Hereditas 90: 195-201 (1979)
Effect of colcemid exposure and methanol acetic acid fixation on human metaphase chromosome structure MOGENS RP)NNE1,OLE ANDERSEN' and MOGENS ERLANDSEN2 The Winslow Institute of H u m a n Anatomy, University of Odense, Denmark The Institute of Mathematics, University of Odense, Denmark
R B N N E , M.,ANDERSEN, 0.and ERLANDSEN, M. 1979. Effect of colcemid exposure and methanol acetic acid fixation on human metaphase chromosome structure. - Hereditas 90: 195-201. Lund, Sweden. ISSN 0018-0661. Received November 10, 1978
Distribution of human lymphoid cells in the mitotic subphases, prophase, prometaphase, metaphase and ana-telophase, was examined, when cells had been cultured and without colcemid exposure. A higher average length of chromosome no. I was found after overnight fixation than after short time fixation with 3: I methanol acetic acid fixative. The probability of observing banded chromosomes on slides that had not been exposed to any postfixational band induction was higher after overnight fixation than after short time fixation. Since an increase in the extraction of histones caused by the methanol acetic acid fixation of metaphase cells led to an increase of average chromosome length as well as more frequent banding of chromosomes, it is suggested that not only non-histone proteins but also histones are involved in the formation of chromosome bands. In cells harvested without having been exposed to colcemid. most of the observed chromosomes were banded prometaphase chromosomes. It is suggested that the chromosome condensation process, which is delayed or partly inhibited when inhibitors of RNA or protein synthesis are added to the growth medium, continues as long as colcemid exposed cells remain arrested at the meta-anaphase border with free -SHgroups available for oxidation. Mogens R m n e . The Winslow Institute of H u m a n Anatomy, University of Odense, Campusvej 55, DK5230 Odense M.Denmark
Induction of specific banding patterns in mammalian metaphase chromosomes has been demonstrated by a variety of postfixation methods. According to the pattern and the technique used to produce these bands, four different categories of bands were defined at the Paris Conference (1971). The four categories are designated Q bands, G bands, C bands, and R bands. Induction methods and experimental data have been the topic of several reviews (HSU 1973; LATT 1976). Recently it was shown (SHAFER 1973; Hsu et al. and RBNNE 1973; RBNNE 1977a,b,c; SANDERMANN 1977; ANDERSEN and RBNNE1978; RBNNE and ANDERSEN 1978; RBNNE and ANDERSEN in press) that agents inhibiting RNA or protein synthesis when added to human lymphoid cells in G2phase either produced G banded or uncoiled chromosomes as those described by OHNUKI (1968) and R B N N E (1977d). The preparation of chromosome slides for light microscopy normally follows the procedure (1965), which involves suggested by HUNGERFORD exposure of a cell population to colcemid. The 14
exposed cell population is then treated with a hypotonic solution of KCI and subsequently fixed with a mixture of methanol and acetic acid. Colcemid inhibits the normal function of the mitotic apparatus but does no interfere with chromosome condensation ( B R I N K L E Yet al. 1967). Thus, the exposed cells are prevented from the normal chromatid separation in anaphase, but the chromosome condensation seems to proceed as long as the cells are exposed to colcemid. Several authors ( S U M N E Ret al. 1973; BOBROW 1973; POTHlER et al. 1975; RETlEF and RUCHEL 1977) have pointed out that the methanol acetic acid fixatives extract a substantial part of histones from the metaphase chromosomes. According to RETIEFand RUCHEL(1977) the H 1 fraction is preferentially extracted. YUNISand SANCHEZ (1975) suggested overnight fixation at 4°C when preparing chromosome slides. RBNNE (1977~)observed that the prolonged fixation seems to favour production of long slightly segmented chromosomes. In this study
196
Hrrediras YO (1979)
M. R 0 N N E ET A L .
Table I . The procedure for colcemid exposure, cold treatment, hypotonic treatment, and fixation for different groups of lymphoid cells cultured for 48 hours Steps in procedure
Group 1
I. 2. 3. 4.
Add colcemid (0.1 pg/ml growth medium) to the cultures 2 h before harvest ( I = 46 h) Cool to 0°C for 30 rnin before harvest (t = 47 1/2 h) Centrifugation at 1000 rpm for 10 min Supernatant decanted off. Leave 1 ml of the growth medium and resuspend the pellet in this volumen. Add 8 mlO.075 M KCI and store at 37.5"C for 5 min 5 . Centrifugation at 1000 rpm for 10 min 6. Supernatant decanted off. leave about 1 ml of the hypotonic salt solution and resuspend the pellet in this volume 7. Add freshly made fixative (3: I methanollacetic acid) drop by drop lo the suspension to a final volume of 5 ml. Store at room temperature for 30 min 8. Centrifugation at I000 rpm for 10 min 9. Change fixative and store at room temperature for 30 min 10. Centrifugation at loo0 rpm for 10 min I I. Change fixative and store at 4°C overnight 12. Centrifugation at loo0 rpm for 10 rnin 13. Change fixative (freshly made) and store at room temperature for 30 min 14. Centrifugation at loo0 rpm for 10 min 15. Supernatant decanted off,resuspend the pellet in 3 ml freshly made fixative and store the suspension at - 20°C
the effect of both fixation time and exposure to colcemid have been investigated with regard to structure and morphology of Giemsa stained chromosomes on the resulting slides.
Materials and methods Culturing conditions and preparation of chromosome slides Lymphoid human cells were cultured for 48 h by adding 0.4 ml of peripheral blood to 10 ml of Eagle's essential minimal medium supplement with 10% fetal calf serum, heparin, antibiotics, and phytohemagglutinin M (DIFCO). The cultures were incubated in a water bath at 373°C. For description of exposure to colcemid, harvest, cold treatment, hypotonic treatment, and fixation, see Table 1. To produce chromosome slides 10 pI of the methanol acetic fixed cell suspension was blown on a dry ethanol cleaned slide held at an angle of 45", and 10 pI of freshly made fixative was then blown at the same slide area. The resulting chromosome slides were air-dried and either directly stained or stored at 4°C.
+
2
+
t t
+
t
3
t t
+
+
t
t
t t
t
+
t
t
t
+ +
t
t t
+ +
+
t
+
t
+
t t
t
+
t
t
t
+ +
Stuining and mounting The slides were stained in 55% Giemsa (Merck) in Sgirensen's phosphate buffer pH 8 for 10-15 min and washed 3 times in the same buffer. The slides were air-dried for 1 h at 40°C under low relative humidity and mounted as described elsewhere (R0NNE e t a ] . 1977). Microscopy and photographic technique Determination of chromosome length was carried out directly by using a calibrated measuring eyepiece. The mitotic subphases were determined directly by microscopy. The chromosomes were grouped in prophase, banded prometaphase, and unbanded metaphase chromosomes. For group 3 cells at ana- and telophase were also scored. Control micrographs were used when needed. The microscope and the photographic technique have been described elsewhere ( R ~ N N 1977d). F.
Results When human lymphoid cells from the same donor were cultured for 48 h and harvested as described, differences in chromosome structure and length within as well as between the groups defined in Table 1 could be observed.
Heredifas 90 (1979)
H U M A N CHROMOSOMESTRUCTURE
Table 2. Length, average length and empirical variance for chromosome no. 1 scored as grouped data in intervals of 1 p m for group 1 and group 2 cells Calculated F, U and P values
Table 3. Log,-transformation and calculations based on group I and group 2 observations
N Intervals in p m
3.0- 4.0 4.0- 5.0 5.0- 6.0 6.0- 7.0
7.0- 8.0 8.0- 9.0 9.0-10.0 10.0-1 1.0 11.0-12.0
No. of observations per interval Group 2 Group 1
3
12 18 12 3 3
6
0
3
0 0
8 13 14
2
so
N i
7.50
S2
2.86
2.86- 2.04 F --
I .40
U
7.50- 5.72
i SZ
Group 1
Group 2
50 1.9885 0.04854
50 1.7190 0.04291
2
0 I
50 5.72 I .40
I%
60% df = 98
s2 0.04573
t
1.9885- 1.7190
d0.04573 (0.02 + 0.02)
=
6.30
P
Effect of colcemid exposure and methanol acetic acid fixation on human metaphase chromosome structure MOGENS RP)NNE1,OLE ANDERSEN' and MOGENS ERLANDSEN2 The Winslow Institute of H u m a n Anatomy, University of Odense, Denmark The Institute of Mathematics, University of Odense, Denmark
R B N N E , M.,ANDERSEN, 0.and ERLANDSEN, M. 1979. Effect of colcemid exposure and methanol acetic acid fixation on human metaphase chromosome structure. - Hereditas 90: 195-201. Lund, Sweden. ISSN 0018-0661. Received November 10, 1978
Distribution of human lymphoid cells in the mitotic subphases, prophase, prometaphase, metaphase and ana-telophase, was examined, when cells had been cultured and without colcemid exposure. A higher average length of chromosome no. I was found after overnight fixation than after short time fixation with 3: I methanol acetic acid fixative. The probability of observing banded chromosomes on slides that had not been exposed to any postfixational band induction was higher after overnight fixation than after short time fixation. Since an increase in the extraction of histones caused by the methanol acetic acid fixation of metaphase cells led to an increase of average chromosome length as well as more frequent banding of chromosomes, it is suggested that not only non-histone proteins but also histones are involved in the formation of chromosome bands. In cells harvested without having been exposed to colcemid. most of the observed chromosomes were banded prometaphase chromosomes. It is suggested that the chromosome condensation process, which is delayed or partly inhibited when inhibitors of RNA or protein synthesis are added to the growth medium, continues as long as colcemid exposed cells remain arrested at the meta-anaphase border with free -SHgroups available for oxidation. Mogens R m n e . The Winslow Institute of H u m a n Anatomy, University of Odense, Campusvej 55, DK5230 Odense M.Denmark
Induction of specific banding patterns in mammalian metaphase chromosomes has been demonstrated by a variety of postfixation methods. According to the pattern and the technique used to produce these bands, four different categories of bands were defined at the Paris Conference (1971). The four categories are designated Q bands, G bands, C bands, and R bands. Induction methods and experimental data have been the topic of several reviews (HSU 1973; LATT 1976). Recently it was shown (SHAFER 1973; Hsu et al. and RBNNE 1973; RBNNE 1977a,b,c; SANDERMANN 1977; ANDERSEN and RBNNE1978; RBNNE and ANDERSEN 1978; RBNNE and ANDERSEN in press) that agents inhibiting RNA or protein synthesis when added to human lymphoid cells in G2phase either produced G banded or uncoiled chromosomes as those described by OHNUKI (1968) and R B N N E (1977d). The preparation of chromosome slides for light microscopy normally follows the procedure (1965), which involves suggested by HUNGERFORD exposure of a cell population to colcemid. The 14
exposed cell population is then treated with a hypotonic solution of KCI and subsequently fixed with a mixture of methanol and acetic acid. Colcemid inhibits the normal function of the mitotic apparatus but does no interfere with chromosome condensation ( B R I N K L E Yet al. 1967). Thus, the exposed cells are prevented from the normal chromatid separation in anaphase, but the chromosome condensation seems to proceed as long as the cells are exposed to colcemid. Several authors ( S U M N E Ret al. 1973; BOBROW 1973; POTHlER et al. 1975; RETlEF and RUCHEL 1977) have pointed out that the methanol acetic acid fixatives extract a substantial part of histones from the metaphase chromosomes. According to RETIEFand RUCHEL(1977) the H 1 fraction is preferentially extracted. YUNISand SANCHEZ (1975) suggested overnight fixation at 4°C when preparing chromosome slides. RBNNE (1977~)observed that the prolonged fixation seems to favour production of long slightly segmented chromosomes. In this study
196
Hrrediras YO (1979)
M. R 0 N N E ET A L .
Table I . The procedure for colcemid exposure, cold treatment, hypotonic treatment, and fixation for different groups of lymphoid cells cultured for 48 hours Steps in procedure
Group 1
I. 2. 3. 4.
Add colcemid (0.1 pg/ml growth medium) to the cultures 2 h before harvest ( I = 46 h) Cool to 0°C for 30 rnin before harvest (t = 47 1/2 h) Centrifugation at 1000 rpm for 10 min Supernatant decanted off. Leave 1 ml of the growth medium and resuspend the pellet in this volumen. Add 8 mlO.075 M KCI and store at 37.5"C for 5 min 5 . Centrifugation at 1000 rpm for 10 min 6. Supernatant decanted off. leave about 1 ml of the hypotonic salt solution and resuspend the pellet in this volume 7. Add freshly made fixative (3: I methanollacetic acid) drop by drop lo the suspension to a final volume of 5 ml. Store at room temperature for 30 min 8. Centrifugation at I000 rpm for 10 min 9. Change fixative and store at room temperature for 30 min 10. Centrifugation at loo0 rpm for 10 min I I. Change fixative and store at 4°C overnight 12. Centrifugation at loo0 rpm for 10 rnin 13. Change fixative (freshly made) and store at room temperature for 30 min 14. Centrifugation at loo0 rpm for 10 min 15. Supernatant decanted off,resuspend the pellet in 3 ml freshly made fixative and store the suspension at - 20°C
the effect of both fixation time and exposure to colcemid have been investigated with regard to structure and morphology of Giemsa stained chromosomes on the resulting slides.
Materials and methods Culturing conditions and preparation of chromosome slides Lymphoid human cells were cultured for 48 h by adding 0.4 ml of peripheral blood to 10 ml of Eagle's essential minimal medium supplement with 10% fetal calf serum, heparin, antibiotics, and phytohemagglutinin M (DIFCO). The cultures were incubated in a water bath at 373°C. For description of exposure to colcemid, harvest, cold treatment, hypotonic treatment, and fixation, see Table 1. To produce chromosome slides 10 pI of the methanol acetic fixed cell suspension was blown on a dry ethanol cleaned slide held at an angle of 45", and 10 pI of freshly made fixative was then blown at the same slide area. The resulting chromosome slides were air-dried and either directly stained or stored at 4°C.
+
2
+
t t
+
t
3
t t
+
+
t
t
t t
t
+
t
t
t
+ +
t
t t
+ +
+
t
+
t
+
t t
t
+
t
t
t
+ +
Stuining and mounting The slides were stained in 55% Giemsa (Merck) in Sgirensen's phosphate buffer pH 8 for 10-15 min and washed 3 times in the same buffer. The slides were air-dried for 1 h at 40°C under low relative humidity and mounted as described elsewhere (R0NNE e t a ] . 1977). Microscopy and photographic technique Determination of chromosome length was carried out directly by using a calibrated measuring eyepiece. The mitotic subphases were determined directly by microscopy. The chromosomes were grouped in prophase, banded prometaphase, and unbanded metaphase chromosomes. For group 3 cells at ana- and telophase were also scored. Control micrographs were used when needed. The microscope and the photographic technique have been described elsewhere ( R ~ N N 1977d). F.
Results When human lymphoid cells from the same donor were cultured for 48 h and harvested as described, differences in chromosome structure and length within as well as between the groups defined in Table 1 could be observed.
Heredifas 90 (1979)
H U M A N CHROMOSOMESTRUCTURE
Table 2. Length, average length and empirical variance for chromosome no. 1 scored as grouped data in intervals of 1 p m for group 1 and group 2 cells Calculated F, U and P values
Table 3. Log,-transformation and calculations based on group I and group 2 observations
N Intervals in p m
3.0- 4.0 4.0- 5.0 5.0- 6.0 6.0- 7.0
7.0- 8.0 8.0- 9.0 9.0-10.0 10.0-1 1.0 11.0-12.0
No. of observations per interval Group 2 Group 1
3
12 18 12 3 3
6
0
3
0 0
8 13 14
2
so
N i
7.50
S2
2.86
2.86- 2.04 F --
I .40
U
7.50- 5.72
i SZ
Group 1
Group 2
50 1.9885 0.04854
50 1.7190 0.04291
2
0 I
50 5.72 I .40
I%
60% df = 98
s2 0.04573
t
1.9885- 1.7190
d0.04573 (0.02 + 0.02)
=
6.30
P
