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Spread chromosomal nucleoli of Chironomus salivary glands
416
Preliminary
notes
not be excluded. However, findings in the syngenic mouse system [2] indicates that antibodies are not essential for the macrophage cytotoxicity [5,6]. The point cannot be proved completely until the human monocytes are demonstrated to act also on autologous red cells. It is interesting to note that the monocytes do not cause release of chromium from mouse red cells in the presence of human serum. It is obvious that this serum contains an opsonin which will stimulate an extensive phagocytosis of the red cells. Chromium is not released under these conditions. Probably isotope that may come loose during lysosomal digestion of the red cells after phagocytosis will subsequently be bound to intracellular monocyte proteins. A more extensive report of our finding is under preparation. References 1. Seljelid, R, Melsom, H & Munthe-Kaas, A, Exptl cell res 75 (1972) 534. 2. Melsom, H & Seljelid, R, J exptl med 137 (1973) 807. 3. Perlmann, P, Perlmann, H & Holm, G, Science 160 (1968) 308. 4. Melsom, H, Munthe-Kaas, A & Seljelid, R. Unpublished observations. 5. Holm, G, Clin exptl immunol 13 (1973) 29. 6. Moller, E, Immunology 16 (1969) 609.
(amplified) nucleoli of amphibian oocytes by electron microscopic techniques. The transcribing rDNA appears in spread preparations as deoxyribonucleoprotein (DNP) axes which are associated, in regular periods, with cascades of lateral, growing rRNP fibrils separated by free (‘spacer’) sections [4-6, 131. The morphological data obtained are in agreement with the corresponding biochemical and physicochemical determinations. Ribosomal RNA cistrons integrated into chromosomes have not yet been clearly identified. Although recently micrographs of spread preparations from total HeLa cell lysates have been presented from Miller’s laboratory which show similar, though not highly repeated, matrix units of laterally attached RNP fibrils on DNP axes [3]; this cannot be considered as a clear demonstration of intrachromosomal rRNA cistrons in transcription. We have chosen for our preparations nuclei of Chironomus salivary glands because (i) of the chromosomal polyteny, (ii) the large size of the nuclei and nucleoli which make them suitable for preparative manipulations, and (iii) the good background of knowledge concerning the nucleolar cytology and biochemistry of these cells (see, e.g., [9, lo]).
Received February 27, 1973 Materials
Spread chromosomal nucleoli of Chironomus salivary glands J. DERKSEN, M. F. TRENDELENBURG, U. SCHEER and W. W. FRANKE, Department of Genetics, Faculty of Sciences, University of Nijmegen, The Netherlands, and Department of Cell Biology, Institute of Biology II, University of Freiburg i.Br., Germany,
BRD
In eukaryotic cells the transcription of ribosomal DNA (rDNA), i.e. of the cistrons of pre-ribosomal RNA (pre-rRNA), has been directly visualized in the extrachromosomal Exptl Cell Res 80 (1973)
and Methods
Salivary glands of Chironomus thummi were prepared and transferred into a medium modified from Robert [ll] containing 0.087 N NaCl, 0.032 N KCI, 0.01 M EDTA and 0.01 M Tris-maleate. nH 6.3. The dissected glands were disrupted by syringmg and the liberated, crude nuclei were transferred into a drop of bidistilled water, adjusted to pH 9.0 with 0.01 M borate buffer and allowed to swell for 15 min. After swelling the cytoplasmic remnants were removed from the nuclei by syringing and the nuclei were transferred into a fresh drop of ‘pH 9-water’. The nuclei were disrupted by hand using watchmakers forceps, and were processed essentially according to the method described bv Miller & Beattv 141and Miller & Bakken f31 as modified by Schee; et k. [13]. Electron micrographs were made either in a Zeiss EM9 or a Siemens 101. and the magnification indicators were controlled using grating replicas.
Preliminary notes
471
Fig. I. Survey electron micrograph of putative transcribing rDNA in spread preparations of nuclei manually isolated from Chironomus thummi salivary glands. The inset shows, at higher magnification, the lateral fibrils
inserting at the DNP-axis with their terminal dense nodules. x 11 700: inset ): 56 000.
Results and Discussion Good nucleolar spreads are much harder to obtain from the salivary gland nuclear contents than with the amphibian oocytes (especially from urodela). This may be largely due to the greater amount of non-nucleolar material present. Fig. 1 shows a survey micrograph of what we interpret as a nucleolar spread. One recognizes DNP axes (magnified in fig. 2) which are for the most part associated with lateral fibrils. These fibrils exhibit at many sites the typical length gradients and reveal the separation of matrix units and ‘free’ segments, i.e. stretches not covered with
fibrils. The matrix units (some startpoints are indicated in fig. 2 by arrows, termini are denoted by bars) were of an average length of 2.2 pm with a relatively wide range (I .9 to 2.6 ,um). The free (‘spacer’) segments (for the divergent terminology of ‘spacer’ see [4, 8, 13, 141) appear to be very short (mean: 0.45 pm; range 0.15 to 0.68 pm). Some situations (e.g. fig. 2) suggest that lateral fibrils can also occur within the ‘spacer’ regions, similar to what has been observed in the extrachromosomal oocyte nucleoli of various amphibia [13]. The lateral fibrils are, when strongly expanded, approx. 150 A thick and, like Exptl Cell Res 80 (1973)
478
Preliminary notes
Fig. 2. Partial magnification from fig. 1 demonstrating matrix units separated by free axial segments. The short
arrows indicate startpoints of matrix units, the bars denote termini. The two long arrows point to long, stretched lateral RNP-fibrils. The double arrow points to a situation which might represent formation of rRNP fibrils within a ‘spacer’ region. x 19 000. -
those described for the amphibian extrachromosomal nucleoli by Miller & Beatty [5] frequently show terminal knobs. The maximal length of such fibrils (in particular those inserting in the terminal parts of matrix units) was measured as 1 pm. The identification of the lateral and axial structures as RNP and DNP, respectively, is still presumptive and based on morphological homology only: The structures observed have essentially the same characteristics as the spread transcription units of rDNA in the extrachromosomal amphibian oocyte nucleoli. Exptl
Cell Res 80 (1973)
They show only two components, namely the axial and the lateral fibrils, a repeat pattern of matrixcovered and non-covered regions, and a length gradient of the lateral fibrils within one matrix unit. The lengths of the matrix units, taken as double-stranded B-form DNA, could code for 1.8 to 2.5 million D RNA, i.e. values which approach the mol wts determined for the pre-rRNA of Chironomus tentans (2.6 x lo6 D, [12]; 3.3 x IO6 D, [9]) and other diptera [2, 71. From the structural homology and from the agreement of the morphological and biochemical data we con-
Preliminary notes 419 elude that our micrographs present the transcribing rDNA of the nucleoli contained in the polytene chromosomes of the Chironomus salivary glands. We have to emphasize, however, that the relative length of free (‘spacer’) axis is much below the corresponding values found in amphibian oocytes [4, 5, 131and in the HeLa cells [I, 31. We thank Dr H. D. Berendes, University of Nijmegen, and Dr H. Falk, University of Freiburg, for various support and Miss Marianne Winter for skilful technical assistance. The study received financial support from the EMBO (short-term fellowship awarded to J. D.) and the Deutsche Forschungsgemeinschaft, SFB 46 (grant awarded to W. W. F.).
References 1. Bakken, A H & Miller, 0 L, J cell biol 55 (1972) 12a. 2. Dalgarno, L, Hosking, D M & Shen, C H, Eur j biochem 24 (1972) 498. 3. Miller, 0 L & Bakken, A H, Acta endocrinol, suppl. 168 (1972) 155. 4. Miller, 0 L & Beatty, B R, Science 164 (1969) 955. 5. - Genetics, suppl. 61 (1969) 134. 6. Miller, 0 L & Hamkalo B A, Int rev cytol 33
(1972) 1. I. Perry, R P, Cheng, T-Y, Freed, J J, Greenberg,
J R. Kellev. D E & Tartof. K D. Proc natl acad sci US 65 ii970) 609. 8. Reeder. R H & Brown./ D D. J mol biol 51 (1970)
361. ' 9. Ringborg, U, Daneholt, B, Edstrom, J-E, Egyhazi,
E & Lambert, B, J mol biol 51 (1970) 327. 10. Ringborg, U & Ryd!ander, L, J cell biol 5 I (1971) 355.
Il. Robert, M, Chromosoma 36 (1971) 1. 12. Rubinstein, L & Clever, U, Biochim biophys acta 246 (1971) 517.
13. Scheer, U; Trendelenburg, M F & Franke, W W, Exptl cell res 80 (1973) 175. 14. Wensink, P C & Brown, D D, J mol biol60 (1971) 235.
Received March 5, 1973 Revised version received May 14, 1973
On the nature and function of yellow aging pigment lipofuscin V. N. KARNAUKHOV, Institute of Biological Physits of the Academy of Sciences of USSR, Pushchino, Moscow Region, USSR, 142292 Summavy. A comparison of lipofuscin granules from
warm-blooded animals and carotenoid-containing granules (cytosomes) from molluscoid neurons was carried out. It was confirmed that the carotenoids are a component of the lipofuscin granules. Data in literature and the experimental data obtained showed that the liuofuscin granules contained carotenoids, myoglobin and some-respiratory enzymes. On the basis of identification of the properties of carotenoid-containing granules (cytosomes) of molluscoid neurones and the lipofuscin granules, it is proposed that the functions of the lipofuscin are those of forming the intracellular oxygen stock and providing the energy requirements of the cells under the conditions of low rate oxygen penetration into tissues.
It has been shown that carotenoids, in common with myoglobin, take part in the oxidative metabolism of molluscoid neurons [l-3]. In the cells the carotenoids and myoglobin are localized in specific granules [4], named cytosomes [5, 61. The cytosomes contain some respiratory enzymes [5, 61 and have the ability of energy-dependent accumulation of SPf under hypoxic conditions [7, 81. Based on the experimental data obtained, it was suggested that in common with myoglobin the carotenoids, having a large number of unsaturated double bonds in their molecules, can function as intracellular oxygen stock [2, 31 and the cytosomes can provide the energy required by the cell under the conditions of low-rate oxygen penetration into tissues [3, 41. When comparing the ultrastructural organization of the molluscoid cytosomes (fig. 1) and lipofuscin granules of warm-blooded animals, we were led to assume that there was a close similarity between the chemical composition and physiological function of these intracellular organoids [9, 10, 111. A considerable oxygen uptake was found [13] in the lipofuscin granule fractions [12]. Exptl Cell Res 80 (1973)
Preliminary
notes
not be excluded. However, findings in the syngenic mouse system [2] indicates that antibodies are not essential for the macrophage cytotoxicity [5,6]. The point cannot be proved completely until the human monocytes are demonstrated to act also on autologous red cells. It is interesting to note that the monocytes do not cause release of chromium from mouse red cells in the presence of human serum. It is obvious that this serum contains an opsonin which will stimulate an extensive phagocytosis of the red cells. Chromium is not released under these conditions. Probably isotope that may come loose during lysosomal digestion of the red cells after phagocytosis will subsequently be bound to intracellular monocyte proteins. A more extensive report of our finding is under preparation. References 1. Seljelid, R, Melsom, H & Munthe-Kaas, A, Exptl cell res 75 (1972) 534. 2. Melsom, H & Seljelid, R, J exptl med 137 (1973) 807. 3. Perlmann, P, Perlmann, H & Holm, G, Science 160 (1968) 308. 4. Melsom, H, Munthe-Kaas, A & Seljelid, R. Unpublished observations. 5. Holm, G, Clin exptl immunol 13 (1973) 29. 6. Moller, E, Immunology 16 (1969) 609.
(amplified) nucleoli of amphibian oocytes by electron microscopic techniques. The transcribing rDNA appears in spread preparations as deoxyribonucleoprotein (DNP) axes which are associated, in regular periods, with cascades of lateral, growing rRNP fibrils separated by free (‘spacer’) sections [4-6, 131. The morphological data obtained are in agreement with the corresponding biochemical and physicochemical determinations. Ribosomal RNA cistrons integrated into chromosomes have not yet been clearly identified. Although recently micrographs of spread preparations from total HeLa cell lysates have been presented from Miller’s laboratory which show similar, though not highly repeated, matrix units of laterally attached RNP fibrils on DNP axes [3]; this cannot be considered as a clear demonstration of intrachromosomal rRNA cistrons in transcription. We have chosen for our preparations nuclei of Chironomus salivary glands because (i) of the chromosomal polyteny, (ii) the large size of the nuclei and nucleoli which make them suitable for preparative manipulations, and (iii) the good background of knowledge concerning the nucleolar cytology and biochemistry of these cells (see, e.g., [9, lo]).
Received February 27, 1973 Materials
Spread chromosomal nucleoli of Chironomus salivary glands J. DERKSEN, M. F. TRENDELENBURG, U. SCHEER and W. W. FRANKE, Department of Genetics, Faculty of Sciences, University of Nijmegen, The Netherlands, and Department of Cell Biology, Institute of Biology II, University of Freiburg i.Br., Germany,
BRD
In eukaryotic cells the transcription of ribosomal DNA (rDNA), i.e. of the cistrons of pre-ribosomal RNA (pre-rRNA), has been directly visualized in the extrachromosomal Exptl Cell Res 80 (1973)
and Methods
Salivary glands of Chironomus thummi were prepared and transferred into a medium modified from Robert [ll] containing 0.087 N NaCl, 0.032 N KCI, 0.01 M EDTA and 0.01 M Tris-maleate. nH 6.3. The dissected glands were disrupted by syringmg and the liberated, crude nuclei were transferred into a drop of bidistilled water, adjusted to pH 9.0 with 0.01 M borate buffer and allowed to swell for 15 min. After swelling the cytoplasmic remnants were removed from the nuclei by syringing and the nuclei were transferred into a fresh drop of ‘pH 9-water’. The nuclei were disrupted by hand using watchmakers forceps, and were processed essentially according to the method described bv Miller & Beattv 141and Miller & Bakken f31 as modified by Schee; et k. [13]. Electron micrographs were made either in a Zeiss EM9 or a Siemens 101. and the magnification indicators were controlled using grating replicas.
Preliminary notes
471
Fig. I. Survey electron micrograph of putative transcribing rDNA in spread preparations of nuclei manually isolated from Chironomus thummi salivary glands. The inset shows, at higher magnification, the lateral fibrils
inserting at the DNP-axis with their terminal dense nodules. x 11 700: inset ): 56 000.
Results and Discussion Good nucleolar spreads are much harder to obtain from the salivary gland nuclear contents than with the amphibian oocytes (especially from urodela). This may be largely due to the greater amount of non-nucleolar material present. Fig. 1 shows a survey micrograph of what we interpret as a nucleolar spread. One recognizes DNP axes (magnified in fig. 2) which are for the most part associated with lateral fibrils. These fibrils exhibit at many sites the typical length gradients and reveal the separation of matrix units and ‘free’ segments, i.e. stretches not covered with
fibrils. The matrix units (some startpoints are indicated in fig. 2 by arrows, termini are denoted by bars) were of an average length of 2.2 pm with a relatively wide range (I .9 to 2.6 ,um). The free (‘spacer’) segments (for the divergent terminology of ‘spacer’ see [4, 8, 13, 141) appear to be very short (mean: 0.45 pm; range 0.15 to 0.68 pm). Some situations (e.g. fig. 2) suggest that lateral fibrils can also occur within the ‘spacer’ regions, similar to what has been observed in the extrachromosomal oocyte nucleoli of various amphibia [13]. The lateral fibrils are, when strongly expanded, approx. 150 A thick and, like Exptl Cell Res 80 (1973)
478
Preliminary notes
Fig. 2. Partial magnification from fig. 1 demonstrating matrix units separated by free axial segments. The short
arrows indicate startpoints of matrix units, the bars denote termini. The two long arrows point to long, stretched lateral RNP-fibrils. The double arrow points to a situation which might represent formation of rRNP fibrils within a ‘spacer’ region. x 19 000. -
those described for the amphibian extrachromosomal nucleoli by Miller & Beatty [5] frequently show terminal knobs. The maximal length of such fibrils (in particular those inserting in the terminal parts of matrix units) was measured as 1 pm. The identification of the lateral and axial structures as RNP and DNP, respectively, is still presumptive and based on morphological homology only: The structures observed have essentially the same characteristics as the spread transcription units of rDNA in the extrachromosomal amphibian oocyte nucleoli. Exptl
Cell Res 80 (1973)
They show only two components, namely the axial and the lateral fibrils, a repeat pattern of matrixcovered and non-covered regions, and a length gradient of the lateral fibrils within one matrix unit. The lengths of the matrix units, taken as double-stranded B-form DNA, could code for 1.8 to 2.5 million D RNA, i.e. values which approach the mol wts determined for the pre-rRNA of Chironomus tentans (2.6 x lo6 D, [12]; 3.3 x IO6 D, [9]) and other diptera [2, 71. From the structural homology and from the agreement of the morphological and biochemical data we con-
Preliminary notes 419 elude that our micrographs present the transcribing rDNA of the nucleoli contained in the polytene chromosomes of the Chironomus salivary glands. We have to emphasize, however, that the relative length of free (‘spacer’) axis is much below the corresponding values found in amphibian oocytes [4, 5, 131and in the HeLa cells [I, 31. We thank Dr H. D. Berendes, University of Nijmegen, and Dr H. Falk, University of Freiburg, for various support and Miss Marianne Winter for skilful technical assistance. The study received financial support from the EMBO (short-term fellowship awarded to J. D.) and the Deutsche Forschungsgemeinschaft, SFB 46 (grant awarded to W. W. F.).
References 1. Bakken, A H & Miller, 0 L, J cell biol 55 (1972) 12a. 2. Dalgarno, L, Hosking, D M & Shen, C H, Eur j biochem 24 (1972) 498. 3. Miller, 0 L & Bakken, A H, Acta endocrinol, suppl. 168 (1972) 155. 4. Miller, 0 L & Beatty, B R, Science 164 (1969) 955. 5. - Genetics, suppl. 61 (1969) 134. 6. Miller, 0 L & Hamkalo B A, Int rev cytol 33
(1972) 1. I. Perry, R P, Cheng, T-Y, Freed, J J, Greenberg,
J R. Kellev. D E & Tartof. K D. Proc natl acad sci US 65 ii970) 609. 8. Reeder. R H & Brown./ D D. J mol biol 51 (1970)
361. ' 9. Ringborg, U, Daneholt, B, Edstrom, J-E, Egyhazi,
E & Lambert, B, J mol biol 51 (1970) 327. 10. Ringborg, U & Ryd!ander, L, J cell biol 5 I (1971) 355.
Il. Robert, M, Chromosoma 36 (1971) 1. 12. Rubinstein, L & Clever, U, Biochim biophys acta 246 (1971) 517.
13. Scheer, U; Trendelenburg, M F & Franke, W W, Exptl cell res 80 (1973) 175. 14. Wensink, P C & Brown, D D, J mol biol60 (1971) 235.
Received March 5, 1973 Revised version received May 14, 1973
On the nature and function of yellow aging pigment lipofuscin V. N. KARNAUKHOV, Institute of Biological Physits of the Academy of Sciences of USSR, Pushchino, Moscow Region, USSR, 142292 Summavy. A comparison of lipofuscin granules from
warm-blooded animals and carotenoid-containing granules (cytosomes) from molluscoid neurons was carried out. It was confirmed that the carotenoids are a component of the lipofuscin granules. Data in literature and the experimental data obtained showed that the liuofuscin granules contained carotenoids, myoglobin and some-respiratory enzymes. On the basis of identification of the properties of carotenoid-containing granules (cytosomes) of molluscoid neurones and the lipofuscin granules, it is proposed that the functions of the lipofuscin are those of forming the intracellular oxygen stock and providing the energy requirements of the cells under the conditions of low rate oxygen penetration into tissues.
It has been shown that carotenoids, in common with myoglobin, take part in the oxidative metabolism of molluscoid neurons [l-3]. In the cells the carotenoids and myoglobin are localized in specific granules [4], named cytosomes [5, 61. The cytosomes contain some respiratory enzymes [5, 61 and have the ability of energy-dependent accumulation of SPf under hypoxic conditions [7, 81. Based on the experimental data obtained, it was suggested that in common with myoglobin the carotenoids, having a large number of unsaturated double bonds in their molecules, can function as intracellular oxygen stock [2, 31 and the cytosomes can provide the energy required by the cell under the conditions of low-rate oxygen penetration into tissues [3, 41. When comparing the ultrastructural organization of the molluscoid cytosomes (fig. 1) and lipofuscin granules of warm-blooded animals, we were led to assume that there was a close similarity between the chemical composition and physiological function of these intracellular organoids [9, 10, 111. A considerable oxygen uptake was found [13] in the lipofuscin granule fractions [12]. Exptl Cell Res 80 (1973)