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Enhancement of non-nuclear DNA synthesis associated with hormone-induced elongation in the cucumber hypocotyl
226 Y. Degani & D. Atsmon to grow in HAT. Cells of one parent would be pretreated with HAT and then mixed with a small number of cells of the other parent. The mixtures would be maintained in normal growth medium, which should prevent the growth of the pretreated parent but which may allow the growth of the hybrids as well as that of the untreated parent. The results presented in this paper can be summarized as follows: (1) More than one out of every 100 T6 cells can fuse spontaneously with an A9 cell to give a viable hybrid; (2) the highest “effective mating rate” is observed when the ratio of T6 to A9 cells is the smallest; (3) the two parental cells can rescue each other; (4) the ability of cells of a given line to form viable hybrids undergoes variations; and (5) HAT-treated cells cannot resume growth in normal medium unless first passed through medium containing HT. This work was supported by a grant from the National Science Foundation, USA. R. D. is a Research Fellow on leave from the Children’s Hospital Medical Center, Boston, Mass. 02115. USA
REFERENCES 1. Coon, H & Weiss, M, F’roc natl acad sci US 62 (1969) 852. 2. Davidson, R, Exptl cell res 55 (1969) 424. 3. Davidson, R & Ephrussi, B, Nature 205 (1965) 1169. 4. Ford, C E, Hamerton, J L, Barnes, D W H & Loutet, J F, Nature 177 (1956) 452. 5. Littlefield, J, Science 145 (1964) 709. 6. - Exptl cell res 41 (1966) 190. 7. Szybalski, W, Szybalska, E H & Ragnie, G, Nat1 cancer inst monogr 7 (1962) 75. Received April 10, 1970
Exptl Cell Res 61
Enhancement of non-nuclear DNA synthesis associated with hormone-induced elongation in the cucumber hypocotyl Y. DEGANI’
and D. ATSMON2, lVolcani
tural Research Institute, ZThe Weizmann Institute Rehovoth, Israel
AgriculNeve Yaar Substation, and of Science (Plant Genetics,)
Summary Gibberellin- and auxin-induced elongation in the cucumber hypocotyl was associated with a substantial increase in DNA content. However, the increase in number of cell nuclei was relatively much smaller than would be expected on that basis. Kadioautographic examinations showed that 3H-thymidine was incorporated into nearly all chloropl&ts, while very few cell nuclei were labelled in the same preparations. Determinations of total DNA content plus measurements of aH-TdR incorporation in the nuclear and non-nuclear fractions indicated that the specific rate of DNA synthesis was 10-20 times greater in the non-nuclear fraction than in the nuclear one. It was concluded, therefore, that most of the hormone-enhanced DNA synthesis in this primarily non-dividing tissue takes place outside the nucleus, in DNA containing organelles, such as chloroplasts and mitochondria.
Recently there have been reports indicating the involvement of DNA synthesis in hormone-regulated plant cell elongation (for recent review see [4]) Since the increase in total DNA during this hormone-induced growth is substantial while the rate of cell division is rather low, it was inferred that the growth is associated with an increase of DNA per cell. Furthermore, it was demonstrated [I, 5, 6, 71 that FUdR, an inhibitor of DNA synthesis, also inhibited the hormone-induced elongation. Therefore it was concluded that cell elongation requires an increase in total DNA per elongating cell. If this addition is nuclear, it would imply polyteny or endopolyploidy. Another possibility is that the newly synthesized DNA in the non-dividing cells will concentrate mainly outside the nucleus, in the DNA-containing organelles, such as mitochondria and chloroplasts. The main object of the present work was to localize this hormone-induced DNA
Plant growth substancesand non-nuclear DNA synthesis 227 synthesis in the elongating cucumber (CUCUmis sativus L.) hypocotyl. This organ responds to two kinds of hormones-gibberellins (GA) and auxins (in the present work, indoleacetic acid, IAA)-and shows enhanced DNA synthesis under the influence of both [l].
Materials and Methods
Table 1. Hormonal effects on DNA increase
in elongating cucumber hypocotyl .a DNA/
Treatment Initial state 48 h later No hormone IAAa GA’
ll;pocoty1 section
% increase over initial state
0.93 1.20 1.53 1.98
29 64 112
Seedlings of cucumbers were grown in soil-filled flats covered with vermiculite and kept in a growth cham- a 10 ~1 drop of 100 ppm (IAA) or 10 ppm (GA4+,) ber at 29”C, 16 h light (Grolux, about 700 f.c. at in 0.05 % Tween 20, per seedling apex at initial state. plant level), 8 h dark. After 96 h, when cotyledons were first fully expanded (the so-called ‘initial state’) a 5 mm section was marked, starting 5 mm below from homogenates according to Smillie & Krotkov cotvledonarv node ~‘HYIJOCO~Y~ section’), and hormones (GA&+ GA,, ‘ca- 1 : 1 mixture, from ICI, 10 [8] and determined by the diphenylamine reaction, mg/l: or IAA from CalBiochem. 100 mnil. in 0.05 % according to Gilles & Myers [2]. Eight hundred Tween 20) were applied in a 10 bl droplet to seedling hypocotyl sections were used for determinations in apex; 0.05 % Tween was applied to controls. Such nuclear vs non-nuclear fractions. One hundred sections were used for each determination of total DNA. hormonal treatments result in a pronounced elongaSH-Thymidine (17.5 CiimM. The Radiochemical tion of the hypocotyl section within 48 h [l]. Centre, kmersham) incorporations were carried out For nuclei counts, hypocotyl sections were fixed on harvested hypocotvl sections. cut into 3 mm long for 12 h in 3 : 1 absolute alcohol and glacial acetic acid, passed through tertiary butyl-alcohol series, segments and shaken continuously in the labeling solution (3H-TdR in distilled water. 10 Ciiml) for embedded in paraffin, cross sectioned at 10 pm and 1 h. The hypocotyl sections were then rinsed in water stained with leucobasic fuchsin (Feulgen reaction). thoroughly, chilled and organelles were separated as Only heterochromatic zones of cell nuclei are thus described previously. The separated fractions of stained. Five seedlings were sectioned for each treatchloroplasts and nuclei were homogenized and washed ment. Preliminary observations indicated that hypoonto membrane filters according 70 Jakob & Bovey cotyl sections were absolutely devoid of any mitotic 131and counted in a Packard Tricarb liquid scintillafigures when examined in squash preparations, and tion spectrometer. Fifty hypocotyl sections were any increase in the number of nuclei could be expectused for each determination. About 80% of the ed only in the bundles of conductive tissue, where counts thus measured were DNase-removable. some cambial activity could take place. Conductive For radioautographic examinations, 3H-TdR inbundles in the hypocotyl are relatively small; therecorporation was carried out as described before, 24 fore, it was practically impossible to obtain median h after initial state. After incorporation, the material sections of such bundles and to make reliable nuclei was fixed in FAA (formalin:glacial acetic acid:70 % counts in longitudinal sections. Instead, hypocotyls were sectioned transversely at 10 pm; three consecu- ethanol, 90 : 5 : 5 v/v/v) for 12 h, transferred to paraffin through the tertiary butyl-alcohol series and sective cross sections were examined at each 1 mm intioned at 5 pm. Mounted sections were washed by terval along the hypocotyl; nuclei were counted in cold 5 % TCA for 15 min, then rinsed in water, dried, these cross sections within one and the same vascular and dipped in emulsion (Ilford Nuclear Research bundle; the total of these counts represented the emulsion K-5), exposed for 6 days and developed. ‘number of nuclei’ for that hypocotyl; the average of The sections were stained with haematoxylin. such counts for five hypocotyls represents the ‘number of nuclei’ for any treatment, as shown in table 2. The senaration of the nuclear fraction from the non-nuclear one was carried out according to Spen- Results and Discussion cer & Wildman [9]. Hypocotyl sections were chopped Previous results [l] as well as the present by razor blade in ice-cold Honda Medium. The work (table 1) demonstrate very clearly that chopped material was placed on a discontinuous sucrose gradient (50, 35, 30, and 20% sucrose in Honda Medium) and centrifuged for 4 h at 22 000 the elongation of the cucumber hypocotyl rpm (Spinco SW-25-l). The chloroplasts formed a section within the experimental period is visible green band between the 30 and 35 % fractions; associated with a substantial increase in the nuclei were in the pellet. Microscopic examination of the fractions showed that while the nuclei fraction total DNA per hypocotyl section. This is contained hardly any chloroplasts, the chloroplast true both for normal growth and for the fraction contained some broken nuclei. hormone (IAA and GA) induced one. The DNA for quantitative determinations was extracted Exptl Cell Res 61
228
Y. Degani & D. Atsmon
Table 2. Effect of hormonal treatments on number of cell nuclei in cross section of a vascular bundle in hypocotyl section 48 h after hormonal treatment
Average no. of nuclei % increase over initial state
cpm/50 hypocotyls
Initial state
Control GA
IAA
308
327
429
6.1
461 49.6
Table 3. Amount of DNA in nuclear and nonnuclear fractions of hypocotyl sections at initial state
Exptl Cell Res 61
Nuclear Non-nuclear
Control
GA
IAA
2 307 2 053
4 338 3 892
5 288 3 926
39.2
increase is largest in the GA-treated material, somewhat smaller in the IAA-treated one, and smallest, although still significant, in the control material. On the other hand, the increase in number of nuclei is much smaller, for all three kinds of seedling (table 2). While in the control plants there is hardly any increase in nuclei, there is a 50 % increase over the initial state in the GA-treated material. This increase was observed in the conductive bundles only, which are also the most probable site for cell division; on the other hand, in the cortical tissue no increase in nuclei number was detected. The number of cells in the conductive bundles, as counted in cross sections, forms only 25-30% of the total number of cells in such a section. Therefore the total increase in number of nuclei per hypocotyl section, as a percentage of initial state, should be a very low one. The results presented in tables 1 and 2
Nuclear Non-nuclear
Table 4. Radioactivity of nuclear- and nonnuclear fraction of hypocotyl sections after 1 h in 3H-thymidine, 12 h after hormonal treatment
rug DNA/hypocotyl section
% of total DNA
0.25 0.03
90.0 10.0
show that during growth of the hypocotyl section the DNA content rises proportionately much more than nuclei number; an increase in DNA content per cell is, therefore, indicated. In order to localize this additional DNA synthesis within the cell, radioautographic studies followed. Radioautographs of tissue sections labelled with 3H-TdR showed that very few nuclei were labelled, mostly around and within the conductive bundles. On the other hand, most of the chloroplasts which were found in all cells throughout the section were labelled. This observation indicates that a great part, if not most, of the DNA synthesis which is associated with hypocotyl elongation takes place in the cytoplasm, rather than in cell nuclei. In order to get some quantitative estimate of the non-nuclear vs nuclear share in the total DNA synthesis, nuclei were separated from chloroplasts and from other cytoplasmic elements on a sucrose gradient, according to Spencer & Wildman [9]. When total DNA was determined in the two fractions by the diphenylamine method it was found (table 3) that not more than 10 % of the total DNA is non-nuclear, while all the other 90 Y0 are nuclear. On the other hand, when 3HTdR incorporation was determined (table 4) it was more or less equally divided between the two fractions; in some more recent data
Peripheral lymphocyte stimulation by Zn2+ the non-nuclear fraction showed twice as much radioactivity, compared with the nuclear fraction. These results imply that the specific activity of the non-nuclear fraction was some lo-20 times higher than that of the nuclei. Since the non-nuclear fraction contained some fragments of nuclei the true ratio of specific activities must have been considerably higher. These results show that in an elongating, predominantly non-dividing, green tissue most of the newly synthesized DNA is nonnuclear. Furthermore, since non-labelled chloroplasts were rather rare in this 1 h pulsed material, it indicates DNA turnover or general increase of it per organelle at all stages of chloroplast development. Such an increase in non-nuclear DNA may be a manifestation of the growth which takes place in this non-dividing tissue rather than a prerequisite for this growth response. Yet it is possible that a small fraction of the newly synthesized DNA, nuclear or non-nuclear, is vital for cell elongation. This study is part of M.Sc. thesis (co-supervised by Professor A. H. Halevy), submitted to The Hebrew University, Department of Floriculture, Rehovoth, Israel, by the senior author.
229
Stimulation of human peripheral lymphocytes by Zn2+ in vitro H. KIRCHNER und H. RiSHL, Haematologische Abteilung, Klinikum Steglitz, Freie Universitiit Berlin, Germany
Human peripheral lymphocytes in shortterm cultures represent a “resting” cell population, but a variety of substances stimulate them to enter S phase, to transform into so-called blast cells and to undergo mitotic divisions. Despite numerous investigations (reviewed by Ling [l]) many of the mechanisms of this activation are unknown. Materials and Methods Our lymphocyte culture system has been previously described [2]. To obtain an optimal response l.O1.5 x lo6 leucocytes/ml were cultivated in Medium TC199 with 30 % autologous plasma, but without antibiotica. For all the experiments the blood of one group of 20 healthy individuals with no history of any allergy was taken. most of them were investigated several t&es. For the autoradiographs the c&ures received 1 &i/ml $H-thymidine (spec. act. 2 Ci/mM) for the final hour and were then washed once with Hanks solution. The further procedure was identical with the method of van Furth & Cohn [3]. Zincsulfate, zinc-chloride, zinc-acetate (Merck p.a.) and zinc-aspartate (kindly provided by F. Kiihler Chemie, AlsbachiBergstraReiBRD) were dissolved in saline, sterilized by filtration, and added at the beginning oi the culture.
Results REFERENCES Degani. Y & Atsmon, D. Unpublished data.
:: Gifies, k W & Myers; A, Nature 206 (1965) 93. 3. Jakob, K M & Bovey, . F, Exptl _ cell res 54 (1969)
118. 4. Key, J L, Ann rev plant physiol 20 (1969) 449. 5. Lang, A & Nitsan, J, Ann NY acad sci 144 (1967) 180. ___.
6. Nitsan, J & Lang, A, Develop biol 21 (1965) 358. 7. - Plant physiol41 (1966) 965. 8. Smillie, R M & Krotkov, G, Canad j bot 38 (1960) 31. 9. Spencer, D & Wildman, S G, Biochemistry 3 (1964) 954. Received April 21, 1970
All of the investigated zinc-compounds induced the development of so-called blasts and mitotic figures in the cultures of all of the investigated persons. The percentage of blasts was between 3 and 15 % in 5 day cultures. They resembled by light microscopy those cells which are found in phytohemagglutinin-stimulated lymphocyte cultures and incorporated 3H-thymidine. In the controls, the spontaneous transformation rate was always less than 0.1 %, and no mitotic figures were found. The optimal dose to induce lymphocyte transformation was 2.5 x 1O-4 M; concentrations below 2.0 x 1O-4 M were ineffective, and concentrations above 3.0 x Exptt Cell Res 61
REFERENCES 1. Coon, H & Weiss, M, F’roc natl acad sci US 62 (1969) 852. 2. Davidson, R, Exptl cell res 55 (1969) 424. 3. Davidson, R & Ephrussi, B, Nature 205 (1965) 1169. 4. Ford, C E, Hamerton, J L, Barnes, D W H & Loutet, J F, Nature 177 (1956) 452. 5. Littlefield, J, Science 145 (1964) 709. 6. - Exptl cell res 41 (1966) 190. 7. Szybalski, W, Szybalska, E H & Ragnie, G, Nat1 cancer inst monogr 7 (1962) 75. Received April 10, 1970
Exptl Cell Res 61
Enhancement of non-nuclear DNA synthesis associated with hormone-induced elongation in the cucumber hypocotyl Y. DEGANI’
and D. ATSMON2, lVolcani
tural Research Institute, ZThe Weizmann Institute Rehovoth, Israel
AgriculNeve Yaar Substation, and of Science (Plant Genetics,)
Summary Gibberellin- and auxin-induced elongation in the cucumber hypocotyl was associated with a substantial increase in DNA content. However, the increase in number of cell nuclei was relatively much smaller than would be expected on that basis. Kadioautographic examinations showed that 3H-thymidine was incorporated into nearly all chloropl&ts, while very few cell nuclei were labelled in the same preparations. Determinations of total DNA content plus measurements of aH-TdR incorporation in the nuclear and non-nuclear fractions indicated that the specific rate of DNA synthesis was 10-20 times greater in the non-nuclear fraction than in the nuclear one. It was concluded, therefore, that most of the hormone-enhanced DNA synthesis in this primarily non-dividing tissue takes place outside the nucleus, in DNA containing organelles, such as chloroplasts and mitochondria.
Recently there have been reports indicating the involvement of DNA synthesis in hormone-regulated plant cell elongation (for recent review see [4]) Since the increase in total DNA during this hormone-induced growth is substantial while the rate of cell division is rather low, it was inferred that the growth is associated with an increase of DNA per cell. Furthermore, it was demonstrated [I, 5, 6, 71 that FUdR, an inhibitor of DNA synthesis, also inhibited the hormone-induced elongation. Therefore it was concluded that cell elongation requires an increase in total DNA per elongating cell. If this addition is nuclear, it would imply polyteny or endopolyploidy. Another possibility is that the newly synthesized DNA in the non-dividing cells will concentrate mainly outside the nucleus, in the DNA-containing organelles, such as mitochondria and chloroplasts. The main object of the present work was to localize this hormone-induced DNA
Plant growth substancesand non-nuclear DNA synthesis 227 synthesis in the elongating cucumber (CUCUmis sativus L.) hypocotyl. This organ responds to two kinds of hormones-gibberellins (GA) and auxins (in the present work, indoleacetic acid, IAA)-and shows enhanced DNA synthesis under the influence of both [l].
Materials and Methods
Table 1. Hormonal effects on DNA increase
in elongating cucumber hypocotyl .a DNA/
Treatment Initial state 48 h later No hormone IAAa GA’
ll;pocoty1 section
% increase over initial state
0.93 1.20 1.53 1.98
29 64 112
Seedlings of cucumbers were grown in soil-filled flats covered with vermiculite and kept in a growth cham- a 10 ~1 drop of 100 ppm (IAA) or 10 ppm (GA4+,) ber at 29”C, 16 h light (Grolux, about 700 f.c. at in 0.05 % Tween 20, per seedling apex at initial state. plant level), 8 h dark. After 96 h, when cotyledons were first fully expanded (the so-called ‘initial state’) a 5 mm section was marked, starting 5 mm below from homogenates according to Smillie & Krotkov cotvledonarv node ~‘HYIJOCO~Y~ section’), and hormones (GA&+ GA,, ‘ca- 1 : 1 mixture, from ICI, 10 [8] and determined by the diphenylamine reaction, mg/l: or IAA from CalBiochem. 100 mnil. in 0.05 % according to Gilles & Myers [2]. Eight hundred Tween 20) were applied in a 10 bl droplet to seedling hypocotyl sections were used for determinations in apex; 0.05 % Tween was applied to controls. Such nuclear vs non-nuclear fractions. One hundred sections were used for each determination of total DNA. hormonal treatments result in a pronounced elongaSH-Thymidine (17.5 CiimM. The Radiochemical tion of the hypocotyl section within 48 h [l]. Centre, kmersham) incorporations were carried out For nuclei counts, hypocotyl sections were fixed on harvested hypocotvl sections. cut into 3 mm long for 12 h in 3 : 1 absolute alcohol and glacial acetic acid, passed through tertiary butyl-alcohol series, segments and shaken continuously in the labeling solution (3H-TdR in distilled water. 10 Ciiml) for embedded in paraffin, cross sectioned at 10 pm and 1 h. The hypocotyl sections were then rinsed in water stained with leucobasic fuchsin (Feulgen reaction). thoroughly, chilled and organelles were separated as Only heterochromatic zones of cell nuclei are thus described previously. The separated fractions of stained. Five seedlings were sectioned for each treatchloroplasts and nuclei were homogenized and washed ment. Preliminary observations indicated that hypoonto membrane filters according 70 Jakob & Bovey cotyl sections were absolutely devoid of any mitotic 131and counted in a Packard Tricarb liquid scintillafigures when examined in squash preparations, and tion spectrometer. Fifty hypocotyl sections were any increase in the number of nuclei could be expectused for each determination. About 80% of the ed only in the bundles of conductive tissue, where counts thus measured were DNase-removable. some cambial activity could take place. Conductive For radioautographic examinations, 3H-TdR inbundles in the hypocotyl are relatively small; therecorporation was carried out as described before, 24 fore, it was practically impossible to obtain median h after initial state. After incorporation, the material sections of such bundles and to make reliable nuclei was fixed in FAA (formalin:glacial acetic acid:70 % counts in longitudinal sections. Instead, hypocotyls were sectioned transversely at 10 pm; three consecu- ethanol, 90 : 5 : 5 v/v/v) for 12 h, transferred to paraffin through the tertiary butyl-alcohol series and sective cross sections were examined at each 1 mm intioned at 5 pm. Mounted sections were washed by terval along the hypocotyl; nuclei were counted in cold 5 % TCA for 15 min, then rinsed in water, dried, these cross sections within one and the same vascular and dipped in emulsion (Ilford Nuclear Research bundle; the total of these counts represented the emulsion K-5), exposed for 6 days and developed. ‘number of nuclei’ for that hypocotyl; the average of The sections were stained with haematoxylin. such counts for five hypocotyls represents the ‘number of nuclei’ for any treatment, as shown in table 2. The senaration of the nuclear fraction from the non-nuclear one was carried out according to Spen- Results and Discussion cer & Wildman [9]. Hypocotyl sections were chopped Previous results [l] as well as the present by razor blade in ice-cold Honda Medium. The work (table 1) demonstrate very clearly that chopped material was placed on a discontinuous sucrose gradient (50, 35, 30, and 20% sucrose in Honda Medium) and centrifuged for 4 h at 22 000 the elongation of the cucumber hypocotyl rpm (Spinco SW-25-l). The chloroplasts formed a section within the experimental period is visible green band between the 30 and 35 % fractions; associated with a substantial increase in the nuclei were in the pellet. Microscopic examination of the fractions showed that while the nuclei fraction total DNA per hypocotyl section. This is contained hardly any chloroplasts, the chloroplast true both for normal growth and for the fraction contained some broken nuclei. hormone (IAA and GA) induced one. The DNA for quantitative determinations was extracted Exptl Cell Res 61
228
Y. Degani & D. Atsmon
Table 2. Effect of hormonal treatments on number of cell nuclei in cross section of a vascular bundle in hypocotyl section 48 h after hormonal treatment
Average no. of nuclei % increase over initial state
cpm/50 hypocotyls
Initial state
Control GA
IAA
308
327
429
6.1
461 49.6
Table 3. Amount of DNA in nuclear and nonnuclear fractions of hypocotyl sections at initial state
Exptl Cell Res 61
Nuclear Non-nuclear
Control
GA
IAA
2 307 2 053
4 338 3 892
5 288 3 926
39.2
increase is largest in the GA-treated material, somewhat smaller in the IAA-treated one, and smallest, although still significant, in the control material. On the other hand, the increase in number of nuclei is much smaller, for all three kinds of seedling (table 2). While in the control plants there is hardly any increase in nuclei, there is a 50 % increase over the initial state in the GA-treated material. This increase was observed in the conductive bundles only, which are also the most probable site for cell division; on the other hand, in the cortical tissue no increase in nuclei number was detected. The number of cells in the conductive bundles, as counted in cross sections, forms only 25-30% of the total number of cells in such a section. Therefore the total increase in number of nuclei per hypocotyl section, as a percentage of initial state, should be a very low one. The results presented in tables 1 and 2
Nuclear Non-nuclear
Table 4. Radioactivity of nuclear- and nonnuclear fraction of hypocotyl sections after 1 h in 3H-thymidine, 12 h after hormonal treatment
rug DNA/hypocotyl section
% of total DNA
0.25 0.03
90.0 10.0
show that during growth of the hypocotyl section the DNA content rises proportionately much more than nuclei number; an increase in DNA content per cell is, therefore, indicated. In order to localize this additional DNA synthesis within the cell, radioautographic studies followed. Radioautographs of tissue sections labelled with 3H-TdR showed that very few nuclei were labelled, mostly around and within the conductive bundles. On the other hand, most of the chloroplasts which were found in all cells throughout the section were labelled. This observation indicates that a great part, if not most, of the DNA synthesis which is associated with hypocotyl elongation takes place in the cytoplasm, rather than in cell nuclei. In order to get some quantitative estimate of the non-nuclear vs nuclear share in the total DNA synthesis, nuclei were separated from chloroplasts and from other cytoplasmic elements on a sucrose gradient, according to Spencer & Wildman [9]. When total DNA was determined in the two fractions by the diphenylamine method it was found (table 3) that not more than 10 % of the total DNA is non-nuclear, while all the other 90 Y0 are nuclear. On the other hand, when 3HTdR incorporation was determined (table 4) it was more or less equally divided between the two fractions; in some more recent data
Peripheral lymphocyte stimulation by Zn2+ the non-nuclear fraction showed twice as much radioactivity, compared with the nuclear fraction. These results imply that the specific activity of the non-nuclear fraction was some lo-20 times higher than that of the nuclei. Since the non-nuclear fraction contained some fragments of nuclei the true ratio of specific activities must have been considerably higher. These results show that in an elongating, predominantly non-dividing, green tissue most of the newly synthesized DNA is nonnuclear. Furthermore, since non-labelled chloroplasts were rather rare in this 1 h pulsed material, it indicates DNA turnover or general increase of it per organelle at all stages of chloroplast development. Such an increase in non-nuclear DNA may be a manifestation of the growth which takes place in this non-dividing tissue rather than a prerequisite for this growth response. Yet it is possible that a small fraction of the newly synthesized DNA, nuclear or non-nuclear, is vital for cell elongation. This study is part of M.Sc. thesis (co-supervised by Professor A. H. Halevy), submitted to The Hebrew University, Department of Floriculture, Rehovoth, Israel, by the senior author.
229
Stimulation of human peripheral lymphocytes by Zn2+ in vitro H. KIRCHNER und H. RiSHL, Haematologische Abteilung, Klinikum Steglitz, Freie Universitiit Berlin, Germany
Human peripheral lymphocytes in shortterm cultures represent a “resting” cell population, but a variety of substances stimulate them to enter S phase, to transform into so-called blast cells and to undergo mitotic divisions. Despite numerous investigations (reviewed by Ling [l]) many of the mechanisms of this activation are unknown. Materials and Methods Our lymphocyte culture system has been previously described [2]. To obtain an optimal response l.O1.5 x lo6 leucocytes/ml were cultivated in Medium TC199 with 30 % autologous plasma, but without antibiotica. For all the experiments the blood of one group of 20 healthy individuals with no history of any allergy was taken. most of them were investigated several t&es. For the autoradiographs the c&ures received 1 &i/ml $H-thymidine (spec. act. 2 Ci/mM) for the final hour and were then washed once with Hanks solution. The further procedure was identical with the method of van Furth & Cohn [3]. Zincsulfate, zinc-chloride, zinc-acetate (Merck p.a.) and zinc-aspartate (kindly provided by F. Kiihler Chemie, AlsbachiBergstraReiBRD) were dissolved in saline, sterilized by filtration, and added at the beginning oi the culture.
Results REFERENCES Degani. Y & Atsmon, D. Unpublished data.
:: Gifies, k W & Myers; A, Nature 206 (1965) 93. 3. Jakob, K M & Bovey, . F, Exptl _ cell res 54 (1969)
118. 4. Key, J L, Ann rev plant physiol 20 (1969) 449. 5. Lang, A & Nitsan, J, Ann NY acad sci 144 (1967) 180. ___.
6. Nitsan, J & Lang, A, Develop biol 21 (1965) 358. 7. - Plant physiol41 (1966) 965. 8. Smillie, R M & Krotkov, G, Canad j bot 38 (1960) 31. 9. Spencer, D & Wildman, S G, Biochemistry 3 (1964) 954. Received April 21, 1970
All of the investigated zinc-compounds induced the development of so-called blasts and mitotic figures in the cultures of all of the investigated persons. The percentage of blasts was between 3 and 15 % in 5 day cultures. They resembled by light microscopy those cells which are found in phytohemagglutinin-stimulated lymphocyte cultures and incorporated 3H-thymidine. In the controls, the spontaneous transformation rate was always less than 0.1 %, and no mitotic figures were found. The optimal dose to induce lymphocyte transformation was 2.5 x 1O-4 M; concentrations below 2.0 x 1O-4 M were ineffective, and concentrations above 3.0 x Exptt Cell Res 61