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Radiation-induced non-random chromosome breakage
Preliminary notes reduced to 2-3 ,ul by dialysis against polyethylene glycol and transferred to cellulose acetate strips prepared for electrophoresis. After 2 hours (barbitone buffer, pH 8.6, 0.05 M; 0.4 mamp/cm width) the strips were dried and stained in 0.1 percent Coomassie Blue. At least two proteins were found moving toward the anode and one toward the cathode with a large proportion, judging by the degree of staining, remaining at the origin. Electrophoretic resolution of the proteins on cellulose acetate, particularly with L. pictus BF, was not clear cut and other methods will be necessary for detailed analysis. As yet, in these studies of the morphogenic inhibitor in BF there has been no clue as to its normal role in the embryo or the mechanism of its inhibitory effects. It may be mentioned here that L. pictus BF injected into the blastocoel of the L. pictus mesenchyme blastula does not inhibit or alter gastrulation. While it is difficult to make such experiments quantitative in terms of the amount injected and retained, nevertheless, the consistently negative results with 15 injected embryos suggest that the inhibitory effects of BF are not due to alteration of some component during preparation. References 1. Berg, W E & Akin, E J, Dev biol 26 (1971) 353. 2. Giudice, G, Dev biol 5 (1962) 402. 3. Motomura, I, Bull mar biol stat Asamushi 10 (1960) 165. 4. Okazaki, K & Miijima, L, Embryologia 8 (1964) 89. Received August 28, 1972
541
Radiation-induced non-random chromosome breakage T. CASPERSSON’ ULLA HAGLUND.’ B. LINDELL2 and LORE ZECH,’ llnstitute fdr Medical Cell Research and Genetics, Medical Nobel Institute, Karolinska Znstitutet, 104 01 Stockholm 60, and 2The National Znstitute of Radiation Protection. Fack. 104 01 Stockholm 60, Sweden
Compared with earlier methods, the recently developed chromosome banding techniques offer improved possibilities for the study of chromosome aberrations. A large statistically evaluated observational material based on the quinacrine fluorescence method demonstrated the stability of the fluorescence patterns of the human chromosomes. This gives a solid basis foi the identification of the location of chromosome breaks [I, 21. Moreover, in studies of the action of mutagens, such as ionizing radiations, this means that one cannot only observe and identify smaller aberrations, but also identify those types of chromosomal disturbances which are difficult or even impossible to observe in mammalian material with conventional techniques. Thus, most of the early studies of radiation-induced chromosomal changes were mainly concerned with the formation of rings and dicentrics, while such aberrations as reciprocal translocations and paracentric inversions were easily overlooked (cf [3]). In most cases the fluorescence technique gives rather clear pictures of such aberrations. Fig. 1 shows nine examples with chromosome patterns so clearly that by comparison with the standard karyotype [l], it is easy to identify directly the chromosome parts involved. The number of metaphases to be analysed in such studies is always much greater than that required in medical clinical work. The routine procedures for karyotyping are quite tedious. It is possible to speed up the work and to analyse reasonably large numbers of metaExptl Cell Res 7.5(1972)
Fig. I. Chromosome aberrations from the materia1 described in the text. Quinacrine mustard technique, x 2 000. a, dicentric chromosome (1; 11); b, dicentric chromosome (3; 9q); c, translocation (Sq; 5q); d, translocation (7; 14q); e, translocation (5q; 7); f, translocation (1Oq; 15); g, duplication (7q); h, acentric fragment, duplication (7q); i, inversion (59).
phases by using a TV-based procedure for rapid identification of chromosomes and chromosome regions [4]. Thus, in such work, one increases the speed about 20 times, mak-
ing statistical studies of chromosome breaks by external mutagens a reasonable proposition. The detailed analysis of aberrations would be facilitated by a technique allowing us to compare small details of the patterns from different chromosomes, e.g. a chromosome with a suspected deletion could be compared with its normal partner. For that purpose the TV-procedure has been further developed so that chromosomes from two different preparations can be projected side by side on the same monitor screen [5]. By this procedure we examined about 500 metaphases corresponding to more than 20 000 chromosomes obtained from X-ray irradiated human lymphocytes from 10 different persons. Two irradiation doses were used, 226 rad and 56 rad, for each dose level
16
17
I8
19
20
21
22
Fig. 2. Location of the chromosome breaks in the material investigated. Exptl Cell Res 75 (1972)
Preliminary notes Table 1. Radiation dose 226 rad
56 rad
114
360
35
294
19
66
Dicentrics
50
11
Translocations Deletions Inversions Duplications
41 48 4
1; 3 2
Fragments Rings
473
2:
Total number of analyzed metaphase ulates Metaphase plates without visible aberrations Metaphase plates with aberrations
using blood cells from 6 persons. The blood cultures were irradiated after 24 h and all cells were harvested after 72 h in culture. Three hours prior to harvesting the cells were treated with colcemid. Table 1 summarizes the results of the analysis of 114 plates (exposed to 226 rads) and 360 plates (exposed to 56 rads). The largest number of aberrations found represents dicentrics, translocations and deletions, while inversions and duplications are comparatively rare. Of special interest is a difference in radiation sensitivity of different chromosomes. These differences between chromosomes are also illustrated by fig. 2 where the locations of about 380 individual breaks are plotted on a schematic chromosome map on which the regions are marked as proposed during the Paris Conference (1971) on Standardization in Human Cytogenetics [6]. Fig. 2 also bears evidence of different sensitivity of different parts of one and the same chromosome. However, any conclusions should be drawn with great caution because the size of the observational material available as yet is relatively small. The resolution of the fluorescence tech-
543
niques is so high that it is often possible to identify precisely the point of breakage. Most of the breaks lie in the paler areas between the more strongly fluorescent bands, in agreement with observations on chromosome breaks in certain mouse tumours [6]. References 1. Caspersson, T, Lomakka, G & Zech, L, Hereditas 67 (1971) 89. 2. MPrller, A, Nilsson, H, Caspersson, T & Lomakka, G, Exptl cell res 70 (1972) 475. 3. Evans, H J, Human radiation cytogenetics (ed H J Evans, W M Court Brown & A S McLean) p. 20. North-Holland, Amsterdam (1967). 4. Caspersson, T, Lindsten, J, Lomakka, G, Wallman, H & Zech, L, Exptl cell res 63 (1970) 477. 5. Caspersson, T, Issler, P & Lomakka, G, Exptl cell res 75 (1972) 543. 6. Paris conference 1971: Standardization in human cytogenetics. Birth defects: Original art. series, vol. 7, p. 7. The National Foundation, NewYork (1972). 7. Zech, L. Unpublished observations. Received October 19, 1972
A TV-based technique for optical analysis of chromosome regions T. CASPERSSON, P. ISSLER and G. LOMAKKA, Institute for Medical CelI Research and Genetics, Medical Nobel Institute, Karolinska Institutet, 104 01 Stockholm 60, Sweden
A TV-based technique for rapid chromosome identification in preparations stained by the quinacrine method or other banding techniques has been described earlier [l]. The equipment proved especially useful in the search for chromosome aberrations caused by various external agents. For instance, in practical work it has been possible to count the number of chromosome breaks in quite large series of metaphases lo-20 times faster than can be done by use of the conventional sorting procedures for banded chromosomes. The first observations indicating different radiosensitivity values in different chromosomes [2] made it desirable to have a procedure by aid of which very detailed comExptl Cell Res 75 (1972)
541
Radiation-induced non-random chromosome breakage T. CASPERSSON’ ULLA HAGLUND.’ B. LINDELL2 and LORE ZECH,’ llnstitute fdr Medical Cell Research and Genetics, Medical Nobel Institute, Karolinska Znstitutet, 104 01 Stockholm 60, and 2The National Znstitute of Radiation Protection. Fack. 104 01 Stockholm 60, Sweden
Compared with earlier methods, the recently developed chromosome banding techniques offer improved possibilities for the study of chromosome aberrations. A large statistically evaluated observational material based on the quinacrine fluorescence method demonstrated the stability of the fluorescence patterns of the human chromosomes. This gives a solid basis foi the identification of the location of chromosome breaks [I, 21. Moreover, in studies of the action of mutagens, such as ionizing radiations, this means that one cannot only observe and identify smaller aberrations, but also identify those types of chromosomal disturbances which are difficult or even impossible to observe in mammalian material with conventional techniques. Thus, most of the early studies of radiation-induced chromosomal changes were mainly concerned with the formation of rings and dicentrics, while such aberrations as reciprocal translocations and paracentric inversions were easily overlooked (cf [3]). In most cases the fluorescence technique gives rather clear pictures of such aberrations. Fig. 1 shows nine examples with chromosome patterns so clearly that by comparison with the standard karyotype [l], it is easy to identify directly the chromosome parts involved. The number of metaphases to be analysed in such studies is always much greater than that required in medical clinical work. The routine procedures for karyotyping are quite tedious. It is possible to speed up the work and to analyse reasonably large numbers of metaExptl Cell Res 7.5(1972)
Fig. I. Chromosome aberrations from the materia1 described in the text. Quinacrine mustard technique, x 2 000. a, dicentric chromosome (1; 11); b, dicentric chromosome (3; 9q); c, translocation (Sq; 5q); d, translocation (7; 14q); e, translocation (5q; 7); f, translocation (1Oq; 15); g, duplication (7q); h, acentric fragment, duplication (7q); i, inversion (59).
phases by using a TV-based procedure for rapid identification of chromosomes and chromosome regions [4]. Thus, in such work, one increases the speed about 20 times, mak-
ing statistical studies of chromosome breaks by external mutagens a reasonable proposition. The detailed analysis of aberrations would be facilitated by a technique allowing us to compare small details of the patterns from different chromosomes, e.g. a chromosome with a suspected deletion could be compared with its normal partner. For that purpose the TV-procedure has been further developed so that chromosomes from two different preparations can be projected side by side on the same monitor screen [5]. By this procedure we examined about 500 metaphases corresponding to more than 20 000 chromosomes obtained from X-ray irradiated human lymphocytes from 10 different persons. Two irradiation doses were used, 226 rad and 56 rad, for each dose level
16
17
I8
19
20
21
22
Fig. 2. Location of the chromosome breaks in the material investigated. Exptl Cell Res 75 (1972)
Preliminary notes Table 1. Radiation dose 226 rad
56 rad
114
360
35
294
19
66
Dicentrics
50
11
Translocations Deletions Inversions Duplications
41 48 4
1; 3 2
Fragments Rings
473
2:
Total number of analyzed metaphase ulates Metaphase plates without visible aberrations Metaphase plates with aberrations
using blood cells from 6 persons. The blood cultures were irradiated after 24 h and all cells were harvested after 72 h in culture. Three hours prior to harvesting the cells were treated with colcemid. Table 1 summarizes the results of the analysis of 114 plates (exposed to 226 rads) and 360 plates (exposed to 56 rads). The largest number of aberrations found represents dicentrics, translocations and deletions, while inversions and duplications are comparatively rare. Of special interest is a difference in radiation sensitivity of different chromosomes. These differences between chromosomes are also illustrated by fig. 2 where the locations of about 380 individual breaks are plotted on a schematic chromosome map on which the regions are marked as proposed during the Paris Conference (1971) on Standardization in Human Cytogenetics [6]. Fig. 2 also bears evidence of different sensitivity of different parts of one and the same chromosome. However, any conclusions should be drawn with great caution because the size of the observational material available as yet is relatively small. The resolution of the fluorescence tech-
543
niques is so high that it is often possible to identify precisely the point of breakage. Most of the breaks lie in the paler areas between the more strongly fluorescent bands, in agreement with observations on chromosome breaks in certain mouse tumours [6]. References 1. Caspersson, T, Lomakka, G & Zech, L, Hereditas 67 (1971) 89. 2. MPrller, A, Nilsson, H, Caspersson, T & Lomakka, G, Exptl cell res 70 (1972) 475. 3. Evans, H J, Human radiation cytogenetics (ed H J Evans, W M Court Brown & A S McLean) p. 20. North-Holland, Amsterdam (1967). 4. Caspersson, T, Lindsten, J, Lomakka, G, Wallman, H & Zech, L, Exptl cell res 63 (1970) 477. 5. Caspersson, T, Issler, P & Lomakka, G, Exptl cell res 75 (1972) 543. 6. Paris conference 1971: Standardization in human cytogenetics. Birth defects: Original art. series, vol. 7, p. 7. The National Foundation, NewYork (1972). 7. Zech, L. Unpublished observations. Received October 19, 1972
A TV-based technique for optical analysis of chromosome regions T. CASPERSSON, P. ISSLER and G. LOMAKKA, Institute for Medical CelI Research and Genetics, Medical Nobel Institute, Karolinska Institutet, 104 01 Stockholm 60, Sweden
A TV-based technique for rapid chromosome identification in preparations stained by the quinacrine method or other banding techniques has been described earlier [l]. The equipment proved especially useful in the search for chromosome aberrations caused by various external agents. For instance, in practical work it has been possible to count the number of chromosome breaks in quite large series of metaphases lo-20 times faster than can be done by use of the conventional sorting procedures for banded chromosomes. The first observations indicating different radiosensitivity values in different chromosomes [2] made it desirable to have a procedure by aid of which very detailed comExptl Cell Res 75 (1972)