Humangenetik 30, 317--323 (1975) © by Springer-Verlag 1975
Sister Chromatid Exchanges and Chromatid Interchanges in Bloom's Syndrome* Traute M. Sehroeder Institut ffir Anthropologie und ttumangenetik der Universitgt Heidelberg Received September 2, 1975 Summary. A comparison is made between the incidences of sister chromatid exchanges (SCE) per chromosome and group of chromosomes and breakage, visible at metaphase like open gaps, breaks, and breaks involved in chromatid interchange formation (CI) in Bloom's syndrome. It can be shown that the two levels of breakage SCE and CI are not correlated as to the locations. The discussion deals with possible interpretations of preferential breakage and reunion at certain homologous chromosomes and the difficulties today to understand SCEs. Introduction
Comings' considerations (1975) on somatic recombinations and sister chromatid exchanges in Bloom's syndrome are in part a reply to a paper published by Vogel and Sehroeder (1974). The latter dealt with an attempt to utilize cytogenetic data of localized chromatid breaks in Bloom's syndrome and other conditions with spontaneous or induced chromosome instability and consequently chromatid interchanges (CI) for first information about the inner order of the chromosomes in the interphase nucleus. The basic idea was to debate on the possibilities that repeated arrangements of certain homologous and non-homologous chromosomes indicate the neighbourhood of the chromosome segments during breakage and recombination. New facts about Bloom's syndrome have been discovered and published in the meantime. Chaganti et al. reported the manifold increase of sister ehromatid exchanges (SCE) in Bloom's syndrome lymphoeytes (1974), Hand and German published their findings on a retarded rate of DNA chain growth in Bloom's syndrome (1975). Comings' paper deals with the question of whether the somatic recombination pattern in Bloom's syndrome and in cells after mitomyein C treatment versus that of Faneoni's anemia indicates somatic pairing of certain homologous and non-homologous chromosome segments or rather is indicative of postreplicative recombination. I-Ie thinks that if the primary defect in Bloom's syndrome results in forming long stretches of single stranded DNA, then this strand can find homologous single strands. This could happen between homologous chromosome segments or non-homologous chromosomes representing exchanges between homologous repetitious DNA segments of certain chromosomes. * Mit Unterstiitzung durch die Deutsche Forschungsgemeinsehaft.
318
T.M. Schroeder
Furthermore, Comings concluded that if his reasoning is correct, then the exchange between homologous chromosomesis not due to somatic pairing during interphase but to a selection of exchange events t h a t occur primarily between homologous stretches of DNA. Thus, sites of homologous and non-homologous ehromatid exchanges m a y indicate homologous DNA segments and might give only distorted information about an arrangement of the chromosomes in the interphase nucleus. The question remains unsolved whether or not the increases in SCE and breakage in Bloom's syndrome are due to one and the same defect. Postreplieative recombination appears to be involved in the Bloom's syndrome (Comings, 1975). H a n d and German (1974) presume t h a t the defect either concerns directly semiconservative DNA replication or the defect results in disturbed cellular metabolism which in turn affects replication. A comparison of the sites of sister chromatid exchanges with the sites of chromatid interchanges per chromosome or group of chromosomes found in Bloom's syndrome m a y therefore elucidate the function of the defect a little further for those who are involved in resolving the biochemical problem or collect cytogenetic data for further consideration. Material The number of sister chromatid exchanges (SCE) per chromosome or group of chromosomes in normal cells ( - c / + ) , cells of heterozygotes (bl/+) and homozygotes (bl/bl) were given in Chaganti et al. (1974). The relative lengths of the chromosomes expected of the quotient observed are used for calculations as published there (Table 1). The lines in Fig. 1 represent deviations from randomness (line 1.0) expressed in values observed : expected in the three conditions. 86 chromatid interchanges (CI) have been found in 5672 cells of 3 male patients with Bloom's syndrome (bl/bl). They were analysed in respect to the chromosome or group of chromosomes respectively involved in the interchange configuration. Gaps and breaks which
2.0 £b
w
8
I--
u
6
~.K w -~.
4
w > n."
1.0
W
2
/ \V /
"~S.\\ /
\
•..-r-~_: / o
'\
~-;~____,~
8
W
u') 03
6
©
4
Chromosome NO/GROUP
b~L
1
2
3
B
C+X
D
E
F
G+Y
Fig. 1. Incidences of SCEs in patients with Bloom's syndrome (bl/bl), heterozygotes (bl/@), and normals ( + / 4 - ) per chromosome or group of chromosomes correlated to their relative lengths (Chaganti et al., 1974)
Sister Chromatid Exchanges in BioonL's Syndrome
319
4.0 ¸
8 6
=--=
GAPS BREAKS AND BREAKS INVOLVED IN INTERCHANGES
/
4 BREAKS INVOKED IN INTERCHANGES
2' 3.0
o
o
/
BREAKS LEADING TO SISTER CHROMATID INTERCHANGES /
/
8 Ld I.-(J h.I Q_ X Ld
w c~ m m O
/
/ / /
6 4' 2
/
2.0
/
//\
8 6 4 2 1.0 8
6 4
bl/bl
r
,"
2
Chromosome N°/GROUP
3
B
C÷X
--,
D
,
E
--
F
@÷Y
Fig. 2. Incidences of SCEs in Bloom's syndrome (bl/bl) compared with incidences of breaks involved in interchanges (CI) and total breakage in Bloom's syndrome per chromosome or group of chromosome correlated to their relative lengths Table 1. Numbers and expectancies of SCEs found in normals, heterozygotes and Bloom's syndrome patients (Chagan~i st al., 1974) correlated to the relative lengths of chromosomes or group of chromosomes Condition: Normal individuals Type of break Chromos. obs.
exp. a
1 2 3 B C+X D E F G+Y
35 36 44 67 169 41 20 3 3
36.41 34.12 28.30 50.84 156.64 42.32 36.62 17.01 16.05
Total
418
418.31
Heterozygot bl/~Sister chromatid exchanges
obs./exp, obs. 0.96 1.05 1.55 1.3t 1.01 0.96 0.54 0.17 0.18
exp. a
34 48 30 72 181 47 8 1 2
35.10 32.64 27.28 48.76 150.72 40.70 35.46 16.52 16.52
403
403.70
Bloom's syndrome bl/bl
obs./exp, obs.
exp. ~
obs./exp.
0.96 1.47 1.09 1.47 1.20 1.15 0.22 0.06 0.12
233 221 197 338 923 257 149 38 50
209.57 196.34 163.89 292.58 900.95 243.50 210.77 97.92 92.39
1.11 1.12 1.20 1.15 1.02 1,05 0,70 0.38 0,54
2406
2406.98
a Based on chromosome measurements given in Chaganti et al. (1974). The quotient observed/expected is used for Fig. 1.
320
T.M. Schroeder
Table 2. Numbers and expectancies of breaks involved in interchanges and numbers of total breaks in Bloom's syndrome correlated to the relative lengths of chromosomes or group of chromosomes Condition: Bloom's syndrome Type ofbreak
chromatid interchanges
Chromos.
obs.
exp. a
obs./exp,
obs.
exp. a
1 2 3 B C-cX D E F G-~Y
10 5 4 9 58 15 31 32 8
13.99 13.29 11.31 20.52 61.99 18.13 15.81 8.67 8.30
0.71 0.37 0.35 0.43 0.93 0.82 1.96 3.69 0.96
51 59 24 45 177 52 58 45 13
44.23 42.01 35.79 64.88 196.06 57.33 50.00 27.41 26.25
Total
172
172.01
544
543.96
gaps and breaks -~ interchanges obs./exp. 1.15 1.40 0.67 0.69 1.00 0.90 1.16 1.64 0.49
a Based on relative lengths of chromosomes given in Paris Conference (1971): Standardization in Human Cytogenetics. Birth Defects- Original Article Series VIII, 7 (1972). The National Foundation, New York; Table 5, data of Lubs et al., calculated for xy diploid cells. The quotient observed/expected is used for Fig. 2.
were found "open" i.e. not involved in reunion--of the same material were also analysed in the numbers per chromosome or group of chromosomes and data were used for further calculations (Table 2). The data published recently by Schroeder and German (1974) are included in this study. The total material is identical with that compiled in Vogel and Schroeder (1974). The number of breaks interchanging, together with the number of "open" gaps and breaks, are thought to give an impression as to whether breaks at different locations within the entire chromosome complement have various probabilities to become involved in interchange formation. Actually, the material published by Vogel and Schroeder (1974) had already suggested such differences. The calculation results in Table 2 are based on chromosome measurements by Lubs et al. (1972) with corrections for the X ¥ diploid karyotype. Deviation from randomness (line 1.0) of both "open" breaks and gaps and interchange breaks are demonstrafed in Fig. 2. In order to obtain a better comparison, the curve of SCE, bl/bl from Fig. 1 is added to this graph.
Results and Discussion T h e 3 c u r v e s in Fig. 1 r e p r e s e n t i n g o b s e r v e d / e x p e c t e d in bl/bl, b l / + a n d ~ - / ~ r u n s i m i l a r l y in t h e i r courses, a t a b o u t t h e s a m e r a n g e , n a m e l y a t e a c h c h r o m o s o m e or g r o u p of c h r o m o s o m e s , e v e n t h o u g h m i n o r differences c a n n o t be e x c l u d e d . E s p e c i a l l y t h e c u r v e f o r m e d b y v a l u e s of b l / b l is n e a r e r t o t h e r a n d o m d i s t r i b u t i o n line w i t h o u t a n y excess of single c h r o m o s o m e s , as is t h e case in t h e o t h e r 2 curves. T h e m a i n i d e a one finally gets f r o m t h i s g r a p h is t h a t t h e i n c r e a s e o f S C E s in B l o o m ' s s y n d r o m e (bl/bl) r e p r e s e n t s a m e r e e n h a n c e m e n t of a process w h i c h also t a k e s p l a c e in cells of n o r m a l c o n t r o l p e r s o n s u n d e r t h e s a m e e x p e r i m e n t a l cond i t i o n s w i t h o u t a n y c h a n g e or p r e f e r e n c e t o c e r t a i n c h r o m o s o m e s . C h a g a n t i et al. discuss t h e l o w e r f r e q u e n c i e s of S C E in t h e s m a l l e r E , F a n d G - g r o u p c h r o m o s o m e s
Sister Chromatid Exchanges in Bloom's Syndrome
321
as a possible result of the rejection of exchanges in the centromere region which constitutes a relatively larger proportion of the length of these chromosomes compared with the longer chromosomes. L a t t (1974) found also fewer SCE's occurring in smaller chromosomes than expected which fact remains to be clarified. The importance here is t h a t actually there is no significant difference between bl/bl, b l / + and + / @ . I t would be most interesting to analyse the sites of SCE in each individual chromosome in the three conditions. Differences m a y still be hidden and escape observation as long as no attention is paid to where preferential loci of SCEs m a y be in single chromosomes. Mechanisms inducing aberrations might be enlightened b y knowing the patterns of SCEs, e.g. mitomyein C as well as Trenimon increases SCEs to a level of Bloom's syndrome's SCEs (Latt, 1974; Hayaehi and 8chmid, 1975; Beck and Obe, 1975) but the breakage induction mechanism certainly differs from each other. I t could well be t h a t special patterns exist which in turn strengthen the argument of Comings (1975) and add a lot of information about the distribution of classes of main band repetitious DNA. On the other hand, precise analyses of SCE loci with improved techniques m a y clarify the lack of SCEs in the smaller chromosomes and alter the present findings of Chaganti et al. (1974) to a more random distribution over the entire karyotype in each condition. Fig. 2 contains again the curve representing the SCEs of Bloom's syndrome bl/bl as given in Fig. 1. The two other curves are formed by ratios observed/expected 1. from total breakage in Bloom's syndrome ("open" breaks, gaps, and breaks involved in interchanges) and 2. the breaks involved in interchanges separately. Actually, the distribution of SCEs on the one hand, and ehromatid breakage and reunion on the other hand, deviate very much from each other almost in every part of the karyotype. Both curves, however, total breakage and breaks involved in interchanges, are similar. The deviation from the SCE line is especially significant in the chromosomes E + F. The particularity of the nonrandom pattern of breakage with or without reunion in various conditions has already been emphasised by Schroeder and German (1974) and Vogel and Sehroeder (1974). Here, this finding once more becomes interesting: it indicates t h a t sister chromatid exchanges and breakage--visible at m e t a p h a s e - - i n Bloom's syndrome m a y not be of one and the same procedure or at least the two levels of breakage m a y not simply be correlated by grades of severity of damage. On the other hand, it is hardly conceivable that the two levels of breakage, SCE and CI are based on different molecular events. Mntagenes like Mitomycin C and Trenimon induce an increase of SCEs, chromatid breaks and CIs. A comparison of the patterns of SCEs, and chromosome breakage and CIs would enlighten some basic questions about a possible order of events as suggested b y Hayashi and Schmid (1975). These authors discussed their findings of increased incidences of SCEs induced by Trenimon in respect to the induction of point mutations in the different conditions. Comparing these results with normal numbers of SCEs in Faneoni's anemia, they concluded that: 1. the increase of SCEs can be used for mutagenecity testing and 2. Faneoni cells do not suffer from point mutations but gross chromosome mutations since they do not show an increase of SCEs. Since, however, mutagenes known to induce point mutations do not in-
322
T.M. Schroeder
Table 3. SCEs, chromatid breakage and chromatid interchanges in various conditions Condition
SCE
CB
C1
HC1
Authors
Bloom's syndrome Fanconi's anemia
+ dn
-4-4-d-
~ + ~
-4--4--
Ataxia telangiectasia
n
Chaganti et al., 1974 Chaganti et al., 1974; Sperling et al., 1975; Hayashi and Schmid, 1975 Chaganti et al., 1974
(+)
(-~)
--
Normal cells treated with: Mitomycin C + qTrenimon -~ +
q- ~ + +
q- q~ +
-}--
Lead acetate
-~
~-
--
n
Kate et al., 1974; Latt, 1974 Beck and Obe, 1975; ttayashi and Sehmid, 1975 Beek and Obe, 1975
SCE = sister chromatid exchange, CB chromatid breakage, C1 ~ chromatid interchange, HC1 = mainly homologous chromosomes involved in interchanges (type I), n = normal, (+), + , + + ~ increased compared with normal, - - = not present.
crease the rate of SCEs (Kate, 1974), it seems too early to draw conclusions like this a b o u t a n y correlation between the two levels of b r e a k a g e - - S C E s a n d CIs. Beck a n d Obe (1975) emphasize the i m p o r t a n c e of first i n f o r m a t i o n on possible repair processes involved i n SCE f o r m a t i o n as shown b y K a t e (1973, 1974). Table 3 compares findings of SCEs breakage a n d types of CIs in various conditions. Bloom's s y n d r o m e m a y serve as a n example for the complexity of events t a k i n g place in the living cell prior to chromosome analyses. The CI p a t t e r n is able to d e m o n s t r a t e a speciality of repair at chromosomal level. Comings' idea t h a t homologous stretches of D N A find each other a n d thus the CIs represent a selection of homologous D N A segments, does n o t entirely explain the p a t t e r n of CIs. The segments of homologous a n d non-homologous chromosomes have in a d d i t i o n to be i n close neighbourhood to be repaired. The picture one gets from these d a t a m a y be distorted since restricted to those chromosome segments which consist of homologous sequencies. However, these points of action can be used v e r y well for the first evidence of neighbouring chromosomes. I n addition, after the defect in Bloom's s y n d r o m e will have been clarified, the points of non-homologous r e c o m b i n a t i o n m a y be indicative of certain repetitive D N A segments.
References Beek, B., 0be, G. : The human leukocyte test system. VI. The use of sister chromatid exchanges as possible indicators for mutagenic activities. Humangenetik 25, 127--134 (1975) Chaganti, l~. S. K., Schonberg, S., German, J. : A manyfold increase in sister chromatid exchange in Bloom's syndrome lymphocytes. Prec. nat. Acad. Sci. (Wash.) 71, 4508--4512 (1974) Comings, D. : Implications of somatic recombinations and sister chromatid exchanges in Bloom's syndrome and cells located with mitomycin C. Humangenetik 28, 191--196 (1975) Hand, g., German, J. : A retarded rate of D~qA chain growth in Bloom's syndrome. Prec. nat. Acad. Sci. (Wash.) 72, 758--762 (1975)
Sister Chromatid Exchanges in Bloom's Syndrome
323
Hayashi, K., Schmid, W. : The rate of sister chromatid exchanges parallel to spontaneous chromosome breakage in Fanconi's anemia and to Trenimon-induced aberrations in human ]ymphocytes and fibroblasts. Humangenetik 29, 201--206 (1975) Kato, H. : Induction of sister chromatid exchanges by UV light and its inhibition by caffeine. Exp. Cell Res. 82, 383--390 (1973) I(ato, H. : Induction of sister chromatid exchanges by chemical mutagencs and its relevance to DNA repair. Exp. Cell Res. 85, 239--247 (1974) Latt, S. A. : Localization of sister chromatid exchanges in human chromosomes. Science 185, 74--76 (1974) Lubs, H., Hostetter, T., Ewing, L. : Paris Conference: Standardization in human cytogenetics. Birth Defects: Original Article Series VIII, No. 7 (1972). The National Foundation, New York Schrocder, T. ~., German, J.: Bloom's syndrome and Fanconi's anemia: Demonstration of two distinctive patterns of chromosome disruption and rearrangement. Humangelletik ~5, 299--306 (1974) Vogel, F., Schroeder, T. M. : The internal order of the interphase nucleus. Humangenetik 25, 265 292 (1974) Profi Dr. med. Trautc M. Schroeder Institut fiir Anthropologic und Humangenetik der Universit~t D-6900 Heidelberg, Im Neuenheimer Feld 328 Federal Republic of Germany
Sister Chromatid Exchanges and Chromatid Interchanges in Bloom's Syndrome* Traute M. Sehroeder Institut ffir Anthropologie und ttumangenetik der Universitgt Heidelberg Received September 2, 1975 Summary. A comparison is made between the incidences of sister chromatid exchanges (SCE) per chromosome and group of chromosomes and breakage, visible at metaphase like open gaps, breaks, and breaks involved in chromatid interchange formation (CI) in Bloom's syndrome. It can be shown that the two levels of breakage SCE and CI are not correlated as to the locations. The discussion deals with possible interpretations of preferential breakage and reunion at certain homologous chromosomes and the difficulties today to understand SCEs. Introduction
Comings' considerations (1975) on somatic recombinations and sister chromatid exchanges in Bloom's syndrome are in part a reply to a paper published by Vogel and Sehroeder (1974). The latter dealt with an attempt to utilize cytogenetic data of localized chromatid breaks in Bloom's syndrome and other conditions with spontaneous or induced chromosome instability and consequently chromatid interchanges (CI) for first information about the inner order of the chromosomes in the interphase nucleus. The basic idea was to debate on the possibilities that repeated arrangements of certain homologous and non-homologous chromosomes indicate the neighbourhood of the chromosome segments during breakage and recombination. New facts about Bloom's syndrome have been discovered and published in the meantime. Chaganti et al. reported the manifold increase of sister ehromatid exchanges (SCE) in Bloom's syndrome lymphoeytes (1974), Hand and German published their findings on a retarded rate of DNA chain growth in Bloom's syndrome (1975). Comings' paper deals with the question of whether the somatic recombination pattern in Bloom's syndrome and in cells after mitomyein C treatment versus that of Faneoni's anemia indicates somatic pairing of certain homologous and non-homologous chromosome segments or rather is indicative of postreplicative recombination. I-Ie thinks that if the primary defect in Bloom's syndrome results in forming long stretches of single stranded DNA, then this strand can find homologous single strands. This could happen between homologous chromosome segments or non-homologous chromosomes representing exchanges between homologous repetitious DNA segments of certain chromosomes. * Mit Unterstiitzung durch die Deutsche Forschungsgemeinsehaft.
318
T.M. Schroeder
Furthermore, Comings concluded that if his reasoning is correct, then the exchange between homologous chromosomesis not due to somatic pairing during interphase but to a selection of exchange events t h a t occur primarily between homologous stretches of DNA. Thus, sites of homologous and non-homologous ehromatid exchanges m a y indicate homologous DNA segments and might give only distorted information about an arrangement of the chromosomes in the interphase nucleus. The question remains unsolved whether or not the increases in SCE and breakage in Bloom's syndrome are due to one and the same defect. Postreplieative recombination appears to be involved in the Bloom's syndrome (Comings, 1975). H a n d and German (1974) presume t h a t the defect either concerns directly semiconservative DNA replication or the defect results in disturbed cellular metabolism which in turn affects replication. A comparison of the sites of sister chromatid exchanges with the sites of chromatid interchanges per chromosome or group of chromosomes found in Bloom's syndrome m a y therefore elucidate the function of the defect a little further for those who are involved in resolving the biochemical problem or collect cytogenetic data for further consideration. Material The number of sister chromatid exchanges (SCE) per chromosome or group of chromosomes in normal cells ( - c / + ) , cells of heterozygotes (bl/+) and homozygotes (bl/bl) were given in Chaganti et al. (1974). The relative lengths of the chromosomes expected of the quotient observed are used for calculations as published there (Table 1). The lines in Fig. 1 represent deviations from randomness (line 1.0) expressed in values observed : expected in the three conditions. 86 chromatid interchanges (CI) have been found in 5672 cells of 3 male patients with Bloom's syndrome (bl/bl). They were analysed in respect to the chromosome or group of chromosomes respectively involved in the interchange configuration. Gaps and breaks which
2.0 £b
w
8
I--
u
6
~.K w -~.
4
w > n."
1.0
W
2
/ \V /
"~S.\\ /
\
•..-r-~_: / o
'\
~-;~____,~
8
W
u') 03
6
©
4
Chromosome NO/GROUP
b~L
1
2
3
B
C+X
D
E
F
G+Y
Fig. 1. Incidences of SCEs in patients with Bloom's syndrome (bl/bl), heterozygotes (bl/@), and normals ( + / 4 - ) per chromosome or group of chromosomes correlated to their relative lengths (Chaganti et al., 1974)
Sister Chromatid Exchanges in BioonL's Syndrome
319
4.0 ¸
8 6
=--=
GAPS BREAKS AND BREAKS INVOLVED IN INTERCHANGES
/
4 BREAKS INVOKED IN INTERCHANGES
2' 3.0
o
o
/
BREAKS LEADING TO SISTER CHROMATID INTERCHANGES /
/
8 Ld I.-(J h.I Q_ X Ld
w c~ m m O
/
/ / /
6 4' 2
/
2.0
/
//\
8 6 4 2 1.0 8
6 4
bl/bl
r
,"
2
Chromosome N°/GROUP
3
B
C÷X
--,
D
,
E
--
F
@÷Y
Fig. 2. Incidences of SCEs in Bloom's syndrome (bl/bl) compared with incidences of breaks involved in interchanges (CI) and total breakage in Bloom's syndrome per chromosome or group of chromosome correlated to their relative lengths Table 1. Numbers and expectancies of SCEs found in normals, heterozygotes and Bloom's syndrome patients (Chagan~i st al., 1974) correlated to the relative lengths of chromosomes or group of chromosomes Condition: Normal individuals Type of break Chromos. obs.
exp. a
1 2 3 B C+X D E F G+Y
35 36 44 67 169 41 20 3 3
36.41 34.12 28.30 50.84 156.64 42.32 36.62 17.01 16.05
Total
418
418.31
Heterozygot bl/~Sister chromatid exchanges
obs./exp, obs. 0.96 1.05 1.55 1.3t 1.01 0.96 0.54 0.17 0.18
exp. a
34 48 30 72 181 47 8 1 2
35.10 32.64 27.28 48.76 150.72 40.70 35.46 16.52 16.52
403
403.70
Bloom's syndrome bl/bl
obs./exp, obs.
exp. ~
obs./exp.
0.96 1.47 1.09 1.47 1.20 1.15 0.22 0.06 0.12
233 221 197 338 923 257 149 38 50
209.57 196.34 163.89 292.58 900.95 243.50 210.77 97.92 92.39
1.11 1.12 1.20 1.15 1.02 1,05 0,70 0.38 0,54
2406
2406.98
a Based on chromosome measurements given in Chaganti et al. (1974). The quotient observed/expected is used for Fig. 1.
320
T.M. Schroeder
Table 2. Numbers and expectancies of breaks involved in interchanges and numbers of total breaks in Bloom's syndrome correlated to the relative lengths of chromosomes or group of chromosomes Condition: Bloom's syndrome Type ofbreak
chromatid interchanges
Chromos.
obs.
exp. a
obs./exp,
obs.
exp. a
1 2 3 B C-cX D E F G-~Y
10 5 4 9 58 15 31 32 8
13.99 13.29 11.31 20.52 61.99 18.13 15.81 8.67 8.30
0.71 0.37 0.35 0.43 0.93 0.82 1.96 3.69 0.96
51 59 24 45 177 52 58 45 13
44.23 42.01 35.79 64.88 196.06 57.33 50.00 27.41 26.25
Total
172
172.01
544
543.96
gaps and breaks -~ interchanges obs./exp. 1.15 1.40 0.67 0.69 1.00 0.90 1.16 1.64 0.49
a Based on relative lengths of chromosomes given in Paris Conference (1971): Standardization in Human Cytogenetics. Birth Defects- Original Article Series VIII, 7 (1972). The National Foundation, New York; Table 5, data of Lubs et al., calculated for xy diploid cells. The quotient observed/expected is used for Fig. 2.
were found "open" i.e. not involved in reunion--of the same material were also analysed in the numbers per chromosome or group of chromosomes and data were used for further calculations (Table 2). The data published recently by Schroeder and German (1974) are included in this study. The total material is identical with that compiled in Vogel and Schroeder (1974). The number of breaks interchanging, together with the number of "open" gaps and breaks, are thought to give an impression as to whether breaks at different locations within the entire chromosome complement have various probabilities to become involved in interchange formation. Actually, the material published by Vogel and Schroeder (1974) had already suggested such differences. The calculation results in Table 2 are based on chromosome measurements by Lubs et al. (1972) with corrections for the X ¥ diploid karyotype. Deviation from randomness (line 1.0) of both "open" breaks and gaps and interchange breaks are demonstrafed in Fig. 2. In order to obtain a better comparison, the curve of SCE, bl/bl from Fig. 1 is added to this graph.
Results and Discussion T h e 3 c u r v e s in Fig. 1 r e p r e s e n t i n g o b s e r v e d / e x p e c t e d in bl/bl, b l / + a n d ~ - / ~ r u n s i m i l a r l y in t h e i r courses, a t a b o u t t h e s a m e r a n g e , n a m e l y a t e a c h c h r o m o s o m e or g r o u p of c h r o m o s o m e s , e v e n t h o u g h m i n o r differences c a n n o t be e x c l u d e d . E s p e c i a l l y t h e c u r v e f o r m e d b y v a l u e s of b l / b l is n e a r e r t o t h e r a n d o m d i s t r i b u t i o n line w i t h o u t a n y excess of single c h r o m o s o m e s , as is t h e case in t h e o t h e r 2 curves. T h e m a i n i d e a one finally gets f r o m t h i s g r a p h is t h a t t h e i n c r e a s e o f S C E s in B l o o m ' s s y n d r o m e (bl/bl) r e p r e s e n t s a m e r e e n h a n c e m e n t of a process w h i c h also t a k e s p l a c e in cells of n o r m a l c o n t r o l p e r s o n s u n d e r t h e s a m e e x p e r i m e n t a l cond i t i o n s w i t h o u t a n y c h a n g e or p r e f e r e n c e t o c e r t a i n c h r o m o s o m e s . C h a g a n t i et al. discuss t h e l o w e r f r e q u e n c i e s of S C E in t h e s m a l l e r E , F a n d G - g r o u p c h r o m o s o m e s
Sister Chromatid Exchanges in Bloom's Syndrome
321
as a possible result of the rejection of exchanges in the centromere region which constitutes a relatively larger proportion of the length of these chromosomes compared with the longer chromosomes. L a t t (1974) found also fewer SCE's occurring in smaller chromosomes than expected which fact remains to be clarified. The importance here is t h a t actually there is no significant difference between bl/bl, b l / + and + / @ . I t would be most interesting to analyse the sites of SCE in each individual chromosome in the three conditions. Differences m a y still be hidden and escape observation as long as no attention is paid to where preferential loci of SCEs m a y be in single chromosomes. Mechanisms inducing aberrations might be enlightened b y knowing the patterns of SCEs, e.g. mitomyein C as well as Trenimon increases SCEs to a level of Bloom's syndrome's SCEs (Latt, 1974; Hayaehi and 8chmid, 1975; Beck and Obe, 1975) but the breakage induction mechanism certainly differs from each other. I t could well be t h a t special patterns exist which in turn strengthen the argument of Comings (1975) and add a lot of information about the distribution of classes of main band repetitious DNA. On the other hand, precise analyses of SCE loci with improved techniques m a y clarify the lack of SCEs in the smaller chromosomes and alter the present findings of Chaganti et al. (1974) to a more random distribution over the entire karyotype in each condition. Fig. 2 contains again the curve representing the SCEs of Bloom's syndrome bl/bl as given in Fig. 1. The two other curves are formed by ratios observed/expected 1. from total breakage in Bloom's syndrome ("open" breaks, gaps, and breaks involved in interchanges) and 2. the breaks involved in interchanges separately. Actually, the distribution of SCEs on the one hand, and ehromatid breakage and reunion on the other hand, deviate very much from each other almost in every part of the karyotype. Both curves, however, total breakage and breaks involved in interchanges, are similar. The deviation from the SCE line is especially significant in the chromosomes E + F. The particularity of the nonrandom pattern of breakage with or without reunion in various conditions has already been emphasised by Schroeder and German (1974) and Vogel and Sehroeder (1974). Here, this finding once more becomes interesting: it indicates t h a t sister chromatid exchanges and breakage--visible at m e t a p h a s e - - i n Bloom's syndrome m a y not be of one and the same procedure or at least the two levels of breakage m a y not simply be correlated by grades of severity of damage. On the other hand, it is hardly conceivable that the two levels of breakage, SCE and CI are based on different molecular events. Mntagenes like Mitomycin C and Trenimon induce an increase of SCEs, chromatid breaks and CIs. A comparison of the patterns of SCEs, and chromosome breakage and CIs would enlighten some basic questions about a possible order of events as suggested b y Hayashi and Schmid (1975). These authors discussed their findings of increased incidences of SCEs induced by Trenimon in respect to the induction of point mutations in the different conditions. Comparing these results with normal numbers of SCEs in Faneoni's anemia, they concluded that: 1. the increase of SCEs can be used for mutagenecity testing and 2. Faneoni cells do not suffer from point mutations but gross chromosome mutations since they do not show an increase of SCEs. Since, however, mutagenes known to induce point mutations do not in-
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Table 3. SCEs, chromatid breakage and chromatid interchanges in various conditions Condition
SCE
CB
C1
HC1
Authors
Bloom's syndrome Fanconi's anemia
+ dn
-4-4-d-
~ + ~
-4--4--
Ataxia telangiectasia
n
Chaganti et al., 1974 Chaganti et al., 1974; Sperling et al., 1975; Hayashi and Schmid, 1975 Chaganti et al., 1974
(+)
(-~)
--
Normal cells treated with: Mitomycin C + qTrenimon -~ +
q- ~ + +
q- q~ +
-}--
Lead acetate
-~
~-
--
n
Kate et al., 1974; Latt, 1974 Beck and Obe, 1975; ttayashi and Sehmid, 1975 Beek and Obe, 1975
SCE = sister chromatid exchange, CB chromatid breakage, C1 ~ chromatid interchange, HC1 = mainly homologous chromosomes involved in interchanges (type I), n = normal, (+), + , + + ~ increased compared with normal, - - = not present.
crease the rate of SCEs (Kate, 1974), it seems too early to draw conclusions like this a b o u t a n y correlation between the two levels of b r e a k a g e - - S C E s a n d CIs. Beck a n d Obe (1975) emphasize the i m p o r t a n c e of first i n f o r m a t i o n on possible repair processes involved i n SCE f o r m a t i o n as shown b y K a t e (1973, 1974). Table 3 compares findings of SCEs breakage a n d types of CIs in various conditions. Bloom's s y n d r o m e m a y serve as a n example for the complexity of events t a k i n g place in the living cell prior to chromosome analyses. The CI p a t t e r n is able to d e m o n s t r a t e a speciality of repair at chromosomal level. Comings' idea t h a t homologous stretches of D N A find each other a n d thus the CIs represent a selection of homologous D N A segments, does n o t entirely explain the p a t t e r n of CIs. The segments of homologous a n d non-homologous chromosomes have in a d d i t i o n to be i n close neighbourhood to be repaired. The picture one gets from these d a t a m a y be distorted since restricted to those chromosome segments which consist of homologous sequencies. However, these points of action can be used v e r y well for the first evidence of neighbouring chromosomes. I n addition, after the defect in Bloom's s y n d r o m e will have been clarified, the points of non-homologous r e c o m b i n a t i o n m a y be indicative of certain repetitive D N A segments.
References Beek, B., 0be, G. : The human leukocyte test system. VI. The use of sister chromatid exchanges as possible indicators for mutagenic activities. Humangenetik 25, 127--134 (1975) Chaganti, l~. S. K., Schonberg, S., German, J. : A manyfold increase in sister chromatid exchange in Bloom's syndrome lymphocytes. Prec. nat. Acad. Sci. (Wash.) 71, 4508--4512 (1974) Comings, D. : Implications of somatic recombinations and sister chromatid exchanges in Bloom's syndrome and cells located with mitomycin C. Humangenetik 28, 191--196 (1975) Hand, g., German, J. : A retarded rate of D~qA chain growth in Bloom's syndrome. Prec. nat. Acad. Sci. (Wash.) 72, 758--762 (1975)
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Hayashi, K., Schmid, W. : The rate of sister chromatid exchanges parallel to spontaneous chromosome breakage in Fanconi's anemia and to Trenimon-induced aberrations in human ]ymphocytes and fibroblasts. Humangenetik 29, 201--206 (1975) Kato, H. : Induction of sister chromatid exchanges by UV light and its inhibition by caffeine. Exp. Cell Res. 82, 383--390 (1973) I(ato, H. : Induction of sister chromatid exchanges by chemical mutagencs and its relevance to DNA repair. Exp. Cell Res. 85, 239--247 (1974) Latt, S. A. : Localization of sister chromatid exchanges in human chromosomes. Science 185, 74--76 (1974) Lubs, H., Hostetter, T., Ewing, L. : Paris Conference: Standardization in human cytogenetics. Birth Defects: Original Article Series VIII, No. 7 (1972). The National Foundation, New York Schrocder, T. ~., German, J.: Bloom's syndrome and Fanconi's anemia: Demonstration of two distinctive patterns of chromosome disruption and rearrangement. Humangelletik ~5, 299--306 (1974) Vogel, F., Schroeder, T. M. : The internal order of the interphase nucleus. Humangenetik 25, 265 292 (1974) Profi Dr. med. Trautc M. Schroeder Institut fiir Anthropologic und Humangenetik der Universit~t D-6900 Heidelberg, Im Neuenheimer Feld 328 Federal Republic of Germany
