- Email: [email protected]
Correlation of Light and Electron Micrographs of Human Metaphase Chromosomes after Incorporation of BUdR and Staining with “33258 Hoechst” and Giemsa
Exp. Path., Bd. 10, S. 220-224 (1975) Institute for Cancer Research, University of Vienna, Vienna, Austria (Head: Prof. DDr. H. WRBA) and University of Münster, Institute for Human Genetics
8hort communication
Correlation of Light and Electron Micrographs of Human Metaphase Chromosomes after Incorporation of BUdR and Staining with "33258 Hoechst" and Giemsa By l\1. BINDER and M. A. KIM With 3 figures (Rereived October 31, 1974) Key-words: Electron microscopy; light microscopy; human metaphase chromosomes; BUdRinrorporation; "33258 Hoechst"; Giemsa
Summary Stained (Giemsa, "33258 Hoechst"l) and "33258 Hoechst" + Giemsa) and unstained metaphase chromosomes from human peripherallymphocytes, after two rounds of replication in the presence of 5-bromodeoxyuridine (BUdR), have been prepared for electron microscopy. There is a positive correlation between light and electron micrographs. The same differential contrast on electron micrographs has been obtained whether the preparations have been stained or not. We attribute this differential contrast primarily to the lesser condensation of the bifilarly substituted chromatid.
Recently a variety of papers have been published dealing with the illcorporation of halogenated base analoques into the DNA of mammalian chromosomes and the subsequent application of various staining procedures(1-6). These all aim at the differential presentation of si ster chromatids and the possibility of demonstrating sister chromatid exehanges. In the present work we have investigated this phenomenon on the eleetronmicroscopic level. We report that in human peripheral lymphoeytes grown for two rounds of replication in a medium containing BUdR, sister chromatids ean exhibit differential contrast on electron micrographs even without subsequent stainillg. Furthermore we compare light micrographs with electron micrographs of chromosome preparations that have been stained after BUdR-incorporation. The staining procedures used were a) Giemsa and b) "33258 Hoechst" alld Giemsa. Finally the possible cause of differential staining is discussed.
JJ aterial and methods Human peripheral lymphocytes were cultured according to the technique by FISCHER and KIM (1). This consisted in short, of pretreating air dried BUdR-treated preparations with "33258 Hoechst", followed by staining with Giemsa. Additionally we used BUdR-treated preparations stained with either Giemsa or "33258 Hoechst" and also BUdR-treated unstained preparations. Slides were coated with a carbon film of about 200 A thickness and areas with suitable metaphase spreads selected under the light microscope and edged with a circular marking devise (REICHERT). Carbon films with attached spreads were then floated onto the surface of dilute hydrofluoric acid 1) "33258 Hoechst" benzimidazol . 3 HOl].
220
=
[2-[2-( 4-hydroxyphenyl)-6-benzimidazolyl]-6-(1-methyl-4-piperazyl)-
I
-a
b
c
d
Fig. la. Light mieroscope preparation of metaphase in second division after "BUdR-33258 HoechstGiemsa" treatment Note differential staining of sister ehromatids. Magnification: X 2,000. Fig. 1 b, 1 c, 1 d. Whole mount electron microscope preparations of the same metaphase as above (arrows fig. 1a). Magnification: X 7,700. b: B group chromosomes with no aberrations. c: Number two chromosome with an interstitial sister chromatid exchange. d: Chromosome number three with a terminal sister chromatid exchange.
15
Exp. Path. Bd. 10, H. 3/4
221
•
a
b
c
d
Fig.2a. Light mieroscope preparation of metaphase in second division after "BUdR-Giemsa" treatment. Note differential staining of sister chromatids. Magnification x 2,200. Fig. 2 b, 2 c, 2 d. Wh oie mount electron microscope preparation of the same metaphase as above (arrows fig. 2a). Ylagnification X 7,700.
222
a
c
b
d
Fig. 3 a, 3 b. Whole mount electron mirroscope prep
Results and discussion Image formation in light microscopy is quite different to that of electron microscopy. In th e latter, the scattering power, and thus the contrast of a particular area of a specimen 15*
223
is directly proportional to its mass density (mass per unit arca = produ<,t of density and thickness). From this one might perhaps expcet different results in eleetron microscopy. But, as ean be seen from the fig. 1 and 2, there is a positive correlation between the light and the electron micrographs irrespective of the stain used (Giemsa or "33258 Ho()chst" and Giemsa). Furthermorc differential contrast is also apparent without staining on electron micrographs (figs. 3 c, 3 d). Staining with dyes for light microscopy obviously does not enhance the mass density of a chromatid and henee the contrast of the electron micrograph will not show any appreciable difference. From this, it could be possiblc that one would not reeognize a differential uptake of dye. In view of all these facts we tend to assume that the differential staining observed in light microscopy is primarily caused by a ('hange in mass dcnsity prior to staining due to BUdR-incorporation. This is in good agreement with ZAKHAROV'S concept of delaycd spiralization (8).
Literature 1. IKUSlIIM.\, T., and S. WOLFF, Sister rhromatid exchanges induced by lightflashes to 5-bromodeoxyuridine- and 5-iododeoxyuridine substituted Chinese hamster ehromosomes. Exp. Cell Res. 81, 15-19 (1974). 2. FISCHER, P., and M. A. KIM, Technique for the visualization of exchange aberrations in human chromosomes. Exp. Path. 10, 216-219 (1975). 3. KATO, H., Spontaneous sisterchromatid exehanges deteeted by a BUdR-labelling method. Nature 251, 70--72 (1974). 4. Kn!, M. A., Chromatid exchange amI heteroehromatin alteration of human chromosomes with BUdR-Iabelling. - Demonstrated by Benzimidazolflnorochrome anel Giemsa-stain. Humangenetik (1974), in the press. 5. LATT, S. A., Microfluorometrie detection of deoxyribonudeie acid replieat.ion in human metaphase chromosomes. Proc. Nat. Acad. Sei. USA 10, 3395--3399 (Hl73). 6. PERHY, P., and S. WOLF, New Giemsa method for the differential staining of sister ehromatids. Nature 251, 156-158 (1974). 7. RUZICKA, F., Einfa('hes Verfahren zur D,ustellung von humanen :\Ietaphasenplatten für das Elektronenmikroskop. Humangenetik 13, 199-204 (1971). 8. ZAKHAROV, A. F., and ;'{. A. EGOLINA, Differential spirali7.ation along mammalian mitotie chromosomes. Chromosoma 38, 341-·3G5 (1972).
Authors' addresses: Dr. 11. BINDER, Institute for Cancer Research, University of Vienna, Borschkegasse 8a, A -1090 Vienna (Austria); Dr. M. A. KIM, University of l\fünster, Institute for Human Genetics, Vesaliusweg 12/14, Münster.
Verantwortlich für die Redaktion: Prof. Dr. F. Bolck, 69 .Tena. Verlag: VEB Gustav Fischer Verlag, 69 Jena, Villengang 2. Telefon 24141, 24142. Alleinige Anzeigenannahme: DEWAG-Werbung Leipzig, 701 Leipzig, Brühl 34-40, Telefon 2 97.40. Für Ausland,.,anzeigen: Interwerbung GmbH, DI:!R -104 Berlin, Tueholskystraße 40, Postlach .230. Satz und Druck: Druckere! "Magnus Poser , 69 Jena. - Veröffentlicht unter LIzenznummer 1546 des Presseamtes beIm Vorsitzenden des MImsterrates der Deutschen Demokratischen Republik. Alle Rechte beim Verlag. Nachdruck (auch auszugsweise) nur mit Genehmigung des Verlages und des Verlassers sowie mit Angabe der Quelle gestattet. Printed in the German Democratic Republic.
224
Tafell
Exp. Path. Dd. 10, H. 3/4
+
I
I
M M
I
M
A
Mi ts chek , IV. Mitt. VEB GUSTAV FISCHER VERLAG JENA
8hort communication
Correlation of Light and Electron Micrographs of Human Metaphase Chromosomes after Incorporation of BUdR and Staining with "33258 Hoechst" and Giemsa By l\1. BINDER and M. A. KIM With 3 figures (Rereived October 31, 1974) Key-words: Electron microscopy; light microscopy; human metaphase chromosomes; BUdRinrorporation; "33258 Hoechst"; Giemsa
Summary Stained (Giemsa, "33258 Hoechst"l) and "33258 Hoechst" + Giemsa) and unstained metaphase chromosomes from human peripherallymphocytes, after two rounds of replication in the presence of 5-bromodeoxyuridine (BUdR), have been prepared for electron microscopy. There is a positive correlation between light and electron micrographs. The same differential contrast on electron micrographs has been obtained whether the preparations have been stained or not. We attribute this differential contrast primarily to the lesser condensation of the bifilarly substituted chromatid.
Recently a variety of papers have been published dealing with the illcorporation of halogenated base analoques into the DNA of mammalian chromosomes and the subsequent application of various staining procedures(1-6). These all aim at the differential presentation of si ster chromatids and the possibility of demonstrating sister chromatid exehanges. In the present work we have investigated this phenomenon on the eleetronmicroscopic level. We report that in human peripheral lymphoeytes grown for two rounds of replication in a medium containing BUdR, sister chromatids ean exhibit differential contrast on electron micrographs even without subsequent stainillg. Furthermore we compare light micrographs with electron micrographs of chromosome preparations that have been stained after BUdR-incorporation. The staining procedures used were a) Giemsa and b) "33258 Hoechst" alld Giemsa. Finally the possible cause of differential staining is discussed.
JJ aterial and methods Human peripheral lymphocytes were cultured according to the technique by FISCHER and KIM (1). This consisted in short, of pretreating air dried BUdR-treated preparations with "33258 Hoechst", followed by staining with Giemsa. Additionally we used BUdR-treated preparations stained with either Giemsa or "33258 Hoechst" and also BUdR-treated unstained preparations. Slides were coated with a carbon film of about 200 A thickness and areas with suitable metaphase spreads selected under the light microscope and edged with a circular marking devise (REICHERT). Carbon films with attached spreads were then floated onto the surface of dilute hydrofluoric acid 1) "33258 Hoechst" benzimidazol . 3 HOl].
220
=
[2-[2-( 4-hydroxyphenyl)-6-benzimidazolyl]-6-(1-methyl-4-piperazyl)-
I
-a
b
c
d
Fig. la. Light mieroscope preparation of metaphase in second division after "BUdR-33258 HoechstGiemsa" treatment Note differential staining of sister ehromatids. Magnification: X 2,000. Fig. 1 b, 1 c, 1 d. Whole mount electron microscope preparations of the same metaphase as above (arrows fig. 1a). Magnification: X 7,700. b: B group chromosomes with no aberrations. c: Number two chromosome with an interstitial sister chromatid exchange. d: Chromosome number three with a terminal sister chromatid exchange.
15
Exp. Path. Bd. 10, H. 3/4
221
•
a
b
c
d
Fig.2a. Light mieroscope preparation of metaphase in second division after "BUdR-Giemsa" treatment. Note differential staining of sister chromatids. Magnification x 2,200. Fig. 2 b, 2 c, 2 d. Wh oie mount electron microscope preparation of the same metaphase as above (arrows fig. 2a). Ylagnification X 7,700.
222
a
c
b
d
Fig. 3 a, 3 b. Whole mount electron mirroscope prep
Results and discussion Image formation in light microscopy is quite different to that of electron microscopy. In th e latter, the scattering power, and thus the contrast of a particular area of a specimen 15*
223
is directly proportional to its mass density (mass per unit arca = produ<,t of density and thickness). From this one might perhaps expcet different results in eleetron microscopy. But, as ean be seen from the fig. 1 and 2, there is a positive correlation between the light and the electron micrographs irrespective of the stain used (Giemsa or "33258 Ho()chst" and Giemsa). Furthermorc differential contrast is also apparent without staining on electron micrographs (figs. 3 c, 3 d). Staining with dyes for light microscopy obviously does not enhance the mass density of a chromatid and henee the contrast of the electron micrograph will not show any appreciable difference. From this, it could be possiblc that one would not reeognize a differential uptake of dye. In view of all these facts we tend to assume that the differential staining observed in light microscopy is primarily caused by a ('hange in mass dcnsity prior to staining due to BUdR-incorporation. This is in good agreement with ZAKHAROV'S concept of delaycd spiralization (8).
Literature 1. IKUSlIIM.\, T., and S. WOLFF, Sister rhromatid exchanges induced by lightflashes to 5-bromodeoxyuridine- and 5-iododeoxyuridine substituted Chinese hamster ehromosomes. Exp. Cell Res. 81, 15-19 (1974). 2. FISCHER, P., and M. A. KIM, Technique for the visualization of exchange aberrations in human chromosomes. Exp. Path. 10, 216-219 (1975). 3. KATO, H., Spontaneous sisterchromatid exehanges deteeted by a BUdR-labelling method. Nature 251, 70--72 (1974). 4. Kn!, M. A., Chromatid exchange amI heteroehromatin alteration of human chromosomes with BUdR-Iabelling. - Demonstrated by Benzimidazolflnorochrome anel Giemsa-stain. Humangenetik (1974), in the press. 5. LATT, S. A., Microfluorometrie detection of deoxyribonudeie acid replieat.ion in human metaphase chromosomes. Proc. Nat. Acad. Sei. USA 10, 3395--3399 (Hl73). 6. PERHY, P., and S. WOLF, New Giemsa method for the differential staining of sister ehromatids. Nature 251, 156-158 (1974). 7. RUZICKA, F., Einfa('hes Verfahren zur D,ustellung von humanen :\Ietaphasenplatten für das Elektronenmikroskop. Humangenetik 13, 199-204 (1971). 8. ZAKHAROV, A. F., and ;'{. A. EGOLINA, Differential spirali7.ation along mammalian mitotie chromosomes. Chromosoma 38, 341-·3G5 (1972).
Authors' addresses: Dr. 11. BINDER, Institute for Cancer Research, University of Vienna, Borschkegasse 8a, A -1090 Vienna (Austria); Dr. M. A. KIM, University of l\fünster, Institute for Human Genetics, Vesaliusweg 12/14, Münster.
Verantwortlich für die Redaktion: Prof. Dr. F. Bolck, 69 .Tena. Verlag: VEB Gustav Fischer Verlag, 69 Jena, Villengang 2. Telefon 24141, 24142. Alleinige Anzeigenannahme: DEWAG-Werbung Leipzig, 701 Leipzig, Brühl 34-40, Telefon 2 97.40. Für Ausland,.,anzeigen: Interwerbung GmbH, DI:!R -104 Berlin, Tueholskystraße 40, Postlach .230. Satz und Druck: Druckere! "Magnus Poser , 69 Jena. - Veröffentlicht unter LIzenznummer 1546 des Presseamtes beIm Vorsitzenden des MImsterrates der Deutschen Demokratischen Republik. Alle Rechte beim Verlag. Nachdruck (auch auszugsweise) nur mit Genehmigung des Verlages und des Verlassers sowie mit Angabe der Quelle gestattet. Printed in the German Democratic Republic.
224
Tafell
Exp. Path. Dd. 10, H. 3/4
+
I
I
M M
I
M
A
Mi ts chek , IV. Mitt. VEB GUSTAV FISCHER VERLAG JENA