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Quinacrine fluorescence of metaphase chromosomes
Experimental
QUINACRINE
FLUORESCENCE OF METAPHASE CHROMOSOMES Identical
TORBJijRN
Cell Research 72 (1972) 56-59
CASPERSSON,l
ALBERT
Patterns in Different de la CHAPELLE,*
Tissues JIM SCHR6DER,Z
and LORE ZECH’
‘Institute for MedicaI CeN Research and Genetics, Medical Nobel Institute, Karolinska Institutet, 10401 Stockholm 60, Sweden, and 2Folkh&an Institute of Genetics, 00100 Helsingfors 10, Finland
SUMMARY The quinacrine mustard fluorescencepatterns of the metaphase chromosomes of different tissues of the same plant specieswere found to be identical. Similar studiesof the
chromosome regions on human material gave the sameresult.
In preparations from human blood cultures extensive studies have been made of the fluorescence patterns of the chromosomes after staining with quinacrine mustard and quinacrine [2-51. Approx. 98% of the total metaphase chromosome length showed very constant and reproducible banding patterns, thus giving a solid basis for chromosome identification. The banding patterns can also be used for identification of chromosome regions, for instance in the study of chromosome rearrangements (see [q). Several chromosomes have very short, often intensely fluorescing regions which show considerable variations from person to person [3, 6-101. The total length of all these regions together is, however, barely 2% of that of the total metaphase chromosome length. Another variability between individuals, concerning some short centromeric regions, has been observed by Craig-Holmes & Shaw [ll] in preparations stained according to Arrighi & Hsu [l] for constitutive heterochromatin. The information which enables somatic Exptl Cell Res 72
cells to differentiate must be carried from cell generation to cell generation but the chemical basis for this process is entirely unknown. The quinacrine and quinacrine mustard patterns reflect not only DNA distribution in the chromosomes but also the accessibility of the DNA in different chromosome regions to base reacting and intercalating agents. The latter factor is determined by the steric relations between the DNA and the chromosomal proteins. As proteins are known to participate in the gene regulation mechanisms it seems reasonable to look for changes in the fluorescence patterns during the progress of differentiation, or to compare the patterns in differently organized tissues. It is of interest to note in this connection that the resolution of fluorescence methods approaches the order of magnitude of genes and gene complexes. The smallest fluorescent bands used in the identification routine (e.g. in chromosome 19) contain the order of lo-l5 g DNA, corresponding to about lo6 nucleotide pairs or 100-l 000 genes. In the present study pattern comparisons
Quinacrine fluorescence of metaphase chromosomes
51
Fig. I. QM-stained metaphase chromosomes from three different tissues: Blood (II), skin (S) and testis (T). x 2 500. The skin and testis cells were from an XX male individual. Chromosomes from (a) the A and B group; (6) the C group; (c) the D, E, F and G group.
between chromosomes from different tissues in the same species of plants and also from different tissues in man will be described. The results show for these different tissues that within the limits of the present day methods the fluorescence patterns are similar. Fluorescence techniques All preparations were stained with quinacrine mustard as described by Caspersson, Zech, Johansson & Modest 121. The uhotoelectric recordings of the fluorescence patterns were made in the wa; described in the same publication.
Results on different types of material
In some plants with large chromosomes, among them Vicia faba and Scilla sibirica, very conspicuous differences in fluorescence intensity have been found between chromo-
some parts in spite of a rather homogeneous distribution of the DNA along the chromosomes [12, 131. These coarse patterns have been found to be quite constant and reproducible in root tip cells. Comparisons between the patterns from root tip cells and endosperm cells in Vicia showed no observable differences. In Scilla identical patterns were found in cells from root tips, endosperm and also pollen tubes. In order to make similar comparisons in human material where the fluorescence patterns show much finer details than in plants, about 50 metaphases were analysed from each of the following tissues: bone marrow, skin, testis and amnion. From blood a very large observational material is already available [3, 61. Exptl Cell Res 72
58
T. Caspersson et al.
Standard methods of cell and tissue culture were used. Blood cells were cultured for 3 days in the presence of phytohemagglutinin. Bone marrow cells were incubated for 2 h in vitro. Primary cultures of the other tissues were subcultured once or twice before harvesting. Colcemid-arrested mitoses were prepared by the air drying method. For each of the approx. 200 cells to be analysed a series of prints of different exposure times were prepared from the fluorescence photographs of the chromosome set. Photographic karyotypes were prepared from the prints showing most details for each chromosome. In this way it was possible to analyse the fine structure of chromosome patterns from each of the different cells and to compare them with the standard karyotype from blood cells. Comparison between the pictures showed, apart from the regions referred to above as showing variations between individualsidentical banding patterns and no differences were observed between the chromosomes from the different tissues. Fig. 1 illustrates the similarity of the fluorescence pattern of human chromosomes from three different tissues (blood, skin and testis). In addition photoelectric recordings were made of the chromosome patterns from the following material: Five metaphases from blood, skin and testis, respectively, of one 46, XX male individual [14, 151 and in addition, from another 46, XX male individual [ 15, 161, five cells from blood and skin respectively. The recorded patterns showed no significant differences between different preparations, outside of the regions known to show individual variations (cf [6]). DISCUSSION Apart from regions known to show variations from individual to individual, the fluoExptl Cell Res 72
rescence patterns were species specific in all the material studied. It is generally assumed that the amounts of chromosomal DNA in somatic cells from different tissues are the same [17]. The QMpattern is, however, not only dependent upon the distribution of the DNA, but greatly influenced by the protein moiety and probably also by the local degree of coiling. It is therefore of special interest to note that no differences could be observed between metaphases from different tissues. As all cells used in this study were obtained after in vitro culture it is conceivable that differences in patterns might be obscured as a result of the conditions of culture. However, bone marrow cells were incubated for only 2 h and in several cultured tissues the morphologic features of the cells showed that they were not dedifferentiated at the time of harvest. In the plant material no culture procedures at all were used. Work by fluorimetry and electron microscopy on the fine details of the patterns of less contracted chromosomes in blood cells is going on and will give still more pattern information. This will permit more penetrating comparisons between different tissues. We are indebted to Mrs K Arrhenius, Mrs A Brolin and Mr J Kudynowski for valuable assistance. These studies were supported by research grants to the Karolinska Institutet from the Swedish Cancer Society and the Swedish Natural Science Research Council and to the Folkhllsan Institute of Genetics from the Sigrid Juselius Foundation, the Finnish National Research Council for Medical Sciences and the Nordisk Insulinfond.
REFERENCES 1. Arrighi, F E & Hsu, T C, Cytogenetics 10 (1971) 81. 2. Casuersson. T. Zech, L. Johansson, C & Modest, E J; Chromosbma 36 (1970) 215. 3. Casoersson. T. Lomakka. G & Zech, L. Hereditas 67 (1971) 85. ’ 4. Caspersson, T, Lomakka, G & MBller, A, Hereditas 67 (1971) 103. 5. Caspersson, T, Lindsten, J, Lomakka, G, Moller, A & Zech, L, Int rev exptl path01 (1972). In press.
Quinacrine 6. Moller, A, Nilsson, H, Caspersson, T & Lomakka, G, Exptl cell res 70 (1972) 475. 7. Caspersson, T, Zech, L, Johansson, C, Lindsten, J & Hulten. M. Exutl cell res 61 (1970) 472. 8. Schnedl, W; Humangenetik 12 (1971) 59. 9. Uchida, I A & Lin, C C, IVth Internatl congr hum genet. Excerpta medica, no. 233 (1971) 180. 10. Manolov, G, Manolova, Y & Levan, A, Herediras 69 (1971) 273. 11. Craig-Holmes, A & Shaw, M, Science 174 (1971) 702.
12. Caspersson, T, Zech, L, Modest, E J, Foley, G E, Wagh, U & Simonsson, E, Exptl cell res 58 (1969) 128.
fluorescence
of metaphase
chromosomes
59
13. Caspersson, T, Zech, L, Modest, E J, Foley, G E, Wagh, U & Simonsson, E, Exptl cell res 58 (1969) 141. 14. de la Chapelle, A, Simila, S, Lanning, M, Kontturi, M & Johansson, C J, Humangenetik 11 (1971) 286. 15. Casnersson. T. de la Chanelle. A. Lindsten. J. Schriider, J &‘Zech, L, Ann g&et 14 (1971) 9: 16. de la Chanelle. A. Hortling, H. Niemi. M & Wennstrijm, J, kcta med Stand, suppl. 412 (1964) 25.
17. Davidson, E H, Gene activity in early development. Academic Press, New York (1968).
Exptl
Cell Res 72
QUINACRINE
FLUORESCENCE OF METAPHASE CHROMOSOMES Identical
TORBJijRN
Cell Research 72 (1972) 56-59
CASPERSSON,l
ALBERT
Patterns in Different de la CHAPELLE,*
Tissues JIM SCHR6DER,Z
and LORE ZECH’
‘Institute for MedicaI CeN Research and Genetics, Medical Nobel Institute, Karolinska Institutet, 10401 Stockholm 60, Sweden, and 2Folkh&an Institute of Genetics, 00100 Helsingfors 10, Finland
SUMMARY The quinacrine mustard fluorescencepatterns of the metaphase chromosomes of different tissues of the same plant specieswere found to be identical. Similar studiesof the
chromosome regions on human material gave the sameresult.
In preparations from human blood cultures extensive studies have been made of the fluorescence patterns of the chromosomes after staining with quinacrine mustard and quinacrine [2-51. Approx. 98% of the total metaphase chromosome length showed very constant and reproducible banding patterns, thus giving a solid basis for chromosome identification. The banding patterns can also be used for identification of chromosome regions, for instance in the study of chromosome rearrangements (see [q). Several chromosomes have very short, often intensely fluorescing regions which show considerable variations from person to person [3, 6-101. The total length of all these regions together is, however, barely 2% of that of the total metaphase chromosome length. Another variability between individuals, concerning some short centromeric regions, has been observed by Craig-Holmes & Shaw [ll] in preparations stained according to Arrighi & Hsu [l] for constitutive heterochromatin. The information which enables somatic Exptl Cell Res 72
cells to differentiate must be carried from cell generation to cell generation but the chemical basis for this process is entirely unknown. The quinacrine and quinacrine mustard patterns reflect not only DNA distribution in the chromosomes but also the accessibility of the DNA in different chromosome regions to base reacting and intercalating agents. The latter factor is determined by the steric relations between the DNA and the chromosomal proteins. As proteins are known to participate in the gene regulation mechanisms it seems reasonable to look for changes in the fluorescence patterns during the progress of differentiation, or to compare the patterns in differently organized tissues. It is of interest to note in this connection that the resolution of fluorescence methods approaches the order of magnitude of genes and gene complexes. The smallest fluorescent bands used in the identification routine (e.g. in chromosome 19) contain the order of lo-l5 g DNA, corresponding to about lo6 nucleotide pairs or 100-l 000 genes. In the present study pattern comparisons
Quinacrine fluorescence of metaphase chromosomes
51
Fig. I. QM-stained metaphase chromosomes from three different tissues: Blood (II), skin (S) and testis (T). x 2 500. The skin and testis cells were from an XX male individual. Chromosomes from (a) the A and B group; (6) the C group; (c) the D, E, F and G group.
between chromosomes from different tissues in the same species of plants and also from different tissues in man will be described. The results show for these different tissues that within the limits of the present day methods the fluorescence patterns are similar. Fluorescence techniques All preparations were stained with quinacrine mustard as described by Caspersson, Zech, Johansson & Modest 121. The uhotoelectric recordings of the fluorescence patterns were made in the wa; described in the same publication.
Results on different types of material
In some plants with large chromosomes, among them Vicia faba and Scilla sibirica, very conspicuous differences in fluorescence intensity have been found between chromo-
some parts in spite of a rather homogeneous distribution of the DNA along the chromosomes [12, 131. These coarse patterns have been found to be quite constant and reproducible in root tip cells. Comparisons between the patterns from root tip cells and endosperm cells in Vicia showed no observable differences. In Scilla identical patterns were found in cells from root tips, endosperm and also pollen tubes. In order to make similar comparisons in human material where the fluorescence patterns show much finer details than in plants, about 50 metaphases were analysed from each of the following tissues: bone marrow, skin, testis and amnion. From blood a very large observational material is already available [3, 61. Exptl Cell Res 72
58
T. Caspersson et al.
Standard methods of cell and tissue culture were used. Blood cells were cultured for 3 days in the presence of phytohemagglutinin. Bone marrow cells were incubated for 2 h in vitro. Primary cultures of the other tissues were subcultured once or twice before harvesting. Colcemid-arrested mitoses were prepared by the air drying method. For each of the approx. 200 cells to be analysed a series of prints of different exposure times were prepared from the fluorescence photographs of the chromosome set. Photographic karyotypes were prepared from the prints showing most details for each chromosome. In this way it was possible to analyse the fine structure of chromosome patterns from each of the different cells and to compare them with the standard karyotype from blood cells. Comparison between the pictures showed, apart from the regions referred to above as showing variations between individualsidentical banding patterns and no differences were observed between the chromosomes from the different tissues. Fig. 1 illustrates the similarity of the fluorescence pattern of human chromosomes from three different tissues (blood, skin and testis). In addition photoelectric recordings were made of the chromosome patterns from the following material: Five metaphases from blood, skin and testis, respectively, of one 46, XX male individual [14, 151 and in addition, from another 46, XX male individual [ 15, 161, five cells from blood and skin respectively. The recorded patterns showed no significant differences between different preparations, outside of the regions known to show individual variations (cf [6]). DISCUSSION Apart from regions known to show variations from individual to individual, the fluoExptl Cell Res 72
rescence patterns were species specific in all the material studied. It is generally assumed that the amounts of chromosomal DNA in somatic cells from different tissues are the same [17]. The QMpattern is, however, not only dependent upon the distribution of the DNA, but greatly influenced by the protein moiety and probably also by the local degree of coiling. It is therefore of special interest to note that no differences could be observed between metaphases from different tissues. As all cells used in this study were obtained after in vitro culture it is conceivable that differences in patterns might be obscured as a result of the conditions of culture. However, bone marrow cells were incubated for only 2 h and in several cultured tissues the morphologic features of the cells showed that they were not dedifferentiated at the time of harvest. In the plant material no culture procedures at all were used. Work by fluorimetry and electron microscopy on the fine details of the patterns of less contracted chromosomes in blood cells is going on and will give still more pattern information. This will permit more penetrating comparisons between different tissues. We are indebted to Mrs K Arrhenius, Mrs A Brolin and Mr J Kudynowski for valuable assistance. These studies were supported by research grants to the Karolinska Institutet from the Swedish Cancer Society and the Swedish Natural Science Research Council and to the Folkhllsan Institute of Genetics from the Sigrid Juselius Foundation, the Finnish National Research Council for Medical Sciences and the Nordisk Insulinfond.
REFERENCES 1. Arrighi, F E & Hsu, T C, Cytogenetics 10 (1971) 81. 2. Casuersson. T. Zech, L. Johansson, C & Modest, E J; Chromosbma 36 (1970) 215. 3. Casoersson. T. Lomakka. G & Zech, L. Hereditas 67 (1971) 85. ’ 4. Caspersson, T, Lomakka, G & MBller, A, Hereditas 67 (1971) 103. 5. Caspersson, T, Lindsten, J, Lomakka, G, Moller, A & Zech, L, Int rev exptl path01 (1972). In press.
Quinacrine 6. Moller, A, Nilsson, H, Caspersson, T & Lomakka, G, Exptl cell res 70 (1972) 475. 7. Caspersson, T, Zech, L, Johansson, C, Lindsten, J & Hulten. M. Exutl cell res 61 (1970) 472. 8. Schnedl, W; Humangenetik 12 (1971) 59. 9. Uchida, I A & Lin, C C, IVth Internatl congr hum genet. Excerpta medica, no. 233 (1971) 180. 10. Manolov, G, Manolova, Y & Levan, A, Herediras 69 (1971) 273. 11. Craig-Holmes, A & Shaw, M, Science 174 (1971) 702.
12. Caspersson, T, Zech, L, Modest, E J, Foley, G E, Wagh, U & Simonsson, E, Exptl cell res 58 (1969) 128.
fluorescence
of metaphase
chromosomes
59
13. Caspersson, T, Zech, L, Modest, E J, Foley, G E, Wagh, U & Simonsson, E, Exptl cell res 58 (1969) 141. 14. de la Chapelle, A, Simila, S, Lanning, M, Kontturi, M & Johansson, C J, Humangenetik 11 (1971) 286. 15. Casnersson. T. de la Chanelle. A. Lindsten. J. Schriider, J &‘Zech, L, Ann g&et 14 (1971) 9: 16. de la Chanelle. A. Hortling, H. Niemi. M & Wennstrijm, J, kcta med Stand, suppl. 412 (1964) 25.
17. Davidson, E H, Gene activity in early development. Academic Press, New York (1968).
Exptl
Cell Res 72