Precursor-product relationships between thymidine nucleotides and DNA in mammalian cells I. Studies on intact cells

107

Biochimica et Biophysica Acta, 565 ( 1 9 7 9 ) 1 0 7 - - 1 1 6 © Elsevier/North-Holland Biomedical Press

BBA 99547 PRECURSOR-PRODUCT RELATIONSHIPS BETWEEN THYMIDINE NUCLEOTIDES AND DNA IN MAMMALIAN CELLS I. STUDIES ON INTACT CELLS

E R N S T A. B A U M A N N * a n d R I C H A R D S C H I N D L E R **

Department of Pathology, University of Bern, 3010 Bern (Switzerland) ( R e c e i v e d M a r c h 21st, 1 9 7 9 )

Key words: Thymidine nucleotide; DNA synthesis; DNA replication; Cell culture; Precursor-product relationship

Summary In an attempt to identify the thymidine nucleotide serving as proximate precursor for DNA synthesis, kinetics of incorporation of [aH]thymidine into individual thymidine nucleotides and into DNA of Chinese hamster ovary cell cultures were analyzed after prelabeling of nucleotides or of DNA with [14C]thymidine. Labeling kinetics of nucleotides were determined in synchronous S-phase cell populations. The rate constant for incorporation of thymidine into dTMP was higher by a factor of approx. 10 than that for incorporation into dTDP and dTTP. On the other hand, kinetics of labeling of dTDP and dTTP were not significantly different from each other, indicating a high rate of interconversion of these two nucleotides. Rate constants for labeling of dTDP and dTTP were in good agreement with that of the proximate DNA precursor as derived from the time course of DNA labeling. This supports the notion that labeled, and possibly also total, intracellular dTDP and dTTP represent single-pool compartments. Due to the rapid interconversion of dTDP and dTTP, these data obtained with intact cells are compatible with the assumption that either dTDP or dTTP, or possibly both of these intermediates, are used as the proximate precursor(s) for DNA synthesis.

* Present address: D e p a r t m e n t of Microbiology and Immunology, McGill University, Montreal, Canada. ** To w h o m correspondence should be addressed.

108 Introduction

Since the work of Kornberg [1] on DNA polymerase I of Escherichia coli, it is widely accepted that 2'-deoxyribonucleoside 5'-triphosphates are the proximate precursors for DNA synthesis. For DNA replication in intact cells, this assumption was, however, questioned by Werner [2] on the basis of kinetic studies with E. coli, and another, as yet unidentified precursor was proposed. Rubinow and Yen [3] concluded that dTDP was more likely than dTTP to be the proximate DNA precursor. Similarly, results obtained with permeabilized E. coli suggested that deoxyribonucleoside diphosphates may be incorporated into DNA without being converted to triphosphates [4]. In cultured human lymphoblasts, only dTTP exhibited labeling kinetics that appeared to be compatible with the function of a proximate precursor of DNA [5]. The results suggested, however, that dTTP was distributed in at least two pool compartments. The assumption of a compartmentation of thymidine nucleotides in mammalian cells was further supported by differences in kinetics of incorporation between thymine and thymidine [6]. Separate pools of thymidine nucleotides synthesized from deoxyuridine monophosphate and from exogenous thymidine were proposed by Kuebbing and Werner [7]. Furthermore, differences in kinetics of nuclear and cytoplasmic deoxyribonucleoside triphosphates have been reported [8]. On the other hand, no evidence for compartmentation of thymidine nucleotides was found by Plagemann [9]. In addition, Wittes and Kidwell [10] concluded that in mouse L cells, all intracellular dTTP served as precursor for DNA synthesis. Since the nature of the proximate precursors for DNA synthesis and their possible compartmentation within mammalian cells do not appear to be unambiguously established as yet, we studied the kinetics of incorporation of thymidine into nucleotides and DNA of cultured cells by means of doublelabeling procedures. For the analysis of the time course of nucleotide labeling, synchronous S-phase cell populations were used. The use of a subcellular system for distinguishing between dTDP and dTTP as proximate DNA precursor will be described in the subsequent paper [11]. Materials and Methods

Cell line and culture techniques. Chinese hamster ovary (CHO) cells [12] were grown as monolayers in culture medium [13] containing 2 . 1 0 - ~ M thymidine. Cultures were frequently tested and found to be free of mycoplasma contamination (tests kindly performed by G. Kronauer). To measure incorporation of labeled precursors, cultures in Petri dishes were incubated as described [13]. Asynchronous cultures were used to measure incorporation of labeled thymidine into DNA, while synchronous cultures were used to measure precursor incorporation into thymidine nucleotides. To prepare synchronous cultures, mitotic cells were collected from monolayers by the mechanical shaking method of Terasima and Tolmach [14] modified by Tobey et al. [15]. The detached cells were suspended in conditioned medium and incubated at 37°C. To determine labeling kinetics of nucleotides, synchronous cultures were

109 brought to 25°C at 6.5 h after incubation of mitotic cells, i.e. at a time when more than 90% of the cells were in S phase. Chemicals. Nucleotides were of analytical grade and purchased from Serva. All radiochemicals were purchased from the Radiochemical Centre, Amersham. Determination of cellular dTTP content. The cellular dTTP content was determined by the method of Lindberg and Skoog [16] as described [13]. Determination of the labeling index in synchronous cultures. Relative numbers of cells engaged in DNA synthesis were determined by incubation of cultures with fresh medium containing [3H]thymidine (5 pCi/ml of medium, 20 Ci/mmol) for 15 min at 37°C. For autoradiography, the cells were fixed, brought onto microscope slides and covered with Kodak NTB-2 emulsion. After the appropriate exposure time, the preparations were developed and stained with Giemsa. The percentage of labeled cells (labeling index) was determined by scoring 300 cells/preparation. Labeling indices obtained by labeling at 37°C were indistinguishable from those obtained by labeling at 25°C. Procedure for measuring incorporation of [3H]thymidine into individual nucleotides. Synchronous cultures were supplied with 2 ml of conditioned medium containing [14C]thymidine (0.1 gCi/ml, 2 • 10 -6 M). After incubation for 1 h at 25°C, 1 ml of conditioned medium containing 0.1 gCi/ml of [14C]thymidine and 6 pCi/ml of [3H]thymidine (total concentration of thymidine: 2 • 10 -4 M) was added (time zero in Fig. 2), and at different times, duplicate cultures were used to determine 3H/~4C cpm ratios of individual thymidine nucleotides as described below. During the 2 h period used for measuring incorporation of [3H]thymidine into nucleotides, 3H radioactivity in the medium decreased by less than 3%, and no significant changes of ~4C radioactivity in the nucleotide fraction (approx. 2500 cpm/culture) were detectable. Counting efficiencies, as determined with 3H and ~4C standards, were 28% and 57%, respectively. Nucleotides were extracted from the cultures at --25°C for 30--120 min with 60% methanol containing 5 • 10 -s M unlabeled dTDP. This procedure provided for extraction of more than 80% of radioactivity present in thymidine nucleotides. The extracts were centrifuged, and the supernatants were lyophilized. The lyophilized extracts were dissolved in 60% methanol, and aliquots were analyzed by thin-layer chromatography on PEI-cellulose plates (Merck) according to Randerath and Randerath [17]. The spots containing thymidine and its nucleotides were scraped off into scintillation vials. After elution for 2 h with 2 ml of 0.02 M Tris-HC1 buffer (pH 6.5) containing 0.7 M MgC12, 18 ml of a xylene/Triton X-100 cocktail were added, and radioactivity attributable to 14C and 3H was determined by scintillation counting. An example of the separation is shown in Fig. 1. In this procedure of extraction and processing for chromatography, the extent of formation, interconversion and degradation of nucleotides was sufficiently small to be negligible, as shown by the following test. Immediately before the addition of 60% methanol to unlabeled cells, 3H-labeled thymidine, dTMP, dTDP, or dTTP, respectively, was added. The samples were extracted, and the distribution of radioactivity in thymidine and its nucleotides was determined. As a control, the individual labeled substances were subjected directly to chromatography without cell extract. The radioactivities recovered

110

200

150

IO0

~50 0 x 0 >-

dTTP

C~ <~ O

dTDP

dTHP

Thy dThd

t

t

START n"

;

FRONT

lb

2o

3'o

RACTION NUMBER Fig. I . Separation of t h y m i n e , t h y m i d i n e and t h y m i d i n e n u c l e o t i d e s by thin-layer c h r o m a t o g r a p h y o n PEI-cellulose. A m i x t u r e of unlabeled cell extract and o~ a s ol ut i on containing 3H-labeled dThd, dTMP, dTDP, dTTP and unlabeled Thy (2.5 m g / m l o f e a c h ) was subjected to c h r o m a t o g r a p h y . The plate was c u t in to 5-mm strips, and radioactivity in each strip was determined. Lower part: spots as s e e n u n d e r ultraviolet light. U p p e r part: distribution of radioactivity.

in the respective chromatography spot (in percent of total radioactivity applied) under the two conditions were nearly identical (+2%). This indicates that thymidine and its nucleotides were n o t phosphorylated or degraded to any significant extent. Measurements of incorporation of [3H]thyrnidine into DNA. Cultures were incubated overnight at 37°C in medium containing [14C]thymidine ( 4 . 1 0 -3 gCi/ml, 2 • 10 -6 M). Subsequently the medium was replaced b y 1.4 ml of conditioned medium containing 2 . 1 0 -6 M unlabeled thymidine, and incubation was continued at 25°C. After 1 h, 0.1 ml of conditioned medium containing [3H]thymidine (30 #Ci/ml, 2 • 10 -6 M) was added. At different time intervals, radioactivity in DNA of duplicate cultures was measured as described [13]. Counting efficiencies, as determined with 3H and 14C standards, were 33% and 54%, respectively. Rates of incorporation of [SH]thymidine into DNA at 25°C were found to be approx. 20% of those at 37°C, and after transfer of cultures from 37 to 25°C, the capacity of cultures to incorporate [3H]thymidine into DNA exhibited a slight decrease with time. To determine the extent of this decrease, five cultures each were incubated at 25°C for 1 h and for 2 h, respectively, before the addition of [3H]thymidine, and incubation was continued for 1 h. Incorporation of the precursor into DNA during the time interval of 2--3 h after transfer to 25°C was found to be 89.3 + 1.7% of that during the time interval of 1--2 h after transfer to 25°C. This decrease in incorporation was used to correct the values obtained for 3H radioactivity in DNA, and corrected values are presented in Fig. 3 and Table I. In similar experiments with syn-

111 chronous S-phase cell cultures, incorporation of labeled thymidine during the time interval of 2--3 h after transfer to 25°C was approx. 95% of that during the time interval of 1--2 h after transfer to 25°C.

Mathematical analysis of labeling kinetics

Incorporation of [aH]thymidine into thymidine nucleotides Mathematical analysis of labeling kinetics of nucleotides was based on the following assumptions: (1) The molar concentration of thymidine, as well as specific activities of [3H]thymidine and of [14C]thymidine in the medium were constant during the period of incorporation; (2) the intracellular concentrations of the three thymidine nucleotides were constant; (3) 14C radioactivity in each of the three intracellular nucleotide pools was constant; (4) intraceUular nucleotide pools were not compartmented; (5) rates of synthesis of dTMP (from thymidine and from deoxyuridine monophosphate), dTDP, dTTP and DNA were constant, and (6) the cell populations were homogeneous, and all cells were engaged in DNA synthesis. With these assumptions, the incorporation of [3H]thymidine into dTMP can be described by the exponential function y = A(1 --e -xt)

(1)

where y, 3H/14C cpm ratio in dTMP (after prelabeling of nucleotides with [14C]thymidine); A, 3H/14C cpm ratio reached after long labeling periods (see Results); t, time after addition of [3H]thymidine, and X, rate constant. As shown in Fig. 2, 3H radioactivity in dTMP increased much more rapidly than that in dTDP and dTTP, while dTDP and dTTP exhibited nearly the same time course of labeling. For reasons of simplicity, the 3H/t4C cpm ratio for dTMP serving as precursor of dTDP and dTTP was, therefore, assumed to be

[3. - ~/

2

~o 3'0

r~

90

120

TIME AFTER ADDITION OF [3H]THYMIDNE, MIN Fig. 2. Kinetics of i n c o r p o r a t i o n o f [ 3 H ] t h y m i d i n e into n u c l e o t i d e s o f s y n c h r o n o u s CHO cultures at 25°C. After prelabeling o f n u e l e o t i d e p o o l s with [ 1 4 C ] t h y m i d i n e for 1 h, [ 3 H ] t h y m i d i n e was added, and 3H/14C c p m ratios w e r e d e t e r m i n e d for t h y m i d i n e (o) and for cellular d T M P (o), d T D P (4), and d T T P (o). - - ~ the calculated best-fit curve for d T T P based o n Eqn. 1; the b r o k e n straight line with the same initial slope as the solid line intersects with t h e plateau level (broken hori z ont a l line) at the t i m e corresponding to 1 / k of d T T P .

112

constant throughout the period of observation, and under this assumption, Eqn. 1 was applied also to dTDP and dTTP. To determine values of k for dTDP and dTTP, the mean of labeling ratios obtained at 90 and 120 min was designated as A (broken horizontal line in Fig. 2), log (A - - y ) was plotted as a function of time, and a linear regression was calculated for values obtained from 0 to 30 min after addition of [3H]thymidine. At later times, values of y usually exceeded 0.9 A and were not used because of larger deviations from a straight line in the semilogarithmic plot. The rate constant ), was derived from the slope of the regression line. As seen in Fig. 2, dTDP and dTTP had not quite reached constant specific activity at 60 min of incubation; due to prelabeling with [*4C]thymidine for only 1 h, 3H/~4C cpm ratios obtained were, therefore, overestimated by up to 3%. This small error was not taken into consideration in calculating values of k.

Incorporation of [3H]thymidine into DNA On the assumption that 3H radioactivity in the proximate precursor of DNA increases according to Eqn. 1, incorporation of [3H]thymidine into DNA may be described by the equation 1

Y= BIt---~(1--e-Xt)l

(2)

as obtained by integration of Eqn. 1, where Y, 3H/14C cpm ratio in DNA (after prelabeling of DNA with ['4C]thymidine); B, rate of incorporation of [3H]thymidine into DNA after the proximate precursor has reached a constant specific activity with respect to 3H; t, time after addition of [3H]thymidine, and k, rate constant for 3H-labeling of the proximate DNA precursor. Using Eqn. 2, values of B and k giving the best fit of calculated Y values to the experimental data were derived as follows: arbitrary values of k were chosen, and for each value of )% a corresponding value of B was calculated as the linear regression in the equation

Y = S × T,

(3)

where T = t--l(1--e

-kt)

(4)

The pairs of values for k and B thus obtained were used in Eqn. 2, and k and B giving the best fit with the experimental values of Y (least squares of differences between the calculated values of Y and measured isotope ratios) were determined. Results

Incorporation of labeled thymidine into nucleotides of S.phase cells Synchronous cultures were prepared by collection of mitotic cells and incubated at 37°C for 6.5 h. At this time, the cultures were brought to 25°C, supplied with [14C]thymidine, and I h later [3H]thymidine was added. At this time, more than 90% of the cells were in S phase, and the labeling index

113 remained at this level for at least the following 120 min. Results obtained in a typical experiment are shown in Fig. 2. At 2 min after the addition of [3H]thymidine the 3H/14C cpm ratio for dTMP already had reached more than 80% of the plateau level. In contrast, the increase in isotope ratio of both dTDP and dTTP occurred considerably more slowly, and 3H/14C cpm ratios observed for these two nucleotides were nearly identical. Maximal 3H/14C cpm ratios of dTDP and dTTP were consistently somewhat lower than that of thymidine. This difference could be accounted for by the presence of impurities in [3H]thymidine [18] as determined by methods described in the following paper [11]. Furthermore, the plateau level of dTMP usually was higher or lower than that of dTDP and dTTP. No such difference was observed, however, if cells were washed prior to extraction, indicating that 3H/~4C cpm ratios of dTMP were affected by materials extracted from the medium. Values of X for dTDP and dTTP were calculated by regression analysis as described above. The X for dTTP thus obtained was used in Eqn. 1, and the resulting function is shown as solid line in Fig. 2. It is seen that the experimental data are in good agreement with the calculated function. The initial slope of the function is represented by the broken line which intersects with the plateau level at the time corresponding to 1/X. The value of 1/X for dTMP may be estimated to be 2 min or less. The values of 1/X for dTDP and dTTP, as determined in three independent experiments, are given in Table I. It is seen that values of 1/~, for dTDP were n o t significantly different from those for dTTP, even though it cannot be excluded that values for dTDP may be slightly lower. To measure cellular dTTP content of S-phase cells, mitotic cells were incubated for 6.5 h at 37°C, brought to 25°C, and incubation was continued in conditioned medium containing 2 • 10 -6 M thymidine. At 1, 2 and 3 h after the onset of incubation at 25°C, the cellular dTTP content was determined and TABLE I R e c i p r o c a l rate constants ( l / X ) for incorporation o f [ 3 H ] t h y m i d J n e into dTDP, dTTP a n d D N A a t 2 5 ° C as obtained b y m a t h e m a t i c a l analysis o f experimental data. I~corporation of [ 3 H ] t h y m i d J n e i n t o d T D P a n d d T T P w a s d e t e r m i n e d after preincubation o f cells w i t h [ 1 4 C ] t h y m i d i n e f o r 1 h, while incorporation o f ' [ 3 H ] t h y m i d i n e i n t o D N A w a s d e t e r m i n e d a f t e r overnight incubation o f cells w i t h [ 1 4 C ] t h y m i d i n e . Rate constants for incorporation into dTDP a n d d T T P w e r e determined b y fitting 3 H / 1 4 C cpm ratios o b t a i n e d for these nucleotides to E q n . 1, and rate constants for incorporation into D N A were o b t a i n e d b y fitting 3 H / 1 4 C cpm ratios of D N A to E q n . 2. Experiment No.

1/~ (min) S - p h a s e cells dTDP

dTTP

DNA

1 2 3 4 5

17.1 17.5 16.4

17.5 18.0 17.6

16.8 18.5 13.8 19.1 18.6

Mean S.E.

17.0 0.3

17.7 0.2

17.8 1.1

114 found to be 135 + 3, 130 + 6, and 128 _+12 pmol/106 cells, respectively (mean o f four measurements + S.E.). Thus, during the time period used to study thymidine nucleotide labeling at 25°C, the cellular dTTP content remained essentially constant.

Distribution of radioactivity between individual nucleotides in S-phase cells Mitotic cells were incubated in medium containing 2 • 10 -6 M thymidine for 6.5 h at 37°C. At this time, [3H]thymidine (2 pCi/ml, 2 • 10 -6 M) was added, and after incubation at 25°C for additional 1, 2, or 3 h, radioactivities in thymidine nucleotides were determined. At all three time points, 87% of total nucleotide radioactivity was associated with dTTP, while 6--7% each were in dTDP and dTMP. As shown above by double-labeling experiments, specific activities of all three nucleotides had attained a nearly constant level at 60 min after addition of [3H]thymidine. It may, therefore, be concluded that the nucleotide pool of S-phase cells consisted of approx. 87% dTTP and 6--7% each o f dTDP and dTMP.

Incorporation of labeled thymidine into DNA Kinetics of incorporation of [3H]thymidine into DNA at 25°C were analyzed in five independent experiments. The results obtained in one of these experiments are shown in Fig. 3. Values for B and ~ in Eqn. 2 giving the best fit of the function with the experimental data were derived as described. The function thus obtained is drawn as a solid line in Fig. 3. It is seen that the experimental data were in good agreement with the values calculated with the best-fit function. The time corresponding to 1/), is represented by the intersection with

6

4

2

Y '

3'o

6'o

~

~o

TIME (MIN) Fig. 3. K i n e t i c s o f i n c o r p o r a t i o n o f [ 3 H ] t h y m i d i n e i n t o D N A . A f t e r o v e r n i g h t i n c u b a t i o n w i t h [ 1 4 C ] t h y m i d i n e , c u l t u r e s w e r e b r o u g h t to 2 5 ° C ; 1 h l a t e r , [ 3 H ] t h y m i d i n e was a d d e d , and 3 H / 1 4 C c p m r a t i o s ( o ) o f D N A w e r e d e t e r m i n e d as a f u n c t i o n o f i n c u b a t i o n tame. T h e f u n c t i o n c o r r e s p o n d i n g t o E q n . 2 as o b t a i n e d b y best-fit analysis is d r a w n as a solid line. The r e c i p r o c a l o f t h e rate c o n s t a n t )~ is r e p r e s e n t e d b y t h e t i m e at w h i c h t h e b r o k e n line w i t h t h e slope B i n t e r s e c t s w i t h t h e z e r o level.

115 the time axis of the broken line with the slope B. The average of 1/k obtained in five experiments was 17.3 min (Table I). In the presence of 2 • 10 -6 M thymidine, not all thymidine residues in newly synthesized DNA were derived from exogenous thymidine. This was shown by comparing incorporation of [3H]thymidine by cultures in which synthesis of thymidine nucleotides from dUMP was blocked by amethopterin. If cultures were incubated for 2 h at 25°C with [3H]thymidine (2 • 10 -6 M, 0.5 Ci/mmol), incorporation into DNA was 62 + 6% of that observed if cultures were supplied with [3H]thymidine ( 2 . 1 0 -s M, 0.5 Ci/mmol), 10-SM amethopterin and 3 • 10 -s M hypoxanthine. Discussion In the study presented, double-labeling procedures were used to determine kinetics of incorporation of thymidine into individual nucleotides and into DNA. Possible variations due to differences in cell numbers/Petri dish were thus eliminated. In addition, no quantitative recovery of labeled nucleotides was required, and conditions of extraction and analysis were chosen under which phosphorylation and dephosphorylation of thymidine and its nucleotides were reduced to insignificant levels. This was achieved by (a) avoiding to wash cells with cold saline prior to extraction; (b) adding unlabeled dTDP to the methanol used to extract the cells; (c) carrying out the extraction at --25°C; (d) limiting the time of extraction, and (e) dissolving the lyophilized extracts in 60% methanol instead of H20. For comparing labeling kinetics of nucleotides with those of DNA, an incubation temperature of 25°C was chosen to provide for a higher resolution along the time axis, and to minimize changes of incubation temperature which may have occurred when individual cultures were removed from the waterbath. Since DNA synthesis at 25°C, as measured by [3H]thymidine incorporation, was approx. 20% of that at 37°C, the 2 h period at 25°C used for studying labeling kinetics corresponded to as little as 4% of the S period of CHO cells which at 37°C is in the order of 10 h. While incorporation of thymidine into DNA is restricted to cells in S phase, labeled thymidine may be taken up and phosphorylated by cells in other phases of the cell cycle as well. For this reason, the rate constant for the proximate DNA precursor as obtained from kinetics of DNA labeling was compared with kinetics of labeling of thymidine nucleotides in synchronous S-phase cell populations. In previous studies attempting to identify the proximate precursor for DNA synthesis in mammalian cells [5], only asynchronous cultures were used. Kinetics of labeling of dTDP were found to be almost identical with those of dTTP (Fig. 2, Table I). On the other hand, the pool size of dTDP was found to be considerably smaller than that of dTTP. If it is accepted that dTTP is formed from dTMP by sequential phosphorylation via dTDP [19], these results indicate that radioactivity was rapidly exchanged between dTDP and dTTP. The difference between the rate constant of dTMP labeling and that of dTDP and dTTP labeling also supports the notion that upon phosphorylation of dTMP, radioactivity was introduced into a pool that was larger by a factor of

116

at least 10 as compared to that of dTMP. Labeling of both dTDP and dTTP was characterized by values of 1/~ which were in good agreement with that of the proximate DNA precursor as derived from the time course of DNA labeling. These results support the notion that intracellular dTDP and dTTP derived from exogenous thymidine each represent a single pool compartment with respect to their function as DNA precursors. Since under the conditions used, not all thymidine residues incorporated into DNA were derived from exogenous thymidine, the data are also compatible with the assumption that thymidine nucleotides formed from endogenous precursors and those synthesized from exogenous thymidine form a single precursor pool. The hypothesis of a single dTTP pool is further supported by the observation that after addition of amethopterin to CHO cells in S phase in thymidine-free medium, cellular dTTP decreased within 10--15 min to nearzero levels [13]. Due to the rapid exchange of radioactivity between dTDP and dTTP, the results obtained in the present study on intact CHO cells do not permit to decide if dTDP or dTTP, or possibly both, are used as the proximate precursor(s) for DNA synthesis. This question was, therefore, further investigated in a subcellular system as described in the following paper [ 11 ].

Acknowledgements This work was supported by the Swiss National Science Foundation. The technical assistance of Miss U. Maurer is gratefully acknowledged. The authors express their gratitude to Prof. H. Cottier for his suggestions and encouragement.

References 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19

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