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Secondary constrictions and nucleolus organizer regions in man
428
Preliminary
notes
induction it may be the manifestation of a specific maturational process. Its relationship to spontaneous regression of human neuroblastoma [ 121and to cell death in solid tumors [13] remains to be determined. In any case, the neuroblastoma system may provide a model for studying the kinetics of tumor cell proliferation [14] in which the rate of cell loss can be manipulated. The conclusion that there is no causal relationship between neurite extension and inhibition of proliferation [3] is reinforced by this study, but certain other data must be reinterpreted. It is especially clear that even though inhibition of DNA synthesis can induce AChE [4], serum deprivation does not induce the enzyme by such a mechanism. If the recent observation that AChE can be induced by two distinct mechanisms [15] is extended to the present case, the effects of serum deprivation are seen to be similar to those of bromodeoxyuridine since both inhibit proliferation relatively little and are sensitive to actinomycin D. Many other studies which rely on the assumption that serum deprivation completely inhibits the proliferation of neuroblastoma cells (e.g. references [ 16, 171)should also be re-evaluated.
8. Lanks, K W, Dorwin, J & Papirmeister, B, J cell bio163 (1974) 824. 9. Vinijchaikul, K & Fitzgerald, P J, Am j path01 66 (1972) 407. 10. Pollack, R, Risser, R, Conlon, S & Rifkin, D, Proc natl acad sci US 71 (1974) 4792. 11. Dulbecco, R, Nature 227 (1970) 802. 12. Aterman, K & Schueller, E F, Am j dis child 120 (1970) 217. 13. Cooper, E H, Bedford, A J & Kenny, T E, Adv cancer res 21 (1975) 59. 14. Janik, P, Briand, P & Hartmann, N R, Cancer res 35 (1975) 3698. 15. Simantov, R & Sachs, L, Dev bio145 (1975) 382. 16. Elul, R, Brons, J & Kravitz, K, Nature 258 (1975) 616. 17. Schubert, D, Brain res 56 (1973) 387. Received June 3, 1976 Accepted October 14, 1976
Secondary constrictions and nucleolus organizer regions in man M. FERRARO, N. ARCHIDIACONO, F. PELLICCIA, M. ROCCHI, A. ROCCHI and A. de CAPGA, Institute of Genetics and the CNR Centre of EVO~Utionary Genetics, University of Rome, 00185 Rome, Italy
A relatively simple technique, “N-banding”, has been shown by Matsui & Sasaki [l], and by Funaki et al. [2] to stain specifically the nucleolus organizer region (NOR) of many organisms, including man. Other authors have recently used different techniques, “AG-SAT” [3] and “Ag-AS” [4], for differential staining of NOR. The results We are indebted to Dr J. Broome for advice and to obtained by these techniques have been E. Pasnikowski and L. Marquez for technical assistance. confirmed in man and other organisms by DNA/i-RNA hybridization experiments: the chromosomal sites differentially stained by References means of N-banding and Ag-AS are the 1. Seeds, N W, Gilman, A G, Amano, T & Nirensame where DNA/rRNA reassociation ocberg, M W, Proc natl acad sci US 66(1970) 160. 2. Blume, A, Gilbert, F, Watson, S, Farber, JI curs [5-71. These studies have shown that Rosenberg, R & Nirenberg, M, Proc natl acad SCI NOR has different locations in the various US 67 (1970) 786. organisms: in the secondary constriction 3. Schubert, D, Humphreys, S, Vitry, E &Jacob, F, Dev bio125 (1971) 514. (SC) as in the rat kangaroo, in the centro4. Lanks, K W, Turnbull, J D, Aloyo, V J, Dorwin, merit region (C), as in mouse, in the teloJ & Papirmeister, B, Exp cell res 93 (1975) 355. 5. Byfield, J E & Karlsson, V, Cell differ 2 (1973) 55. meres (T) as in the Chinese hamster, and 6. Schubert, D & Jacob, F, Proc natl acad sci US 67 in the satellite (SAT) as in the chimpanzee, (1970) 247. f. Helson, L, Ca 25 (1975) 264. the African green monkey, and in man. In Exp Cell Res 104 (1977)
Preliminary notes
429
Fig. 1. D- and G-group chromosomes from different cells. (a) Chromosomes stained in duplicate with
Giemsa or quinacrine and N-banding. (6) Giemsa and Ag-AS.
particular, as far as man is concerned, some authors [ l-3,8] consider that both N-banding and Ag-AS techniques differentially stain the satellite bodies of acrocentric chromosomes, in contrast to the well accepted theory [9-lo] that in man the sites of ribosomal genes are located in the secondary constrictions. On the other hand, the level of resolution of the DNA/r-RNA hybridization technique does not permit discrimination between the two regions of the labelled area: i.e. the satellite and its constriction, except for cases with enlarged satellite stalks [6]. The above mentioned data have led some authors [ 1,2] to the conclusion that in man the sites of ribosomal genes are on the satellites of acrocentric chromosomes, and other authors [8] have come to quite the opposite conclusion, that N-banding techniques is not always specific for the NOR. The use of both N-banding and Ag-AS on human chromosomes has caused us to draw conclusions which are in contrast to those of the above-mentioned authors. We have in fact been able to demonstrate that
both these techniques selectively stain the secondary constrictions which are located on the short arms of the acrocentric chromosomes of man. Normal D- and G-group chromosomes from peripheral blood cultures of four phenotypically normal individuals have been analysed. The preparations were stained with standard Giemsa, photographed and then processed with either the N-banding or the Ag-AS technique, and rephotographed. A direct comparison of the same chromosomes stained first with Giemsa and then with the specific staining techniques allowed us to establish that the differentially stained sites were located on the short arms of all acrocentric chromosomes, well below the satellite (figs 1, 2).
Fig. 2. Some D and G-group chromosomes stained
with Ag-AS. The satellite appears clearly visible above the stained constriction. Exp Cell Res 104 (1977)
430
Preliminary
notes
It has to be mentioned that after N-banding the chromosomes are faintly stained and the satellites generally invisible; this might explain the misinterpretation of data by some of the above-mentioned authors. However, the use of the Ag-AS technique, which does not alter the morphology and gives a better staining of the chromosomes, together with the direct examination of the photographs of the same chromosomes before and after banding, confirmed our opinion that both techniques selectively stain the secondary constrictions of acrocentric chromosomes, and not the satellite. Our results therefore support the theory that in man ribosomal genes are located in the secondary constrictions of the acrocentric chromosomes. These data also demonstrate the effectiveness of both the N-banding and the Ag-AS techniques in selectively staining the NOR. Further studies on NOR staining of variant D- and G-group chromosomes are in progress.
Enzyme loading of nucleated chicken erythrocytes ESTRELLA ANG, R. GLEW and G. IHLER,’ Department of Biochemistry, School of Medicine, University of Pittsburgh, Pittsburgh, PA 15261, USA Summary. Exogenous enzymes can be loaded into nucleated chicken erythrocytes by hypotonic hemolysis. The hemolysed and reseated cells retain the ability to activate transport ions and to survive while circulating in the blood of chickens. It is proposed that these cells may be useful for the investigation of several aspects of cellular biochemistry.
1. Matsui, S & Sasaki, M, Nature 246 (1973) 148. 2. Funaki, K, Matsui, S & Sasaki, M, Chromosoma 49 (1975) 357. 3. Howell, W M, Denton, T E & Diamond, J R, Experientia 3 1(1975) 260. 4. Goodpasture, C & Bloom S E, Chromosoma 53 (1975) 37. 5. Henderson,A, Warburton, D & Atwood, K C, Proc natl acad sci US 69 (1972) 3394. 6. Evans, H J, Buckland, R A & Pardue, M L, Chromosoma48(1974)405. 7. Hsu, T C, Spirito, S E & Pardue, M L, Chromosoma 53 (1975) 25. 8. Faust, J & Vogel, W, Nature 249 (1974) 352. 9. Ferguson-Smith, M A & Handmaker, S D, Lancet i (1961) 638. 10. Ohno, S, Kaplan, W D, Trujillo, J M & Kinosita, R, Lancet ii (1961) 123.
Holes or pores can be reversibly opened in erythrocyte membranes by dilution of the cells into hypotonic medium, allowing either ions and small molecules or macromolecules such as enzymes to be trapped in the cells [ 1, 21. Enzyme-loaded human erythrocytes may be medically useful for delivering enzymes to lysosomes of erythrophagocytic cells for treatment of storage disorders such as Gaucher’s disease or for degrading substances by circulating enzyme-loaded cells [2, 3, 41. Enzyme-loaded erythrocytes could also be useful for studying erythrocyte biochemistry and physiology either by introducing non-erythrocyte enzymes into the cells or altering the levels of authentic erythrocyte enzymes. However, the possible range of experiments is seriously limited by the fact that mammalian erythrocytes lack DNA and mitochondria, thus eliminating many of the interesting problems of cellular and molecular biology We have therefore examined chicken erythrocytes which are nucleated and contain mitochondria to determine if it is possible to load these cells with foreign enzymes in a manner similar to mammalian erythrocytes. In this work we show that chicken erythrocytes can be loaded with exogenous proteins and that the resealed
Received June 30, 1976 Accepted October 15, 1976
’ To whom reprint requests should be addressed.
References
Exp CeNRes 104 (1977)
Preliminary
notes
induction it may be the manifestation of a specific maturational process. Its relationship to spontaneous regression of human neuroblastoma [ 121and to cell death in solid tumors [13] remains to be determined. In any case, the neuroblastoma system may provide a model for studying the kinetics of tumor cell proliferation [14] in which the rate of cell loss can be manipulated. The conclusion that there is no causal relationship between neurite extension and inhibition of proliferation [3] is reinforced by this study, but certain other data must be reinterpreted. It is especially clear that even though inhibition of DNA synthesis can induce AChE [4], serum deprivation does not induce the enzyme by such a mechanism. If the recent observation that AChE can be induced by two distinct mechanisms [15] is extended to the present case, the effects of serum deprivation are seen to be similar to those of bromodeoxyuridine since both inhibit proliferation relatively little and are sensitive to actinomycin D. Many other studies which rely on the assumption that serum deprivation completely inhibits the proliferation of neuroblastoma cells (e.g. references [ 16, 171)should also be re-evaluated.
8. Lanks, K W, Dorwin, J & Papirmeister, B, J cell bio163 (1974) 824. 9. Vinijchaikul, K & Fitzgerald, P J, Am j path01 66 (1972) 407. 10. Pollack, R, Risser, R, Conlon, S & Rifkin, D, Proc natl acad sci US 71 (1974) 4792. 11. Dulbecco, R, Nature 227 (1970) 802. 12. Aterman, K & Schueller, E F, Am j dis child 120 (1970) 217. 13. Cooper, E H, Bedford, A J & Kenny, T E, Adv cancer res 21 (1975) 59. 14. Janik, P, Briand, P & Hartmann, N R, Cancer res 35 (1975) 3698. 15. Simantov, R & Sachs, L, Dev bio145 (1975) 382. 16. Elul, R, Brons, J & Kravitz, K, Nature 258 (1975) 616. 17. Schubert, D, Brain res 56 (1973) 387. Received June 3, 1976 Accepted October 14, 1976
Secondary constrictions and nucleolus organizer regions in man M. FERRARO, N. ARCHIDIACONO, F. PELLICCIA, M. ROCCHI, A. ROCCHI and A. de CAPGA, Institute of Genetics and the CNR Centre of EVO~Utionary Genetics, University of Rome, 00185 Rome, Italy
A relatively simple technique, “N-banding”, has been shown by Matsui & Sasaki [l], and by Funaki et al. [2] to stain specifically the nucleolus organizer region (NOR) of many organisms, including man. Other authors have recently used different techniques, “AG-SAT” [3] and “Ag-AS” [4], for differential staining of NOR. The results We are indebted to Dr J. Broome for advice and to obtained by these techniques have been E. Pasnikowski and L. Marquez for technical assistance. confirmed in man and other organisms by DNA/i-RNA hybridization experiments: the chromosomal sites differentially stained by References means of N-banding and Ag-AS are the 1. Seeds, N W, Gilman, A G, Amano, T & Nirensame where DNA/rRNA reassociation ocberg, M W, Proc natl acad sci US 66(1970) 160. 2. Blume, A, Gilbert, F, Watson, S, Farber, JI curs [5-71. These studies have shown that Rosenberg, R & Nirenberg, M, Proc natl acad SCI NOR has different locations in the various US 67 (1970) 786. organisms: in the secondary constriction 3. Schubert, D, Humphreys, S, Vitry, E &Jacob, F, Dev bio125 (1971) 514. (SC) as in the rat kangaroo, in the centro4. Lanks, K W, Turnbull, J D, Aloyo, V J, Dorwin, merit region (C), as in mouse, in the teloJ & Papirmeister, B, Exp cell res 93 (1975) 355. 5. Byfield, J E & Karlsson, V, Cell differ 2 (1973) 55. meres (T) as in the Chinese hamster, and 6. Schubert, D & Jacob, F, Proc natl acad sci US 67 in the satellite (SAT) as in the chimpanzee, (1970) 247. f. Helson, L, Ca 25 (1975) 264. the African green monkey, and in man. In Exp Cell Res 104 (1977)
Preliminary notes
429
Fig. 1. D- and G-group chromosomes from different cells. (a) Chromosomes stained in duplicate with
Giemsa or quinacrine and N-banding. (6) Giemsa and Ag-AS.
particular, as far as man is concerned, some authors [ l-3,8] consider that both N-banding and Ag-AS techniques differentially stain the satellite bodies of acrocentric chromosomes, in contrast to the well accepted theory [9-lo] that in man the sites of ribosomal genes are located in the secondary constrictions. On the other hand, the level of resolution of the DNA/r-RNA hybridization technique does not permit discrimination between the two regions of the labelled area: i.e. the satellite and its constriction, except for cases with enlarged satellite stalks [6]. The above mentioned data have led some authors [ 1,2] to the conclusion that in man the sites of ribosomal genes are on the satellites of acrocentric chromosomes, and other authors [8] have come to quite the opposite conclusion, that N-banding techniques is not always specific for the NOR. The use of both N-banding and Ag-AS on human chromosomes has caused us to draw conclusions which are in contrast to those of the above-mentioned authors. We have in fact been able to demonstrate that
both these techniques selectively stain the secondary constrictions which are located on the short arms of the acrocentric chromosomes of man. Normal D- and G-group chromosomes from peripheral blood cultures of four phenotypically normal individuals have been analysed. The preparations were stained with standard Giemsa, photographed and then processed with either the N-banding or the Ag-AS technique, and rephotographed. A direct comparison of the same chromosomes stained first with Giemsa and then with the specific staining techniques allowed us to establish that the differentially stained sites were located on the short arms of all acrocentric chromosomes, well below the satellite (figs 1, 2).
Fig. 2. Some D and G-group chromosomes stained
with Ag-AS. The satellite appears clearly visible above the stained constriction. Exp Cell Res 104 (1977)
430
Preliminary
notes
It has to be mentioned that after N-banding the chromosomes are faintly stained and the satellites generally invisible; this might explain the misinterpretation of data by some of the above-mentioned authors. However, the use of the Ag-AS technique, which does not alter the morphology and gives a better staining of the chromosomes, together with the direct examination of the photographs of the same chromosomes before and after banding, confirmed our opinion that both techniques selectively stain the secondary constrictions of acrocentric chromosomes, and not the satellite. Our results therefore support the theory that in man ribosomal genes are located in the secondary constrictions of the acrocentric chromosomes. These data also demonstrate the effectiveness of both the N-banding and the Ag-AS techniques in selectively staining the NOR. Further studies on NOR staining of variant D- and G-group chromosomes are in progress.
Enzyme loading of nucleated chicken erythrocytes ESTRELLA ANG, R. GLEW and G. IHLER,’ Department of Biochemistry, School of Medicine, University of Pittsburgh, Pittsburgh, PA 15261, USA Summary. Exogenous enzymes can be loaded into nucleated chicken erythrocytes by hypotonic hemolysis. The hemolysed and reseated cells retain the ability to activate transport ions and to survive while circulating in the blood of chickens. It is proposed that these cells may be useful for the investigation of several aspects of cellular biochemistry.
1. Matsui, S & Sasaki, M, Nature 246 (1973) 148. 2. Funaki, K, Matsui, S & Sasaki, M, Chromosoma 49 (1975) 357. 3. Howell, W M, Denton, T E & Diamond, J R, Experientia 3 1(1975) 260. 4. Goodpasture, C & Bloom S E, Chromosoma 53 (1975) 37. 5. Henderson,A, Warburton, D & Atwood, K C, Proc natl acad sci US 69 (1972) 3394. 6. Evans, H J, Buckland, R A & Pardue, M L, Chromosoma48(1974)405. 7. Hsu, T C, Spirito, S E & Pardue, M L, Chromosoma 53 (1975) 25. 8. Faust, J & Vogel, W, Nature 249 (1974) 352. 9. Ferguson-Smith, M A & Handmaker, S D, Lancet i (1961) 638. 10. Ohno, S, Kaplan, W D, Trujillo, J M & Kinosita, R, Lancet ii (1961) 123.
Holes or pores can be reversibly opened in erythrocyte membranes by dilution of the cells into hypotonic medium, allowing either ions and small molecules or macromolecules such as enzymes to be trapped in the cells [ 1, 21. Enzyme-loaded human erythrocytes may be medically useful for delivering enzymes to lysosomes of erythrophagocytic cells for treatment of storage disorders such as Gaucher’s disease or for degrading substances by circulating enzyme-loaded cells [2, 3, 41. Enzyme-loaded erythrocytes could also be useful for studying erythrocyte biochemistry and physiology either by introducing non-erythrocyte enzymes into the cells or altering the levels of authentic erythrocyte enzymes. However, the possible range of experiments is seriously limited by the fact that mammalian erythrocytes lack DNA and mitochondria, thus eliminating many of the interesting problems of cellular and molecular biology We have therefore examined chicken erythrocytes which are nucleated and contain mitochondria to determine if it is possible to load these cells with foreign enzymes in a manner similar to mammalian erythrocytes. In this work we show that chicken erythrocytes can be loaded with exogenous proteins and that the resealed
Received June 30, 1976 Accepted October 15, 1976
’ To whom reprint requests should be addressed.
References
Exp CeNRes 104 (1977)