- Email: [email protected]
Segregation of chromosomal DNA in chinese hamster fibroblasts in vitro
132
L. L. Deaven & E. Stubblefield
throughout the fractions. This was not the case and therefore our findings of higher specific activities in the heavier fractions and lower ones in the lighter fractions indicate that differentsized classes of nucleoli exist in situ, which have different metabolic rates of RNA turnover as well as different activities of selected enzymes [l]. Our present results confirm other data indicating that the nucleolus is the most active part of the nucleus involved in RNA synthesis [3, 61. Table 1 reveals that in spite of the large absolute amounts of RNA in the fractions containing nuclear debris and chromatin (fractions 13-l 5), there is a sharp decrease in specific activity when the gradient reaches this region, and in most nucleolar fractions the specific activities are higher than in the respective nuclei. In one experiment a sample of isolated nucleoli of fractions 5 + 6 of a preparation from a regenerating liver was taken for examination by electron microscopy. Fig. 1 reveals that the preparation contained nucleoli of a high degree of purity and integrity of nucleolar structure. The work reoortedin this vaver was undertaken during the tenure oi a Public Heaitd Service Fellowship of the National Cancer Institute and a travel grant of the American Cancer Society awarded by the International Union against Cancer. The technical assistance of Miss Claire Horowitz is gratefully acknowledged.
REFERENCES 1. Kaufmann, E, Traub, A & Teitz, Y, Exptl cell res 49 (1968) 215. 2. Munro, H N & Fleck, A, Analyst 91 (1966) 78. 3. Muramatsu, M, Hodnett, J L & Busch, H, Biochim biophys acta 91 (1964) 592. 4. Shinozuka, H, Verney, E & Sidransky, H, Lab invest 18 (1968) 72. 5. Sidransky, H & Farber, E, Arch path01 66 (1958) 119. 6. Tsukada, K & Liebeman, I, J biol them 239 (1964). Received December 8, 1968
Exptl
Cell
Res 55
SEGREGATION OF CHROMOSOMAL DNA IN CHINESE HAMSTER FIBROBLASTS IN VITRO L. L. DEAVEN
and E. STUBBLEFIELD,
Cell Biology, Department of Biology, M. D. Anderson Hospital and Tumor Houston, Tex. 77025, USA
Section of
University of Texas, Institute at Houston,
The segregation of labeled DNA in chromosomes of higher organisms was first studied by Taylor, Woods & Hughes in 1957 [9]. They observed that after 3HTdR incorporation into the DNA of Vicia fuba, the chromosomes were labeled in both chromatids at the first metaphase (M 1) following isotope uptake, but were labeled in only one chromatid in the second metaphase (M 2). This semiconservative distribution of label could be interpreted as either a support of the Watson-Crick model of replication of a single DNA molecule or as a semiconservative distribution of several DNA strands within a chromatid [9]. In more recent work, Taylor [8] concluded that anaphase chromosomes were in a singlestranded (unineme) condition. Subsequent experiments by Peacock [4], however, are not in agreement with a unineme structure for chromosomal DNA. In Peacock’s autoradiographs most chromosomes in the M2 division were labeled in only one chromatid, but some were labeled over homologous regions of both chromatids. He considered that the occurrence of these “isolabeled” chromosomes or chromosomal segments in M2 provided critical evidence in favor of a multistranded (polyneme) condition. Prescott & Bender [5] studied the segregation of labeled chromosomal DNA in partially (50%) synchronized cultures of Chinese hamster fibroblasts and in human leucocytes in vitro. They studied four consecutive cell divisions after isotope addition and failed to find any case of isolabeling. This paper reports the results of autoradiographic experiments on chromosomal DNA segregation in Chinese hamster fibroblasts. These results are not in agreement with the observations of Prescott & Bender. In order to study the distribution of the DNA synthesized during an entire S period, cell populations were
Chromosomal
synchronized by the Colcemid reversal technique [7]. These synchronous populations could be labeled continuously for 8 to 10 h without labeling the cells for more than one S period. Small numbers of mitotic cells trypsinized from stock cultures without exposure to Colcemid and grown on glass slides for several generations were used as controls. Methods
DNA
segregation
in fibroblasts
133
Table 1. Distribution of label in Chinese hamster chromosomes following 3HTdR incorporation for one S period” AU chromosomes Generation Ml Ma
No. chromosomes Percentage scored labeled 500 500 500
100 100
Persentage isolabeled 100 34
56 13 MS This study utilized two types of male Chinese hamster fibroblasts (line Don and strain Don C) X and Y only 50 100 100 which have a generation time of approx. 12 h Ml 50 100 MS (Gl, 2; S, 7; G2, 2.5; M, 0.5) [7]. The cells were MS 50 70 ii synchronized by selective trypsinization from a Pooled data from two expts. stock cultures after a 2 h Colcemid (0.06 pg/ml) block. In all experiments the initial synchrony was greater than 90 %. The synchronized cell populations were incubated in 3HTdR (0.5 their DNA synthesis and utilization of the la&ml) mixed with nonradioactive TdR (5 rug/ beled precursor before being exposed to Colceml) for 10 h. The cultures were then rinsed twice mid, whereas in the other technique the cells with warm Hanks solution, and the isotope was were exposed to Colcemid before 3HTdR inchased in conditioned medium containing noncorporation. In this investigation the results radioactive TdR (5 pg/ml). Cultures were again were the same regardless of the time of Colcemid blocked with Colcemid (0.09 ,ug/ml) at 10 or 12 introduction. This is in agreement with the obh intervals, collected with 0.1 % pronase, hyposervations of Woods & Schairer [lo] and Peatonic treated with 1 % sodium citrate for six min, cock [4] that colchicine does not affect the distriand fixed in 50 % acetic acid. Autoradiographs bution of newly synthesized DNA. were prepared over squash preparations with AR-10 stripping film [6]. One set of slides was Results developed after a 15-day exposure, and others The autoradiographs of the Ml cells indicate at lo-day intervals thereafter. -that all labeled chromosomes had isotope inThe experiments by La Cour & Pelt [2] indicorporated into both chromatids (fig. 1, table 1). cate that the presence of colchicine during Grain counts over these chromosomes fit the 3HTdR uptake influences the labeling patterns expected 1: 1 hypothesis for label distribution of Ml and M2 chromosomes. Since cells syn- between sister chromatids. In M2 cells 34% of chronized with Colcemid may retain the drug the labeled chromosomes were isolabeled in during the following S phase, a second series of portions or for the entire length of the chromoexperiments was run using an alternative method some, while others were labeled in one chromatid of label incorporation. The 3HTdR was added only (fig. 2). Isolabeling occurred in the X and to stock cultures which were allowed to grow for in the Y chromosomes at M2 with a very high 10.5 h. At this time Colcemid (0.06 pug/ml) was frequency (80 “/o). One of the most striking obserintroduced to block mitosis for 2 h. This provations of this study was the presence of isolabeled chromosomes in cells for at least eight cedure was so designed that all of the metaphases generations after isotope uptake. Fig. 3, 4 and 5 accumulated during the 2 h Colcemid block had been in the presence of isotope for one and only are examples of isolabeled chromosomes from one complete S period. These cells had completed M3, M4 and M5 cells respectively. Exptl Cell Res 55
134 L. L. Deaven & E. Stubblefield
Figs 1-5. Cells from five consecutive divisions following DNA labeling for one S period. x 1950. Fig. 1. All chromatids are labeled indicating the cell incorporated SHTdR for the entire S period.
A No. 1 chromosome is labeled in only one chromatid with the exception of a terminal exchange, while most of the others are isolabeled either segmentally or for the entire length of the chromosome. Fig. 2.
Exptl
Cell Res 55
3. M3 cell with unlabeled chromosomes some isolabeled for the entire length.
Fig.
as well as
Fig. 4. M4 cell with two chromosomes showing terminal isolabeling and one entirely isolabeled. Fig.
5. M5 cell with one isolabeled chromosome.
Chromosomal Discussion
In addition to a polyneme condition for chromosomal DNA, several other hypotheses have been offered to explain the origin of isolabeled chromosomes after Ml. A few of these ideas will be considered: (1) Utilization of residual isotope: Incorporation of residual isotope in the M2 interphase can be ruled out because all M2 cells had at least one unlabeled chromatid in an early replicating chromosome (fig. 2). These unlabeled regions would have incorporated 3HTdR if it had been present in the medium. (2) Multiple chromatid exchanges: Multiple exchanges between sister chromatids beyond the resolving power of light microscopy cannot be eliminated as an alternative explanation; however, there is no evidence to support it. In previous studies with Chinese hamster cells sister chromatid exchanges occurred with a frequency of one per three chromosomes for each cell cycle [3]. The exchange rate was not influenced by variations in the amount of incorporated 3HTdR. In the present experiments the isotope was diluted with nonradioactive TdR to minimize radiation effects, and chromosome aberrations were not observed. (3) Interchromosomal exchange: In some cells the possibility of chromatid exchange between homologous chromosomes cannot be eliminated. However, this mechanism, requires the homolog of the isolabeled chromosome to be “iso-unlabeled”, a criterion not always met in our autoradiographs. Isolabeling occurred frequently in the sex chromosomes where chromatid exchange with a homolog is impossible. The disagreement between the results of this investigation and those reported previously are probably due to differences in experimental procedures. Prescott & Bender labeled stock cultures for 15 min, washed the cells with fresh medium, allowed them to grow for 8 h, and at this time shook off the mitotic cells. The synchronous cell populations obtained by this method were not exposed to isotope during late S. In our study the late replicating chromosomes, especially the X and the Y, were frequently isolabeled at M2. In other experiments
DNA
segregation
in fibroblasts
135
Prescott & Bender pulse-labeled stock cultures for 10 or 30 min, while in the present studies the cells were labeled for the entire S period (6-8 h). In 1964 [l] Hsu demonstrated the presence of many simultaneous replication points in mammalian chromosomes. With the exception of the late replicating areas, chromosomes continue to incorporate precursor throughout most of the S period. The duration of isotope application would not be expected to affect the DNA segregation pattern in a unineme chromosome. However, DNA segregation patterns in polyneme chromosomes might be altered by varying the time of exposure to isotope if the strands replicated in tandem. In the latter case a longer exposure would result in more labeled strands than a short exposure and thereby increase the probability of observing strand segregation at M2. The simplest interpretation of these observations is that Chinese hamster chromosomes are multistranded. The data also suggest that in at least some sites all of the strands in a chromatid do not replicate simultaneously. We wish to express our appreciation to Dr T. C. Hsu for his interest and assistancethroughout the investigation and in the ureuaration of this manuscrint. Predoctoral fellow, USPHS Grant NC1 5 Tl CA-5047. Supported in part by Research Grants E-286 from the American Cancer Society and DRG-269 from the Damon Rynyon Memorial Fund for Cancer Research.
REFERENCES 1. 2. 3. 4. 5. 6. 7. ;: 10.
Hsu, T C, J cell bio123 (1964) 53. La Cour, L F & Pelt, S R, Nature 183 (1959) 1455. Marin, G & Prescott, D M, J cell biol21 (1964) 159. Peacock, W J, Proc natl acad sci US 49 (1963) 793. Prescott, D M & Bender, M A, Exptl cell res 29 (1963) 430. Schmid, W, Human chromosome methodology (ed J Yunis) p. 91. Academic Press, New York (1965). Stubblefield, E, Klevecz, R & Deaven, L, J cell physiol 69 (1967) 345. Taylor, J H, Genetics 43 (1958) 515. Taylor, J H, Woods, P S & Hughes, W L, Proc natl acad sci US 43 (1957) 122. Woods, P S & Schairer, M U, Nature 183 (1959) 303.
Received December 16, 1968
Exptl Cell Res 55
L. L. Deaven & E. Stubblefield
throughout the fractions. This was not the case and therefore our findings of higher specific activities in the heavier fractions and lower ones in the lighter fractions indicate that differentsized classes of nucleoli exist in situ, which have different metabolic rates of RNA turnover as well as different activities of selected enzymes [l]. Our present results confirm other data indicating that the nucleolus is the most active part of the nucleus involved in RNA synthesis [3, 61. Table 1 reveals that in spite of the large absolute amounts of RNA in the fractions containing nuclear debris and chromatin (fractions 13-l 5), there is a sharp decrease in specific activity when the gradient reaches this region, and in most nucleolar fractions the specific activities are higher than in the respective nuclei. In one experiment a sample of isolated nucleoli of fractions 5 + 6 of a preparation from a regenerating liver was taken for examination by electron microscopy. Fig. 1 reveals that the preparation contained nucleoli of a high degree of purity and integrity of nucleolar structure. The work reoortedin this vaver was undertaken during the tenure oi a Public Heaitd Service Fellowship of the National Cancer Institute and a travel grant of the American Cancer Society awarded by the International Union against Cancer. The technical assistance of Miss Claire Horowitz is gratefully acknowledged.
REFERENCES 1. Kaufmann, E, Traub, A & Teitz, Y, Exptl cell res 49 (1968) 215. 2. Munro, H N & Fleck, A, Analyst 91 (1966) 78. 3. Muramatsu, M, Hodnett, J L & Busch, H, Biochim biophys acta 91 (1964) 592. 4. Shinozuka, H, Verney, E & Sidransky, H, Lab invest 18 (1968) 72. 5. Sidransky, H & Farber, E, Arch path01 66 (1958) 119. 6. Tsukada, K & Liebeman, I, J biol them 239 (1964). Received December 8, 1968
Exptl
Cell
Res 55
SEGREGATION OF CHROMOSOMAL DNA IN CHINESE HAMSTER FIBROBLASTS IN VITRO L. L. DEAVEN
and E. STUBBLEFIELD,
Cell Biology, Department of Biology, M. D. Anderson Hospital and Tumor Houston, Tex. 77025, USA
Section of
University of Texas, Institute at Houston,
The segregation of labeled DNA in chromosomes of higher organisms was first studied by Taylor, Woods & Hughes in 1957 [9]. They observed that after 3HTdR incorporation into the DNA of Vicia fuba, the chromosomes were labeled in both chromatids at the first metaphase (M 1) following isotope uptake, but were labeled in only one chromatid in the second metaphase (M 2). This semiconservative distribution of label could be interpreted as either a support of the Watson-Crick model of replication of a single DNA molecule or as a semiconservative distribution of several DNA strands within a chromatid [9]. In more recent work, Taylor [8] concluded that anaphase chromosomes were in a singlestranded (unineme) condition. Subsequent experiments by Peacock [4], however, are not in agreement with a unineme structure for chromosomal DNA. In Peacock’s autoradiographs most chromosomes in the M2 division were labeled in only one chromatid, but some were labeled over homologous regions of both chromatids. He considered that the occurrence of these “isolabeled” chromosomes or chromosomal segments in M2 provided critical evidence in favor of a multistranded (polyneme) condition. Prescott & Bender [5] studied the segregation of labeled chromosomal DNA in partially (50%) synchronized cultures of Chinese hamster fibroblasts and in human leucocytes in vitro. They studied four consecutive cell divisions after isotope addition and failed to find any case of isolabeling. This paper reports the results of autoradiographic experiments on chromosomal DNA segregation in Chinese hamster fibroblasts. These results are not in agreement with the observations of Prescott & Bender. In order to study the distribution of the DNA synthesized during an entire S period, cell populations were
Chromosomal
synchronized by the Colcemid reversal technique [7]. These synchronous populations could be labeled continuously for 8 to 10 h without labeling the cells for more than one S period. Small numbers of mitotic cells trypsinized from stock cultures without exposure to Colcemid and grown on glass slides for several generations were used as controls. Methods
DNA
segregation
in fibroblasts
133
Table 1. Distribution of label in Chinese hamster chromosomes following 3HTdR incorporation for one S period” AU chromosomes Generation Ml Ma
No. chromosomes Percentage scored labeled 500 500 500
100 100
Persentage isolabeled 100 34
56 13 MS This study utilized two types of male Chinese hamster fibroblasts (line Don and strain Don C) X and Y only 50 100 100 which have a generation time of approx. 12 h Ml 50 100 MS (Gl, 2; S, 7; G2, 2.5; M, 0.5) [7]. The cells were MS 50 70 ii synchronized by selective trypsinization from a Pooled data from two expts. stock cultures after a 2 h Colcemid (0.06 pg/ml) block. In all experiments the initial synchrony was greater than 90 %. The synchronized cell populations were incubated in 3HTdR (0.5 their DNA synthesis and utilization of the la&ml) mixed with nonradioactive TdR (5 rug/ beled precursor before being exposed to Colceml) for 10 h. The cultures were then rinsed twice mid, whereas in the other technique the cells with warm Hanks solution, and the isotope was were exposed to Colcemid before 3HTdR inchased in conditioned medium containing noncorporation. In this investigation the results radioactive TdR (5 pg/ml). Cultures were again were the same regardless of the time of Colcemid blocked with Colcemid (0.09 ,ug/ml) at 10 or 12 introduction. This is in agreement with the obh intervals, collected with 0.1 % pronase, hyposervations of Woods & Schairer [lo] and Peatonic treated with 1 % sodium citrate for six min, cock [4] that colchicine does not affect the distriand fixed in 50 % acetic acid. Autoradiographs bution of newly synthesized DNA. were prepared over squash preparations with AR-10 stripping film [6]. One set of slides was Results developed after a 15-day exposure, and others The autoradiographs of the Ml cells indicate at lo-day intervals thereafter. -that all labeled chromosomes had isotope inThe experiments by La Cour & Pelt [2] indicorporated into both chromatids (fig. 1, table 1). cate that the presence of colchicine during Grain counts over these chromosomes fit the 3HTdR uptake influences the labeling patterns expected 1: 1 hypothesis for label distribution of Ml and M2 chromosomes. Since cells syn- between sister chromatids. In M2 cells 34% of chronized with Colcemid may retain the drug the labeled chromosomes were isolabeled in during the following S phase, a second series of portions or for the entire length of the chromoexperiments was run using an alternative method some, while others were labeled in one chromatid of label incorporation. The 3HTdR was added only (fig. 2). Isolabeling occurred in the X and to stock cultures which were allowed to grow for in the Y chromosomes at M2 with a very high 10.5 h. At this time Colcemid (0.06 pug/ml) was frequency (80 “/o). One of the most striking obserintroduced to block mitosis for 2 h. This provations of this study was the presence of isolabeled chromosomes in cells for at least eight cedure was so designed that all of the metaphases generations after isotope uptake. Fig. 3, 4 and 5 accumulated during the 2 h Colcemid block had been in the presence of isotope for one and only are examples of isolabeled chromosomes from one complete S period. These cells had completed M3, M4 and M5 cells respectively. Exptl Cell Res 55
134 L. L. Deaven & E. Stubblefield
Figs 1-5. Cells from five consecutive divisions following DNA labeling for one S period. x 1950. Fig. 1. All chromatids are labeled indicating the cell incorporated SHTdR for the entire S period.
A No. 1 chromosome is labeled in only one chromatid with the exception of a terminal exchange, while most of the others are isolabeled either segmentally or for the entire length of the chromosome. Fig. 2.
Exptl
Cell Res 55
3. M3 cell with unlabeled chromosomes some isolabeled for the entire length.
Fig.
as well as
Fig. 4. M4 cell with two chromosomes showing terminal isolabeling and one entirely isolabeled. Fig.
5. M5 cell with one isolabeled chromosome.
Chromosomal Discussion
In addition to a polyneme condition for chromosomal DNA, several other hypotheses have been offered to explain the origin of isolabeled chromosomes after Ml. A few of these ideas will be considered: (1) Utilization of residual isotope: Incorporation of residual isotope in the M2 interphase can be ruled out because all M2 cells had at least one unlabeled chromatid in an early replicating chromosome (fig. 2). These unlabeled regions would have incorporated 3HTdR if it had been present in the medium. (2) Multiple chromatid exchanges: Multiple exchanges between sister chromatids beyond the resolving power of light microscopy cannot be eliminated as an alternative explanation; however, there is no evidence to support it. In previous studies with Chinese hamster cells sister chromatid exchanges occurred with a frequency of one per three chromosomes for each cell cycle [3]. The exchange rate was not influenced by variations in the amount of incorporated 3HTdR. In the present experiments the isotope was diluted with nonradioactive TdR to minimize radiation effects, and chromosome aberrations were not observed. (3) Interchromosomal exchange: In some cells the possibility of chromatid exchange between homologous chromosomes cannot be eliminated. However, this mechanism, requires the homolog of the isolabeled chromosome to be “iso-unlabeled”, a criterion not always met in our autoradiographs. Isolabeling occurred frequently in the sex chromosomes where chromatid exchange with a homolog is impossible. The disagreement between the results of this investigation and those reported previously are probably due to differences in experimental procedures. Prescott & Bender labeled stock cultures for 15 min, washed the cells with fresh medium, allowed them to grow for 8 h, and at this time shook off the mitotic cells. The synchronous cell populations obtained by this method were not exposed to isotope during late S. In our study the late replicating chromosomes, especially the X and the Y, were frequently isolabeled at M2. In other experiments
DNA
segregation
in fibroblasts
135
Prescott & Bender pulse-labeled stock cultures for 10 or 30 min, while in the present studies the cells were labeled for the entire S period (6-8 h). In 1964 [l] Hsu demonstrated the presence of many simultaneous replication points in mammalian chromosomes. With the exception of the late replicating areas, chromosomes continue to incorporate precursor throughout most of the S period. The duration of isotope application would not be expected to affect the DNA segregation pattern in a unineme chromosome. However, DNA segregation patterns in polyneme chromosomes might be altered by varying the time of exposure to isotope if the strands replicated in tandem. In the latter case a longer exposure would result in more labeled strands than a short exposure and thereby increase the probability of observing strand segregation at M2. The simplest interpretation of these observations is that Chinese hamster chromosomes are multistranded. The data also suggest that in at least some sites all of the strands in a chromatid do not replicate simultaneously. We wish to express our appreciation to Dr T. C. Hsu for his interest and assistancethroughout the investigation and in the ureuaration of this manuscrint. Predoctoral fellow, USPHS Grant NC1 5 Tl CA-5047. Supported in part by Research Grants E-286 from the American Cancer Society and DRG-269 from the Damon Rynyon Memorial Fund for Cancer Research.
REFERENCES 1. 2. 3. 4. 5. 6. 7. ;: 10.
Hsu, T C, J cell bio123 (1964) 53. La Cour, L F & Pelt, S R, Nature 183 (1959) 1455. Marin, G & Prescott, D M, J cell biol21 (1964) 159. Peacock, W J, Proc natl acad sci US 49 (1963) 793. Prescott, D M & Bender, M A, Exptl cell res 29 (1963) 430. Schmid, W, Human chromosome methodology (ed J Yunis) p. 91. Academic Press, New York (1965). Stubblefield, E, Klevecz, R & Deaven, L, J cell physiol 69 (1967) 345. Taylor, J H, Genetics 43 (1958) 515. Taylor, J H, Woods, P S & Hughes, W L, Proc natl acad sci US 43 (1957) 122. Woods, P S & Schairer, M U, Nature 183 (1959) 303.
Received December 16, 1968
Exptl Cell Res 55