65
Mutation Research, 42 (1977) 65-70
© Elsevier/North-Holland Biomedical Press
INDUCTION OF DNA SINGLE-STRAND BREAKS IN BARLEY BY SODIUM AZIDE APPLIED AT pH 3
J. VELEMINSKY, T. GICHNER and V. POKORNY Institute of Experimental Botany, Flemingouo 2, Prague 6 (Czechoslovakia) (Received April 4th, 1976) (Revision received August 17th, 1976) (Accepted September 6th, 1976)
Summary Sodium azide (1 to 50 mM), adjusted to pH 3 and applied for 2 h to presoaked barley seeds, induced a dose-dependent frequency of single-strand breaks in DNA of non-germinating embryos. This was demonstrated by sedimentation analyses of isolated DNA samples in alkaline sucrose gradients and in neutral sucrose gradients with 80% formamide. The doses applied also inhibited dose dependently the root length, seed germination and partially the seedling height. Only the sub-lethal doses (10 and 12.5 mM) induced a low frequency of chromatid breaks and translocations in the root tip metaphases. The sedimentation rate (in alkaline sucrose gradients) of calf thymus DNA treated with sodium azide at pH 3, was similar to that of the control DNA treated with buffer (pH 3) alone.
Introduction Sodium azide, an inhibitor of respiration, is mutagenic in bacteria [3] as well as in barley [8,9]. In Salmonella it induced mutation of the base substitution type but no frame shifts [3,9]. In barley the mutagenic action (scored by "gene" mutations in the M2 generation) is especially strong when azide is applied at pH 3 either to dry or briefly soaked seeds [3,8]. Doses inducing the highest frequency of mutations, comparable e.g. with those produced by ethyl methanesulphonate, are reported not to increase the spontaneous rate of chromosomal aberrations in root tips or in meiosis [11]. Several hypotheses on the molecular nature of azide mutagenesis have been postulated, based on the interaction with DNA [2,10} in genetical experiments with bacteria in vitro [3,4,20], on measurement of enzyme inhibition in barley [12], and on the presence or absence of synergism in combined treatment of azide with ionizing radiation (11], N-methyl-N-nitrosourea, diethylsulphate
66
[5], etc. However, none of these hypotheses can explain all results presented in the literature on azide action. Moreover, in bacteria and in plant or animal cells, no types of azide-induced DNA lesions in vivo have been described. In this paper we present results on the sedimentation properties of DNA, influenced in vivo (barley seeds) or in vitro (calf thymus DNA) by azide adjusted to pH 3. Material and methods Seeds of barley variety Amethyst were soaked for 10 h in water and treated for 2 h in 1 to 50 mM azide dissolved in 0.2 M NaH2 P0 4 and adjusted immediately before the start of treatment to pH 3 by phosphoric acid. After 30 min of post-wash, part of the seeds (about 100) was sown on wet sand in petri dishes for measurement of germination, root length and chromosomal aberrations in the root tips; another part (about 100 seeds) was sown on soil in boxes in the greenhouse for the measurement of seedling height; and the major part was used for the immediate isolation of DNA from the seed embryos. Root length and germination were measured after 3 days, the seedling height after 14 days. For scoring the chromosomal aberrations (chromatid breaks and translocations) in root tip cells, squash preparations stained by the Feulgen method were used. 200 metaphases were evaluated for each azide concentration and each of the 3 restitution times. DNA was isolated from non-germinating seed embryos (about 600-800) by the modified method of Marmur as previously described [17,18]. This procedure enables one to get the double-stranded DNA with molecular weight "'10 7 daltons. Treatment in vitro was carried out with high molecular calf thymus DNA dissolved in 0.15 M NaCl and 0.015 M sodium citrate (SSC) acidified with formic acid to pH 3.2. After 2 h of incubation with azide at 25°C, DNA was precipitated with ethanol and dissolved in SSC. Sedimentation of DNA samples was performed in alkaline sucrose gradient (5--20% sucrose w/v in 0.9 M NaCl, 1 mM EDTA and 0.3 M NaOH), neutral sucrose gradient (5-20% sucrose w]» in 0.9 M NaCl and 1 mM EDTA pH 7), and in neutral sucrose gradient containing 80% formamide (1-15% sucrose w/v dissolved in 80 vol formamide:20 vol H20, pH 7.4). The last-mentioned neutral sucrose-formamide gradient was used to distinguish the formed single strand breaks (SSB) in vivo from alkali-labile sites [13]. Before being layered on this gradient, DNA samples were dissolved in 80% formamide and incubated for 30 min at 37°C. According to hyperchromicity measurement, in these conditions DNA was denatured before, and remained denatured during centrifugation (unpubl.). After the centrifugation in a Spinco L2-65 B ultracentrifuge, rotor SW 27.1 (gradient length 9.2 em) or SW 56 Ti (gradient length 5.5 em), gradients were removed from the top by a Buchler Auto-Densi-Flow II, and DNA was measured continuously in a differential UV analyzer. The position of the peak centre of the DNA sedimentation curve was also taken for the expression of the sedimentation rate. In Fig. 3 the distance of this peak centre, from the peak centre of control (untreated) DNA is expressed by the percentage of the total gradient length.
67
Results Azide applied at pH 3 induced a dose-dependent inhibition of seed germination, root length and seedling height reduction, and at higher doses, a weak increase of chromosomal aberrations in the root tips. The most sensitive characters seem to be root length reduction and inhibition of germination, whereas the seedling height was depressed at a sub-lethal dose (12.5 mM) only to 60% of the control. The highest frequency of breaks and rearrangements in metaphases (9.0%) was reached after the same sub-lethal dose of azide (Fig. 1). The values given in the curve for chromosomal aberrations represent the highest frequency obtained after various periods of restitution, i.e. for 5 mM NaN 3 it was 36 h restitution; for 7.5 mM it was 48 h; for 10 mM, 72 h and for 12.5 mM, 96 h of restitution. In alkaline sucrose gradients, sedimentation rates of DNA samples decreased with dose of azide (Fig. 2a). This dependency on the dose of azide is expressed by comparing the distance of the peak centres of DNA sedimentation curves from the control DNA (Fig. 3, full line; each point on a line represents a mean value from three independent experiments). It is evident that the amount of SSB or alkali-labile sites increased nearly linearly even at lethal doses (25 and 50 mM) of azide. The alkaline gradient cannot be used to distinguish between SSB and alkalilabile sites, since alkaline conditions convert the alkali-labile sites to singlestrand breaks in vitro. For this purpose, aliquots of the same DNA samples,
100T""-_ _
180
80
20
------ ~..
.0••••
O'
,
o
1
5 10 mM NaN 3
12.5
15
Fig. 1. Effects of sodium azide (NaN 3) applied at pH 3 for 2 h to IG-h pre-soaked barley seeds. ( e - - e ) Seed germination; ( _ _ ) seedling height (% of control); ( 0 - - 0 ) root length (% of control); (0- - - - - -0) chromosomal aberrations in metaphases of root tips.
68
a
b
123456
1
36
1.0
1.0
-:0.5
0.5
>u
c
J.
oIII
• ~
'!"
o
20
40
60
+-top
80
100
20
1. of gradient length
40
60
80
100
bottom
Fig. 2. (a) Sedimentation curves of DNA in alkaline sucrose gradient. 19 h, 25000 r.p.m., 5°C, rotor SW 27.1. Azide concentrations: 1, 50 mM; 2, 25 mM; 3, 10 mM; 4,5 mM; 5, 1 mM; 6, O. (b) Sedimentation curves of DNA in neutral sucrose formamide gradient, 6 h, 55,000 r.p.rn., 25°C, rotor SW 56 Ti. Azide concentrations: 1,50 mM; 3,10 mM; 6, O.
which had been analyzed in alkaline sucrose gradients, were denatured in 80% fonnamide and centrifuged in neutral conditions of 80% formamide in the sucrose gradient. The comparison of the sedimentation pattern of the same DNA samples in alkaline sucrose (Fig. 2a) and in neutral sucrose gradient with 80% fonnamide (Fig. 2b) indicates similar differences from the control DNA. We may suppose, therefore, that at least a part if not all the SSB was formed in vivo in the course of the 2·h treatment of seeds with azide at pH 3.
.ll20
"I
cr z c
"015
::c
ou E
,g
.
~10
lit
c
.!
~
• ~ 5 ...o
;;
III
X
~.... 0 1
5
....... -- --_ 10
.... ----- ------------.
25 mM NaN3
50
Fig. 3. Dependence of DNA sedimentation rate in alkaline sucrose gradient (full line) and in neutral sucrose gradient (dashed line) on dose of sodium azide (NaN3) applied in vivo at pH 3.
69
In contrast with the different sedimentation properties of denatured, azidetreated DNA, the size of the double-stranded form of the same samples, as analyzed in neutral sucrose gradient, seems not to be influenced by the action of azide. This is documented by the position of the peak centre of the sedimentation curves in the gradient relative to the untreated control barley DNA (Fig. 3, dashed line). We can thus conclude that azide induced in vivo a dose-dependent frequency of SSB in barley DNA, without inducing double-strand breaks. Application of five sodium azide concentrations at pH 3.2 (2-100 mM, 2 h, 25°C) to calf thymus DNA in vitro resulted in a similar lack of dose-dependent changes of the sedimentation patterns on an alkaline sucrose gradient. Differences of the peak centre distance from the untreated DNA varied from 8 to 0.7%. The heating of azide-treated DNA samples for 10 min at 100 0 e before they were layered on the alkaline sucrose gradient did not change these values. Thus, in our experimental conditions and dose range, azide induced neither SSB, alkali labile sites (apurinic or apyrimidinic sites) nor heat-labile sites in vitro. Discussion Detection of DNA breaks is mostly performed on native DNA according to the method of McGrath and Williams [7] by lysing the bacterial or mammalian cells on the top of the alkaline sucrose gradient. This method requires DNA that had been pre-labelled in the course of the preceding cell cycle before the action of break-inducing agents. This was impossible to do in the conditions of our experiments in which the non-growing tissues of barley embryos were treated before seed germination. Moreover, tissues and cells of higher plants cannot be lysed on the gradient unless protoplasts are obtained. The DNA isolation according to Marmur, used in our experiments, certainly lowered the sensitivity of sedimentation analysis. In spite of this disadvantage the decreasing
sedimentation rate of DNA in alkaline sucrose gradient with increasing dose of azide obtained in all our experiments, as well as the results obtained in neutral formamide gradients, indicates that SSB are formed in the embryonic cells of barley seeds as a consequence of azide treatment at pH 3 in vivo. Although in low amount, SSB were induced even by concentrations usually used for mutation induction [6,7,10,11]. This implies that SSB, or the DNA lesions from which they originate, can be considered as one of the possible pre-mutational lesions in barley. How these lesions are induced and what is the nature of the mechanism leading to the mutation induction in barley, however, cannot be decided from the results presented here. Kleinhofs and Smith [4] had demonstrated that azide mutagenesis in bacteria is dependent on DNA lesions excisable by the uvr system, and mutations are produced by the error-prone ree-dependent repair pathways. Base damage which leads to the base loss (comparable e.g. to that known after ')'-rays [6] or after the action of alkylating agents [19]) can offer a good substrate for both an error-prone pathway and backbone breakage. Endonuclease, specific for apurinic and apyrimidinic sites and which produces SSB, has now been isolated from barley [14]. Both excision repair pathway [16,17] and post-replication
70
repair [15] were demonstrated in barley for DNA damaged induced by alkylating agents. Enzymes, acting in these processes, can contribute to the origin of breaks or mutations even after the action of azide. Moreover, the spontaneous origin of SSB caused by lowering the cellular pH due to the uptake of hydrazoic acid cannot be excluded. The inability to find any DNA lesion induced by azide at pH 3 in vitro [2] supports the assumption that, in vivo, azide is activated by a yet unknown interaction of hydrazoic acid with cellular components. References
2 3
4 5
6 7 8 9 10 11
12
13
14 15
16 17
18
19 20
Howland, G.P., R.W. Hart and M.L. Yette, Repair of DNA strand breaks after gamma-irradiation of protoplast isolated from cultured wild carrot cells, Mutation Res., 27 (1975) 81-89. Kleinhofs, A.M., M. Kleinschmidt, D. Sciaky and S. von Broernbsen, Azide mutagenesis. In vitro studies, Mutation Res., 29 (1975) 497-500. Kleinhofs, A., C. Sander, R.A. Nilan and C.F. Konzak, Azide mutagenicity-mechanism and nature of mutants produced, Polyploidy and Induced Mutations in Plant Breeding, IAEA, Vienna, 1974, pp. 195-199. Kleinhofs, A. and J.A. Smith, Effect of excision repair on azide-induced rnutagensis, Mutation Res., 41 (1976) 233-240. Konzak, C.F., M. Nikneiad, I. Wickham and E. Donaldson, Mutagenic interaction of sodium azide on mutations induced in barley seeds treated with diethyl sulfate or N-methyl-N'-nitrosourea, Mutation Res., 30 (1975) 55-62. Mattern, M.R., P. V. Hariharan and P.A. Cerutti, Selective excision of gamma ray damaged thymine from the DNA of cultured mammalian cells, Biochim. Biophys. Acta, 395 (1975) 48-55. McGrath, R.A. and R.W. Williams, Reconstruction in vivo of irradiated Escherichia coli deoxyribonucleic acid. The rejoining of broken pieces, Nature, 212 (1966) 534-535. Nilan, R.A. and A. Kleinhofs, Azide mutagenesis in barley, HIth Int. Barley Genetics Svmp., July 712, 1975, p. 87 (abstract). Nilan, R.A., E.G. Sideris, A. Kleinhofs, C. Sander and C.F. Konzak, Azide, a potent mutagen, Mutation Res., 17 (1973) 142-144. Sideris, E.G. and M. Argv rakis, Chemical alterations induced in DNA and DNA components by the mutagenic agent azide, Biochim. Biophys, Acta, 366 (1974) 367~373. Sideris, E .• R.A. Nilan and T.P. Bogvo, Differential effect of sodium azide on the frequency of radiation-induced chromosome aberrations vs the frequency of radiation-induced chlorophyll mutations in Hordeum vulgare, Radiation Botany, 13 (1973) 315-322. Sideris, E.G., R.A. Nilan and C.F. Konzak, Relationship of radiation-induced damage in barley seeds to the inhibition of certain oxidoreductases by sodium azide, Induced Mutations in Plants, IAEA, Vienna, 1969, pp. 313-322. Strauss, B.S. and M. Robbins, DNA methylated in vitro by a monofunctional alkvlating agent as a substrate for a specific nuclease from Micrococcus Ivsodeih ticus, Biochim. Biophvs. Acta, 161 (1968) 68-75. Svachulovli, J., J. Satava and J. Veleminskv, Endonuclease specific for apu rirric sites, isolated from barley leaves, FEBS Lett., in press. Veleminskv, J., T. Gichner and V. Pokorny, Caffeine enhancement of alkylating agent-induced injury in barley: its connection to DNA single strand breaks and their repair, Mutation Res., 28 (1975) 7986. Velemfnskv, J., S. Zadrazil and V. Pokorny, Repair synthesis in barley embryos treated with methyl nitrosourea, Second Symposium on DNA, Liblice Castle near Prague, 1976 (abstract). Velemfnskv, J., S. Zadra~i1, V. Pokorny, T. Gichner and J. Svachulova, Repair of single-strand breaks and fate of N-7-methylguanine in DNA during the recovery from genetical damage induced by Nmethyl-rv-nitrosourea in barley seeds, Mutation Res., 17 (1973) 49-58. Veleminskv, J., S. Zadra~il, V. Pokorny, T. Gichner and J. Svachulova, Storage effect in barley. Changes in the amount of DNA lesions induced by methyl and ethyl methanesulphonate, Mutation Res., 19 (1973) 73-81. Verly, W.G. and E. Rassart, Purification of Escherichia coli endonuclease specific for apurinic sites in DNA, J. BioI. Chern., 250 (1975) 8214-8219. WySS, 0., J. Clark, F. Haas and W.S. Stone, The role of peroxide in the biological effects of irradiated broth, J. Bacteriol., 56 (1948) 51-57.
Mutation Research, 42 (1977) 65-70
© Elsevier/North-Holland Biomedical Press
INDUCTION OF DNA SINGLE-STRAND BREAKS IN BARLEY BY SODIUM AZIDE APPLIED AT pH 3
J. VELEMINSKY, T. GICHNER and V. POKORNY Institute of Experimental Botany, Flemingouo 2, Prague 6 (Czechoslovakia) (Received April 4th, 1976) (Revision received August 17th, 1976) (Accepted September 6th, 1976)
Summary Sodium azide (1 to 50 mM), adjusted to pH 3 and applied for 2 h to presoaked barley seeds, induced a dose-dependent frequency of single-strand breaks in DNA of non-germinating embryos. This was demonstrated by sedimentation analyses of isolated DNA samples in alkaline sucrose gradients and in neutral sucrose gradients with 80% formamide. The doses applied also inhibited dose dependently the root length, seed germination and partially the seedling height. Only the sub-lethal doses (10 and 12.5 mM) induced a low frequency of chromatid breaks and translocations in the root tip metaphases. The sedimentation rate (in alkaline sucrose gradients) of calf thymus DNA treated with sodium azide at pH 3, was similar to that of the control DNA treated with buffer (pH 3) alone.
Introduction Sodium azide, an inhibitor of respiration, is mutagenic in bacteria [3] as well as in barley [8,9]. In Salmonella it induced mutation of the base substitution type but no frame shifts [3,9]. In barley the mutagenic action (scored by "gene" mutations in the M2 generation) is especially strong when azide is applied at pH 3 either to dry or briefly soaked seeds [3,8]. Doses inducing the highest frequency of mutations, comparable e.g. with those produced by ethyl methanesulphonate, are reported not to increase the spontaneous rate of chromosomal aberrations in root tips or in meiosis [11]. Several hypotheses on the molecular nature of azide mutagenesis have been postulated, based on the interaction with DNA [2,10} in genetical experiments with bacteria in vitro [3,4,20], on measurement of enzyme inhibition in barley [12], and on the presence or absence of synergism in combined treatment of azide with ionizing radiation (11], N-methyl-N-nitrosourea, diethylsulphate
66
[5], etc. However, none of these hypotheses can explain all results presented in the literature on azide action. Moreover, in bacteria and in plant or animal cells, no types of azide-induced DNA lesions in vivo have been described. In this paper we present results on the sedimentation properties of DNA, influenced in vivo (barley seeds) or in vitro (calf thymus DNA) by azide adjusted to pH 3. Material and methods Seeds of barley variety Amethyst were soaked for 10 h in water and treated for 2 h in 1 to 50 mM azide dissolved in 0.2 M NaH2 P0 4 and adjusted immediately before the start of treatment to pH 3 by phosphoric acid. After 30 min of post-wash, part of the seeds (about 100) was sown on wet sand in petri dishes for measurement of germination, root length and chromosomal aberrations in the root tips; another part (about 100 seeds) was sown on soil in boxes in the greenhouse for the measurement of seedling height; and the major part was used for the immediate isolation of DNA from the seed embryos. Root length and germination were measured after 3 days, the seedling height after 14 days. For scoring the chromosomal aberrations (chromatid breaks and translocations) in root tip cells, squash preparations stained by the Feulgen method were used. 200 metaphases were evaluated for each azide concentration and each of the 3 restitution times. DNA was isolated from non-germinating seed embryos (about 600-800) by the modified method of Marmur as previously described [17,18]. This procedure enables one to get the double-stranded DNA with molecular weight "'10 7 daltons. Treatment in vitro was carried out with high molecular calf thymus DNA dissolved in 0.15 M NaCl and 0.015 M sodium citrate (SSC) acidified with formic acid to pH 3.2. After 2 h of incubation with azide at 25°C, DNA was precipitated with ethanol and dissolved in SSC. Sedimentation of DNA samples was performed in alkaline sucrose gradient (5--20% sucrose w/v in 0.9 M NaCl, 1 mM EDTA and 0.3 M NaOH), neutral sucrose gradient (5-20% sucrose w]» in 0.9 M NaCl and 1 mM EDTA pH 7), and in neutral sucrose gradient containing 80% formamide (1-15% sucrose w/v dissolved in 80 vol formamide:20 vol H20, pH 7.4). The last-mentioned neutral sucrose-formamide gradient was used to distinguish the formed single strand breaks (SSB) in vivo from alkali-labile sites [13]. Before being layered on this gradient, DNA samples were dissolved in 80% formamide and incubated for 30 min at 37°C. According to hyperchromicity measurement, in these conditions DNA was denatured before, and remained denatured during centrifugation (unpubl.). After the centrifugation in a Spinco L2-65 B ultracentrifuge, rotor SW 27.1 (gradient length 9.2 em) or SW 56 Ti (gradient length 5.5 em), gradients were removed from the top by a Buchler Auto-Densi-Flow II, and DNA was measured continuously in a differential UV analyzer. The position of the peak centre of the DNA sedimentation curve was also taken for the expression of the sedimentation rate. In Fig. 3 the distance of this peak centre, from the peak centre of control (untreated) DNA is expressed by the percentage of the total gradient length.
67
Results Azide applied at pH 3 induced a dose-dependent inhibition of seed germination, root length and seedling height reduction, and at higher doses, a weak increase of chromosomal aberrations in the root tips. The most sensitive characters seem to be root length reduction and inhibition of germination, whereas the seedling height was depressed at a sub-lethal dose (12.5 mM) only to 60% of the control. The highest frequency of breaks and rearrangements in metaphases (9.0%) was reached after the same sub-lethal dose of azide (Fig. 1). The values given in the curve for chromosomal aberrations represent the highest frequency obtained after various periods of restitution, i.e. for 5 mM NaN 3 it was 36 h restitution; for 7.5 mM it was 48 h; for 10 mM, 72 h and for 12.5 mM, 96 h of restitution. In alkaline sucrose gradients, sedimentation rates of DNA samples decreased with dose of azide (Fig. 2a). This dependency on the dose of azide is expressed by comparing the distance of the peak centres of DNA sedimentation curves from the control DNA (Fig. 3, full line; each point on a line represents a mean value from three independent experiments). It is evident that the amount of SSB or alkali-labile sites increased nearly linearly even at lethal doses (25 and 50 mM) of azide. The alkaline gradient cannot be used to distinguish between SSB and alkalilabile sites, since alkaline conditions convert the alkali-labile sites to singlestrand breaks in vitro. For this purpose, aliquots of the same DNA samples,
100T""-_ _
180
80
20
------ ~..
.0••••
O'
,
o
1
5 10 mM NaN 3
12.5
15
Fig. 1. Effects of sodium azide (NaN 3) applied at pH 3 for 2 h to IG-h pre-soaked barley seeds. ( e - - e ) Seed germination; ( _ _ ) seedling height (% of control); ( 0 - - 0 ) root length (% of control); (0- - - - - -0) chromosomal aberrations in metaphases of root tips.
68
a
b
123456
1
36
1.0
1.0
-:0.5
0.5
>u
c
J.
oIII
• ~
'!"
o
20
40
60
+-top
80
100
20
1. of gradient length
40
60
80
100
bottom
Fig. 2. (a) Sedimentation curves of DNA in alkaline sucrose gradient. 19 h, 25000 r.p.m., 5°C, rotor SW 27.1. Azide concentrations: 1, 50 mM; 2, 25 mM; 3, 10 mM; 4,5 mM; 5, 1 mM; 6, O. (b) Sedimentation curves of DNA in neutral sucrose formamide gradient, 6 h, 55,000 r.p.rn., 25°C, rotor SW 56 Ti. Azide concentrations: 1,50 mM; 3,10 mM; 6, O.
which had been analyzed in alkaline sucrose gradients, were denatured in 80% fonnamide and centrifuged in neutral conditions of 80% formamide in the sucrose gradient. The comparison of the sedimentation pattern of the same DNA samples in alkaline sucrose (Fig. 2a) and in neutral sucrose gradient with 80% fonnamide (Fig. 2b) indicates similar differences from the control DNA. We may suppose, therefore, that at least a part if not all the SSB was formed in vivo in the course of the 2·h treatment of seeds with azide at pH 3.
.ll20
"I
cr z c
"015
::c
ou E
,g
.
~10
lit
c
.!
~
• ~ 5 ...o
;;
III
X
~.... 0 1
5
....... -- --_ 10
.... ----- ------------.
25 mM NaN3
50
Fig. 3. Dependence of DNA sedimentation rate in alkaline sucrose gradient (full line) and in neutral sucrose gradient (dashed line) on dose of sodium azide (NaN3) applied in vivo at pH 3.
69
In contrast with the different sedimentation properties of denatured, azidetreated DNA, the size of the double-stranded form of the same samples, as analyzed in neutral sucrose gradient, seems not to be influenced by the action of azide. This is documented by the position of the peak centre of the sedimentation curves in the gradient relative to the untreated control barley DNA (Fig. 3, dashed line). We can thus conclude that azide induced in vivo a dose-dependent frequency of SSB in barley DNA, without inducing double-strand breaks. Application of five sodium azide concentrations at pH 3.2 (2-100 mM, 2 h, 25°C) to calf thymus DNA in vitro resulted in a similar lack of dose-dependent changes of the sedimentation patterns on an alkaline sucrose gradient. Differences of the peak centre distance from the untreated DNA varied from 8 to 0.7%. The heating of azide-treated DNA samples for 10 min at 100 0 e before they were layered on the alkaline sucrose gradient did not change these values. Thus, in our experimental conditions and dose range, azide induced neither SSB, alkali labile sites (apurinic or apyrimidinic sites) nor heat-labile sites in vitro. Discussion Detection of DNA breaks is mostly performed on native DNA according to the method of McGrath and Williams [7] by lysing the bacterial or mammalian cells on the top of the alkaline sucrose gradient. This method requires DNA that had been pre-labelled in the course of the preceding cell cycle before the action of break-inducing agents. This was impossible to do in the conditions of our experiments in which the non-growing tissues of barley embryos were treated before seed germination. Moreover, tissues and cells of higher plants cannot be lysed on the gradient unless protoplasts are obtained. The DNA isolation according to Marmur, used in our experiments, certainly lowered the sensitivity of sedimentation analysis. In spite of this disadvantage the decreasing
sedimentation rate of DNA in alkaline sucrose gradient with increasing dose of azide obtained in all our experiments, as well as the results obtained in neutral formamide gradients, indicates that SSB are formed in the embryonic cells of barley seeds as a consequence of azide treatment at pH 3 in vivo. Although in low amount, SSB were induced even by concentrations usually used for mutation induction [6,7,10,11]. This implies that SSB, or the DNA lesions from which they originate, can be considered as one of the possible pre-mutational lesions in barley. How these lesions are induced and what is the nature of the mechanism leading to the mutation induction in barley, however, cannot be decided from the results presented here. Kleinhofs and Smith [4] had demonstrated that azide mutagenesis in bacteria is dependent on DNA lesions excisable by the uvr system, and mutations are produced by the error-prone ree-dependent repair pathways. Base damage which leads to the base loss (comparable e.g. to that known after ')'-rays [6] or after the action of alkylating agents [19]) can offer a good substrate for both an error-prone pathway and backbone breakage. Endonuclease, specific for apurinic and apyrimidinic sites and which produces SSB, has now been isolated from barley [14]. Both excision repair pathway [16,17] and post-replication
70
repair [15] were demonstrated in barley for DNA damaged induced by alkylating agents. Enzymes, acting in these processes, can contribute to the origin of breaks or mutations even after the action of azide. Moreover, the spontaneous origin of SSB caused by lowering the cellular pH due to the uptake of hydrazoic acid cannot be excluded. The inability to find any DNA lesion induced by azide at pH 3 in vitro [2] supports the assumption that, in vivo, azide is activated by a yet unknown interaction of hydrazoic acid with cellular components. References
2 3
4 5
6 7 8 9 10 11
12
13
14 15
16 17
18
19 20
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