Hereditas 80: 83-90
(1975)
Heterochromatin and chromosome aberrations. A comparative study of three mouse cell lines with different karyotype and heterochromatin distribution A. T. NATARAJAN and T. RAPOSA Wallenberg Laboratory, Stockholm University, Stockholm, Sweden
NATARAJAN, A. T. and RAPOSA,T. 1975. Heterochromatin and chromosome aberrations. A comparative study of three mouse cell lines with different karyotype and heterochromatin distribution. - Hereditas 80: 83-90. Lund, Sweden. ISSN 0018-0661. Received January 24, 1975 Mitomycin C induced chromosome aberrations in mouse cells differing in chromosome number and karyotype were studied. The material included (1) laboratory mouse (2n = 40), all telocentric chromosomes possessing centromeric heterochromatin, (2) laboratory mouse X tobacco mouse hybrid (2n = 33) with 7 metacentrics and 26 telocentrics, and (3) ascites tumour cells (MSWBS) (2n = 28--29), with 9 to 10 biarmed chromosomes, with heterochromatin localised to the centromeric regions in all, except two marker chromosomes - one telocentric with 4 intercalary blocks, and one biarmed chromosome with two terminal blocks. Both chromatid intrachanges and interchanges were scored. It was found: (a) In laboratory mouse about 80 % of the abberations are localised to the heterochromatic regions; (b) In the tobacco mouse X laboratory mouse hybrid, the exchanges are between two telocentrics or a telocentric and a metacentric, but seldom between two metacentrics; (c) In the MSWBS tumour cells, the centromeric heterochromatin was involved more often in interchanges than the intercalary blocks, without any restriction between two biarmed chromosomes to form exchanges. A. T. Natarajan, Walfenberg Laboratory, Lilla Frescati, S-10405 Stockholm 50, Sweden
Spontaneously occurring chromosome aberrations as well as those induced by chemical agents and viruses are localized to the heterochromatic regions in several plants and mammals (NATARAJAN and AHNSTROM 1973). After an evaluation of the factors which may be responsible for the observed extreme localization in different material with varying amount and distribution of heterochromatin, it was concluded that
To elucidate this problem further, we have used mouse as a model system for the following reasons:
(a) In mouse, the constitutive heterochromatic regions are very rich in repetitive DNA (JONES 1970; PARDUE and GALL1970; NATARAJAN et al. 1973), (b) different karyotypes within the genus different species or cell lines - are available
-
as
(a) the concentration of highly repetitive DNA in these regions must be the most important one (NATARAJAN and AHNSTROM 1970, 1973), and
(c) normal and neoplastic cells with similar karyotypes allow a comparative study and
(b) the manner in which the heterochromatic regions organize themselves to form chromocentres in the interphase plays an important role in determining the types of aberrations realised (NATARAJAN et al. 1974).
(d) this system allows the study of similar cells e.g. ascites tumour cells - in vitro and in vivo for their response to different agents. In the present communication, a comparative study of the patterns of the alkylating antibiotic mitomycin Hereditas 80, 19i 5
84
A. T. NATARAJAN A N D T. RAPOSA
C induced chromosome aberrations in mouse lines having different karyotypes and distribution of heterochromatin is presented.
RAJAN and SCHMID 1971; NATARAJAN et al. 1974). The dosage of mitomycin varied, however, from 0.04 to 0.5 pg/ml. In the case of MSWBS, the animals carrying tumours were injected with mitomycin C: in isotonic saline (4 pg/ml of ascitic fluid), and the preparations were made according Material and methods to the schedule described earlier (NATARAJAN et al. 1973). Cell lines and their chromosome characteristics Staining. All preparations were made from (1) Laboratory mouse (Mus musculus). 2n= 40, colchicine-blocked metaphases. Some preparaall telocentric chromosomes with centromeric tions were processed by G banding technique heterochromatic blocks except the Y (Fig. 1). and others by C banding technique (Paris ConPrimary cultures were set up from 12 to 14 days ference, 1972). Only chromatid aberrations were old embryos and the growing fibroblast cells encountered. The different types of aberrations were classified and their localization was recorded. were used in their second passage. Since the heterochromatin in mouse chromosomes (2) Laboratory mousex tobacco mouse ( M . pois localised to the centromeric regions, it is schiuvinus) F, hybrids: 21-1233 with 7 metarelatively easy to recognize these regions in abercentrics and 26 telocentrics, all having heterorations (see Fig. 2, 3, 7 to 11). But, in the case of chromatin proximal to the centromeric regions MSWBS line, there was intercalary heterochroexcept the Y. Primary cultures were set up from matin, as well as heterochromatic block near the 12 to 14 days old embryos (Fig. 4), centromere of only one of the arms of the (3) MSWBS tumour line: 2nn=28 or 29, with biarmed chromosomes. To get a more precise 9 to 10 biarmed chromosomes (LEVANet al. 1972), scoring, both C banding as well as benzimidazole with heterochromatin around the centrorneric banding (RAPOSA and NATARAJAN 1974) were regions of all the chromosomes. In addition, one performed. telocentric has 4 blocks of intercalary heterochromatin and one biarmed chromosome has two additional heterochromatic blocks in the long arm (Fig. 6). These tumour cells in passages Results and discussion 160 to 178, were made available through the 1 . Laboratory mouse courtesy of Professor G. Klein. The frequency of different types of aberrations produced by mitomycin C is presented in Table 1 . Mitomycin C treatment It can be seen that about 74 % of the chromatid In vitro cultures of fibroblasts were treated breaks occurs at the centromeric heterochromatic according to the schedule described earlier (NATA- regions. Different types of chromatid exchanges
Table 1. Localisation of mitomycin-induced chromatid aberrations in Mus musculus The cells were scored at 22 and 32 hours, following treatment with 0.04 pg/ml of mitomycin C
No. of cells analysed
300
No. of abnormal cells
155
Chromatid interchanges localised in
N o . of chromatid breaks localised in centromeric region
telomeric region
other regions
cc
CT
CM
TT
TM
MM
81
8
22
66(36)1
21
9
21
5
8
C = centrorneric region; T = telorneric region; M = other regions Number of exchanges involving both the chromatids of both the chromosomes
1
Hereditas 80, 1975
HETEROCHROMATIN AND CHROMOSOME ABERRATIONS
85
1
t
\ &
0 2
I H
I
/ rr
Fig. 1-3. - Fig. 1. C-banded chromosomes of laboratory mouse showing the centromeric heterochromatin in all the chromosomes except Y (arrow). - Fig. 2 and 3. Mitomycin C treated laboratory mouse cells, showing different types of exchanges involving centromeric regions (arrows).
Hereditas 80,1975
86
A. T. NATARAJAN AND T. RAPOSA
are noticed with this treatment, which are il- Table 2. Mitomycin C induced chromatid exlustrated in Fig. 2 and 3. About 70 % of exchanges changes in F, hybrid cells ( M . musciilus X M . are localized to the centromeric heterochromatic poschiavinus) regions. An interesting feature is that more than Total number of exchanges analysed was 114, from 250 half of the exchanges involving the centromeric cells regions of two chromosomes, has both the chromatids united or joined together in the aberra- Localisation Exchanges between tions (Fig. 3). It is estimated that about 10 % telometatelocentrics of the mouse-chromosome regions is heterochrocentrics centrics and matic (NATARAJAN and AHNSTROM1973), and metacentrics these regions account for the localization of about 7 0 % of the aberrations. As pointed out Centromeric 18 0 40 earlier, this preferential occurrence of aberrations region 8 0 10 is most probably due to the enrichment of repeti- Telomeric tive DNA in these regions which makes it easier region 4 3 3 for aberration formation during repair of lesions Other regions induced by chemicals or radiations (NATARAJAN and AHNSTROM 1973). Though there are 39 or 40 heterochromatic regions proximal to the cen- regions. Though there were large proportions of tromere in mouse, there are usually a lower aberrations involving two telocentrics, or one number of chromocentres in the interphase. The telo- and one metacentric (Fig. 5 ) , there were no range is between 7 to 26 with an average of 14.22 aberrations involving two metacentrics. This per cell (NATARAJAN and GROPP1972). This would strongly suggests that there must be a specific explain the very high frequency of centromeric control of the association of chromocentres in exchanges in the laboratory-mouse chromosomes the interphase and there is a homologous association between the telocentric and the metacentric following mitomycin C treatment. involving that telocentric chromosome in the Robertsonian fusion. 2. Laboratory mouse X tobacco mouse hybrid In an earlier study (NATARAJAN and GROPP1972) it was reported that the number of chromocentres 3. MSWBS tumour line which was formed in fibroblasts of tobacco mouse The normal distribution of heterochromatin in ranges from 2 to 16, with an average of 9.4. this line is illustrated in Fig. 6 and the types of This was expected in view of the fact that tobacco aberrations recovered following mitomycin C mouse has a diploid chromosome number of 26, treatment in this material are illustrated in Fig. 7 of which 14 are metacentrics arising from Ro- to 11. The quantitative data are presented in bertsonian fusion (GROPPet al. 1970; NATARAJANTable 3. About 75 % of the exchanges involves and GROPP1972). When the behaviour of the F, the heterochromatic regions. While exchanges hybrid cells (between tobacco mouse and labora- between biarmed chromosomes and telocentrics tory mouse) was studied with regard to the chro- are common, the exchanges between the biarmed mocentre formation, it was found that the hybrid chromosomes are less than expected on the basis follows the tobacco-mouse pattern and not an of random exchanges between heterochromatic intermediate pattern, indicating a possible as- regions. This frequency is, however, higher when sociation between metacentrics and the homolo- compared to the hybrid material between tobacco gOUS teIOCentriCS (NATARAJAN and GROPP1972). mouse and laboratory mouse. Since the MSWBS Mitomycin C induced aberrations which reflect tumour has gone through many structural to a great extent the association of different changes, there is obviously no rigorous restricchromosomes or chromosomal regions in in- tion on the association of heterochromatic regions terphase, were analysed in this material. The to form chromocentres. An analysis of the chroresults are presented in Table 2. The results mocentre frequency in this material indicated that indicate that the aberrations are localised prefer- it ranged between 6 to 19 with an average of 14, entially to the centromeric heterochromatic which is higher than in the tobacco mouse and Hereditas 80,1975
HETEROCHROMATIN AND CHROMOSOME ABERRATIONS
m
87
'
6 Fig. 4-7. - Fig. 4 and 5. Chromosome complement from a lab. mouseX tobacco mouse hybrid, showing 7 metacentrics and 26 telocentrics (2n= 33). A mitomycin C induced centromeric exchange between a telocentric and metacentric is indicated by an arrow in Fig. 5. - Fig. 6 and 7. C-banded chromosomes of MSWBS tumour cells. Fig. 6. Normal complement. The two marker chromosomes with extra heterochromatic blocks are indicated by arrows. - Fig. 7. Mitomycin C treated cell. The arrow indicates an aberration involving the marker chromosome.
its hybrid with the laboratory mouse. The telocentric marker chromosome was very often involved in aberrations - both in exchanges as well as intrachanges (Fig. 7, 8, 10, 11). In exchanges,
only the telomeric and not the intercalary heterochromatic blocks of this chromosome was involved. On the other hand, the second marker chromosome, i.e. the biarmed one, did not involve Hereditas 80. 1975
88
A. T. NATARAJAN A N D T. RAPOSA
8
9 't
u
0
11
Fig. 8-11. C-banded chromosomes of MSWBS tumour cells treated with mitomycin C. Arrows indicate aberrations involving the marker chromosome 1 except in Fig. 9 where it indicates a centromeric exchange.
in any intrachanges. Out of 23 exchanges involving this chromosome which were recorded, only 3 were related to the two extra heterochromatic blocks, while the rest were associated with the centromeric heterochromatic blocks. This Hereditas
80, 1975
indicates that in addition to the presence of heterochromatin, a region should be in close proximity with a similar region in order to form aberrations. Therefore, the centromeric heterochromatin of this biarmed marker chromosome
HETEROCHROMATIN AND CHROMOSOME ABERRATIONS
89
Table 3. Localisation of mitomycin C induced chromatid aberrations in mouse tumour line MSWBS in vivo No. of cells analysed
250
No. of fragments in Het.
Eu.
12
6
No. of exchanges
356
Exchanges between
Exchanges between
Exchanges in marker chromosome
TT
BB
BT
Het/Het
Eu/Eu
HetjEu
1
2
183
28
146
245
58
53
33
25
T = telocentric; B = biarmed chromosomes; Het. = heterochromatic regions; Eu. = euchromatic regions; Marker 1: telomeric chromosome with 4 bands of heterochromatin Marker 2: biarmed chromosome with two extra blocks of terminal heterochromatin in addition to the two centromeric blocks
is more often associated with exchanges than the distal heterochromatic blocks. Centromeric heterochromatic regions come close together during anaphase movement and remain associated so in and NATARAJAN 1966). the interphase too (KUMAR An interesting observation in the MSWBS tumour line treated with mitomycin C is the low frequency of chromatid breaks, which are less than 5 % of the total aberrations (Table 3). In the normal mouse fibroblast cultures treated with mitomycin C, the chromatid breaks contribute to about 45 % of the total aberratiosn (Table 1). This difference in the pattern of chromosome aberration formation is very interesting and this would be an excellent system to study the relationship between DNA repair synthesis and its relevance to chromosome aberration formation. A tentative explanation for the low frequency of breaks in the tumour line is that these cells are extremely efficient in repair of DNA damage and that they make several misrepairs during this process, leading to exchanges. Acknowledgments. -These investigations were supported by the Swedish Natural Research Council, the Swedish Atomic Research Council and the Swedish Cancer Society. We are grateful to Professor A. Gropp for providing laboratory mouse tobacco x mouse hybrid material.
Literature cited GROPP,A., TETTENBORN, U. and VON LEHMANN, E. 1970. Chromosomenvariation vom Roberton’schen Typus bei der Tabakmaus, M. poschiavinus, und ihren Hybriden mit der Laboratoriumsmaus. - Cytogenetics 9: 9-23. JONES,K. W. 1970. Chromosomal and nuclear location of mouse satellite DNA in individual cells. - Mature 225: 91 2-91 5 .
KUMAR, S . and NATARAJAN, A. T. 1966. Kinetics of twobreak chromosome exchanges and the spatial arrangement of chromosome strands in interphase nucleus. -Ibid. 209: 796-797. LEVAN, A., BREGULA, U. and KLEIN,G. 1972. The stemline idiogram of the MSWBS tumour of the mouse and the problem of centric fusion. - Hereditas 70: 283- 294. N A T A R A I A N , A. T. and AHNSTROM, G . 1970. The IOCahation of radiation induced chromosome aberrations in relation to the distribution of heterochromatin in Secale cereafe. - Chromosoma 30: 250-257. - 1973. Induced chromosome aberrations and heterochromatin. - In Modern Aspects of Cytogenetics Constitutive Heterochromatin in Man. Symp. Med. Hoechst 6.F.K. Schattauer Veriag, Stuttgart, New York, p. 215-223. NATARAJAN, A. T., AHNSTROM, G. and RAPOSA, T. 1973. Distribution of constitutive heterochromatin in the J. Nut. chromosomes of MSWBS ascites tumor cells. Cancer Inst. 50: 1721-1726. NATARAJAN, A. T., AHNSTROM, G. and SHARMA, R. P. 1974. Heterochromatin and chromosome aberrations in Microtus agrestis. Role of chromosome association. Mutat. Res. 22: 73-79. NATARAJAN, A. T. and GROPP,A. 1972. A fluorescent study of the heterochromatin and nucleolar organization of the laboratory mouse and tobacco mouse. - Exp. Cell Res. 74: 245-250. NATARAIAN, A. T. and SCHMID, w. 1971. Differential response of constitutive and facultative heterochromatin in the manifestation of mitymycin C induced chromosome aberrations in Chinese hamster cells in vitro. Chromosoma 33: 48-52. Paris Conference 1971. Standardization in Human Cytogenetics. - Birth Defects: Original Article Series, VIll: 7 , 1972. The National Science Foundation, New York. PARDUE, M. L.and GALL,J. G.1970. Chromosomal localization of mouse satellite DNA. - Science 168: 1356 1358. RAPOSA,T. and N A T A R A I A N , A. T. 1974. Fluorescence banding pattern of human and mouse chromosomes with a benzimidazol derivative (Hoechst 33258). Humangenetik 21: 221-226. ~
Hereditas 80, 1975
(1975)
Heterochromatin and chromosome aberrations. A comparative study of three mouse cell lines with different karyotype and heterochromatin distribution A. T. NATARAJAN and T. RAPOSA Wallenberg Laboratory, Stockholm University, Stockholm, Sweden
NATARAJAN, A. T. and RAPOSA,T. 1975. Heterochromatin and chromosome aberrations. A comparative study of three mouse cell lines with different karyotype and heterochromatin distribution. - Hereditas 80: 83-90. Lund, Sweden. ISSN 0018-0661. Received January 24, 1975 Mitomycin C induced chromosome aberrations in mouse cells differing in chromosome number and karyotype were studied. The material included (1) laboratory mouse (2n = 40), all telocentric chromosomes possessing centromeric heterochromatin, (2) laboratory mouse X tobacco mouse hybrid (2n = 33) with 7 metacentrics and 26 telocentrics, and (3) ascites tumour cells (MSWBS) (2n = 28--29), with 9 to 10 biarmed chromosomes, with heterochromatin localised to the centromeric regions in all, except two marker chromosomes - one telocentric with 4 intercalary blocks, and one biarmed chromosome with two terminal blocks. Both chromatid intrachanges and interchanges were scored. It was found: (a) In laboratory mouse about 80 % of the abberations are localised to the heterochromatic regions; (b) In the tobacco mouse X laboratory mouse hybrid, the exchanges are between two telocentrics or a telocentric and a metacentric, but seldom between two metacentrics; (c) In the MSWBS tumour cells, the centromeric heterochromatin was involved more often in interchanges than the intercalary blocks, without any restriction between two biarmed chromosomes to form exchanges. A. T. Natarajan, Walfenberg Laboratory, Lilla Frescati, S-10405 Stockholm 50, Sweden
Spontaneously occurring chromosome aberrations as well as those induced by chemical agents and viruses are localized to the heterochromatic regions in several plants and mammals (NATARAJAN and AHNSTROM 1973). After an evaluation of the factors which may be responsible for the observed extreme localization in different material with varying amount and distribution of heterochromatin, it was concluded that
To elucidate this problem further, we have used mouse as a model system for the following reasons:
(a) In mouse, the constitutive heterochromatic regions are very rich in repetitive DNA (JONES 1970; PARDUE and GALL1970; NATARAJAN et al. 1973), (b) different karyotypes within the genus different species or cell lines - are available
-
as
(a) the concentration of highly repetitive DNA in these regions must be the most important one (NATARAJAN and AHNSTROM 1970, 1973), and
(c) normal and neoplastic cells with similar karyotypes allow a comparative study and
(b) the manner in which the heterochromatic regions organize themselves to form chromocentres in the interphase plays an important role in determining the types of aberrations realised (NATARAJAN et al. 1974).
(d) this system allows the study of similar cells e.g. ascites tumour cells - in vitro and in vivo for their response to different agents. In the present communication, a comparative study of the patterns of the alkylating antibiotic mitomycin Hereditas 80, 19i 5
84
A. T. NATARAJAN A N D T. RAPOSA
C induced chromosome aberrations in mouse lines having different karyotypes and distribution of heterochromatin is presented.
RAJAN and SCHMID 1971; NATARAJAN et al. 1974). The dosage of mitomycin varied, however, from 0.04 to 0.5 pg/ml. In the case of MSWBS, the animals carrying tumours were injected with mitomycin C: in isotonic saline (4 pg/ml of ascitic fluid), and the preparations were made according Material and methods to the schedule described earlier (NATARAJAN et al. 1973). Cell lines and their chromosome characteristics Staining. All preparations were made from (1) Laboratory mouse (Mus musculus). 2n= 40, colchicine-blocked metaphases. Some preparaall telocentric chromosomes with centromeric tions were processed by G banding technique heterochromatic blocks except the Y (Fig. 1). and others by C banding technique (Paris ConPrimary cultures were set up from 12 to 14 days ference, 1972). Only chromatid aberrations were old embryos and the growing fibroblast cells encountered. The different types of aberrations were classified and their localization was recorded. were used in their second passage. Since the heterochromatin in mouse chromosomes (2) Laboratory mousex tobacco mouse ( M . pois localised to the centromeric regions, it is schiuvinus) F, hybrids: 21-1233 with 7 metarelatively easy to recognize these regions in abercentrics and 26 telocentrics, all having heterorations (see Fig. 2, 3, 7 to 11). But, in the case of chromatin proximal to the centromeric regions MSWBS line, there was intercalary heterochroexcept the Y. Primary cultures were set up from matin, as well as heterochromatic block near the 12 to 14 days old embryos (Fig. 4), centromere of only one of the arms of the (3) MSWBS tumour line: 2nn=28 or 29, with biarmed chromosomes. To get a more precise 9 to 10 biarmed chromosomes (LEVANet al. 1972), scoring, both C banding as well as benzimidazole with heterochromatin around the centrorneric banding (RAPOSA and NATARAJAN 1974) were regions of all the chromosomes. In addition, one performed. telocentric has 4 blocks of intercalary heterochromatin and one biarmed chromosome has two additional heterochromatic blocks in the long arm (Fig. 6). These tumour cells in passages Results and discussion 160 to 178, were made available through the 1 . Laboratory mouse courtesy of Professor G. Klein. The frequency of different types of aberrations produced by mitomycin C is presented in Table 1 . Mitomycin C treatment It can be seen that about 74 % of the chromatid In vitro cultures of fibroblasts were treated breaks occurs at the centromeric heterochromatic according to the schedule described earlier (NATA- regions. Different types of chromatid exchanges
Table 1. Localisation of mitomycin-induced chromatid aberrations in Mus musculus The cells were scored at 22 and 32 hours, following treatment with 0.04 pg/ml of mitomycin C
No. of cells analysed
300
No. of abnormal cells
155
Chromatid interchanges localised in
N o . of chromatid breaks localised in centromeric region
telomeric region
other regions
cc
CT
CM
TT
TM
MM
81
8
22
66(36)1
21
9
21
5
8
C = centrorneric region; T = telorneric region; M = other regions Number of exchanges involving both the chromatids of both the chromosomes
1
Hereditas 80, 1975
HETEROCHROMATIN AND CHROMOSOME ABERRATIONS
85
1
t
\ &
0 2
I H
I
/ rr
Fig. 1-3. - Fig. 1. C-banded chromosomes of laboratory mouse showing the centromeric heterochromatin in all the chromosomes except Y (arrow). - Fig. 2 and 3. Mitomycin C treated laboratory mouse cells, showing different types of exchanges involving centromeric regions (arrows).
Hereditas 80,1975
86
A. T. NATARAJAN AND T. RAPOSA
are noticed with this treatment, which are il- Table 2. Mitomycin C induced chromatid exlustrated in Fig. 2 and 3. About 70 % of exchanges changes in F, hybrid cells ( M . musciilus X M . are localized to the centromeric heterochromatic poschiavinus) regions. An interesting feature is that more than Total number of exchanges analysed was 114, from 250 half of the exchanges involving the centromeric cells regions of two chromosomes, has both the chromatids united or joined together in the aberra- Localisation Exchanges between tions (Fig. 3). It is estimated that about 10 % telometatelocentrics of the mouse-chromosome regions is heterochrocentrics centrics and matic (NATARAJAN and AHNSTROM1973), and metacentrics these regions account for the localization of about 7 0 % of the aberrations. As pointed out Centromeric 18 0 40 earlier, this preferential occurrence of aberrations region 8 0 10 is most probably due to the enrichment of repeti- Telomeric tive DNA in these regions which makes it easier region 4 3 3 for aberration formation during repair of lesions Other regions induced by chemicals or radiations (NATARAJAN and AHNSTROM 1973). Though there are 39 or 40 heterochromatic regions proximal to the cen- regions. Though there were large proportions of tromere in mouse, there are usually a lower aberrations involving two telocentrics, or one number of chromocentres in the interphase. The telo- and one metacentric (Fig. 5 ) , there were no range is between 7 to 26 with an average of 14.22 aberrations involving two metacentrics. This per cell (NATARAJAN and GROPP1972). This would strongly suggests that there must be a specific explain the very high frequency of centromeric control of the association of chromocentres in exchanges in the laboratory-mouse chromosomes the interphase and there is a homologous association between the telocentric and the metacentric following mitomycin C treatment. involving that telocentric chromosome in the Robertsonian fusion. 2. Laboratory mouse X tobacco mouse hybrid In an earlier study (NATARAJAN and GROPP1972) it was reported that the number of chromocentres 3. MSWBS tumour line which was formed in fibroblasts of tobacco mouse The normal distribution of heterochromatin in ranges from 2 to 16, with an average of 9.4. this line is illustrated in Fig. 6 and the types of This was expected in view of the fact that tobacco aberrations recovered following mitomycin C mouse has a diploid chromosome number of 26, treatment in this material are illustrated in Fig. 7 of which 14 are metacentrics arising from Ro- to 11. The quantitative data are presented in bertsonian fusion (GROPPet al. 1970; NATARAJANTable 3. About 75 % of the exchanges involves and GROPP1972). When the behaviour of the F, the heterochromatic regions. While exchanges hybrid cells (between tobacco mouse and labora- between biarmed chromosomes and telocentrics tory mouse) was studied with regard to the chro- are common, the exchanges between the biarmed mocentre formation, it was found that the hybrid chromosomes are less than expected on the basis follows the tobacco-mouse pattern and not an of random exchanges between heterochromatic intermediate pattern, indicating a possible as- regions. This frequency is, however, higher when sociation between metacentrics and the homolo- compared to the hybrid material between tobacco gOUS teIOCentriCS (NATARAJAN and GROPP1972). mouse and laboratory mouse. Since the MSWBS Mitomycin C induced aberrations which reflect tumour has gone through many structural to a great extent the association of different changes, there is obviously no rigorous restricchromosomes or chromosomal regions in in- tion on the association of heterochromatic regions terphase, were analysed in this material. The to form chromocentres. An analysis of the chroresults are presented in Table 2. The results mocentre frequency in this material indicated that indicate that the aberrations are localised prefer- it ranged between 6 to 19 with an average of 14, entially to the centromeric heterochromatic which is higher than in the tobacco mouse and Hereditas 80,1975
HETEROCHROMATIN AND CHROMOSOME ABERRATIONS
m
87
'
6 Fig. 4-7. - Fig. 4 and 5. Chromosome complement from a lab. mouseX tobacco mouse hybrid, showing 7 metacentrics and 26 telocentrics (2n= 33). A mitomycin C induced centromeric exchange between a telocentric and metacentric is indicated by an arrow in Fig. 5. - Fig. 6 and 7. C-banded chromosomes of MSWBS tumour cells. Fig. 6. Normal complement. The two marker chromosomes with extra heterochromatic blocks are indicated by arrows. - Fig. 7. Mitomycin C treated cell. The arrow indicates an aberration involving the marker chromosome.
its hybrid with the laboratory mouse. The telocentric marker chromosome was very often involved in aberrations - both in exchanges as well as intrachanges (Fig. 7, 8, 10, 11). In exchanges,
only the telomeric and not the intercalary heterochromatic blocks of this chromosome was involved. On the other hand, the second marker chromosome, i.e. the biarmed one, did not involve Hereditas 80. 1975
88
A. T. NATARAJAN A N D T. RAPOSA
8
9 't
u
0
11
Fig. 8-11. C-banded chromosomes of MSWBS tumour cells treated with mitomycin C. Arrows indicate aberrations involving the marker chromosome 1 except in Fig. 9 where it indicates a centromeric exchange.
in any intrachanges. Out of 23 exchanges involving this chromosome which were recorded, only 3 were related to the two extra heterochromatic blocks, while the rest were associated with the centromeric heterochromatic blocks. This Hereditas
80, 1975
indicates that in addition to the presence of heterochromatin, a region should be in close proximity with a similar region in order to form aberrations. Therefore, the centromeric heterochromatin of this biarmed marker chromosome
HETEROCHROMATIN AND CHROMOSOME ABERRATIONS
89
Table 3. Localisation of mitomycin C induced chromatid aberrations in mouse tumour line MSWBS in vivo No. of cells analysed
250
No. of fragments in Het.
Eu.
12
6
No. of exchanges
356
Exchanges between
Exchanges between
Exchanges in marker chromosome
TT
BB
BT
Het/Het
Eu/Eu
HetjEu
1
2
183
28
146
245
58
53
33
25
T = telocentric; B = biarmed chromosomes; Het. = heterochromatic regions; Eu. = euchromatic regions; Marker 1: telomeric chromosome with 4 bands of heterochromatin Marker 2: biarmed chromosome with two extra blocks of terminal heterochromatin in addition to the two centromeric blocks
is more often associated with exchanges than the distal heterochromatic blocks. Centromeric heterochromatic regions come close together during anaphase movement and remain associated so in and NATARAJAN 1966). the interphase too (KUMAR An interesting observation in the MSWBS tumour line treated with mitomycin C is the low frequency of chromatid breaks, which are less than 5 % of the total aberrations (Table 3). In the normal mouse fibroblast cultures treated with mitomycin C, the chromatid breaks contribute to about 45 % of the total aberratiosn (Table 1). This difference in the pattern of chromosome aberration formation is very interesting and this would be an excellent system to study the relationship between DNA repair synthesis and its relevance to chromosome aberration formation. A tentative explanation for the low frequency of breaks in the tumour line is that these cells are extremely efficient in repair of DNA damage and that they make several misrepairs during this process, leading to exchanges. Acknowledgments. -These investigations were supported by the Swedish Natural Research Council, the Swedish Atomic Research Council and the Swedish Cancer Society. We are grateful to Professor A. Gropp for providing laboratory mouse tobacco x mouse hybrid material.
Literature cited GROPP,A., TETTENBORN, U. and VON LEHMANN, E. 1970. Chromosomenvariation vom Roberton’schen Typus bei der Tabakmaus, M. poschiavinus, und ihren Hybriden mit der Laboratoriumsmaus. - Cytogenetics 9: 9-23. JONES,K. W. 1970. Chromosomal and nuclear location of mouse satellite DNA in individual cells. - Mature 225: 91 2-91 5 .
KUMAR, S . and NATARAJAN, A. T. 1966. Kinetics of twobreak chromosome exchanges and the spatial arrangement of chromosome strands in interphase nucleus. -Ibid. 209: 796-797. LEVAN, A., BREGULA, U. and KLEIN,G. 1972. The stemline idiogram of the MSWBS tumour of the mouse and the problem of centric fusion. - Hereditas 70: 283- 294. N A T A R A I A N , A. T. and AHNSTROM, G . 1970. The IOCahation of radiation induced chromosome aberrations in relation to the distribution of heterochromatin in Secale cereafe. - Chromosoma 30: 250-257. - 1973. Induced chromosome aberrations and heterochromatin. - In Modern Aspects of Cytogenetics Constitutive Heterochromatin in Man. Symp. Med. Hoechst 6.F.K. Schattauer Veriag, Stuttgart, New York, p. 215-223. NATARAJAN, A. T., AHNSTROM, G. and RAPOSA, T. 1973. Distribution of constitutive heterochromatin in the J. Nut. chromosomes of MSWBS ascites tumor cells. Cancer Inst. 50: 1721-1726. NATARAJAN, A. T., AHNSTROM, G. and SHARMA, R. P. 1974. Heterochromatin and chromosome aberrations in Microtus agrestis. Role of chromosome association. Mutat. Res. 22: 73-79. NATARAJAN, A. T. and GROPP,A. 1972. A fluorescent study of the heterochromatin and nucleolar organization of the laboratory mouse and tobacco mouse. - Exp. Cell Res. 74: 245-250. NATARAIAN, A. T. and SCHMID, w. 1971. Differential response of constitutive and facultative heterochromatin in the manifestation of mitymycin C induced chromosome aberrations in Chinese hamster cells in vitro. Chromosoma 33: 48-52. Paris Conference 1971. Standardization in Human Cytogenetics. - Birth Defects: Original Article Series, VIll: 7 , 1972. The National Science Foundation, New York. PARDUE, M. L.and GALL,J. G.1970. Chromosomal localization of mouse satellite DNA. - Science 168: 1356 1358. RAPOSA,T. and N A T A R A I A N , A. T. 1974. Fluorescence banding pattern of human and mouse chromosomes with a benzimidazol derivative (Hoechst 33258). Humangenetik 21: 221-226. ~
Hereditas 80, 1975
