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Surface topography of human chromosomes examined at each stage during ASG banding procedure
Preliminary motes 585 useful tool in the study of muscle development. We thank the Muscular Dystrophy Group of Great Britain for financial support and Mr Colin Atherton for taking the photographs. A. C. is a postdoctoral fellow of the Medical Research Council; G. E. M. is a Beit Memorial Fellow.
References 1. Dawson, D M, Eppenberger, H M & Kaplan, N 0, J biol them 242 (1967) 210. 2. Eppenberger, H M, Eppenberger, M, Richterich, R & Aebi, H, Dev biol 10 (1964) 1. 3. Morris, G E & Cole, R J, Exptl cell res 74 (1972) 191 ___.
4. Coleman, J R & Coleman, A W, J cell physiol 72, suppl. 1 (1968) 19. 5. Shainberg, A, Yagil, G & Yaffe, D, Dev biol 25 (1971) 1. 6. Werner, I, Ph.D. Thesis, Brown University, Providence, R. 1. (1966). 7. Konigsberg, I R, McElvain, N, Tootle, M & Herrmann, H, J biophys biochem cytol 8 (1960) 333. 8. Dawson. D M. Eooenberger, H M & Epoenberger, M E, Ann’NYacad sci J51 (1968) 6<6. 9. Campell, P N & Sargent, J R, Techniques in protein biosynthesis, vol. 1, p. 310. Academic Press. New York (1967). 10. Ingwall, J S, Morales,‘M F & Stockdale, F E, Proc natl acad sci US 69 (1972) 2250. 11. Morris, G E, Rourke, A &’ Heywood, S M. Unpublished work. 12. Cahn, R D, The stability of the differentiated state (ed H Ursprung) p. 58. Springer-Verlag, New York (1968). Received April 27, 1972 Revised version received July 3, 1972
examined by electron microscopy, showed a collapse of the chromosomes after 2 x SSC treatment. On subsequent staining with Giemsa a marked alteration of chromosomal morphology occurred resulting in a fibrillar appearance and transverse ridging of the chromatids.
The recent introduction of chromosomal banding techniques [I, 51 has greatly facilitated the identification of individual metaphase chromosomes. As the ASG technique [5] has been routinely used in this laboratory. we have examined the surface morphology of chromosomes, at each stage of the banding procedure, by light and electron microscopy. Materials and Methods Metaphase preparations of peripheral blood lymphocvtes were oreoared 131and then treated as follows: (;1>untreated cbntrol~ {b) 2 x SSC for 1 h at 60°C; (c) 2 x SSC for 1 h at 60°C followed bv a 1: 50 dilution of Giemsa in pH 6.8 buffer for 14 h. These slides were examined to ensure banding was present. Preparations, for examination of the chromosomal surface topography by light microscopy, were shadowed at an angle of 45” with a heavy coating of gold/ palladium. These preparations were mounted in DePeX and examined under oil immersion using bright field transmitted illumination. Electron microscope specimens were oreoared bv removing sinelestage carbon replicas from the slides using 4 Nsodkm hydroxide at 60°C. To ensure removal of all bioloaical material the carbon replicas were left floating on the sodium hydroxide at 60°C overnight. The films were then washed by transferring them to a bath of distilled water. Portions of the replicas were picked up on grids, dried, shadowed at an angle of 30” with gold/ palladium and examined with a GEC-AEI EM6B electron microscope.
Results and Discussion Surface topography of human chromosomes examined at each stage during ASG banding procedure I. P. GORMLEY
and A. ROSS, Medical Research Council, Clinical and Population Cytogenetics Unit, Western General Hospital, Edinburgh, Scotland
Summary The surface topography of human chromosomes has been examined before, during, and after the ASG banding procedure. A new. simple technique for exami&@ the surface topography-of unstained preparations, by light microscopy, is described. This technique together with single-stage carbon replicas,
The untreated chromosomes (figs 1,2) showed a smooth surface topography. However well defined replicas of metaphase chromosomes are difficult to obtain due to the presence of cytoplasmic material [2, 41. After treatment with 2 X SSC (figs 3, 4) the centres of the chromatids appeared to collapse leaving a ridge around their perimeter and no obvious surface structure was observed. The cytoplasmic material seen in the untreated preparations was not observed in these preparations. Exptl Cell Res 74 (1972)
586 Preliminary notes
Fig. Fig. Fig. Fig.
I. Light microscope preparation of an untreated cell in metaphase. 2. Replica of untreated chromosomes. 3. Light microscope preparation treated with 2 x SSC. 4. Replica of chromosomes treated with 2 x SSC.
Chromosomes treated with 2 x SSC and stained with Giemsa as in the standard ASG technique [5] (fig. 5) showed no surface topography in the light microscope preparations shadowed with a heavy metal, probably due to the increased opacity of the chromosomes. The replicas (fig. 6) however showed a marked change in the structure of the chromoExptl Cd Res 74 (1972)
somes. The surface of the chromosomes was fibrillar with fibres extending out from their periphery and marked transverse ridging occurred on adjacent sides of each chromatid. The light microscope technique which we have described provides a simple rapid method of observing the gross surface topography of unstained material and can com-
Preliminary
Fig. 5. Light microscope preparation treated with 2 x SSC and Giemsa. Fig. 6. Replica of chromosomes, treated with 2 x SSC and Giemsa, showing ridging
plement results obtained by the more difficult and time consuming replica techniques. The results showed that the chromosomes collapsed after treatment with 2 x SSC which could be due to a rearrangement and/or a loss of chromosomal material. Cytoplasmic material appeared to be removed from the cell and collapse of the chromosomes may be a consequence of the loss of surrounding cytoplasmic material or more probably to a loss of material from the chrome. somes themselves. When chromosomes are stained with Giemsa after 2 x SSC treatment banding patterns are observed with the light microscope [5]. Replicas of these chromosomes showed a marked alteration of the chromosomal material leading to transverse ridging of the surface and to the appearance of fibrils. It is suggested from these results that the banding patterns obtained with Giemsa using light microscopy are due to the ridges absorbing more dye than the other parts of the chromosome. Work is currently in progress to determine whether these ridges on the chromosomes correspond to the band-
n ?tes
587
(arrowed).
ing observed by light microscopy using standard banding techniques. We are grateful to Dr A. T. Summer for discussion and assistance with the banding technique, to Mr N. Davidson for photographic assistance-and to Professor H. J. Evans for advice and encouragement. References 1. Caspersson, T, Lomakka, G & Zech, L, Hereditas 67 (1971) 89. 2. Christenhuss. R. Biichner. Th & Pfeiffer. R A. Nature 216 (1967) 379. ’ 3. Hunaerford. D A. Stain technol40 (1965) 333. 4. Net&&h, P’ W, Ampola, M G & ‘Vet&r, H G, Lancet ii (1967) 1366. 5. Sumner, AT, Evans, H J & Buckland, R A, Nature new biol 232 (1971) 31. Received April 27, 1972
Unique and repetitive DNA sequences in the genome of Chironomus tentans R. I. SACHS and U. CLEVER, Department of Biological Sciences, Purdue University, Lafayette, Ind. 47907, USA Summary It is estimated that 95.5 9/, of the DNA represents single-copy sequences. Sequences in the redundant Exptl Cell Res 74 (1972)
References 1. Dawson, D M, Eppenberger, H M & Kaplan, N 0, J biol them 242 (1967) 210. 2. Eppenberger, H M, Eppenberger, M, Richterich, R & Aebi, H, Dev biol 10 (1964) 1. 3. Morris, G E & Cole, R J, Exptl cell res 74 (1972) 191 ___.
4. Coleman, J R & Coleman, A W, J cell physiol 72, suppl. 1 (1968) 19. 5. Shainberg, A, Yagil, G & Yaffe, D, Dev biol 25 (1971) 1. 6. Werner, I, Ph.D. Thesis, Brown University, Providence, R. 1. (1966). 7. Konigsberg, I R, McElvain, N, Tootle, M & Herrmann, H, J biophys biochem cytol 8 (1960) 333. 8. Dawson. D M. Eooenberger, H M & Epoenberger, M E, Ann’NYacad sci J51 (1968) 6<6. 9. Campell, P N & Sargent, J R, Techniques in protein biosynthesis, vol. 1, p. 310. Academic Press. New York (1967). 10. Ingwall, J S, Morales,‘M F & Stockdale, F E, Proc natl acad sci US 69 (1972) 2250. 11. Morris, G E, Rourke, A &’ Heywood, S M. Unpublished work. 12. Cahn, R D, The stability of the differentiated state (ed H Ursprung) p. 58. Springer-Verlag, New York (1968). Received April 27, 1972 Revised version received July 3, 1972
examined by electron microscopy, showed a collapse of the chromosomes after 2 x SSC treatment. On subsequent staining with Giemsa a marked alteration of chromosomal morphology occurred resulting in a fibrillar appearance and transverse ridging of the chromatids.
The recent introduction of chromosomal banding techniques [I, 51 has greatly facilitated the identification of individual metaphase chromosomes. As the ASG technique [5] has been routinely used in this laboratory. we have examined the surface morphology of chromosomes, at each stage of the banding procedure, by light and electron microscopy. Materials and Methods Metaphase preparations of peripheral blood lymphocvtes were oreoared 131and then treated as follows: (;1>untreated cbntrol~ {b) 2 x SSC for 1 h at 60°C; (c) 2 x SSC for 1 h at 60°C followed bv a 1: 50 dilution of Giemsa in pH 6.8 buffer for 14 h. These slides were examined to ensure banding was present. Preparations, for examination of the chromosomal surface topography by light microscopy, were shadowed at an angle of 45” with a heavy coating of gold/ palladium. These preparations were mounted in DePeX and examined under oil immersion using bright field transmitted illumination. Electron microscope specimens were oreoared bv removing sinelestage carbon replicas from the slides using 4 Nsodkm hydroxide at 60°C. To ensure removal of all bioloaical material the carbon replicas were left floating on the sodium hydroxide at 60°C overnight. The films were then washed by transferring them to a bath of distilled water. Portions of the replicas were picked up on grids, dried, shadowed at an angle of 30” with gold/ palladium and examined with a GEC-AEI EM6B electron microscope.
Results and Discussion Surface topography of human chromosomes examined at each stage during ASG banding procedure I. P. GORMLEY
and A. ROSS, Medical Research Council, Clinical and Population Cytogenetics Unit, Western General Hospital, Edinburgh, Scotland
Summary The surface topography of human chromosomes has been examined before, during, and after the ASG banding procedure. A new. simple technique for exami&@ the surface topography-of unstained preparations, by light microscopy, is described. This technique together with single-stage carbon replicas,
The untreated chromosomes (figs 1,2) showed a smooth surface topography. However well defined replicas of metaphase chromosomes are difficult to obtain due to the presence of cytoplasmic material [2, 41. After treatment with 2 X SSC (figs 3, 4) the centres of the chromatids appeared to collapse leaving a ridge around their perimeter and no obvious surface structure was observed. The cytoplasmic material seen in the untreated preparations was not observed in these preparations. Exptl Cell Res 74 (1972)
586 Preliminary notes
Fig. Fig. Fig. Fig.
I. Light microscope preparation of an untreated cell in metaphase. 2. Replica of untreated chromosomes. 3. Light microscope preparation treated with 2 x SSC. 4. Replica of chromosomes treated with 2 x SSC.
Chromosomes treated with 2 x SSC and stained with Giemsa as in the standard ASG technique [5] (fig. 5) showed no surface topography in the light microscope preparations shadowed with a heavy metal, probably due to the increased opacity of the chromosomes. The replicas (fig. 6) however showed a marked change in the structure of the chromoExptl Cd Res 74 (1972)
somes. The surface of the chromosomes was fibrillar with fibres extending out from their periphery and marked transverse ridging occurred on adjacent sides of each chromatid. The light microscope technique which we have described provides a simple rapid method of observing the gross surface topography of unstained material and can com-
Preliminary
Fig. 5. Light microscope preparation treated with 2 x SSC and Giemsa. Fig. 6. Replica of chromosomes, treated with 2 x SSC and Giemsa, showing ridging
plement results obtained by the more difficult and time consuming replica techniques. The results showed that the chromosomes collapsed after treatment with 2 x SSC which could be due to a rearrangement and/or a loss of chromosomal material. Cytoplasmic material appeared to be removed from the cell and collapse of the chromosomes may be a consequence of the loss of surrounding cytoplasmic material or more probably to a loss of material from the chrome. somes themselves. When chromosomes are stained with Giemsa after 2 x SSC treatment banding patterns are observed with the light microscope [5]. Replicas of these chromosomes showed a marked alteration of the chromosomal material leading to transverse ridging of the surface and to the appearance of fibrils. It is suggested from these results that the banding patterns obtained with Giemsa using light microscopy are due to the ridges absorbing more dye than the other parts of the chromosome. Work is currently in progress to determine whether these ridges on the chromosomes correspond to the band-
n ?tes
587
(arrowed).
ing observed by light microscopy using standard banding techniques. We are grateful to Dr A. T. Summer for discussion and assistance with the banding technique, to Mr N. Davidson for photographic assistance-and to Professor H. J. Evans for advice and encouragement. References 1. Caspersson, T, Lomakka, G & Zech, L, Hereditas 67 (1971) 89. 2. Christenhuss. R. Biichner. Th & Pfeiffer. R A. Nature 216 (1967) 379. ’ 3. Hunaerford. D A. Stain technol40 (1965) 333. 4. Net&&h, P’ W, Ampola, M G & ‘Vet&r, H G, Lancet ii (1967) 1366. 5. Sumner, AT, Evans, H J & Buckland, R A, Nature new biol 232 (1971) 31. Received April 27, 1972
Unique and repetitive DNA sequences in the genome of Chironomus tentans R. I. SACHS and U. CLEVER, Department of Biological Sciences, Purdue University, Lafayette, Ind. 47907, USA Summary It is estimated that 95.5 9/, of the DNA represents single-copy sequences. Sequences in the redundant Exptl Cell Res 74 (1972)