Experimental
STUDIES
Cell Research
ON DNA CONTENT,
DNA TEMPLATE
ACTIVITY
PARAMECIUM M. R. KLASS Department
98 (1976) 63-72
RNA SYNTHESIS,
AND
IN AGING
OF
CELLS
AURELIA
and JOAN SMITH-SONNEBORN
of Zoology and Physiology,
University of Wyoming, Laramie,
WY 82071, USA
SUMMARY Investigations by Feulgen microspectrophotometry in Paramecium au&a indicated that as fission age increased the amount of macronuclear DNA decreased. It was also found that the amount of RNA synthesis as determined by the in vivo incorporation of [aH]uridine decreased as the fission age increased. An alternative in situ assay of the DNA template activity determined by the RNA polymerase-catalyzed incorporation of rH]UTP is described. The DNA template activity of older cells was shown to be significantly lower on a per cell basis than that of younger cells. The majority of this reduction was shown to be due to the gradual loss of DNA template with an increase in fission age. The specific activity of the DNA template, however, does show a small but significant decrease as the fission age of the cell increases.
The ciliate Paramecium aurelia has long served as a model system for the study of cellular aging [17]. The life cycle of P. aurelia is characterized by: (1) sexual immaturity, when the cell cannot mate or undergo autogamy; (2) maturity, when the cell is capable of sexual reorganization; and (3) senescence [ 11, 171. Associated with senescence are a number of age-correlated phenotypes: (1) reduction in daily fission rate [17, 181; (2) increased number of micronuclear mutations [18]; (3) increased mortality immediately following autogamy [7, 18, 191; (4) increased sensitivity to UV light [12]; (5) increased sensitivity to caffeine [13]; (6) changes in the DNA synthesis pattern [14]; and (7) an increase in the probability of cellular death [17. 18, 191. ’ Present address: Molecular, Cellular and Developmental Biology Department, Boulder, CO 80302, USA. 5-761819
University
of Colorado,
The Paramecium system offers the unique advantage of rejuvenation, i.e., reinitiating a new life cycle by mating or autogamy. During either of these sexual processes a new macronucleus develops from a micronucleus and the cell is rejuvenated. The diploid micronucleus is considered the germ line of the cell while the macronucleus is highly polyploid (800 n) and is considered the somatic line responsible for the phenotype of the cell. Parent clones undergoing successive mitotic divisions age and die, but the parents can initiate progeny clones by undergoing autogamy or mating, thus assuring the “immortality of the species”. In the present study, age in P. aurelia is defined as the number of divisions the cell has undergone since its last sexual process, either autogamy or conjugation. Previous studies in this laboratory have indicated ExprlCell Res 98 (1976)
64
Klcrss crnd Smith-Sonnebortl
significant changes in the DNA synthesis pattern as the fission age of the cell increased [14]. Results indicated that the maximum amount of incorporation of [“HIthymine per unit area of the macronucleus was always less in older cells. The present study was a composite investigation of agecorrelated changes in: (I) DNA content of the macronucleus by Feulgen microspectrophotometry; (2) amount of RNA synthesis by incorporation of r3H]uridine; and (3) DNA template activity by RNA polymerase-catalyzed incorporation of r3H]UTP. Significant changes in DNA content, RNA synthesis and specific activity of the DNA template were found. MATERIALS
AND METHODS
conditions. Cells of Paramecium aurelia, syngen 4, stock 51 (d4-186), mating type 7, were kindly supplied by Professor T. M. Sonneborn, Indiana University. Syngen 4 is used because autogamy can be induced at about 20 fissions after the previous autoaamv but rarelv occurs in dailv isolation lines grown :m a- favorable medium [17]. -The cells were maintained at 27°C in Ceronhvl medium. inoculated 24 h before use with Enterohacter aerogenes and adjusted to pH 6.7. Culture
For pulse-labeling studies, synchronizrd cells of P. trrrrrlicr were incubated at 27°C for I h periods in [:‘H]uridine-supplemented medium. After 1 h, the labeled cells were removed. washed 4 times and collected on Millipore filter\ for counting in the scintillation counter.
Sc~intillafioncorrrrtine. Labeled cells were washed 4 times in cold medium and samples of IO cells were collected on 25 mm Millipore filters. The filters were washed with 5 c/r TCA followed by 95 55 ethanol. After thorough drying. each filter was placed in a liquid scintillation viayin IO ml of scintillation fluid consisting of toluene plus 5 g/l PPO and 0.5 g/l POPOP was added. The vials were then counted in a Beckman LS100 liquid scintillation counter. To control against the transfer of exogenous label during the washing procedure. a comparable volume of the fourth wash was counted after all labeled cells had been washed and subtracted from the total counts. and grain counting. Slides were coated with Kodak NTB-2 liquid emulsion, exposed for the appropriate time and developed with Kodak D-19. Autoradiographic preparations were observed using a Zeiss 40x oil-immersion phase contrast objective. Grain counts were made over the entire macronucleus and the total number of grains/macronucleus was recorded.
Autoradiography
Feulgen microspectrophotometry
At subsequent intervals post-division synchronized cells were removed from the pooled sample, fixed with 4 % buffered formalin (pH 7. I) and flattened with a coverslin. After the coverslins had been removed by dipping in liquid nitrogen,’ slides were passed through 100, 95 and 75% ethanols and Daily isolation lines. The procedure of Sonneborn [ 171 subsequently to distilled water. The cells were then hvdrolvzed in I N HCI (60°C) for 9 min and nlaced in was used for conducting daily isolation lines to obtain aging cells and has been published in detail elsewhere Schiffs reagent for 15 min. After bleaching-in dilute sulfurous acid the slides were rinsed in distilled water, [141. dehydrated and mounted in Euparal (GBI Labs Limited). Microspectrophotometric determinations were [3H]Uridine incorporation made at 546 nm for the whole macronucleus using the Barr & Stroud Integrating Microdensitometer. Method of labeling cells in viva. Two labeling procedures were used: (1) cells were labeled for one en- The total extinction values corrected for background tire interfission period; (2) cells were pulse-labeled for were recorded. I h periods at subsequent hourly intervals post division. DNA template activity Cells were synchronized by individually selecting Cell preparation for in situ studies. At subsequent individing cells from a mass culture with a micropipette under a dissectine microscooe. Svnchronized cells of tervals post-division synchronized cells were removed from the pooled sample and fixed with a drop of 45 % P. aurelia were iicubated in culture medium supplemented with raHlt.uidine (Schwa&Mann snec. act. 28 acetic acid on a gelatinized slide and a coverslip added. CilmMole) f& ihe entire interfission period, after The cells were then squashed by exerting sufficient which the cells were removed, washed 4 times in non- pressure to rupture the nuclear membrane while observing under a dissecting microscope. After fixation, radioactive medium and collected on Millipore filters for counting in the scintillation counter as described coversiips were removed by freezing in liquid nitrobelow. As a control, samples of cells labeled for the gen. Slides were then passed through 100, 95 and 75 % entire interfission period were processed for auto- ethanols, and finally‘ transferred to distilled water. Slides were then washed for 30 min in cold 5 % TCA radiography. These cells were washed 4 times, fixed to remove any native pools of ribonucleoside triphoswith 4% buffered formalin (pH 7.1) on gelatinized slides and processed for autoradiography as described phates and were then washed in cold tap water, dehydrated and air-dried. below. Exptl Cell Res 98 (1976)
Cellprrparation.
DNA content, RNA synthesis, template activity in P. aurelia
Table 1. Specificity
of [3H]uridine
incor-
poration Synchronized cells were labeled for the entire interfission period, washed, and fixed for autoradiography as described in Materials and Methods. Control slides were subjected to either DNase or RNase digestion. Data represent the mean number of grains/U area f95% CIMd (n=20). Actinomycin D controls were carried out by adding actinomycin D (IS wg/ml) to the [3H]uridine-supplemented culture medium. After labeling, cells were washed and collected on Millipore filters as described in Materials and Methods. Data are represented as the mean number of cpm/cell ?95 % CIM for 5 samples of IO cells each. ([3H]uridine, IO &i/ml, spec. act. 28 CilmMole) Age (in fissions) 58
II6
No. of grains per unit area No treatment DNase
RNase
"20.8L3.6 b20.8k2.8
"19.0f2.2 *18.5?2.4
"2.220.3 "2.150.2
"IS.Of3.1 b14.6t2.6
a13.0f2.0 *13.5+2.4
"3.220.7 "3.0f0.5
cpm/cell Actinomycin-D+ [3H]uridine c44.0k-2.0
65
treatments and inhibitors. Enzymatic digestions were carried out over 4 h periods at 3PC. Deoxyribonuclease (Worthington, electrophoretically purified) in 4~10~~ M MgSO, and 0.01 M sodium acetate, and ribonuclease (Worthington) were used at a concentration of 0.3 mg/ml. The RNase, in 0.1 M phosphate buffer, was freed of possible DNase activity by heating for 20 min at 100°C. After the respective enzymatic treatments, slides were washed in 3 changes of water, rinsed for IS min in cold 5 % TCA, washed again in water, dehydrated in ethanol and airdried. Actinomycin D (Calbiochem, lyophylized, mol. wt 1255.5), when used for the in situ procedure, was used at a concentration of 200 pg/ml. A 0.05 ml drop was placed on the slide, followed by a coverslip to insure uniform spreading. After IO min the coverslip was washed off with water and the slides were washed 3 times in distilled water and air-dried. For the in vivo procedure, actinomycin D was added to the culture medium to give a final concentration of 15 wg/ml which is not lethal to the cells [6].
Enzymatic
RESULTS In vivo incorporation
of [Hluridine
Evidence for the specificity of the label used for RNA is shown in table 1. RNase removed 79-89% of the label and 90% of
4.0io.9
D Mean number of grains/U area (81 Fm*) of the macronucleus. b Mean number of grains/U area (81 pm2) of the cytoplasm. ’ Mean number of cpmlcell from 5 samples of 10 cells each, counted in the scintillation counter. d CIM, Confidence Interval of the Mean. of labeling cells in situ. A reaction mixture was prepared, consisting of 0.1 mM ATP, 0.1 mM CTP, 0.1 mM GTP, 0.003 PM [SH]UTP (Schwarzl Mann spec. act. 19 CilmMole, 0.5 mCi/ml), 0.001 M MnCI,, 0.004 M MgC&, 0.01 M 2-mercaptoethanol, 0.04 M Tris-HCI pH 7.9 and 6.67 U/ml of E. coli RNA polymerase (BoehringerlMannheim, prepared from E. coli MRE 600 by the technique of Burgess & Travers, contains sigma factor and is essentially free of DNA polymerase, RNase, DNase, and polynucleotide phosphorylase). A 0.05 ml drop of this reaction mixture was placed on each slide and covered with a no. 1 coverslip. The slides were placed in special moisture chambers and incubated at 3PC for 30 min. The reaction was then stopped by rinsing off the coverslip with cold 5 % TCA followed by 3 washes in cold 5 % TCA. Slides were then rinsed in running tap water for 20 min. dehydrated, and air-dried. The slides were coated with Kodak NTB-2 nuclear track emulsion, exposed for 2 days, developed in Kodak D-19, followed by fixation in Kodak Rapid Fixer. Method
5i I
10
20
30
40
50
m
70
80
90
1001101~0130140
,
I. Abscissa: fission age; ordinate: cpmlcell. [SH]Uridine incorporation. Data represent the total amount of [3H]uridine (IO &i/ml spec. act. 28 Ci/ mMole) incorporated during the entire interfission period. Each point is the mean cpmlcell +95% CIM from 5 samples of IO cells each collected on Millipore filters and counted in the scintillation counter as described in Materials and Methods. (r, -0.87; slope, -0.23). Cells were labeled continuously until they were observed entering the next division.
Fig.
Exptl Cell Res 98 (1976)
66
Kluss and Smith-Sonnehorn
Table 2. [3H]Uridine incorporation ly intervals during the cell cycle
at hour-
Synchronized cells of different sees were obtained as described in Materials and Methods. At subsequent intervals post division, cells were removed from the pooled sample and given 1 h pulses of [3H]uridine (50 &i/ml, spec. act. 28 CilmMole) The data represent the mean cpmlcell from .s samples of IO cells each i-95% CIM. Cells were prepared and counted in the scintillation counter as described in Materials and Methods. Those means underlined by the same line are not significantly different from one another at the 95 % level of confidence using the Mann-Whitney U Statistic [ 161 The dashed line indicates fission and represents the time when the first cell in the population was observed in the next division. Ninety percent of the cells divided within the subsequent 0.5 h interval Time (hours)
6 tissions
70 tissions
IS0 tissions
0-I l-2 2-3 3-4
205+25 230+30 4llk45 342 +40
lO4f IS l46+20 179k25 170+20
65f IO lO6+ I5 lO9k I6 149&22
4-5
275535
139t20
l24+
5-6
387k50
323 +40
136k20
l60+20 -----
121~18 94-t 14
6-7 7-8 Total
----I 850
I221
I8
904
the uptake was inhibited by actinomycin D (I5 pg/ml). As described previously [6], the majority of C3H]uridine is directly incorporated into RNA and is not in the form of unincorporated RNA precursors in the cytoplasm. The total amount of incorporation of r3H]uridine measured over the entire interfission period is shown in fig. 1. The results show reduction in the total uptake of [“HIuridine in older cells. Results from pulse-labeled studies of the amount of in vivo incorporation of [3H]~ridine during hourly intervals after division are summarized in tables 2 and 4. It is clear that the amount of incorporation of [3H]uridine at each hourly interval during the Exptl Cell Res 98 (1976)
cell cycle is significantly @
STUDIES
Cell Research
ON DNA CONTENT,
DNA TEMPLATE
ACTIVITY
PARAMECIUM M. R. KLASS Department
98 (1976) 63-72
RNA SYNTHESIS,
AND
IN AGING
OF
CELLS
AURELIA
and JOAN SMITH-SONNEBORN
of Zoology and Physiology,
University of Wyoming, Laramie,
WY 82071, USA
SUMMARY Investigations by Feulgen microspectrophotometry in Paramecium au&a indicated that as fission age increased the amount of macronuclear DNA decreased. It was also found that the amount of RNA synthesis as determined by the in vivo incorporation of [aH]uridine decreased as the fission age increased. An alternative in situ assay of the DNA template activity determined by the RNA polymerase-catalyzed incorporation of rH]UTP is described. The DNA template activity of older cells was shown to be significantly lower on a per cell basis than that of younger cells. The majority of this reduction was shown to be due to the gradual loss of DNA template with an increase in fission age. The specific activity of the DNA template, however, does show a small but significant decrease as the fission age of the cell increases.
The ciliate Paramecium aurelia has long served as a model system for the study of cellular aging [17]. The life cycle of P. aurelia is characterized by: (1) sexual immaturity, when the cell cannot mate or undergo autogamy; (2) maturity, when the cell is capable of sexual reorganization; and (3) senescence [ 11, 171. Associated with senescence are a number of age-correlated phenotypes: (1) reduction in daily fission rate [17, 181; (2) increased number of micronuclear mutations [18]; (3) increased mortality immediately following autogamy [7, 18, 191; (4) increased sensitivity to UV light [12]; (5) increased sensitivity to caffeine [13]; (6) changes in the DNA synthesis pattern [14]; and (7) an increase in the probability of cellular death [17. 18, 191. ’ Present address: Molecular, Cellular and Developmental Biology Department, Boulder, CO 80302, USA. 5-761819
University
of Colorado,
The Paramecium system offers the unique advantage of rejuvenation, i.e., reinitiating a new life cycle by mating or autogamy. During either of these sexual processes a new macronucleus develops from a micronucleus and the cell is rejuvenated. The diploid micronucleus is considered the germ line of the cell while the macronucleus is highly polyploid (800 n) and is considered the somatic line responsible for the phenotype of the cell. Parent clones undergoing successive mitotic divisions age and die, but the parents can initiate progeny clones by undergoing autogamy or mating, thus assuring the “immortality of the species”. In the present study, age in P. aurelia is defined as the number of divisions the cell has undergone since its last sexual process, either autogamy or conjugation. Previous studies in this laboratory have indicated ExprlCell Res 98 (1976)
64
Klcrss crnd Smith-Sonnebortl
significant changes in the DNA synthesis pattern as the fission age of the cell increased [14]. Results indicated that the maximum amount of incorporation of [“HIthymine per unit area of the macronucleus was always less in older cells. The present study was a composite investigation of agecorrelated changes in: (I) DNA content of the macronucleus by Feulgen microspectrophotometry; (2) amount of RNA synthesis by incorporation of r3H]uridine; and (3) DNA template activity by RNA polymerase-catalyzed incorporation of r3H]UTP. Significant changes in DNA content, RNA synthesis and specific activity of the DNA template were found. MATERIALS
AND METHODS
conditions. Cells of Paramecium aurelia, syngen 4, stock 51 (d4-186), mating type 7, were kindly supplied by Professor T. M. Sonneborn, Indiana University. Syngen 4 is used because autogamy can be induced at about 20 fissions after the previous autoaamv but rarelv occurs in dailv isolation lines grown :m a- favorable medium [17]. -The cells were maintained at 27°C in Ceronhvl medium. inoculated 24 h before use with Enterohacter aerogenes and adjusted to pH 6.7. Culture
For pulse-labeling studies, synchronizrd cells of P. trrrrrlicr were incubated at 27°C for I h periods in [:‘H]uridine-supplemented medium. After 1 h, the labeled cells were removed. washed 4 times and collected on Millipore filter\ for counting in the scintillation counter.
Sc~intillafioncorrrrtine. Labeled cells were washed 4 times in cold medium and samples of IO cells were collected on 25 mm Millipore filters. The filters were washed with 5 c/r TCA followed by 95 55 ethanol. After thorough drying. each filter was placed in a liquid scintillation viayin IO ml of scintillation fluid consisting of toluene plus 5 g/l PPO and 0.5 g/l POPOP was added. The vials were then counted in a Beckman LS100 liquid scintillation counter. To control against the transfer of exogenous label during the washing procedure. a comparable volume of the fourth wash was counted after all labeled cells had been washed and subtracted from the total counts. and grain counting. Slides were coated with Kodak NTB-2 liquid emulsion, exposed for the appropriate time and developed with Kodak D-19. Autoradiographic preparations were observed using a Zeiss 40x oil-immersion phase contrast objective. Grain counts were made over the entire macronucleus and the total number of grains/macronucleus was recorded.
Autoradiography
Feulgen microspectrophotometry
At subsequent intervals post-division synchronized cells were removed from the pooled sample, fixed with 4 % buffered formalin (pH 7. I) and flattened with a coverslin. After the coverslins had been removed by dipping in liquid nitrogen,’ slides were passed through 100, 95 and 75% ethanols and Daily isolation lines. The procedure of Sonneborn [ 171 subsequently to distilled water. The cells were then hvdrolvzed in I N HCI (60°C) for 9 min and nlaced in was used for conducting daily isolation lines to obtain aging cells and has been published in detail elsewhere Schiffs reagent for 15 min. After bleaching-in dilute sulfurous acid the slides were rinsed in distilled water, [141. dehydrated and mounted in Euparal (GBI Labs Limited). Microspectrophotometric determinations were [3H]Uridine incorporation made at 546 nm for the whole macronucleus using the Barr & Stroud Integrating Microdensitometer. Method of labeling cells in viva. Two labeling procedures were used: (1) cells were labeled for one en- The total extinction values corrected for background tire interfission period; (2) cells were pulse-labeled for were recorded. I h periods at subsequent hourly intervals post division. DNA template activity Cells were synchronized by individually selecting Cell preparation for in situ studies. At subsequent individing cells from a mass culture with a micropipette under a dissectine microscooe. Svnchronized cells of tervals post-division synchronized cells were removed from the pooled sample and fixed with a drop of 45 % P. aurelia were iicubated in culture medium supplemented with raHlt.uidine (Schwa&Mann snec. act. 28 acetic acid on a gelatinized slide and a coverslip added. CilmMole) f& ihe entire interfission period, after The cells were then squashed by exerting sufficient which the cells were removed, washed 4 times in non- pressure to rupture the nuclear membrane while observing under a dissecting microscope. After fixation, radioactive medium and collected on Millipore filters for counting in the scintillation counter as described coversiips were removed by freezing in liquid nitrobelow. As a control, samples of cells labeled for the gen. Slides were then passed through 100, 95 and 75 % entire interfission period were processed for auto- ethanols, and finally‘ transferred to distilled water. Slides were then washed for 30 min in cold 5 % TCA radiography. These cells were washed 4 times, fixed to remove any native pools of ribonucleoside triphoswith 4% buffered formalin (pH 7.1) on gelatinized slides and processed for autoradiography as described phates and were then washed in cold tap water, dehydrated and air-dried. below. Exptl Cell Res 98 (1976)
Cellprrparation.
DNA content, RNA synthesis, template activity in P. aurelia
Table 1. Specificity
of [3H]uridine
incor-
poration Synchronized cells were labeled for the entire interfission period, washed, and fixed for autoradiography as described in Materials and Methods. Control slides were subjected to either DNase or RNase digestion. Data represent the mean number of grains/U area f95% CIMd (n=20). Actinomycin D controls were carried out by adding actinomycin D (IS wg/ml) to the [3H]uridine-supplemented culture medium. After labeling, cells were washed and collected on Millipore filters as described in Materials and Methods. Data are represented as the mean number of cpm/cell ?95 % CIM for 5 samples of IO cells each. ([3H]uridine, IO &i/ml, spec. act. 28 CilmMole) Age (in fissions) 58
II6
No. of grains per unit area No treatment DNase
RNase
"20.8L3.6 b20.8k2.8
"19.0f2.2 *18.5?2.4
"2.220.3 "2.150.2
"IS.Of3.1 b14.6t2.6
a13.0f2.0 *13.5+2.4
"3.220.7 "3.0f0.5
cpm/cell Actinomycin-D+ [3H]uridine c44.0k-2.0
65
treatments and inhibitors. Enzymatic digestions were carried out over 4 h periods at 3PC. Deoxyribonuclease (Worthington, electrophoretically purified) in 4~10~~ M MgSO, and 0.01 M sodium acetate, and ribonuclease (Worthington) were used at a concentration of 0.3 mg/ml. The RNase, in 0.1 M phosphate buffer, was freed of possible DNase activity by heating for 20 min at 100°C. After the respective enzymatic treatments, slides were washed in 3 changes of water, rinsed for IS min in cold 5 % TCA, washed again in water, dehydrated in ethanol and airdried. Actinomycin D (Calbiochem, lyophylized, mol. wt 1255.5), when used for the in situ procedure, was used at a concentration of 200 pg/ml. A 0.05 ml drop was placed on the slide, followed by a coverslip to insure uniform spreading. After IO min the coverslip was washed off with water and the slides were washed 3 times in distilled water and air-dried. For the in vivo procedure, actinomycin D was added to the culture medium to give a final concentration of 15 wg/ml which is not lethal to the cells [6].
Enzymatic
RESULTS In vivo incorporation
of [Hluridine
Evidence for the specificity of the label used for RNA is shown in table 1. RNase removed 79-89% of the label and 90% of
4.0io.9
D Mean number of grains/U area (81 Fm*) of the macronucleus. b Mean number of grains/U area (81 pm2) of the cytoplasm. ’ Mean number of cpmlcell from 5 samples of 10 cells each, counted in the scintillation counter. d CIM, Confidence Interval of the Mean. of labeling cells in situ. A reaction mixture was prepared, consisting of 0.1 mM ATP, 0.1 mM CTP, 0.1 mM GTP, 0.003 PM [SH]UTP (Schwarzl Mann spec. act. 19 CilmMole, 0.5 mCi/ml), 0.001 M MnCI,, 0.004 M MgC&, 0.01 M 2-mercaptoethanol, 0.04 M Tris-HCI pH 7.9 and 6.67 U/ml of E. coli RNA polymerase (BoehringerlMannheim, prepared from E. coli MRE 600 by the technique of Burgess & Travers, contains sigma factor and is essentially free of DNA polymerase, RNase, DNase, and polynucleotide phosphorylase). A 0.05 ml drop of this reaction mixture was placed on each slide and covered with a no. 1 coverslip. The slides were placed in special moisture chambers and incubated at 3PC for 30 min. The reaction was then stopped by rinsing off the coverslip with cold 5 % TCA followed by 3 washes in cold 5 % TCA. Slides were then rinsed in running tap water for 20 min. dehydrated, and air-dried. The slides were coated with Kodak NTB-2 nuclear track emulsion, exposed for 2 days, developed in Kodak D-19, followed by fixation in Kodak Rapid Fixer. Method
5i I
10
20
30
40
50
m
70
80
90
1001101~0130140
,
I. Abscissa: fission age; ordinate: cpmlcell. [SH]Uridine incorporation. Data represent the total amount of [3H]uridine (IO &i/ml spec. act. 28 Ci/ mMole) incorporated during the entire interfission period. Each point is the mean cpmlcell +95% CIM from 5 samples of IO cells each collected on Millipore filters and counted in the scintillation counter as described in Materials and Methods. (r, -0.87; slope, -0.23). Cells were labeled continuously until they were observed entering the next division.
Fig.
Exptl Cell Res 98 (1976)
66
Kluss and Smith-Sonnehorn
Table 2. [3H]Uridine incorporation ly intervals during the cell cycle
at hour-
Synchronized cells of different sees were obtained as described in Materials and Methods. At subsequent intervals post division, cells were removed from the pooled sample and given 1 h pulses of [3H]uridine (50 &i/ml, spec. act. 28 CilmMole) The data represent the mean cpmlcell from .s samples of IO cells each i-95% CIM. Cells were prepared and counted in the scintillation counter as described in Materials and Methods. Those means underlined by the same line are not significantly different from one another at the 95 % level of confidence using the Mann-Whitney U Statistic [ 161 The dashed line indicates fission and represents the time when the first cell in the population was observed in the next division. Ninety percent of the cells divided within the subsequent 0.5 h interval Time (hours)
6 tissions
70 tissions
IS0 tissions
0-I l-2 2-3 3-4
205+25 230+30 4llk45 342 +40
lO4f IS l46+20 179k25 170+20
65f IO lO6+ I5 lO9k I6 149&22
4-5
275535
139t20
l24+
5-6
387k50
323 +40
136k20
l60+20 -----
121~18 94-t 14
6-7 7-8 Total
----I 850
I221
I8
904
the uptake was inhibited by actinomycin D (I5 pg/ml). As described previously [6], the majority of C3H]uridine is directly incorporated into RNA and is not in the form of unincorporated RNA precursors in the cytoplasm. The total amount of incorporation of r3H]uridine measured over the entire interfission period is shown in fig. 1. The results show reduction in the total uptake of [“HIuridine in older cells. Results from pulse-labeled studies of the amount of in vivo incorporation of [3H]~ridine during hourly intervals after division are summarized in tables 2 and 4. It is clear that the amount of incorporation of [3H]uridine at each hourly interval during the Exptl Cell Res 98 (1976)
cell cycle is significantly @
