199
Mutation Research, 52 (1978) 199--206 © Elsevier/North-Holland Biomedical Press
SCANNING-ELECTRON MICROSCOPY OF CHROMOSOME ABERRATIONS
MYLES L. MACE JR. a, YERACH DASKAL b and WAYNE WRAY a
Departments o f a Ce'll Biology and b Pharmacology, Bay lor College o f Medicine, Houston, Texas 77030 (U.S.A.) (Received 7 July 1977) (Revision received 9 May 1978) (Accepted 5 June 1978)
Summary This study is the first report of scanning-electron microscopy of isolated and purified metaphase chromosomes containing drug-induced aberrations. The technique reported allows high resolution topological examination of chromosomal aberrations which may pass undetected with conventional techniques.
Introduction
This study is the first report of scanning-electron microscopy of isolated and purified metaphase chromosomes containing drug-induced aberrations. It was undertaken to evaluate the ultrastructural nature of chromosomal aberrations in order to gain further insight into chromosomal architecture and regions of lesions containing drug-induced aberrations. Evaluation of the morphology of chromosome damage has been largely to assess the frequency of chromosome aberrations on a per cell basis at the resolution level of light microscopy although the additional resolution afforded by transmission electron microscopy has been utilized in some cases [5,6]. There are also several relevant ultrastructural studies on the nature of chromosome gaps and breaks which utilize transmission or scanning-electron microscopy of water-spread or airdried chromosome preparations [4,7,9,13]. Adriamycin was used to induce aberrations in this study. Adriamycin is an anthracycline antineoplastic agent which acts by intercalation into DNA, thereby inhibiting DNA-dependent DNA and RNA polymerases [16]. It binds specifically with DNA by intercalation between adjacent base pairs of the double helical structure, inducing stereoAbbreviations: CHO, Chinese monohydrate.
sodium
hamster
ovary; PIPES, piperazine-N,N'-bis(2-ethane sulfonic
acid) mono-
200 chemical chromatin disordering or damage which may be later expressed in metaphase as chromosome aberrations [11] ranging from simple deletions to extensive fragmentation. Materials and methods Chinese hamster ovary (CHO) cells grown in tissue culture were incubated in media with 0.4 pg/ml of adriamycin for 1 h, allowed to recover 14 h in media without drug, and metaphase cells collected after a 2-h Colcemid arrest. Cells were prepared for examination with light microscopy by conventional techniques. Air-dried metaphase plates were made on glass slides, stained with 2% Giemsa, and scored for the frequency of chromosomal aberrations. Isolated chromosomes were prepared by nitrogen cavitation by the method of Wray and Stubblefield [15]. The chromosome isolation buffer contained 1.0 M hexylene glycol (2-methyl-2,4-pentane diol, Eastman Organic), 0.5 mM CaC12, and 0.1 mM piperazine-N,N'-bis(2-ethane sulfonic acid) monosodium m o n o h y d r a t e (PIPES, Cal Biochem) at a pH of 6.8. Chromosomes were further purified by centrifugation through a 10--40% sucrose step gradient in order to remove cytoplasmic debris. Isolated and purified chromosomes were either deposited directly onto Formvar-coated 200-mesh copper grids or centrifuged onto grids, as described by Miller [10], through chromosome buffer at 1000 × g for 10 min. Specimens were stabilized en grid by immersion in 2% aqueous uranyl acetate followed by dehydration in graded series (25--100%) of either ethanol or hexylene glycol. Amyl acetate was used as the transition fluid prior to drying at the critical point of CO2, as described by Anderson [3], in a Denton Critical Point drier. After drying, specimens were transferred to a Kinney (KSM-2) high vacuum evaporator, maintained at a pressure of 2 × 10 -s Torr for a minimum of 30 min, and then purified argon was admitted into the chamber to a pressure of 175 ~. After purging the bell jar for 2 min with argon, specimens were coated with a gold-palladium alloy (60 : 40) for 50 sec and 10 mA in an argon atmosphere with a D.C. sputtering module (Denton Vacuum). The thickness of deposited material was determined by diameter measurements of coated and uncoated latex spheres (E. Fullam Inc.) with TEM and SEM. Samples were examined with a JEOL 100C electron microscope equipped with an ASID-4D at 40 kV using a side entry goniometer at 30--35 angle of tilt. Chromosomes were photographed using Polaroid 105 P/N film from a 2000 line CRT. Results Exposure of CHO cells to adriamycin doses greater than 1.0 pg/ml causes a significant inhibitory effect on the entry of cells into mitosis. At a level of 0.4 pg/ml, the mitotic index 2 h after Colcemid addition was 21%. 42% of the metaphase spreads showed some aberrations, ranging from simple deletions to extensive chromosomal fragmentation (Table 1). Moderate chromosomal aberrations were predominantly the chromatidal type, including simple or multiple chromatid breaks and deletions. In addition, other abnormalities such as chromosome elongation and centomere separation were observed.
201 TABLE 1 FREQUENCY
OF ADRIAMYCIN-INDUCED CHROMOSOMAL
T o t a l m e t a p h a s e plates
ABERRATIONS
104
Metaphase plates w i t h aberrations
44
T y p e o f aberrations observed breaks gaps fragments deletions rings multiple translocations
8 13 11 19 1 32
Fig. 1 shows a scanning-electron micrograph of an untreated isolated and purified metaphase chromosome. The chromatids consist of numerous microconvules, ranging in size from 2 5 0 - - 6 5 0 / ~ (net measurements, with the thickness of the metal coating deducted), which appear to be randomly distributed over the entire chromosome surface. The centromeric region is well defined, chromatids are closely aligned with a furrow between them, and frequently nuclear membranes are observed at the telomeres. Chromosomes which have been isolated and purified from cells treated with adriamycin show the general morphological details seen in control specimens.
Fig. 1. Scanning-electron m i c r o g r a p h o f an isolated untreated CHO m e t a p h a s e c h r o m o s o m e . B a r , 1/~.
202
Fig. 2. D r u g - i n d u c e d gap (pointers) in a CHO m e t a p h a s e c h r o m o s o m e . Bar, 1 /~.
The following figures have been selected as representative of chromosomal aberrations which have been observed. Fig. 2 is an example of a chromosomal gap in which there apparently is a loss of material; however, continuity within the chromatid remains. Although less material is missing in the sister chromatid, and would probably not be distinguished as a gap by Giemsa staining, this region may be considered a homologous gap within the chromatid. Fig. 3 is an example of a single chromatid gap with almost complete cleavage of the arm (arrows), probably at the centromere region. The cleaved portion still retains association with the sister chromatid. A complete break of one chromatid together with an incomplete break of the sister chromatid is shown in Fig. 4, where the broken-off fragment is connected to one arm by a thin cord of chromatin with sister chromatids tightly paired in the fragment. Frequently it is difficult to distinguish, in Giemsa-stained preparations, whether a lightly stained constriction between chromosomal arms represents an actual chromosome or is the result of a random or sticky association. Fig. 5 is a scanning-electron micrograph of such a situation. Therefore this example represents a single chromosome with either stretching of the centromere or a gap at the centromeric region. Fig. 6 is an example of a rare ring chromosome with continuity along the entire chromatid. It is n o t possible with scanning-electron microscopy of isolated chromosomes to determine whether this example represents a single or pair of chromatids which have been joined.
203
F i g . 3. C H O m e t a p h a s e c h r o m o s o m e w i t h a c h r o m a t i d g a p ( a r r o w s ) . B a r , 1 /~.
Fig. 4 . C H O m e t a p h a s e c h r o m o s o m e w i t h a n a l m o s t c o m p l e t e c h r o m o s o m a l b r e a k . B a r , 1/~.
204
Fig. 5. C H O m e t a p h a s e c h r o m o s o m e w i t h a c e n t r o m e r i c gap. Bax, 1 ~.
Fig. 6. C H O m e t a p h a s e c h r o m o s o m e in a ring c o n f i g u r a t i o n . P o i n t e d i n d i c a t e d m e m b r a n o u s p a t c h . Bar, 1/~.
205 The presence of membrane which is presumably nuclear {pointer) should be noted, which may represent former telomeres as previously described [8]. Discussion Scanning-electron microscopy offers the advantage of high resolution of topological details in the examination of chromosome lesions. Small chromatid gaps such as in Fig. 2 may pass unnoticed with light microscopy and those similax to Fig. 3 scored as breaks. Typical gaps observed with scanning-electron microscopy range from almost complete breaks connected by a few discrete chromatin fibers to slight depressions with the chromatids. Frequently, on the chromatid adjacent to the gap there is also some loss or damage at the identical region b u t n o t necessarily with the same degree of involvement. Brogger [7] states that the average length of the proximal and distal parts of a chromatid with a break or gap is a b o u t 9% shorter than the length of the intact sister chromatid. It has also been reported that the length of a chromatid with a gap often exceeds, by the size of the gap, the length of the sister chromatid [5]. This would suggest that the gap is only a stretched region of the chromatid, resulting n o t from chromatin loss but an error in either the folding or the packing. Our results generally support this second concept b u t there are exceptions where there seems to be selective loss of material in only one chromatid. The difference in these results could well be explained due to waterspreading techniques used by Brogger [7] contrasted with a critical point drying of isolated chromosomes used in this study. A chromatid break is characterized by the complete absence of chromatin fibers between the severed parts. With transmission-electron microscopy, there appears to be no difference between a normal telomere and the region exhibiting a complete chromatid break [5]. This observation suggests that the membranous patches associated with some telomeres represent a specific association, and are not produced b y either isolation or purification of chromosomes. Generally our results support this observation with t w o exceptions. The first is that the membrane patches, presumably nuclear in origin, are frequently associated with telomeres b u t have n o t been observed at a chromatid or chromosome break. In addition, telomeres appear as a somewhat bulbous end, uniformly covered with microconvules, while a break is a fiat cut across the chromatid where the surface of the break may be eroded in some regions. However, no ultrastructural features are observed which would explain why broken ends of chromosomes are sticky and therefore tend to combine with other broken chromosome ends. Especially obscure at the present time is the relation of chromosome breaks and gaps to the chromosome " c o r e " or chromosome "scaffold" [1,2,12,14]. Further studies to elucidate the relationship of chromosome architecture with biochemical and genetic approaches are in progress. The technique reported here allows high resolution topological examination of chromosomal aberrations which may pass undetected with conventional techniques. This technique may also be extended to include cell preparations which have been fixed in methanol--acetic acid, thereby allowing analysis of complete karyotypes.
206
Acknowledgements These studies were supported by NSF PCM 75-05622, American Cancer Society VC-163, National Cancer Institute CA-18455, Cancer Research Center Grant CA-10893 P. 5 from the National Cancer Institute, the National Institutes of Health Grant HD-00444, and PHS Award 5 F32 HD 01861.02. References 1 Adolph, K.W., S.M. Cheng, J.R. Paulson and U.K. Laemmli, Isolation of a prot e i n scaffold from m i t o t i c HeLa cell chromosomes, Proc. Natl. Acad. Sci. (U.S.A.), 74 (1977) 4937--4941. 2 Adolph, K.W., S.M. Cheng and U.K. Laemmli, Role of nonhi s t one proteins in metaphase c hromos ome structure, Cell, 12 (1977) 805--816. 3 Anderson, T.F., Techniques for the preservation of three-dimensional structure in preparing specimens for the electron microscope, Trans. N.Y. Acad. Sci., 13 (1951) 130--134. 4 Brecher, S., Ultrastructural observations of X-ray induce d c h r o m a t i d gaps, Mutation Res., 42 (1977) 249--268. 5 Brinkley, B.R., and W.N. Hittelman, Ultrastructure of m a m m a l i a n c h r o m o s o m e aberrations, Int. Rev. Cytochem., 42 (1975) 49--101. 6 Brinkley, B.R., and M.N. Shaw, Ultrastructural aspects of c h r o m o s o m e damage, Genetic concepts and neoplasia, Williams and Wllkins, Baltimore, 1969, pp. 313--345. 7 Brogger, A., Apparently spontaneous c h r o m o s o m e damage in h u m a n l e u k o c y t e s and the nature of c h r o m a t i d gaps, Humangenetik, 13 (1971) 1--14. 8 Daskal, Y., M.L. Mace Jr. and H. Busch, High resolution scaning electron mi c ros c opy of isolated Chinese h a m s t e r chromosomes, IITRI/SEM (1976) 163--170. 9 Fisher, K.M., T.F. Budinger, A.T. Foin and T.E. Everhart, Spontaneous c h r o m o s o m e gaps in Marmosa mitis, Mutation Res., 22 (1974) 299--303. 10 Miller Jr., O.L., and A.H. Bakken, Morphological studies of transcription, Karolinska Symp., 5 (1972) 155--177. 11 Newsome, Y.L., and L.G. Littlefield, Adriamycin induced c h r o m o s o m e aberrations in h u m a n fibroblasts, J. Natl. Cancer Inst., 55 (1975) 1061--1064. 12 Paulson, J.R., and U.K. Laemmli, The structure of histone-dcpleted metaphase chromosomes, Cell, 12 (1977) 817--828. 13 Scheid, W., and H. Traut, Visualization by scanning electron microscopy of chromatic lesions ( " g a p s " ) induced by X-rays in chromosomes of Vicia faba, Mutation Res., 11 (1971) 253--255. 14 Stubblefield, E., and W. Wray, Architecture of the Chinese ha ms t e r metaphase chromosome, Chromosoma, 32 (1971) 262--294. 15 Wray, W., and E. Stubblefield, A new m e t h o d for the rapid isolation of chromosomes, m i t o t i c apparatus, or nuclei from m a m m a l i a n fibroblasts at near neutral pH, Exptl. Cell Res.0 59 (1970) 469--478. 16 Zunino, F., R.A. G a m b e t t a and A. Zaccara, Interaction of d a u n o m y c i n w i t h DNA, Biochim. Biophys. Acta, 277 (1972) 498--498.
Mutation Research, 52 (1978) 199--206 © Elsevier/North-Holland Biomedical Press
SCANNING-ELECTRON MICROSCOPY OF CHROMOSOME ABERRATIONS
MYLES L. MACE JR. a, YERACH DASKAL b and WAYNE WRAY a
Departments o f a Ce'll Biology and b Pharmacology, Bay lor College o f Medicine, Houston, Texas 77030 (U.S.A.) (Received 7 July 1977) (Revision received 9 May 1978) (Accepted 5 June 1978)
Summary This study is the first report of scanning-electron microscopy of isolated and purified metaphase chromosomes containing drug-induced aberrations. The technique reported allows high resolution topological examination of chromosomal aberrations which may pass undetected with conventional techniques.
Introduction
This study is the first report of scanning-electron microscopy of isolated and purified metaphase chromosomes containing drug-induced aberrations. It was undertaken to evaluate the ultrastructural nature of chromosomal aberrations in order to gain further insight into chromosomal architecture and regions of lesions containing drug-induced aberrations. Evaluation of the morphology of chromosome damage has been largely to assess the frequency of chromosome aberrations on a per cell basis at the resolution level of light microscopy although the additional resolution afforded by transmission electron microscopy has been utilized in some cases [5,6]. There are also several relevant ultrastructural studies on the nature of chromosome gaps and breaks which utilize transmission or scanning-electron microscopy of water-spread or airdried chromosome preparations [4,7,9,13]. Adriamycin was used to induce aberrations in this study. Adriamycin is an anthracycline antineoplastic agent which acts by intercalation into DNA, thereby inhibiting DNA-dependent DNA and RNA polymerases [16]. It binds specifically with DNA by intercalation between adjacent base pairs of the double helical structure, inducing stereoAbbreviations: CHO, Chinese monohydrate.
sodium
hamster
ovary; PIPES, piperazine-N,N'-bis(2-ethane sulfonic
acid) mono-
200 chemical chromatin disordering or damage which may be later expressed in metaphase as chromosome aberrations [11] ranging from simple deletions to extensive fragmentation. Materials and methods Chinese hamster ovary (CHO) cells grown in tissue culture were incubated in media with 0.4 pg/ml of adriamycin for 1 h, allowed to recover 14 h in media without drug, and metaphase cells collected after a 2-h Colcemid arrest. Cells were prepared for examination with light microscopy by conventional techniques. Air-dried metaphase plates were made on glass slides, stained with 2% Giemsa, and scored for the frequency of chromosomal aberrations. Isolated chromosomes were prepared by nitrogen cavitation by the method of Wray and Stubblefield [15]. The chromosome isolation buffer contained 1.0 M hexylene glycol (2-methyl-2,4-pentane diol, Eastman Organic), 0.5 mM CaC12, and 0.1 mM piperazine-N,N'-bis(2-ethane sulfonic acid) monosodium m o n o h y d r a t e (PIPES, Cal Biochem) at a pH of 6.8. Chromosomes were further purified by centrifugation through a 10--40% sucrose step gradient in order to remove cytoplasmic debris. Isolated and purified chromosomes were either deposited directly onto Formvar-coated 200-mesh copper grids or centrifuged onto grids, as described by Miller [10], through chromosome buffer at 1000 × g for 10 min. Specimens were stabilized en grid by immersion in 2% aqueous uranyl acetate followed by dehydration in graded series (25--100%) of either ethanol or hexylene glycol. Amyl acetate was used as the transition fluid prior to drying at the critical point of CO2, as described by Anderson [3], in a Denton Critical Point drier. After drying, specimens were transferred to a Kinney (KSM-2) high vacuum evaporator, maintained at a pressure of 2 × 10 -s Torr for a minimum of 30 min, and then purified argon was admitted into the chamber to a pressure of 175 ~. After purging the bell jar for 2 min with argon, specimens were coated with a gold-palladium alloy (60 : 40) for 50 sec and 10 mA in an argon atmosphere with a D.C. sputtering module (Denton Vacuum). The thickness of deposited material was determined by diameter measurements of coated and uncoated latex spheres (E. Fullam Inc.) with TEM and SEM. Samples were examined with a JEOL 100C electron microscope equipped with an ASID-4D at 40 kV using a side entry goniometer at 30--35 angle of tilt. Chromosomes were photographed using Polaroid 105 P/N film from a 2000 line CRT. Results Exposure of CHO cells to adriamycin doses greater than 1.0 pg/ml causes a significant inhibitory effect on the entry of cells into mitosis. At a level of 0.4 pg/ml, the mitotic index 2 h after Colcemid addition was 21%. 42% of the metaphase spreads showed some aberrations, ranging from simple deletions to extensive chromosomal fragmentation (Table 1). Moderate chromosomal aberrations were predominantly the chromatidal type, including simple or multiple chromatid breaks and deletions. In addition, other abnormalities such as chromosome elongation and centomere separation were observed.
201 TABLE 1 FREQUENCY
OF ADRIAMYCIN-INDUCED CHROMOSOMAL
T o t a l m e t a p h a s e plates
ABERRATIONS
104
Metaphase plates w i t h aberrations
44
T y p e o f aberrations observed breaks gaps fragments deletions rings multiple translocations
8 13 11 19 1 32
Fig. 1 shows a scanning-electron micrograph of an untreated isolated and purified metaphase chromosome. The chromatids consist of numerous microconvules, ranging in size from 2 5 0 - - 6 5 0 / ~ (net measurements, with the thickness of the metal coating deducted), which appear to be randomly distributed over the entire chromosome surface. The centromeric region is well defined, chromatids are closely aligned with a furrow between them, and frequently nuclear membranes are observed at the telomeres. Chromosomes which have been isolated and purified from cells treated with adriamycin show the general morphological details seen in control specimens.
Fig. 1. Scanning-electron m i c r o g r a p h o f an isolated untreated CHO m e t a p h a s e c h r o m o s o m e . B a r , 1/~.
202
Fig. 2. D r u g - i n d u c e d gap (pointers) in a CHO m e t a p h a s e c h r o m o s o m e . Bar, 1 /~.
The following figures have been selected as representative of chromosomal aberrations which have been observed. Fig. 2 is an example of a chromosomal gap in which there apparently is a loss of material; however, continuity within the chromatid remains. Although less material is missing in the sister chromatid, and would probably not be distinguished as a gap by Giemsa staining, this region may be considered a homologous gap within the chromatid. Fig. 3 is an example of a single chromatid gap with almost complete cleavage of the arm (arrows), probably at the centromere region. The cleaved portion still retains association with the sister chromatid. A complete break of one chromatid together with an incomplete break of the sister chromatid is shown in Fig. 4, where the broken-off fragment is connected to one arm by a thin cord of chromatin with sister chromatids tightly paired in the fragment. Frequently it is difficult to distinguish, in Giemsa-stained preparations, whether a lightly stained constriction between chromosomal arms represents an actual chromosome or is the result of a random or sticky association. Fig. 5 is a scanning-electron micrograph of such a situation. Therefore this example represents a single chromosome with either stretching of the centromere or a gap at the centromeric region. Fig. 6 is an example of a rare ring chromosome with continuity along the entire chromatid. It is n o t possible with scanning-electron microscopy of isolated chromosomes to determine whether this example represents a single or pair of chromatids which have been joined.
203
F i g . 3. C H O m e t a p h a s e c h r o m o s o m e w i t h a c h r o m a t i d g a p ( a r r o w s ) . B a r , 1 /~.
Fig. 4 . C H O m e t a p h a s e c h r o m o s o m e w i t h a n a l m o s t c o m p l e t e c h r o m o s o m a l b r e a k . B a r , 1/~.
204
Fig. 5. C H O m e t a p h a s e c h r o m o s o m e w i t h a c e n t r o m e r i c gap. Bax, 1 ~.
Fig. 6. C H O m e t a p h a s e c h r o m o s o m e in a ring c o n f i g u r a t i o n . P o i n t e d i n d i c a t e d m e m b r a n o u s p a t c h . Bar, 1/~.
205 The presence of membrane which is presumably nuclear {pointer) should be noted, which may represent former telomeres as previously described [8]. Discussion Scanning-electron microscopy offers the advantage of high resolution of topological details in the examination of chromosome lesions. Small chromatid gaps such as in Fig. 2 may pass unnoticed with light microscopy and those similax to Fig. 3 scored as breaks. Typical gaps observed with scanning-electron microscopy range from almost complete breaks connected by a few discrete chromatin fibers to slight depressions with the chromatids. Frequently, on the chromatid adjacent to the gap there is also some loss or damage at the identical region b u t n o t necessarily with the same degree of involvement. Brogger [7] states that the average length of the proximal and distal parts of a chromatid with a break or gap is a b o u t 9% shorter than the length of the intact sister chromatid. It has also been reported that the length of a chromatid with a gap often exceeds, by the size of the gap, the length of the sister chromatid [5]. This would suggest that the gap is only a stretched region of the chromatid, resulting n o t from chromatin loss but an error in either the folding or the packing. Our results generally support this second concept b u t there are exceptions where there seems to be selective loss of material in only one chromatid. The difference in these results could well be explained due to waterspreading techniques used by Brogger [7] contrasted with a critical point drying of isolated chromosomes used in this study. A chromatid break is characterized by the complete absence of chromatin fibers between the severed parts. With transmission-electron microscopy, there appears to be no difference between a normal telomere and the region exhibiting a complete chromatid break [5]. This observation suggests that the membranous patches associated with some telomeres represent a specific association, and are not produced b y either isolation or purification of chromosomes. Generally our results support this observation with t w o exceptions. The first is that the membrane patches, presumably nuclear in origin, are frequently associated with telomeres b u t have n o t been observed at a chromatid or chromosome break. In addition, telomeres appear as a somewhat bulbous end, uniformly covered with microconvules, while a break is a fiat cut across the chromatid where the surface of the break may be eroded in some regions. However, no ultrastructural features are observed which would explain why broken ends of chromosomes are sticky and therefore tend to combine with other broken chromosome ends. Especially obscure at the present time is the relation of chromosome breaks and gaps to the chromosome " c o r e " or chromosome "scaffold" [1,2,12,14]. Further studies to elucidate the relationship of chromosome architecture with biochemical and genetic approaches are in progress. The technique reported here allows high resolution topological examination of chromosomal aberrations which may pass undetected with conventional techniques. This technique may also be extended to include cell preparations which have been fixed in methanol--acetic acid, thereby allowing analysis of complete karyotypes.
206
Acknowledgements These studies were supported by NSF PCM 75-05622, American Cancer Society VC-163, National Cancer Institute CA-18455, Cancer Research Center Grant CA-10893 P. 5 from the National Cancer Institute, the National Institutes of Health Grant HD-00444, and PHS Award 5 F32 HD 01861.02. References 1 Adolph, K.W., S.M. Cheng, J.R. Paulson and U.K. Laemmli, Isolation of a prot e i n scaffold from m i t o t i c HeLa cell chromosomes, Proc. Natl. Acad. Sci. (U.S.A.), 74 (1977) 4937--4941. 2 Adolph, K.W., S.M. Cheng and U.K. Laemmli, Role of nonhi s t one proteins in metaphase c hromos ome structure, Cell, 12 (1977) 805--816. 3 Anderson, T.F., Techniques for the preservation of three-dimensional structure in preparing specimens for the electron microscope, Trans. N.Y. Acad. Sci., 13 (1951) 130--134. 4 Brecher, S., Ultrastructural observations of X-ray induce d c h r o m a t i d gaps, Mutation Res., 42 (1977) 249--268. 5 Brinkley, B.R., and W.N. Hittelman, Ultrastructure of m a m m a l i a n c h r o m o s o m e aberrations, Int. Rev. Cytochem., 42 (1975) 49--101. 6 Brinkley, B.R., and M.N. Shaw, Ultrastructural aspects of c h r o m o s o m e damage, Genetic concepts and neoplasia, Williams and Wllkins, Baltimore, 1969, pp. 313--345. 7 Brogger, A., Apparently spontaneous c h r o m o s o m e damage in h u m a n l e u k o c y t e s and the nature of c h r o m a t i d gaps, Humangenetik, 13 (1971) 1--14. 8 Daskal, Y., M.L. Mace Jr. and H. Busch, High resolution scaning electron mi c ros c opy of isolated Chinese h a m s t e r chromosomes, IITRI/SEM (1976) 163--170. 9 Fisher, K.M., T.F. Budinger, A.T. Foin and T.E. Everhart, Spontaneous c h r o m o s o m e gaps in Marmosa mitis, Mutation Res., 22 (1974) 299--303. 10 Miller Jr., O.L., and A.H. Bakken, Morphological studies of transcription, Karolinska Symp., 5 (1972) 155--177. 11 Newsome, Y.L., and L.G. Littlefield, Adriamycin induced c h r o m o s o m e aberrations in h u m a n fibroblasts, J. Natl. Cancer Inst., 55 (1975) 1061--1064. 12 Paulson, J.R., and U.K. Laemmli, The structure of histone-dcpleted metaphase chromosomes, Cell, 12 (1977) 817--828. 13 Scheid, W., and H. Traut, Visualization by scanning electron microscopy of chromatic lesions ( " g a p s " ) induced by X-rays in chromosomes of Vicia faba, Mutation Res., 11 (1971) 253--255. 14 Stubblefield, E., and W. Wray, Architecture of the Chinese ha ms t e r metaphase chromosome, Chromosoma, 32 (1971) 262--294. 15 Wray, W., and E. Stubblefield, A new m e t h o d for the rapid isolation of chromosomes, m i t o t i c apparatus, or nuclei from m a m m a l i a n fibroblasts at near neutral pH, Exptl. Cell Res.0 59 (1970) 469--478. 16 Zunino, F., R.A. G a m b e t t a and A. Zaccara, Interaction of d a u n o m y c i n w i t h DNA, Biochim. Biophys. Acta, 277 (1972) 498--498.
