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In situ microfluorometry of kinetoplast and nuclear DNAs in Trypanosoma gambiense unusual repairment of DNA after treatment with bleomycin
Zbl. Bakt. Hyg. A 264, 386-391 (1987)
In situ Microfluorometry of Kinetoplast and Nuclear DNAs in Trypanosoma gambiense Unusual Repairment of DNA After Treatment with Bleomycin*
no
2 3 SHOZO INOKI 1 , YOSHIHIRO , TSUTOMU ARAKI , TSUNE]I 2 4 ARAKI!, MIKIO OKA , HUMIO OSAKI , and MASA-OKI YAMADA 3
Department of Parasitology, Nara Medical University, Kashihara, Nara 634, Japan Department of Parasitology, School of Medicine, The University of Tokushima, Tokushi. rna 770, Japan 3 Department of Anatomy, School of Medicine, The University of Tokushima, Tokushima 770,Japan 4 Kochi Medical School, Nankoku, Kochi 781-51, Japan 1
2
With 4 Figures· Received March 20, 1986
Summary The blood stream form of Trypanosoma gambiense was smeared on a nonfluorescent slide glass with 1 [tg/ml of Hoechst 33258 in 1mM Tris-Hel buffer (pH 7.2) containing 1% 2-mercaptoethanol and subjected to the in situ microfluorometry. Effects of bleomycin (BL) on the kinetoplast (K)-DNA and nuclear (N)-DNA of T. gambiense were examined in the time course to 6 h after injection of BL into the infected mice. An enhancement of fluorescence occurred 30 min after the injection and then slowed down. This enhancement was due to DNA synthesis both in the K-DNA and N-DNA. This suggests that the strong repairment occurs in both DNAs after treatment with BL. Introduction The kinetoplast DNA (K-DNA) was revealed under electron microscopy by many authors (Riou and Pantrizel, 1967; Riou, 1968; Riou and Pantrizel, 1971; Ono et al., 1971; Ozeki et al., 1970, 1971) and its in situ quantification has been studied on microfluorometry by Inoki (1976,1981) and Dvorak (1980, 1982). In determination of K-DNA, a fluorochrome such as Hoechst 33258 (Ho) and DAPI bound specifically
'f Presented at the 5th German-Japanese Cooperative Symposium on Protozoan Diseases, Tokyo, Japan, Sept. 25-28, 1985. This study was supplemented to the preliminary experiments initiated by late Prof. Dr. Shozo Inoki who died suddenly at the end of March 1985. We would like to dedicate the supplemental data to him with our heartily regret.
Microfluorometry of Kinetoplast
387
with AT base pairs was effective because K-DNA contained enough amount of AT. But, troublesome errors of fluorescence quenching on the smear specimen of trypanosomes were unavoidable (Inoki et a!', 1981). This study was made on effects of bleomycin (BL) which reduced the fluoresence (Inoki et a!., 1984 a). Undesirable quenching in fluoresence intensity was eliminated by using 2-mercaptoethanol in the staining medium. The fluorescence of DNA-Ho was temporarily enhanced after in vivo administration of BL. The enhancement depends on the increase of DNA synthesis. The hypothetical concept of stimulating DNA synthesis is discussed in the paper. Materials and Methods
1. Isolation oftrypanosomes. A clone of Trypanosoma gambiense (strain Wellcome) used for series of experiments was given by courtesy of Dr. Max C. McCohen, the Eli Lilly Laboratories, Indianapolis, Indiana, U.S.A. It has been passed through mice at the Department of Protozoology, Research Institute for Microbial Diseases, Osaka University. Mice (ddY strain) weighing 20 g were inoculated with this clone and used for the study. The trypanosome cells were collected from the infected mouse blood through the DEAE cellulose column (Lanham and Godfrey, 1970). 2. Microfluorometry. The infected mice were bled from the tail end. The blood was smeared on a nonfluorescent slide glass and then stained with 1 I-lg/ml of Ho in 1 mM TrisHCI buffer, pH 7.2, containing 1% 2-mercaptoethanol and sealed with nail lacquer. The stained specimens were subjected to the fluorescence microscope. (Nikon XF-EF) equipped with a photon counter. 3. Chemical analyses. Isolated parasites were adjusted at a concentration of 10 9 cells/ml in phosphate-buffered saline, pH 7.4, and then homogenized with a teflon grinder. The homogenate was fractionated into RNA, DNA and protein fractions according to Schneider's method (1946). The content of RNA in the parasites was measured by the methods of Freck and Munro (1962) and Ceriotti (1955), respectively. 4. Administration of Bleomycin. Bleomycin (Nippon Kayaku Pharmaceutical Co., Ltd.) dissolved in 0.85% NaCl was administered intraperitoneally into the heavily infected mice at a dose of 1 mg/kg. Results Until 4.5 h after intraperitoneal administration of a single dose of BL into mice, trypanosomes in the peripheral blood appeared to be unchanged in number per ml blood (Table 1). The DNA amount of I-lg/10 7 cells was nearly doubled after 4.5 h lapse Table 1. Number of cells and DNA content after in vivo treatment with BL Time after treatment with BL (30 I-lg, i.p.)
Number of cells in the mouse blood
DNA
hr
10 9 1.1 1.2 1.0 1.1 1.0
I-lgll0 7 cells 8.3 13.2 10.2 11.1 16.2
o
0.5 1 3 4.5
cells/ml 0.1 0.1 0.1 0.2 0.2
± ± ± ± ±
388
S.lnoki et al.
in parallel with the increase of RNA. However, a temporal rise of DNA occurred 30 min after the administration. The increase of DNA was also observed in a short term microfluorometrically in both the cells which contained one (IK-IN) and two (2K-IN) kinetoplasts with a single nucleus (Fig. I). The population kinetics of both IK-IN and 2K-IN types did not change in the initial 2 h on the level of 55% of the IK-IN population, then changed to the level of 65% after 3 h or more (Fig. 2). This means that the changing time of the 2K-IN type to the 2 (lK-IN) type may be suppressed at least for 2 h after administration of BL, and then about 5% of the 2K-IN type may be allowed to change to the IK-IN type. So that, the population ratio of the IK-IN type to the 2K-IN was not changed noticeably in the time course till 6 h after the administration. In the microfluotometric assay of K-DNA (Fig. 3), a
FUD
1500
.. ,
,~. ,, ,
,
: ,
/ ,
.,
1000
(Nucleus)
"
''
~ ...
2K-1N
........ .,
\---~
500
o~'
o
.,
! ! !
1 2 3 4 time after treatment with BL
5
6h
Fig. 1. Microfluorometry of nuclear DNA in lK-lN and 2K-2N cells after treatment with
BL.
%
.
100
(2K -1Nl -(2K -2Nl -2(1K-1N)
r -----_,
50r
0'"
o
1K-1N
! .
1
2
3 4 time after treatment with BL
5
6h
Fig. 2. Percent incidence of lK-lN and 2K-IN cells after treatment with BL.
Microfluorometry of Kinetoplast
389
temporal increase of fluorescence occurred 30 min after the adminstration simultaneously in the kinetoplasts of both lK-lN and 2K-IN types, whereas the 2K-IN type was estimated totally as the two kinetoplasts. The ratio of K-DNA to N-DNA was followed after treatment with BL, and the ratios between kinetoplasts of both lK-lN and 2K-IN types were compared (Fig. 4). There was a considerable fall of the ratio after a 30 min interval and a good agreement between both types. The fall was estimated as about 3%. The value seems to be great in comparison with that in the initial control. So, the temporal DNA synthesis found in both kinetoplasts is not as much as that in the nuclei on the basis of DNA assay with the fluorescence parameter of AT base pairs. Thus, the fluorescence enhancement of both N- and K·DNAs occurred in parallel with the total increase of DNA found in the chemical assay. The enhancement did not occur in pure calf thymus DNA treated in vitro with BL.
Fuo
100
,.
(Kinetoplast)
"
80
60'
0'
...
"
2K-1N
40
20'
~c~
oL' o
!
,
!
!
!
1 2 3 4 time after treatment with BL
5
6h
Fig, 3. Microfluorometry of kinetoplast DNA in lK-lN and 2K-IN cells after treatment with BL.
%(',)
81-\~ H
' ,
....__ ~ K-1N
--.-~~·~;N----..
,r········
2
,
,
1
I
!
2
3
!
4
,
5
time after treatment with BL
Fig. 4. Ratio of K-DNA to N-DNA after treatment with BL.
I
6h
390
S.Inoki et al.
Discussion The data indicate that the results of chemical DNA assay agree appreciably with those of microfluoroassay of DNA by using such a specific fluorescent probe as Ho. Concerning the base content in trypanosomes, the AT-base content proceeds the GCcontent of DNA, especially the AT occupies up to 70% in the K-DNA of trypanosomes (Steinert and Steinert, 1962; Riou, 1968) assayed with ethidium bromide. According to the high content of AT with Ho, the K-DNA is regarded as much as DNA than usual DNA which contains an equivalent amount of AT to Gc. In the advantage of high sensitivity of Ho to AT, Ho was effectively applied for the assay of trypanosome DNA in the present paper. A sudden increment of Ho fluorescence occurred in both K- and N-DNAs in a short term of 30 min after in vivo administration of BL. The increase reached 30% or more, and agreed with the increase of DNA amount estimated by an orthodox chemical assay suggesting a sudden increase of DNA itself. When pure calf thymus DNA was treated in vitro with BL, and then Ho fluorescence was measured, no increase was found in the fluorescence. The increase seems to be depending mostly on DNA synthesis, because the increase is not seemingly depending on the modification of Ho bound to AT after treatment with BL. If the increase of fluorescence intensity was due to DNA synthesis caused by BL, a 30% increase was too big for the repairment of DNA injury possibly affected by BL. However, the increased amount was absolutely responsible for the DNA synthesis on the basis of both chemical and fluorometric assays. This sort of temporal increase is known as the repairment after DNA injury. When DNA is destructed by enough amount of BL, the repairment system may not work. The DNA destruction is seemingly not enough and then repairing activity is kept working at the dose we used here. When specific inhibitors of the repairment enzyme system known such as 3-aminobenzamide (Ohnishi et aI., 1982, 1983) was injected together with BL, the fluorescence was suppressed ([noki et al., 1984 b). If it was reasonably fitted for the present data, it is highly possible to induce an overrepairment occurred only after the DNA injury by BL. Acknowledgements. The authors are indebted to Prof. Keiichi Nozu and Dr. Takeo Ohnishi, Department of Biology, Nara Medical University, for their advice and discussion in preparing the data.
References
1. Ceriotti, G.: Determination of nucleic acid in animal tissue. ]. BioI. Chern. 214 (1955) 59~70
2. Dvorik, ]. A., D. 1. Hartman, and M. A. Miles: Trypanosoma cruzi: Correlation of growth kinetics to zymodeme type in clones derived from various sources. ]. Protozoal. 27 (1980) 472-474 3. Dvorak, ]. A., T. E. Hall, M. St. ]. Crane, ]. C. Engel, J. P. McDaniel, and R. Uriegas: Trypanosoma cruzi: Flow cytometric analyses. I. Analysis of total DNA/organism by means of mithramycin-induced fluorescence. J. Protozoal. 29 (1982) 430-437 4. Freck, A. and H. A. Munro: The precision of ultraviolet absorption measurements in the Schmidt-Thannhauser procedure for nucleic acid estimation. Biochem. Biophys. Acta 55 (1962) 571-583
Microfluorometry of Kinetoplast
391
5. Inoki, S., H. Osaki, and M. Furuya: In situ microspectrofluorometry of nuclear and kinetoplast DNA in Trypanosoma cruzi. Resumenes de Trabajos Libres. (IV Congreso Centroamericano de Microbiologia y Parasitologia. San jose, Costa Rica), p. 256 (1976) 6. Inoki, S., H. Osaki, and M. Furuya: In situ microfluorometry of whole kinetoplast and nuclear DNA of Trypanosoma gambiense and Trypanosoma cruzi. Zbl. Bakt. Hyg., 1. Abt. Orig. A 250 (1981) 182-190 7. Inoki, S., H. Osaki, and M. Furuya: Effects of bleomycin on kinetoplast DNA and nuclear DNA in Trypanosoma gambiense using in situ microfluorometry technique. Zbl. Bakt. Hyg., A 256 (1984 a) 300-304 8. Inoki, S., H. Osaki, M. Furuya, Y. Ito, and M. Oka: In situ microfluorometry of kinetoplast DNA and nuclear DNA in Trypanosoma gambiense. Papers presented at 3rd lnternatl. Congr. on Cell Biology in Tokyo, 4137 (1984 b) p.465 9. Lanham, S. M. and D. G. Godfrey: Isolation of salivarian trypanosomes from man and other mammals using DEAE-cellulose. Exp. Parasit. 28 (1970) 521-534 10. Ohnishi, T., H. Eimoto, and K. Okaichi: Enhancement of ultraviolet or N-methyl-N'nitro-N-nitrosoguanidine sensitivity of Dicytostelium discoideum by 3-aminobenzamide. Photochem. Photo bioI. 35 (1982) 515-519 11. Ohnishi, T., Y. Ohashi, K. Nozu, and S. Inoki: Mutagenicity of anti-trypanosomal drug. Ro 7-1051, in E. coli. jap.]. Gen. 58 (1983) 505-509 12. Ono, T., Y. Ozeki, S. Okubo, and S. Inoki: Characterization of nuclear and satellite DNA from trypanosomes. Biken j. 14 (1971) 203-215 13. Ozeki, Y., T. Ono, S. Okubo, and S. Inoki: Electron microscopy of DNA released from ruptured kinetoplasts of Trypanosoma gambiense. Biken]. 13 (1970) 387-393 14. Ozeki, Y., V. Sooksri, T. Ono, and S. Inoki: Studies on the ultrastructure of kinetoplasts of Trypanosoma cruzi and Trypanosoma gambiense by autoradiography and enzymatic digestion. Biken]. 14 (1971) 97-118 15. Riou, G. and R. Pantrizel: Fractionnement et caracterisation de deux bandes satellites de DNA chez Trypanosoma gambiense. C. R. Acad. Sci. Ser. P 265 (1967) 61-63 16. Riou, G.: Disparition de l'ADN du kinetoplaste de Trypanosoma cruzi cultive en presence de bromure d'ethidium. C. R. Acad. Sci. Ser. P 266 (1968) 250-252 17. Riou, G. and R. Pantrizel: Nuclear and kinetoplast DNA from trypanosomes. j. Protozool. 16 (1969) 509-513 18. Schneider, W. Phosphorus compounds in animal tissues. III. A comparison of methods for the estimation of nucleic acids. ]. BioI. Chern. 164 (1946) 741-751 19. Steinert, M. and Steinert, G.: La synthese de l'acide desoxyribo-nucleique au cours du cycle de division de Trypanosoma mega. ]. Protozoal. 9 (1962) 203-211
c.:
Dr. Shozo Inoki, Dept. of Parasitology, Nara Medical University, Kashihara, Nara 634, japan
26 Zbl. Bakt. Hyg. A 264/3-4
In situ Microfluorometry of Kinetoplast and Nuclear DNAs in Trypanosoma gambiense Unusual Repairment of DNA After Treatment with Bleomycin*
no
2 3 SHOZO INOKI 1 , YOSHIHIRO , TSUTOMU ARAKI , TSUNE]I 2 4 ARAKI!, MIKIO OKA , HUMIO OSAKI , and MASA-OKI YAMADA 3
Department of Parasitology, Nara Medical University, Kashihara, Nara 634, Japan Department of Parasitology, School of Medicine, The University of Tokushima, Tokushi. rna 770, Japan 3 Department of Anatomy, School of Medicine, The University of Tokushima, Tokushima 770,Japan 4 Kochi Medical School, Nankoku, Kochi 781-51, Japan 1
2
With 4 Figures· Received March 20, 1986
Summary The blood stream form of Trypanosoma gambiense was smeared on a nonfluorescent slide glass with 1 [tg/ml of Hoechst 33258 in 1mM Tris-Hel buffer (pH 7.2) containing 1% 2-mercaptoethanol and subjected to the in situ microfluorometry. Effects of bleomycin (BL) on the kinetoplast (K)-DNA and nuclear (N)-DNA of T. gambiense were examined in the time course to 6 h after injection of BL into the infected mice. An enhancement of fluorescence occurred 30 min after the injection and then slowed down. This enhancement was due to DNA synthesis both in the K-DNA and N-DNA. This suggests that the strong repairment occurs in both DNAs after treatment with BL. Introduction The kinetoplast DNA (K-DNA) was revealed under electron microscopy by many authors (Riou and Pantrizel, 1967; Riou, 1968; Riou and Pantrizel, 1971; Ono et al., 1971; Ozeki et al., 1970, 1971) and its in situ quantification has been studied on microfluorometry by Inoki (1976,1981) and Dvorak (1980, 1982). In determination of K-DNA, a fluorochrome such as Hoechst 33258 (Ho) and DAPI bound specifically
'f Presented at the 5th German-Japanese Cooperative Symposium on Protozoan Diseases, Tokyo, Japan, Sept. 25-28, 1985. This study was supplemented to the preliminary experiments initiated by late Prof. Dr. Shozo Inoki who died suddenly at the end of March 1985. We would like to dedicate the supplemental data to him with our heartily regret.
Microfluorometry of Kinetoplast
387
with AT base pairs was effective because K-DNA contained enough amount of AT. But, troublesome errors of fluorescence quenching on the smear specimen of trypanosomes were unavoidable (Inoki et a!', 1981). This study was made on effects of bleomycin (BL) which reduced the fluoresence (Inoki et a!., 1984 a). Undesirable quenching in fluoresence intensity was eliminated by using 2-mercaptoethanol in the staining medium. The fluorescence of DNA-Ho was temporarily enhanced after in vivo administration of BL. The enhancement depends on the increase of DNA synthesis. The hypothetical concept of stimulating DNA synthesis is discussed in the paper. Materials and Methods
1. Isolation oftrypanosomes. A clone of Trypanosoma gambiense (strain Wellcome) used for series of experiments was given by courtesy of Dr. Max C. McCohen, the Eli Lilly Laboratories, Indianapolis, Indiana, U.S.A. It has been passed through mice at the Department of Protozoology, Research Institute for Microbial Diseases, Osaka University. Mice (ddY strain) weighing 20 g were inoculated with this clone and used for the study. The trypanosome cells were collected from the infected mouse blood through the DEAE cellulose column (Lanham and Godfrey, 1970). 2. Microfluorometry. The infected mice were bled from the tail end. The blood was smeared on a nonfluorescent slide glass and then stained with 1 I-lg/ml of Ho in 1 mM TrisHCI buffer, pH 7.2, containing 1% 2-mercaptoethanol and sealed with nail lacquer. The stained specimens were subjected to the fluorescence microscope. (Nikon XF-EF) equipped with a photon counter. 3. Chemical analyses. Isolated parasites were adjusted at a concentration of 10 9 cells/ml in phosphate-buffered saline, pH 7.4, and then homogenized with a teflon grinder. The homogenate was fractionated into RNA, DNA and protein fractions according to Schneider's method (1946). The content of RNA in the parasites was measured by the methods of Freck and Munro (1962) and Ceriotti (1955), respectively. 4. Administration of Bleomycin. Bleomycin (Nippon Kayaku Pharmaceutical Co., Ltd.) dissolved in 0.85% NaCl was administered intraperitoneally into the heavily infected mice at a dose of 1 mg/kg. Results Until 4.5 h after intraperitoneal administration of a single dose of BL into mice, trypanosomes in the peripheral blood appeared to be unchanged in number per ml blood (Table 1). The DNA amount of I-lg/10 7 cells was nearly doubled after 4.5 h lapse Table 1. Number of cells and DNA content after in vivo treatment with BL Time after treatment with BL (30 I-lg, i.p.)
Number of cells in the mouse blood
DNA
hr
10 9 1.1 1.2 1.0 1.1 1.0
I-lgll0 7 cells 8.3 13.2 10.2 11.1 16.2
o
0.5 1 3 4.5
cells/ml 0.1 0.1 0.1 0.2 0.2
± ± ± ± ±
388
S.lnoki et al.
in parallel with the increase of RNA. However, a temporal rise of DNA occurred 30 min after the administration. The increase of DNA was also observed in a short term microfluorometrically in both the cells which contained one (IK-IN) and two (2K-IN) kinetoplasts with a single nucleus (Fig. I). The population kinetics of both IK-IN and 2K-IN types did not change in the initial 2 h on the level of 55% of the IK-IN population, then changed to the level of 65% after 3 h or more (Fig. 2). This means that the changing time of the 2K-IN type to the 2 (lK-IN) type may be suppressed at least for 2 h after administration of BL, and then about 5% of the 2K-IN type may be allowed to change to the IK-IN type. So that, the population ratio of the IK-IN type to the 2K-IN was not changed noticeably in the time course till 6 h after the administration. In the microfluotometric assay of K-DNA (Fig. 3), a
FUD
1500
.. ,
,~. ,, ,
,
: ,
/ ,
.,
1000
(Nucleus)
"
''
~ ...
2K-1N
........ .,
\---~
500
o~'
o
.,
! ! !
1 2 3 4 time after treatment with BL
5
6h
Fig. 1. Microfluorometry of nuclear DNA in lK-lN and 2K-2N cells after treatment with
BL.
%
.
100
(2K -1Nl -(2K -2Nl -2(1K-1N)
r -----_,
50r
0'"
o
1K-1N
! .
1
2
3 4 time after treatment with BL
5
6h
Fig. 2. Percent incidence of lK-lN and 2K-IN cells after treatment with BL.
Microfluorometry of Kinetoplast
389
temporal increase of fluorescence occurred 30 min after the adminstration simultaneously in the kinetoplasts of both lK-lN and 2K-IN types, whereas the 2K-IN type was estimated totally as the two kinetoplasts. The ratio of K-DNA to N-DNA was followed after treatment with BL, and the ratios between kinetoplasts of both lK-lN and 2K-IN types were compared (Fig. 4). There was a considerable fall of the ratio after a 30 min interval and a good agreement between both types. The fall was estimated as about 3%. The value seems to be great in comparison with that in the initial control. So, the temporal DNA synthesis found in both kinetoplasts is not as much as that in the nuclei on the basis of DNA assay with the fluorescence parameter of AT base pairs. Thus, the fluorescence enhancement of both N- and K·DNAs occurred in parallel with the total increase of DNA found in the chemical assay. The enhancement did not occur in pure calf thymus DNA treated in vitro with BL.
Fuo
100
,.
(Kinetoplast)
"
80
60'
0'
...
"
2K-1N
40
20'
~c~
oL' o
!
,
!
!
!
1 2 3 4 time after treatment with BL
5
6h
Fig, 3. Microfluorometry of kinetoplast DNA in lK-lN and 2K-IN cells after treatment with BL.
%(',)
81-\~ H
' ,
....__ ~ K-1N
--.-~~·~;N----..
,r········
2
,
,
1
I
!
2
3
!
4
,
5
time after treatment with BL
Fig. 4. Ratio of K-DNA to N-DNA after treatment with BL.
I
6h
390
S.Inoki et al.
Discussion The data indicate that the results of chemical DNA assay agree appreciably with those of microfluoroassay of DNA by using such a specific fluorescent probe as Ho. Concerning the base content in trypanosomes, the AT-base content proceeds the GCcontent of DNA, especially the AT occupies up to 70% in the K-DNA of trypanosomes (Steinert and Steinert, 1962; Riou, 1968) assayed with ethidium bromide. According to the high content of AT with Ho, the K-DNA is regarded as much as DNA than usual DNA which contains an equivalent amount of AT to Gc. In the advantage of high sensitivity of Ho to AT, Ho was effectively applied for the assay of trypanosome DNA in the present paper. A sudden increment of Ho fluorescence occurred in both K- and N-DNAs in a short term of 30 min after in vivo administration of BL. The increase reached 30% or more, and agreed with the increase of DNA amount estimated by an orthodox chemical assay suggesting a sudden increase of DNA itself. When pure calf thymus DNA was treated in vitro with BL, and then Ho fluorescence was measured, no increase was found in the fluorescence. The increase seems to be depending mostly on DNA synthesis, because the increase is not seemingly depending on the modification of Ho bound to AT after treatment with BL. If the increase of fluorescence intensity was due to DNA synthesis caused by BL, a 30% increase was too big for the repairment of DNA injury possibly affected by BL. However, the increased amount was absolutely responsible for the DNA synthesis on the basis of both chemical and fluorometric assays. This sort of temporal increase is known as the repairment after DNA injury. When DNA is destructed by enough amount of BL, the repairment system may not work. The DNA destruction is seemingly not enough and then repairing activity is kept working at the dose we used here. When specific inhibitors of the repairment enzyme system known such as 3-aminobenzamide (Ohnishi et aI., 1982, 1983) was injected together with BL, the fluorescence was suppressed ([noki et al., 1984 b). If it was reasonably fitted for the present data, it is highly possible to induce an overrepairment occurred only after the DNA injury by BL. Acknowledgements. The authors are indebted to Prof. Keiichi Nozu and Dr. Takeo Ohnishi, Department of Biology, Nara Medical University, for their advice and discussion in preparing the data.
References
1. Ceriotti, G.: Determination of nucleic acid in animal tissue. ]. BioI. Chern. 214 (1955) 59~70
2. Dvorik, ]. A., D. 1. Hartman, and M. A. Miles: Trypanosoma cruzi: Correlation of growth kinetics to zymodeme type in clones derived from various sources. ]. Protozoal. 27 (1980) 472-474 3. Dvorak, ]. A., T. E. Hall, M. St. ]. Crane, ]. C. Engel, J. P. McDaniel, and R. Uriegas: Trypanosoma cruzi: Flow cytometric analyses. I. Analysis of total DNA/organism by means of mithramycin-induced fluorescence. J. Protozoal. 29 (1982) 430-437 4. Freck, A. and H. A. Munro: The precision of ultraviolet absorption measurements in the Schmidt-Thannhauser procedure for nucleic acid estimation. Biochem. Biophys. Acta 55 (1962) 571-583
Microfluorometry of Kinetoplast
391
5. Inoki, S., H. Osaki, and M. Furuya: In situ microspectrofluorometry of nuclear and kinetoplast DNA in Trypanosoma cruzi. Resumenes de Trabajos Libres. (IV Congreso Centroamericano de Microbiologia y Parasitologia. San jose, Costa Rica), p. 256 (1976) 6. Inoki, S., H. Osaki, and M. Furuya: In situ microfluorometry of whole kinetoplast and nuclear DNA of Trypanosoma gambiense and Trypanosoma cruzi. Zbl. Bakt. Hyg., 1. Abt. Orig. A 250 (1981) 182-190 7. Inoki, S., H. Osaki, and M. Furuya: Effects of bleomycin on kinetoplast DNA and nuclear DNA in Trypanosoma gambiense using in situ microfluorometry technique. Zbl. Bakt. Hyg., A 256 (1984 a) 300-304 8. Inoki, S., H. Osaki, M. Furuya, Y. Ito, and M. Oka: In situ microfluorometry of kinetoplast DNA and nuclear DNA in Trypanosoma gambiense. Papers presented at 3rd lnternatl. Congr. on Cell Biology in Tokyo, 4137 (1984 b) p.465 9. Lanham, S. M. and D. G. Godfrey: Isolation of salivarian trypanosomes from man and other mammals using DEAE-cellulose. Exp. Parasit. 28 (1970) 521-534 10. Ohnishi, T., H. Eimoto, and K. Okaichi: Enhancement of ultraviolet or N-methyl-N'nitro-N-nitrosoguanidine sensitivity of Dicytostelium discoideum by 3-aminobenzamide. Photochem. Photo bioI. 35 (1982) 515-519 11. Ohnishi, T., Y. Ohashi, K. Nozu, and S. Inoki: Mutagenicity of anti-trypanosomal drug. Ro 7-1051, in E. coli. jap.]. Gen. 58 (1983) 505-509 12. Ono, T., Y. Ozeki, S. Okubo, and S. Inoki: Characterization of nuclear and satellite DNA from trypanosomes. Biken j. 14 (1971) 203-215 13. Ozeki, Y., T. Ono, S. Okubo, and S. Inoki: Electron microscopy of DNA released from ruptured kinetoplasts of Trypanosoma gambiense. Biken]. 13 (1970) 387-393 14. Ozeki, Y., V. Sooksri, T. Ono, and S. Inoki: Studies on the ultrastructure of kinetoplasts of Trypanosoma cruzi and Trypanosoma gambiense by autoradiography and enzymatic digestion. Biken]. 14 (1971) 97-118 15. Riou, G. and R. Pantrizel: Fractionnement et caracterisation de deux bandes satellites de DNA chez Trypanosoma gambiense. C. R. Acad. Sci. Ser. P 265 (1967) 61-63 16. Riou, G.: Disparition de l'ADN du kinetoplaste de Trypanosoma cruzi cultive en presence de bromure d'ethidium. C. R. Acad. Sci. Ser. P 266 (1968) 250-252 17. Riou, G. and R. Pantrizel: Nuclear and kinetoplast DNA from trypanosomes. j. Protozool. 16 (1969) 509-513 18. Schneider, W. Phosphorus compounds in animal tissues. III. A comparison of methods for the estimation of nucleic acids. ]. BioI. Chern. 164 (1946) 741-751 19. Steinert, M. and Steinert, G.: La synthese de l'acide desoxyribo-nucleique au cours du cycle de division de Trypanosoma mega. ]. Protozoal. 9 (1962) 203-211
c.:
Dr. Shozo Inoki, Dept. of Parasitology, Nara Medical University, Kashihara, Nara 634, japan
26 Zbl. Bakt. Hyg. A 264/3-4