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Retention of tritiated thymidine in grasshopper neuroblasts
201 RETENTION
OF TRITIATED
GRASSHOPPER
THYMIDINE
IN
NEUROBLASTS
W. M. LEACH Institute
of Radiation Biology, Department of Zoology and Entomology, The University of Tennessee, Knoxville, Term., U.S.A. Received April 1, 1964
Intracellular retention of thymidine and/or its derivatives other than deoxyribonucleic acid (DNA) has been demonstrated in sea urchin eggs [I] and the bacterium Alcaligenes fecalis [5]. This study was undertaken to determine whether tritiated thymidine was retained in grasshopper neuroblasts that were exposed to this precursor during a period of no DNA synthesis. Living neuroblasts can be observed by bright-field microscopy, all stages of the mitotic cycle can be identified, duration of the stages of the cell cycle is known [2], and the period of DNA synthesis has been established in relation to the cell cycle [4, 71. Materials and methods.-Embryos of the grasshopper Chortophaga viridifasciata (De Geer) at an age equivalent to 13-14 days of development at 26°C were removed from eggs and freed from surrounding membranes and yolk in Shaw’s [9] culture medium. Embryos were incubated at 26°C in culture medium which contained tritiated thymidine (3H-TdR)1 at concentrations of 1 or 10 PC/ml. Hanging drop preparations were made and neuroblasts in selected mitotic stages were mapped for subsequent reidentification in sections (techniques reviewed by Carlson and Gaulden [3]). Mitotic progress of cells was observed by bright-field microscopy. At the end of the observation period tissue was fixed for 5 min in 50 per cent acetic acid, dehydrated and embedded in paraffin. Sections 5 p thick were cut and autoradiograms were prepared [8]. Silver grains were counted in emulsion over sections of neuroblasts. Additional technical details are given as necessary in the text. Results and conclusions.-The following experiment was done to test the possibility that 3H-TdR may enter and be retained within neuroblasts which were not in a period of DNA synthesis. Embryos were incubated for 20 min in culture medium which contained 3H-TdR, then rinsed 5 min in medium which contained unlabeled thymidine (100 times more concentrated than the 3H-TdR), then transferred to medium without thymidine for 10 min, then again to medium without thymidine for approximately 1 min. Neuroblasts in metaphase and anaphase were mapped within 10 min after hanging drop preparations were made. Cells were observed until they reached late telophase or interphase (see Fig. 1 A). Tissue was fixed and autoradiograms were prepared. The number of grains over reidentified neuroblast nuclei is shown in Fig. 2. These data show that neuroblasts exposed to 3H-TdR when DNA synthesis was not 1 Tritiated thymidine was obtained from New England Nuclear Corp. at specific activities of 0.5 to 5.4 C/mM. Samples of each lot were tested for radioactive contamination by ascending chromatography (n-butanol : water, 86 : 14) followed by gas flow counting. Lots with more than 5 per cent contamination were purified chromatographically. Experimental
Cell Rt%nrch
35
W. M. Leach
202
in progress incorporate 3H into DNA during the subsequent DNA synthetic peri0d.l In order to determine whether neuroblasts could incorporate 3H into DNA before the start of DNA synthesis, the previous experiment was repeated with the following modifications: 5 min after hanging drop preparations were made neuroblasts in
Fig. l.-Relative duration of cell stages of the grasshopper neuroblast at 26°C. P, time during which embryos were incubated in 3H-TdR. C, Time during which 3H-TdR was “chased” with excess unlabeled TdR. S, Period of DNA synthesis [5, 81. EP, Early prophase; MP, middle prophase; LP, late prophase; VLP, very late prophase; lzI, metaphase; A, anaphase; El’, early telophase; Xl T, middle telophase; L T, late telophase; IN T, interphase; VEP, very early prophase. A, Embryos fixed in late telophase or interphase after end of incubation in 3H-TdR. B, Embryos fixed in metaphase at end of incubation in 3H-TdR. Total time of cell cycle at 26°C about 8 hr. Relative duration of cell stages and time of complete cell cycle from Carlson [2].
middle prophase were identified and mapped. Mapped cells were timed to metaphase. Tissue was fixed and autoradiograms were prepared (see Fig. 1 B). Mean grain number with 95 per cent confidence limits, over neuroblasts reidentified in autoradiograms was 1.3 k 0.7. Mean grain number, with 95 per cent confidence limits, of areas (380~2) of emulsion not over tissue was 1.0 +0.6. Since neuroblasts exposed to 3H-TdR between middle prophase and metaphase were not labeled it is clear that incorporation of 3H into DNA does not occur under these experimental conditions. The average time from middle prophase to metaphase, with 95 per cent confidence limits, was 94 26 min at 26°C. The interval between the start of incubation in 3H-TdR and mapping of cells in the previous experiment was less than 50 min, therefore neuroblasts mapped initially in metaphase or anaphase were well into, or beyond, middle prophase at the start of incubation. The following experiment was designed to test whether sufficient 3H-TdR was transferred through rinse (or “chase”) solutions to account for the observed labeling. Embryos were incubated in used “chase” solutions for 40-120 min. Hanging drop preparations were made and maintained for approximately 4 hr. Embryos were fixed and autoradiograms were prepared. Silver grains were scored over sections of neuroblasts in interphase and very early prophase. Grains over randomly selected tissue were scored within an area of 380 p2 at the plane of the image. Background grain numbers were estimated by grain counts per 380 p2 over randomly selected areas of emulsion near, but not over, tissue. These data are presented in Fig. 3. About 60 1 Before application of emulsion two preparations were treated with DNase for 2 hr at 38°C. Nuclei in these digested preparations were Feulgen negative, and were unlabeled. Experimental
Cell Research 35
Retention of tritiated thymidine in grasshopper neuroblasts
203
per cent of grain numbers were less than 2. From these data it was concluded that transfer of 3H-TdR in rinse solutions was negligible. The following points stand out as results of this study: (1) 3H-TdR which entered cells during a period of no DNA synthesis may be incorporated into DNA during a subsequent period of DNA synthesis; and (2) solutions which contained excess
..l
I-,
IL Fig. 2.
Fig. Z.--Grain numbers over nuclei of neuroblasts incubated in 3H-TdH during a period of no DNA synthesis plotted against concentration of isotope. Tissue was fixed after selected cells Ninety-five per cent confidence intervals on entered DNA synthesis. Means indicated by -. means indicated by vertical lines. Emulsion exposure time, 1 pC/ml, 10 days; 10 $/ml, 5 days. Fig. 3.-Distribution of grain numbers in autoradiograms of embryos incubated in used “chase” solutions. A, Over neuroblasts in interphase and very early prophase; H, over randomly selected tissue; C, in emulsion near, but not over, tissue. Emulsion exposure time, 10 days. See text for additional details.
unlabeled thymidine were not effective in removing all unincorporated 3H-TdR derivatives from neuroblasts. In experiments similar to those of Taylor et al. [lo] autoradiograms were prepared of neuroblasts which were approaching the second cytokinesis after end of incubation in 3H-TdR during a period of DNA synthesis. Chromosomes of these cells were labeled in cytologically identical regions of both chromatids [6]. This result may reflect capacities of neuroblasts to retain 3H-TdR between periods of DNA synthesis and to utilize its derivatives during DNA synthesis. The comments and criticisms of Drs J. G. Carlson, M. E. Gaulden, R. A. McGrath, D. M. Prescott and R. B. Setlow and discussions with them of various aspects of this work are gratefully acknowledged. This study was supported in part by the Atomic Energy Commission under Contract No. AT-(40-I)-2575. Experimentnl
Cell Reseurch 35
M. De Vincentiis, F. Salvatore and V. Zappia REFERENCES 1. BUCHER, N. L. R. and MAZIA, 1)., J. Biophys. Biochem. Cytol. 7, 651 (1960). 2. CARLSON, J. G., Cold Spring Harbor Symp. Quant. Biol. 9, 104 (1941). 3. CARLSON, J. G. and GAULDEN, M. E., in PRESCOTT, D. M. (ed.) Methods in Cell Physiology. Academic Press, New York. 1964. 4. GAULDEN, M. E., Genetics 41, 645 (1956). 5. LARK, K. G., Biochim. Biophys. Acta 51, 107 (1961). 6. LEACH, W. M., Ass. Southeast. Biol. Bull. 10, 32 (1963). 7. MCGRATH, R. A., Rudiation Res. 19, 526 (1963). 8. MCGRATH, R. A., LEACH, W. M. and CARLSON, J. G., J. Roy. ;Ilicroscop. Sot. 82, 55 (1963). 9. SHAW, E. I., Exptl Cell Res. 11, 580 (1956). 10. TAYLOR, J. H., WOODS, P. S. and HUGHES, W. L., Proc. AT&l. Acad. Sci. C.S. 43, 122 (1957).
NITROGEN
CATABOLISM
STUDIES
of Biological University
PROTEUS
ON THE IDENTIFICATION OF END PRODUCTS’
M. DE VINCENTIIS, Institute
IN AMOEBA
F. SALVATORE
and V. ZAPPIA
Chemistry, Medical School, and Institute of General Biology and Genetics, of Naples; Stazione Zoologica, Naples, and Chair of Histology and Embryology, University of Camerino, Italy Received April 7, 1964
Although numerous studies on the nitrogen catabolism of various protozoa (Tetrahymena, Trypanosomida, Plasmodia, etc.) [7] are on record, data are lacking on “free living” the only study on this subject was the one amebae [l]. To our knowledge performed by Cailleau in 1934 [2] on cultures of Acanthamoeba Castellanii in peptone media, but it offered no conclusions as to what might be the pathways of nitrogen excretion. On the other hand, a unique compound, carbamildiurea (triuret), not previously found in living organisms, has since been characterized [3-51 in the cytoplasm of Amoeba proteus; it was considered to be a nitrogen excretion product. With a view toward an extensive study on the different aspects of nitrogen catawhat nitrogenous end bolism in Amoeba proteus, we have begun by determining products are to be found in the extracellular medium of this organism. In this paper evidence
is presented
which
indicates
tion products of nitrogen catabolism. Experimenfal.-Cultures of Amoeba Laboratoire de Morphologie Animale,
ammonia
as one of the most
important
excre-
proteus (kindly supplied by Prof. J. Brachet, UniversitC Libre de Bruxelles, Belgium) were
1 This work was aided by a fellowship of the Consiglio Nazionale delle Ricerche to the Stazione Zoologica of Naples and by a grant of the Impresa Enzimologia of the Consiglio Nazionale delle Ricerche, Italy. Experimental
Cell Research 35
OF TRITIATED
GRASSHOPPER
THYMIDINE
IN
NEUROBLASTS
W. M. LEACH Institute
of Radiation Biology, Department of Zoology and Entomology, The University of Tennessee, Knoxville, Term., U.S.A. Received April 1, 1964
Intracellular retention of thymidine and/or its derivatives other than deoxyribonucleic acid (DNA) has been demonstrated in sea urchin eggs [I] and the bacterium Alcaligenes fecalis [5]. This study was undertaken to determine whether tritiated thymidine was retained in grasshopper neuroblasts that were exposed to this precursor during a period of no DNA synthesis. Living neuroblasts can be observed by bright-field microscopy, all stages of the mitotic cycle can be identified, duration of the stages of the cell cycle is known [2], and the period of DNA synthesis has been established in relation to the cell cycle [4, 71. Materials and methods.-Embryos of the grasshopper Chortophaga viridifasciata (De Geer) at an age equivalent to 13-14 days of development at 26°C were removed from eggs and freed from surrounding membranes and yolk in Shaw’s [9] culture medium. Embryos were incubated at 26°C in culture medium which contained tritiated thymidine (3H-TdR)1 at concentrations of 1 or 10 PC/ml. Hanging drop preparations were made and neuroblasts in selected mitotic stages were mapped for subsequent reidentification in sections (techniques reviewed by Carlson and Gaulden [3]). Mitotic progress of cells was observed by bright-field microscopy. At the end of the observation period tissue was fixed for 5 min in 50 per cent acetic acid, dehydrated and embedded in paraffin. Sections 5 p thick were cut and autoradiograms were prepared [8]. Silver grains were counted in emulsion over sections of neuroblasts. Additional technical details are given as necessary in the text. Results and conclusions.-The following experiment was done to test the possibility that 3H-TdR may enter and be retained within neuroblasts which were not in a period of DNA synthesis. Embryos were incubated for 20 min in culture medium which contained 3H-TdR, then rinsed 5 min in medium which contained unlabeled thymidine (100 times more concentrated than the 3H-TdR), then transferred to medium without thymidine for 10 min, then again to medium without thymidine for approximately 1 min. Neuroblasts in metaphase and anaphase were mapped within 10 min after hanging drop preparations were made. Cells were observed until they reached late telophase or interphase (see Fig. 1 A). Tissue was fixed and autoradiograms were prepared. The number of grains over reidentified neuroblast nuclei is shown in Fig. 2. These data show that neuroblasts exposed to 3H-TdR when DNA synthesis was not 1 Tritiated thymidine was obtained from New England Nuclear Corp. at specific activities of 0.5 to 5.4 C/mM. Samples of each lot were tested for radioactive contamination by ascending chromatography (n-butanol : water, 86 : 14) followed by gas flow counting. Lots with more than 5 per cent contamination were purified chromatographically. Experimental
Cell Rt%nrch
35
W. M. Leach
202
in progress incorporate 3H into DNA during the subsequent DNA synthetic peri0d.l In order to determine whether neuroblasts could incorporate 3H into DNA before the start of DNA synthesis, the previous experiment was repeated with the following modifications: 5 min after hanging drop preparations were made neuroblasts in
Fig. l.-Relative duration of cell stages of the grasshopper neuroblast at 26°C. P, time during which embryos were incubated in 3H-TdR. C, Time during which 3H-TdR was “chased” with excess unlabeled TdR. S, Period of DNA synthesis [5, 81. EP, Early prophase; MP, middle prophase; LP, late prophase; VLP, very late prophase; lzI, metaphase; A, anaphase; El’, early telophase; Xl T, middle telophase; L T, late telophase; IN T, interphase; VEP, very early prophase. A, Embryos fixed in late telophase or interphase after end of incubation in 3H-TdR. B, Embryos fixed in metaphase at end of incubation in 3H-TdR. Total time of cell cycle at 26°C about 8 hr. Relative duration of cell stages and time of complete cell cycle from Carlson [2].
middle prophase were identified and mapped. Mapped cells were timed to metaphase. Tissue was fixed and autoradiograms were prepared (see Fig. 1 B). Mean grain number with 95 per cent confidence limits, over neuroblasts reidentified in autoradiograms was 1.3 k 0.7. Mean grain number, with 95 per cent confidence limits, of areas (380~2) of emulsion not over tissue was 1.0 +0.6. Since neuroblasts exposed to 3H-TdR between middle prophase and metaphase were not labeled it is clear that incorporation of 3H into DNA does not occur under these experimental conditions. The average time from middle prophase to metaphase, with 95 per cent confidence limits, was 94 26 min at 26°C. The interval between the start of incubation in 3H-TdR and mapping of cells in the previous experiment was less than 50 min, therefore neuroblasts mapped initially in metaphase or anaphase were well into, or beyond, middle prophase at the start of incubation. The following experiment was designed to test whether sufficient 3H-TdR was transferred through rinse (or “chase”) solutions to account for the observed labeling. Embryos were incubated in used “chase” solutions for 40-120 min. Hanging drop preparations were made and maintained for approximately 4 hr. Embryos were fixed and autoradiograms were prepared. Silver grains were scored over sections of neuroblasts in interphase and very early prophase. Grains over randomly selected tissue were scored within an area of 380 p2 at the plane of the image. Background grain numbers were estimated by grain counts per 380 p2 over randomly selected areas of emulsion near, but not over, tissue. These data are presented in Fig. 3. About 60 1 Before application of emulsion two preparations were treated with DNase for 2 hr at 38°C. Nuclei in these digested preparations were Feulgen negative, and were unlabeled. Experimental
Cell Research 35
Retention of tritiated thymidine in grasshopper neuroblasts
203
per cent of grain numbers were less than 2. From these data it was concluded that transfer of 3H-TdR in rinse solutions was negligible. The following points stand out as results of this study: (1) 3H-TdR which entered cells during a period of no DNA synthesis may be incorporated into DNA during a subsequent period of DNA synthesis; and (2) solutions which contained excess
..l
I-,
IL Fig. 2.
Fig. Z.--Grain numbers over nuclei of neuroblasts incubated in 3H-TdH during a period of no DNA synthesis plotted against concentration of isotope. Tissue was fixed after selected cells Ninety-five per cent confidence intervals on entered DNA synthesis. Means indicated by -. means indicated by vertical lines. Emulsion exposure time, 1 pC/ml, 10 days; 10 $/ml, 5 days. Fig. 3.-Distribution of grain numbers in autoradiograms of embryos incubated in used “chase” solutions. A, Over neuroblasts in interphase and very early prophase; H, over randomly selected tissue; C, in emulsion near, but not over, tissue. Emulsion exposure time, 10 days. See text for additional details.
unlabeled thymidine were not effective in removing all unincorporated 3H-TdR derivatives from neuroblasts. In experiments similar to those of Taylor et al. [lo] autoradiograms were prepared of neuroblasts which were approaching the second cytokinesis after end of incubation in 3H-TdR during a period of DNA synthesis. Chromosomes of these cells were labeled in cytologically identical regions of both chromatids [6]. This result may reflect capacities of neuroblasts to retain 3H-TdR between periods of DNA synthesis and to utilize its derivatives during DNA synthesis. The comments and criticisms of Drs J. G. Carlson, M. E. Gaulden, R. A. McGrath, D. M. Prescott and R. B. Setlow and discussions with them of various aspects of this work are gratefully acknowledged. This study was supported in part by the Atomic Energy Commission under Contract No. AT-(40-I)-2575. Experimentnl
Cell Reseurch 35
M. De Vincentiis, F. Salvatore and V. Zappia REFERENCES 1. BUCHER, N. L. R. and MAZIA, 1)., J. Biophys. Biochem. Cytol. 7, 651 (1960). 2. CARLSON, J. G., Cold Spring Harbor Symp. Quant. Biol. 9, 104 (1941). 3. CARLSON, J. G. and GAULDEN, M. E., in PRESCOTT, D. M. (ed.) Methods in Cell Physiology. Academic Press, New York. 1964. 4. GAULDEN, M. E., Genetics 41, 645 (1956). 5. LARK, K. G., Biochim. Biophys. Acta 51, 107 (1961). 6. LEACH, W. M., Ass. Southeast. Biol. Bull. 10, 32 (1963). 7. MCGRATH, R. A., Rudiation Res. 19, 526 (1963). 8. MCGRATH, R. A., LEACH, W. M. and CARLSON, J. G., J. Roy. ;Ilicroscop. Sot. 82, 55 (1963). 9. SHAW, E. I., Exptl Cell Res. 11, 580 (1956). 10. TAYLOR, J. H., WOODS, P. S. and HUGHES, W. L., Proc. AT&l. Acad. Sci. C.S. 43, 122 (1957).
NITROGEN
CATABOLISM
STUDIES
of Biological University
PROTEUS
ON THE IDENTIFICATION OF END PRODUCTS’
M. DE VINCENTIIS, Institute
IN AMOEBA
F. SALVATORE
and V. ZAPPIA
Chemistry, Medical School, and Institute of General Biology and Genetics, of Naples; Stazione Zoologica, Naples, and Chair of Histology and Embryology, University of Camerino, Italy Received April 7, 1964
Although numerous studies on the nitrogen catabolism of various protozoa (Tetrahymena, Trypanosomida, Plasmodia, etc.) [7] are on record, data are lacking on “free living” the only study on this subject was the one amebae [l]. To our knowledge performed by Cailleau in 1934 [2] on cultures of Acanthamoeba Castellanii in peptone media, but it offered no conclusions as to what might be the pathways of nitrogen excretion. On the other hand, a unique compound, carbamildiurea (triuret), not previously found in living organisms, has since been characterized [3-51 in the cytoplasm of Amoeba proteus; it was considered to be a nitrogen excretion product. With a view toward an extensive study on the different aspects of nitrogen catawhat nitrogenous end bolism in Amoeba proteus, we have begun by determining products are to be found in the extracellular medium of this organism. In this paper evidence
is presented
which
indicates
tion products of nitrogen catabolism. Experimenfal.-Cultures of Amoeba Laboratoire de Morphologie Animale,
ammonia
as one of the most
important
excre-
proteus (kindly supplied by Prof. J. Brachet, UniversitC Libre de Bruxelles, Belgium) were
1 This work was aided by a fellowship of the Consiglio Nazionale delle Ricerche to the Stazione Zoologica of Naples and by a grant of the Impresa Enzimologia of the Consiglio Nazionale delle Ricerche, Italy. Experimental
Cell Research 35