- Email: [email protected]
Sister chromatid exchange in patients with viral disease
450
Preliminary
notes
induced phase separation of phospholipids which is speculated to occur during fusion [22]. Recently, Chen was able to demonstrate a major redistribution of the LETS protein during and after fusion by immunofluorescence. He reported that when fusion was blocked, the redistribution of LETS did not occur. Furthermore, recognition of LETS was significantly reduced after fusion as determined by radioimmunoassay. Our observation of the 245000 iodinated material which appeared as a double band (M,A-M,B) at prefusion and a single band (M,) at mid- and postfusion might reflect specific modification or replacement of these surface proteins which may be necessary in order for fusion to occur. Modification of cell surface proteins appears to be important for cell-cell interaction in other systems. Hynes [lS] reported that proteolysis may reveal cryptic sites in the membranes of normal cells much like those seen in transformed cells. Pouyssegur & Pastan [ 191 observed that mutant fibroblasts with substrate adhesion deficiencies lacked many of the iodinated surface proteins found on nonmutant fibroblasts. Rutishauser et al. [23] proposed a model in which specific proteolytic cleavage of surface proteins may be involved in recognition and adhesion of embryonic chick neural retinal cells. Therefore, the observed increase in iodinated material in the 10500 M, band (table 1) may be suggestive of an increase in proteolytic activity associated with fusing muscle cells. It is tempting to speculate that specific proteolytic cleavage sites might exist on prefusion myoblasts which are modified in order to bring about fusion. Substantiation of increased protease activity associated with fusing muscle cells remains to be conclusively demonstrated. This possibility is currently under investigation in our laboratory. E-v Cell Res I I3 (1978)
This research was supported by the Muscular Dystrophy Association of America.
References 1. Yaffe, D & Feldman, M, Dev biol 11 (1965) 300. 2. Holtzer, H, Bischoff, R & Chacko, S, Cellular recognition, pp. 19-25. Appleton, New York (1969). 3. Reporter, M & Norris, G, Differentiation 1 (1973) 83. 4. Reporter, M & Raveed, D, Science lSl(1973) 863. 5. Kent, C, Schimmel, S & Vagelos, P, Biochim biophys acta 360 (1974) 312. 6. Winand, R & Luzzati, D, Biochimie 57 (1975) 764. 7. Hynes, R 0, Martin, G S, Shearer, M, Critchley, D R & Epstein, C J, Dev biol48 (1976) 35. 8. Chen, LB, Cell 10 (1977) 393. 9. Yaffe, D, Proc natl acad sci US 61 (1968) 477. IO. Konigsberg, I R, Methods in developmental biology, pp. 520-521. T. Y. Crowell Co., New York (1967). Il. Hauschka, S D, Growth, nutrition and metabolism of cells in culture, pp. 84-85. Academic Press, New York (1972). 12. Fishbach. G D. Dev biol28 (1972) 407. 13. Moss, P & Strdhman, R, Ceil 10 (1977) 265. 14. Ames Co. Catalog on disc electrophoresis, p. 14. Elkhart, Indiana (1976). 15. Laemmli, U D, Nature 227 (1970) 680. 16. Eisen, H N, Immunology, p. 375. Harper & Row, New York (1974). 17. Hogg, N M, Proc natl acad sci US 71 (1974) 489. 18. Hynes, R 0, Proc natl acad sci US 70 (1973) 3170. 19. Pouyssegur, J M & Pastan, I, Proc natl acad sci US 73 (1976) 544. 20. Teng, N & Chen, LB, Nature 259 (1976) 578. 21. Richler, C & Yaffe, D, Dev bio123 (1970) 1. 22. Weidekamm, E, Schudt, C & Brdiczka, D, Biochim biophys acta 433 (1976) 169. 23. Rutishauser, U, Thiery, J P, Brackenbury, R, Amisela, B & Edelman, G, Proc natl acad sci US 73 (1976) 577. Received August 18, 1977 Revised version received January 4, 1978 Accepted January 20, 1978
Sister chromatid viral disease
exchange in patients with
KAREN KURVINK,’ CLARA D. BLOOMFIELD* and JAROSLAV CERVENKA,’ ‘Divisions of Oral Patholoav and of Human and Oral Genetics. and zDepartment of kedicine, University of Minnesota Health Sciences Center. Minneauolis. MN 55455. USA
Sister chromatid exchange (SCE) frequencies were studied in differentially stained chromosomes from lymphocytes of 17 patients with viral disease. The mean SCE score for the patients was 8.7+
Summary.
Preliminary
Table 1. SCE in lymphocytes from individuals
Disease
IlO.
Time from onset of symptoms (days)
Herpes simplex
11 30 43 83 86 85 101 70
1 7 2 2 >l mo. 3 2 2
Patient
Mean SCE + S.D.
22 9 29
9.2f3.5 7.8k3.7 6.3k2.2 10.6f2.9 13.5f4.9 9.Ot4.2 7.3k2.6 5.5f2.4 8.7k2.6
3-16 3-15 3-12 4-18 3-22 3-17 3-14 2-13
15 34 41 20 56 13
4.5k2.1 11.8f4.2 6.9k3.4 10.8k3.9 9.0f3.3 5.9k2.5 8.2k2.9
2-10 4-21 2-19 4-19 3-21 3-11
41 39 52
11.4f4.4 13.924.7 4.8k2.3 lO.Of4.7
6-23 7-33 2-12
Total Cold/flu (unspecified)
12 34 40 2 42
4 4 3 3 3 7
Total Hepatitis
10 33 80
>2 mo. >2 mo. 30
Total
45 1
with viral disease
No. cells scored
:: 16 19 42
notes
Range
Normal (control) SCE mean is 6.052.1 based on scores from 1564 mitoses from 44 healthy individuals. 2.9 standard deviations. SCE scores were signiticantly elevated in the patients compared with the controls @
Viruses are known to alter chromosome structure to varying degrees depending on the type of virus, duration of exposure and dose [ 1, 21. Sister chromatid exchange (SCE) has been studied in virally infected human fibroblast cell lines [3] but not in lymphocytes from patients with viral disease. In the following study, SCE frequencies were scored in chromosomes from lymphocytes of individuals with viral disease and in long term cultured B lymphocytes positive for Epstein Barr virus (EBV). Materials
and Methods
Peripheral lymphocytes from 44 controls and 17 patients were cultured for 72 h at 37°C in equal amounts 30-781812
of autologous serum and TC 199 medium (Difco). Phytohemagglutinin (PHA) (0.2 ml, Gibco), penicillinstreptomycin (0.1 ml) and a 10d4M final concentration of bromodeoxyuridine (BUdR) were added to each 8 ml culture. Colchicine (0.8 Mg/ml) was added for the final 3 h. At all times cultures were kept in total darkness. Harvesting included hypotonic treatment in 0.075 M KC1 and fixation in -3 : 1 methanol/glacial acetic acid. Slides were differentiallv stained for SCE with Giemsa according to the procedure of Korenberg & Freedlender [4]. The number of mitoses scored per patient varied between 9 and 56 depending upon the availability of well spread and differentially stained mitoses. Centromeric exchanges were included in the total SCE counts in both controls and patients. Patients with the following viral diseases were studied: herpes simplex, infectious hepatitis and nonspecific upper respiratory infections (see table 1). All patients were untreated at the time of study. Control individuals had no known disease or recent viral, chemical or radiation exposure. In addition, long-term cultured B lvmphocvtes (cell line 1788) were cultured without mitogdn in- RPM1 1640 medium. Harvesting and chromosome staining were identical to the 72 h culture procedure used for peripheral lymphocytes. The B lymphocytes were positive for the Epstein Barr virus. The cell line 1788 was a gift from Dr R. Condie of the University of Minnesota, Department of Surgery. Nick Hydrukovich assisted in their culturing and Err, Cc// Res 113 (1978)
452
Preliminary
notes
analysis. The cell line 1788was initially from Associate Biomedical Systems in Buffalo, N.Y. The cells were obtained from a normal donor in 1968and were not experimentally infected. The EBV genome has been found in the cell (27 EBV eenome/cell). The cell line tests were negative for EBV particles, negative for EBV capsid antigen, and positive for EBV nuclear antigen.
Results and Discussion
was 391. The adjusted mean SCE/chromosome was 0.295 and the mean SCE/diploid mitosis, 13.6. This is the first report of SCE in cultured human B lymphocytes. Studies are needed on SCE frequencies in B lymphocytes from normal individuals. Previous in vitro studies have suggested that viral infection causes elevated SCE rates. Brown & Crossen [5] observed significant increases in SCEs in Rauscher leukemia virus infected mouse embryo cells in vitro. Wolff et al. [3] found that SV40 transformed human cell lines had slightly elevated SCE values compared with normal cell lines. They suggested that the presence of an integrated viral genome increases SCE frequency. Our preliminary results on SCE frequency in human lymphocytes support these in vitro studies and suggest that lymphocytes from individuals with viral disease have slightly elevated SCE rates compared with those from normal controls.
The mean SCE frequency for the 17 patients with viral illness was 8.7k2.9 standard deviations (SD.) (range in mean scores: 4.5-13.9) (table 1). This was significantly higher (p
Preliminary
notes
453
11. Chaganti, R S K, Schonberg, S & German, J, Proc natl acad sci US 71 (1974) 4508. 12. Schroeder, T M & German, J, Human genet 25 (1974) 299. 13. German, J, Schonberg, S, Louie, E & Chaganti, R S K, Am j human genet 29 (1977) 248. Received August 30, 1977 Revised version received January 17, 1978 Accepted January 23, 1978
Asymmetric decondensation of the L cell heterochromatin by Hoechst 33258
Fig. 2. Cut-out chromosomes of L cells with decondensation at constitutive heterochromatin bv the treatment of the cultures with 40 pg/ml Hoechst-33258 for 8 h. A marker chromosome with interstitial heterochromatin on its long arm, bi-armed chromosomes after centric fusion, and acrocentric chromosomes are presented, from left to right.
S. MATSUKUMA and T. UTAKOJI, Department of Cell Biology, Cancer Institute, Toshima-ku, Tokyo 170, Japan
only in the mouse heterochromatin and not in the human chromosomes of human/ Summary. A benzimidazole derivative, Hoechst 33258 mouse somatic cell hybrids [2]. can induce decondensation of constitutive heterochromatin in the mouse derived L cell chromosomes when We report here an observation which imthe compound is given in sufficiently high concentration (40 pg/ml) to the L cell culture. Hoechst 33258 at plies that the decondensation of the L cell low concentration (1 pg/ml, 16 h) cannot produce this constitutive heterochromatin is induced by effect on L cell chromosomes. Bromodeoxyuridine the binding of Hoechst 33258 molecules to (BUdR) incorporation for one cell cycle simultaneous with the Hoechst 33258treatment at low concentration A-T base pairs of the mouse satellite DNA. could decondense heterochromatin segments in metaphase chromosomes. The heterochromatin decondensation, however, was asymmetric; it was observed only on one chromatid and the other of a chromosome remained in condensed state. The observation of asymmetric decondensation of heterochromatin by Hoechst 33258 after BUdR incornoration for one cell cycle, the association of A-T r&h satellite DNA to mouse heterochromatin, and available data on the specific binding of Hoechst 33258 to A-T base pairs of DNA and on the higher affinity of the compound to BUdR substituted DNA than to ordinary DNA implied that the binding of Hoechst 33258 molecules to A-T rich satellite DNA is the cause of heterochromatin decondensation.
Decondensation of particular segments of the L cell chromosomes has been observed after the treatment of the cell with a bisbenzimidazole derivative, Hoechst 33258 [l]. Decondensed segments of the L cell chromosome after Hoechst 33258 treatment were stained deeply by C-staining, revealing that the decondensed regions were the constitutive heterochromatin of the mouse [ 11.The decondensation of heterochromatin by Hoechst 33258, however, is unique to mouse heterochromatin and its satellite DNA, since the decondensation was found
Materials
and Methods
Mouse L cells were cultured with Eagle’s Minimal Essential Medium with Earle’s salts supplemented with 10% fetal bovine serum at 37°C. The cultures in exponential growth phase were treated with Hoechst 33258, dissolved in the medium, for 8 and 16 h. (Hoechst 33258 was a gift of Dr H. Loewe, Farbwerke Hoechst AG, Frankfurt/Main.) In some experiments the cultures were subjected to bromodeoxyuridine (BUdR, Sigma Corp., Calif., 100 pg/ml medium) incorooration for 24 h, and treated with Hoechst 33258 forthe last 16 h of the BUdR treatment. The chromosome slides of the treated cultures and of the controls were prepared by the ordinary air-drying method. The chromosome preparations were stained with the Giemsa solution diluted to l/SO with l/75 M SGrensen phosphate buffer at pH 6.8 for 20 min.
Results and Discussion
Chromosomes of the L cells treated with 40 pg/ml Hoechst 33258 for 16 h were decondensed at centromeric regions in almost every metaphase cell. The decondensation was remarkable in a marker chromosome in which four interstitial heterochromatin segments were elongated, and at centromeric regions of several bi-armed chromosomes (fig. 1). Such decondensation of defined Exp Cd Res 113 (1978)
Preliminary
notes
induced phase separation of phospholipids which is speculated to occur during fusion [22]. Recently, Chen was able to demonstrate a major redistribution of the LETS protein during and after fusion by immunofluorescence. He reported that when fusion was blocked, the redistribution of LETS did not occur. Furthermore, recognition of LETS was significantly reduced after fusion as determined by radioimmunoassay. Our observation of the 245000 iodinated material which appeared as a double band (M,A-M,B) at prefusion and a single band (M,) at mid- and postfusion might reflect specific modification or replacement of these surface proteins which may be necessary in order for fusion to occur. Modification of cell surface proteins appears to be important for cell-cell interaction in other systems. Hynes [lS] reported that proteolysis may reveal cryptic sites in the membranes of normal cells much like those seen in transformed cells. Pouyssegur & Pastan [ 191 observed that mutant fibroblasts with substrate adhesion deficiencies lacked many of the iodinated surface proteins found on nonmutant fibroblasts. Rutishauser et al. [23] proposed a model in which specific proteolytic cleavage of surface proteins may be involved in recognition and adhesion of embryonic chick neural retinal cells. Therefore, the observed increase in iodinated material in the 10500 M, band (table 1) may be suggestive of an increase in proteolytic activity associated with fusing muscle cells. It is tempting to speculate that specific proteolytic cleavage sites might exist on prefusion myoblasts which are modified in order to bring about fusion. Substantiation of increased protease activity associated with fusing muscle cells remains to be conclusively demonstrated. This possibility is currently under investigation in our laboratory. E-v Cell Res I I3 (1978)
This research was supported by the Muscular Dystrophy Association of America.
References 1. Yaffe, D & Feldman, M, Dev biol 11 (1965) 300. 2. Holtzer, H, Bischoff, R & Chacko, S, Cellular recognition, pp. 19-25. Appleton, New York (1969). 3. Reporter, M & Norris, G, Differentiation 1 (1973) 83. 4. Reporter, M & Raveed, D, Science lSl(1973) 863. 5. Kent, C, Schimmel, S & Vagelos, P, Biochim biophys acta 360 (1974) 312. 6. Winand, R & Luzzati, D, Biochimie 57 (1975) 764. 7. Hynes, R 0, Martin, G S, Shearer, M, Critchley, D R & Epstein, C J, Dev biol48 (1976) 35. 8. Chen, LB, Cell 10 (1977) 393. 9. Yaffe, D, Proc natl acad sci US 61 (1968) 477. IO. Konigsberg, I R, Methods in developmental biology, pp. 520-521. T. Y. Crowell Co., New York (1967). Il. Hauschka, S D, Growth, nutrition and metabolism of cells in culture, pp. 84-85. Academic Press, New York (1972). 12. Fishbach. G D. Dev biol28 (1972) 407. 13. Moss, P & Strdhman, R, Ceil 10 (1977) 265. 14. Ames Co. Catalog on disc electrophoresis, p. 14. Elkhart, Indiana (1976). 15. Laemmli, U D, Nature 227 (1970) 680. 16. Eisen, H N, Immunology, p. 375. Harper & Row, New York (1974). 17. Hogg, N M, Proc natl acad sci US 71 (1974) 489. 18. Hynes, R 0, Proc natl acad sci US 70 (1973) 3170. 19. Pouyssegur, J M & Pastan, I, Proc natl acad sci US 73 (1976) 544. 20. Teng, N & Chen, LB, Nature 259 (1976) 578. 21. Richler, C & Yaffe, D, Dev bio123 (1970) 1. 22. Weidekamm, E, Schudt, C & Brdiczka, D, Biochim biophys acta 433 (1976) 169. 23. Rutishauser, U, Thiery, J P, Brackenbury, R, Amisela, B & Edelman, G, Proc natl acad sci US 73 (1976) 577. Received August 18, 1977 Revised version received January 4, 1978 Accepted January 20, 1978
Sister chromatid viral disease
exchange in patients with
KAREN KURVINK,’ CLARA D. BLOOMFIELD* and JAROSLAV CERVENKA,’ ‘Divisions of Oral Patholoav and of Human and Oral Genetics. and zDepartment of kedicine, University of Minnesota Health Sciences Center. Minneauolis. MN 55455. USA
Sister chromatid exchange (SCE) frequencies were studied in differentially stained chromosomes from lymphocytes of 17 patients with viral disease. The mean SCE score for the patients was 8.7+
Summary.
Preliminary
Table 1. SCE in lymphocytes from individuals
Disease
IlO.
Time from onset of symptoms (days)
Herpes simplex
11 30 43 83 86 85 101 70
1 7 2 2 >l mo. 3 2 2
Patient
Mean SCE + S.D.
22 9 29
9.2f3.5 7.8k3.7 6.3k2.2 10.6f2.9 13.5f4.9 9.Ot4.2 7.3k2.6 5.5f2.4 8.7k2.6
3-16 3-15 3-12 4-18 3-22 3-17 3-14 2-13
15 34 41 20 56 13
4.5k2.1 11.8f4.2 6.9k3.4 10.8k3.9 9.0f3.3 5.9k2.5 8.2k2.9
2-10 4-21 2-19 4-19 3-21 3-11
41 39 52
11.4f4.4 13.924.7 4.8k2.3 lO.Of4.7
6-23 7-33 2-12
Total Cold/flu (unspecified)
12 34 40 2 42
4 4 3 3 3 7
Total Hepatitis
10 33 80
>2 mo. >2 mo. 30
Total
45 1
with viral disease
No. cells scored
:: 16 19 42
notes
Range
Normal (control) SCE mean is 6.052.1 based on scores from 1564 mitoses from 44 healthy individuals. 2.9 standard deviations. SCE scores were signiticantly elevated in the patients compared with the controls @
Viruses are known to alter chromosome structure to varying degrees depending on the type of virus, duration of exposure and dose [ 1, 21. Sister chromatid exchange (SCE) has been studied in virally infected human fibroblast cell lines [3] but not in lymphocytes from patients with viral disease. In the following study, SCE frequencies were scored in chromosomes from lymphocytes of individuals with viral disease and in long term cultured B lymphocytes positive for Epstein Barr virus (EBV). Materials
and Methods
Peripheral lymphocytes from 44 controls and 17 patients were cultured for 72 h at 37°C in equal amounts 30-781812
of autologous serum and TC 199 medium (Difco). Phytohemagglutinin (PHA) (0.2 ml, Gibco), penicillinstreptomycin (0.1 ml) and a 10d4M final concentration of bromodeoxyuridine (BUdR) were added to each 8 ml culture. Colchicine (0.8 Mg/ml) was added for the final 3 h. At all times cultures were kept in total darkness. Harvesting included hypotonic treatment in 0.075 M KC1 and fixation in -3 : 1 methanol/glacial acetic acid. Slides were differentiallv stained for SCE with Giemsa according to the procedure of Korenberg & Freedlender [4]. The number of mitoses scored per patient varied between 9 and 56 depending upon the availability of well spread and differentially stained mitoses. Centromeric exchanges were included in the total SCE counts in both controls and patients. Patients with the following viral diseases were studied: herpes simplex, infectious hepatitis and nonspecific upper respiratory infections (see table 1). All patients were untreated at the time of study. Control individuals had no known disease or recent viral, chemical or radiation exposure. In addition, long-term cultured B lvmphocvtes (cell line 1788) were cultured without mitogdn in- RPM1 1640 medium. Harvesting and chromosome staining were identical to the 72 h culture procedure used for peripheral lymphocytes. The B lymphocytes were positive for the Epstein Barr virus. The cell line 1788 was a gift from Dr R. Condie of the University of Minnesota, Department of Surgery. Nick Hydrukovich assisted in their culturing and Err, Cc// Res 113 (1978)
452
Preliminary
notes
analysis. The cell line 1788was initially from Associate Biomedical Systems in Buffalo, N.Y. The cells were obtained from a normal donor in 1968and were not experimentally infected. The EBV genome has been found in the cell (27 EBV eenome/cell). The cell line tests were negative for EBV particles, negative for EBV capsid antigen, and positive for EBV nuclear antigen.
Results and Discussion
was 391. The adjusted mean SCE/chromosome was 0.295 and the mean SCE/diploid mitosis, 13.6. This is the first report of SCE in cultured human B lymphocytes. Studies are needed on SCE frequencies in B lymphocytes from normal individuals. Previous in vitro studies have suggested that viral infection causes elevated SCE rates. Brown & Crossen [5] observed significant increases in SCEs in Rauscher leukemia virus infected mouse embryo cells in vitro. Wolff et al. [3] found that SV40 transformed human cell lines had slightly elevated SCE values compared with normal cell lines. They suggested that the presence of an integrated viral genome increases SCE frequency. Our preliminary results on SCE frequency in human lymphocytes support these in vitro studies and suggest that lymphocytes from individuals with viral disease have slightly elevated SCE rates compared with those from normal controls.
The mean SCE frequency for the 17 patients with viral illness was 8.7k2.9 standard deviations (SD.) (range in mean scores: 4.5-13.9) (table 1). This was significantly higher (p
Preliminary
notes
453
11. Chaganti, R S K, Schonberg, S & German, J, Proc natl acad sci US 71 (1974) 4508. 12. Schroeder, T M & German, J, Human genet 25 (1974) 299. 13. German, J, Schonberg, S, Louie, E & Chaganti, R S K, Am j human genet 29 (1977) 248. Received August 30, 1977 Revised version received January 17, 1978 Accepted January 23, 1978
Asymmetric decondensation of the L cell heterochromatin by Hoechst 33258
Fig. 2. Cut-out chromosomes of L cells with decondensation at constitutive heterochromatin bv the treatment of the cultures with 40 pg/ml Hoechst-33258 for 8 h. A marker chromosome with interstitial heterochromatin on its long arm, bi-armed chromosomes after centric fusion, and acrocentric chromosomes are presented, from left to right.
S. MATSUKUMA and T. UTAKOJI, Department of Cell Biology, Cancer Institute, Toshima-ku, Tokyo 170, Japan
only in the mouse heterochromatin and not in the human chromosomes of human/ Summary. A benzimidazole derivative, Hoechst 33258 mouse somatic cell hybrids [2]. can induce decondensation of constitutive heterochromatin in the mouse derived L cell chromosomes when We report here an observation which imthe compound is given in sufficiently high concentration (40 pg/ml) to the L cell culture. Hoechst 33258 at plies that the decondensation of the L cell low concentration (1 pg/ml, 16 h) cannot produce this constitutive heterochromatin is induced by effect on L cell chromosomes. Bromodeoxyuridine the binding of Hoechst 33258 molecules to (BUdR) incorporation for one cell cycle simultaneous with the Hoechst 33258treatment at low concentration A-T base pairs of the mouse satellite DNA. could decondense heterochromatin segments in metaphase chromosomes. The heterochromatin decondensation, however, was asymmetric; it was observed only on one chromatid and the other of a chromosome remained in condensed state. The observation of asymmetric decondensation of heterochromatin by Hoechst 33258 after BUdR incornoration for one cell cycle, the association of A-T r&h satellite DNA to mouse heterochromatin, and available data on the specific binding of Hoechst 33258 to A-T base pairs of DNA and on the higher affinity of the compound to BUdR substituted DNA than to ordinary DNA implied that the binding of Hoechst 33258 molecules to A-T rich satellite DNA is the cause of heterochromatin decondensation.
Decondensation of particular segments of the L cell chromosomes has been observed after the treatment of the cell with a bisbenzimidazole derivative, Hoechst 33258 [l]. Decondensed segments of the L cell chromosome after Hoechst 33258 treatment were stained deeply by C-staining, revealing that the decondensed regions were the constitutive heterochromatin of the mouse [ 11.The decondensation of heterochromatin by Hoechst 33258, however, is unique to mouse heterochromatin and its satellite DNA, since the decondensation was found
Materials
and Methods
Mouse L cells were cultured with Eagle’s Minimal Essential Medium with Earle’s salts supplemented with 10% fetal bovine serum at 37°C. The cultures in exponential growth phase were treated with Hoechst 33258, dissolved in the medium, for 8 and 16 h. (Hoechst 33258 was a gift of Dr H. Loewe, Farbwerke Hoechst AG, Frankfurt/Main.) In some experiments the cultures were subjected to bromodeoxyuridine (BUdR, Sigma Corp., Calif., 100 pg/ml medium) incorooration for 24 h, and treated with Hoechst 33258 forthe last 16 h of the BUdR treatment. The chromosome slides of the treated cultures and of the controls were prepared by the ordinary air-drying method. The chromosome preparations were stained with the Giemsa solution diluted to l/SO with l/75 M SGrensen phosphate buffer at pH 6.8 for 20 min.
Results and Discussion
Chromosomes of the L cells treated with 40 pg/ml Hoechst 33258 for 16 h were decondensed at centromeric regions in almost every metaphase cell. The decondensation was remarkable in a marker chromosome in which four interstitial heterochromatin segments were elongated, and at centromeric regions of several bi-armed chromosomes (fig. 1). Such decondensation of defined Exp Cd Res 113 (1978)