand SINGH (18) for the algae Chara and )v'itella. SPARROW eta/. (2°) postulated that species with low chromosome numbers are more sensitive to irradiation than species with high numbers. Our data agree with these observations: S. melanosporum with 6 large chromosomes received greater damage than S. sticticum with 50 small dodike chromosomes. X-irradiation and gamma-irradiation have been used to demonstrate the centromeric condition of chromosomes with parallel separation of chromatids. (3'6' ta,21,23) In these investigations chromosomes were fragmented and chromatid movement was examined at anaphase. Cells of S. melanosporum irradiated with 3.0 kR and 15.0kR contained chromosome fragments which moved to the poles during anaphase. Large fragments always continued to separate in parallel fashion. Since no fragments were left behind, they apparently have diffuse centromeres or are polycentric. Such centromeric distribution has already been reported for several species. MUGHAL and GODWARD(12) demonstrated a polycentric condition for the green alga Spirogyra majuscuta. Certain hemipteran insects, Rhodnium prolixus and Oncopeltus fasciatus, have been reported by BucK {2) and by COMINGS and OKODA (4) tO possess a diffuse type centromere over the entire chromosomal surface. BRASELTON(1} found several blocks of centromerle material at intervals along chromosomes for the flowering plants Luzula purpurea and Cyperus alternifolius. Even though our evidence and that of others suggest that chromosomes of diverse species of plants and animals have diffuse centromeres or are polycentric, observations at the ultrastructural level might clarify' this interpretation in S. melanosporum. Sirogonium melanosporum appears to be highly resistant to chromosomal fragmentation compared to the closely related alga Spirog~ra crassa which has 12 chromosomes. GODWARD(?) ir-
44
CHARLES V. WELLS and ROBERT W. HOSHAW
radiated filaments of S. crassa with 15.0 kR and found 90 chromosome fragments per damaged nucleus. By contrast, Sirogonium melanosporum with its six chromosomes had less than 10 fragments. While this alga may possess greater resistance to chromosomal fragmentation, it is possible that numerous fragments were not observed because badly damaged cells failed to survive irradiation. O u r inability to observe chromosomal fragments in S. sticticum subjected to either 3.0 or 15.0 kR may result from the difficulty in identifying fragments a m o n g dotlike chromosomes. However, no acentric fragments were seen during the movement of chromosomes at anaphase. This suggests that the entire chromosome has centromeric activity. Also, fragments m a y become surrounded by the nucleolar substance which is known to assist chromosome movement during mitosis. ~1°' 22) A conspicuous irradiation effect in S. sticticum was the formation of giant cells. U p to 26.0% of the cells in S. sticticum were giant cells following irradiation during periods of active cell division. Although the mechanism of origin is unknown, giant cell formation from ionizing radiation has also been reported for three algae: Mougeotia, ~16) Chlorella ~8) and ZygnemaJ TM Additionally, in the filamentous green alga Oedogonium cardiacum, HORSLEY et al. 0~ reported that the G 2 period of the cell cycle was most sensitive to irradiation damage, based on giantcell occurrence. The unicellular green alga Chlorella is most sensitive to irradiation prior to D N A synthesisJ s~ PAINTER et al. 113~ report the probability following X-irradiation of H e L a S 3 cells surviving to produce giant cells, independent of a cell's position in the D N A metabolic cycle. Although giant cells did not undergo mitosis, they evidently were able to continue the synthesis of R N A and the enzymes required for extensive cell wall elongation. The large nuclei of giant cells may represent polyploids as suggested by GODWARD (7) for ~VIougeotia. Since giant cells do not form cross walls that cut chloroplasts in two and distribute them to the two division products, they contain chloroplasts which constrict and accumulate around the nucleus. This behavior is difficult to interpret
because the constriction is not associated with any organellar movement. However, PICKETTHEAPS~xs) has reported in Spirogyra that constricdon of chloroplasts is mediated by the plasma membrane. Algal structure and behavior are subject to major alteration by gamma-irradiation. In addition, by selecting species of Sirogonium with rodlike and dotlike chromosome types, markedly altered behavior and structural aberrations may be demonstrated in different species of a single genus.
Acknowledgements--A portion of this research was supported by NSF grants GB-25130 and DEB76-82624 to R.W.H. The authors thank Dr. HERBERT M. HULL for his critical review of the manuscript.
RE]FEII.]KNI21~-~ 1. BRASELTONJ. P. (1971) The ultrastructure of the non-localized kinetochores in Luzula and Cyperus. Chromosoma ~ 89-99.. 2. Buck R. C. (1967) Mitosis and meiosis in Rhodnius prolixus: the fine structure of the spindle and diffuse kinetochore. 07. Ultrastruct. l~s. 18, 489-501. 3. CASTRO D. DE, CAMARA A. and MALHEIROSN. (1949) X-rays in the centromere problem of Luzula purpurea Link. Genet. Iber. 1, 49-54. 4. COMINOS D. E. and OKODA T. A. (1972) Holocentric chromosomes in Oncopeltus: kinetochore plates are present in mitosis but absent in meiosis. Chroraosoma 37, 177-i 92. 5. FREY'rAGA. H. (1964) Use of commercial bleach to improve separation of orchid chromosomes. Stain Technol. 39~ 167-169. 6. GODWARD M. B. E. (1954) Irradiation of Spirogyra chromosomes. H~;~dity ~ 293 (Abstr.). 7. GOt)WARDM. B. E. (1962) Invisible irradiation. Pages 551-566 in R. A. LEWlN, ed. Physiology and biochemistry of algae. Academic, New York. 8. HAMPL W., ALTMANN H. and BIEBL R. (1971) Unterschiede und Beeinflussung der Erholungskapazit/it von Chlorellazellen in verschiedenen Stadien des Zellzyklus. Radiat. Bot. 11, 201-207. 9. HOgSLEYR. J., BANERJEES. N. and BANERJEEN. (1967) Analysis of lethal responses in Oedogonium cardiacum irradiated at different cell stages. Radiat. Bot. 7, 465-476.
GAMMA IRRADIATION EFFECTS ON TWO SPECIES OF GREEN ALGA SIROGO~TUM 10. HosnAw R. W. and WAER R. D. (1967) Polycentric chromosomes in Sirogonium melanosporum. Can. J. Bot. 45~ 1169-1171. 11. HUOHEs-ScHR~a)ER S. and RIs H. (1941) The diffuse spindle attachment of coccids, verified by the mitotic behavior of induced chromosome fragments. J. Exp. Zool. M, 429-456. 12. MUOnAL S. and GODWARD M. B. E. (1973) K_inetochore and microtubules in two members of Chlorophyceae, Cladophorafracta and Spirogyra majuscula. Chromosoma14~ 213-229. 13. PAINTER R. B., McA~PXNE V. W. R. and GERMAmS M. (1961) The relationship of the metabolic state of deoxyribonucleic acid during X-irradiation to HeLa S3 giant cell formation. Radiat. Res. 14, 653-661. 14. PF.TROVAJ. (1962) The 'direct' and 'indirect' effects of ionizing radiation on the alga Zygnema. Folia Biol. 9~ 51-58. 15. PICKETT-HEAPSJ. D. (1975) Green algae: structure, reproduction and evolution in selected genera. Sinauer, Sunderland, Massachusetts. 606 p. 16. PRASAD B. N. and GODWARD M. B. E. (1968) Cytology of irradiated Mougeotia sp. .Nucleus (Calcutta) 11, 43-49. 17. PRINCSHmM E. G. (1946) The biphasic or soil-
18.
19.
20.
21.
22.
23.
45
water culture method for growing algae and flagellata, j . Ecol. 33, 193-204. SARMA Y. S. R. K. and SINGH S. B. (1977) Irradiation studies on the karyology of Charophyta--I. X-rays and gamma-rays. Cytologia 42, 279-290. SPARROW A. H. and CHRISTENSEN E. (1954) Tolerance of certain higher plants to chronic exposure to gamma radiation from cobalt-60. Science 118, 697-698. SPARROW A. H., UNDERBRINK A. G. and SP^RROW R. C. (1967) Chromosomes and cellular radiosensitivity--I. The relationship of D o to chromosome volume and complexity in seventy-nine different organisms. Radiat. Res. 32, 915-945. TANAKAN. and TANAKAN. (1977) Chromosome studies in Chionographis (Liliaceae) I. On the holokinetic nature of chromosomes in Chionographis japonica Maxim. Cytologia 42, 753763. WELLS C. V. and HOSnAW R. W. (1971) The nuclear cytology of Sirogonium. J. Phycol. 7, 279284. WroTE M. J. D. (1936) Chromosome cycle of Ascaris megalocephala. Nature 137, 783.