DNA-dependent RNA polymerase levels during the response of human peripheral lymphocytes to phytohemagglutinin

Cell, Vol . 4, 5 1 -57, January 1975, Copyright© by MIT

DNA-Dependent RNA Polymerase Levels during the Response of Human Peripheral Lymphocytes to Phytohemagglutinin Judith A . Jaehning, Carleton C . Stewart, and Robert G . Roeder Department of Biological Chemistry Division of Biology and Biomedical Sciences Washington University, and Section of Cancer Biology Division of Radiation Oncology Mallinckrodt Institute of Radiology Washington University School of Medicine St . Louis, Missouri 63110

Summary The cellular levels of the various RNA polymerases have been monitored in resting human peripheral lymphocytes and in lymphocytes stimulated by phytohemagglutinin . Activity was measured in the presence of exogenous templates following solubilization and chromatographic resolution of the different RNA polymerases . Resting lymphocytes contain Class I, II, and III RNA polymerases, although the respective levels of activity are very low compared to the levels in metabolically active cell types . During the PHA-induced transformation of resting lymphocytes, the Class I, II, and III enzyme levels rise dramatically . During four days exposure to PHA, the levels of RNA polymerases I and III (which synthesize, respectively, rRNA and the transfer and 5S RNAs) increase 17 fold, while the level of RNA polymerase II (which synthesizes heterogeneous nuclear RNA) increases 8 fold . The possible relationship between enzyme levels and the regulation of gene expression is discussed . Introduction Peripheral blood lymphocytes are a resting, metabolically inactive population of cells that can be stimulated to proliferate in culture by treatment with the plant lectin phytohemagglutinin (PHA), (Nowell, 1960) . This process of blast transformation involves rapid and dramatic increases in the rates of synthesis of both RNA and protein in addition to increases in several other metabolic activities (Cooper, 1970) . An increase in DNA synthesis is evident on about the second day of culture, with cell division beginning approximately a day later (Loeb, Ewald, and Agarwal, 1970) . Although the apparent rates of synthesis of all species of RNA increase, it appears that the rates of synthesis of rRNA and tRNA increase earlier and to a greater extent than does the rate of synthesis of DNA-like (heterogeneous nuclear) RNA (Kay and Cooper, 1969 ; Cooper, 1969) . These conclusions are reinforced by other studies of the rates of synthesis of presumptive rRNA and DNAlike RNA species by the endogenous RNA poly-

merase activities in nuclei isolated from human PHA stimulated lymphocytes (Cooke and Kay, 1973 ; Cooke and Brown, 1973 : Pogo, 1972) . The mechanism by which these dramatic alterations in the rates of synthesis of the major classes of RNA are effected in response to PHA is not understood . The cellular activity levels of specific RNA polymerases could be altered, specific templates could be activated, or a combination of these changes might be involved if both RNA polymerases and activated (derepressed) templates were limiting . The first possibility appears tenable since eucaryotic cells contain at least three classes of RNA polymerases (Roeder and Rutter, 1969) which are functionally distinct . RNA polymerase I transcribes the rRNA genes ; RNA polymerase 11 transcribes the DNA sequences for heterogeneous nuclear RNA (hnRNA) ; and RNA polymerase III transcribes the tRNA and 5S RNA genes (reviewed in Weinmann and Roeder, 1974 ; Roeder et al ., 1974) . The Class I, Il, and III RNA polymerases also differ structurally (Schwartz and Roeder, 1974, 1975 ; Roeder et al ., 1974 ; Sklar, Schwartz, and Roeder, 1975 ; Kedinger, Gissinger, and Chambon, 1974), compatible with the idea that these enzymes and the genes which they transcribe may be regulated independently . Cooke and Brown (1973) have demonstrated increased levels of solubilized RNA polymerase activity (dependent upon exogenous templates) in crude extracts from PHA stimulated porcine lymphocytes . However, the levels of the Class I, II, and III RNA polymerases were not clearly distinguished, nor was it clear whether yields of solubilized RNA polymerases were quantitative . The present report describes the solubilization, chromatographic resolution, and determination of the cellular activity levels of Class I, II, and Ill RNA polymerases in PHAstimulated human lymphocytes . These studies have also employed a new cell culture technique (Stewart, Cramer, and Steward, 1975) which provides better growth and viability of cultured lymphocytes and which has permitted analyses of stimulated lymphocytes over longer periods of exposure to PHA . Results Physiological and Biochemical Characteristics of Cultured Lymphocytes The cell culture techniques described in Experimental Procedures have been developed to provide maximum viability and growth of human peripheral lymphocytes during PHA-induced blast transformation (Stewart et al ., 1975) . In addition, the use of the pronase-cetrimide cell counting procedure (Stewart et al ., 1975) allows the precise determi-



Cell 52

nation of viable cell numbers at all times during culture . Figure 1 A shows a typical experiment in which cell numbers during the course of the culture period are plotted as a percentage of initial values . In the absence of PHA the lymphocyte cell number shows only a gradual decrease over the 6 day culture period . In the presence of PHA the cell number initially drops to approximately 50% of the day 0 value . Cell division begins between days 2 and 3, and nearly exponential growth continues until day 5 . Cell numbers stop increasing at this time, and after day 6 begin to decrease (data not shown) . The DNA content of the PHA-stimulated cells, as shown in Figure 1B, increases rapidly between day 1 and 2 as would be expected for a population of cells preparing to divide . During the exponential growth phase of days 3 and 4, the DNA content is approximately twice that of the unstimulated cells, while on day 5 it approaches the value of the resting cells and reflects the cessation of division by these cells . The incorporation of tritiated thymidine into the DNA of control and stimulated lymphocytes during a 1 hr pulse provides a rough estimate

of DNA synthesis activity . The data in Figure 1 C indicate that in these PHA-stimulated lymphocytes DNA synthesis begins between days 1 and 2, peaks on day 3, and then begins to fall off as the cells return to a resting phase on day 5 . These rate changes correlate well with the observed changes in DNA content over the same period . The rates of incorporation of tritiated uridine into RNA in control and stimulated lymphocytes during a 1 hr pulse provide crude estimates of the RNA synthetic activities of these cells . However, since neither nucleotide precursor pool specific activities nor RNA turnover rates are considered, they do not necessarily reflect true rates of RNA synthesis . It can be seen that 24 hr after exposure to PHA the rate of incorporation of tritiated uridine has increased approximately 4 fold . This increase is similar to that observed in other studies of PHAstimulated lymphocytes (Loeb et al ., 1970 ; Cooke and Kay, 1973), but is clearly much smaller than the nearly 100 fold increase in incorporation observed on days 2 and 3 . By days 4 and 5 the rates of labeled uridine incorporation have decreased,

C

1200

U

800

0

I

400

M

E 00

V

0 6000

0 1 .2

_ T

a) U

4) U

O

0 .8-

4000

0 D

Q Z

I M

0.4-

E 0-

2000

0

0.01 0

2

3 Days

4

5

6

0

0

2

3

4

5

6

Days

Figure 1 . Characteristics of Human Peripheral Lymphocytes Cultured in the Presence and Absence of PHA Peripheral lymphocytes were isolated and cultured in the presence and absence of PHA, and all measurements were performed as described in Experimental Procedures . (A) cell numbers; (B) cellular DNA content ; (C) 3H-thymidine incorporation ; (D) 3 H-uridine incorporation . (B), (C), and (D) are values for 10 5 cells .

Levels of Lymphocyte RNA Polymerase 53

but they are still greatly elevated relative to the resting cell level . Fractionation of RNA Polymerases From the above information on the temporal sequence of biochemical changes during lymphocyte transformation, the period from days 0 to 4 was chosen to monitor possible changes in RNA polymerase levels during the conversion of the lymphocytes from the resting to the dividing state . RNA polymerases were solubilized (as described in Experimental Procedures) in order to render the enzymes dependent on exogenous templates for activity . Because only small quantities of material were available, certain precautions were necessary to avoid losses of enzyme activity due to dilution . The addition of exogenous protein to protect the enzyme (see Experimental Procedures) and the maintenance of high enzyme concentrations during solubilization minimized these problems . In addition, all operations were performed on the same day to prevent losses of activity on storage . Table 1 shows the results of a typical experiment where these precautions were taken, and it can be seen that there is no loss during solubilization of either RNA polymerase II activity or the combined RNA polymerase I plus III activity . Activities of Class I plus III RNA polymerase are determined by their resistance to low concentrations of a-amanitin, while class II activity is measured by its sensitivity to low concentrations of a-amanitin (Schwartz et al ., 1974b) . To accurately determine the relative proportions of the RNA polymerase activities, crude extracts were subjected to chromatography on DEAESephadex, which resolves the Class I, II, and III RNA

polymerases (Roeder and Rutter, 1969) . Figure 2 shows two representative experiments, in which extracts from a day 0 control and from cells exposed to PHA for four days were examined . In both cases two major peaks of activity are evident . The first peak corresponds to RNA polymerase I on the basis of its elution position at 0 .09-0 .11 M ammonium sulfate and its insensitivity to a-amanitin . The second major peak corresponds to RNA polymerase II on the basis of its elution position at 0 .19-0 .20 M ammonium sulfate and its complete sensitivity to 0 .5 jig a-amanitin per ml . When the chromatographic

0.4 800

0.2

400

a a

0 0

GO

2000

1000

Table 1 . Recovery of RNA Polymerase Activity after Solubilization and DEAE-Sephadex Chromatography Units of RNA Polymerase Activity 0 Fraction

I + III

II

Fl A

200

155

F1 B

193

197

F2

170

164

DEAE-Sephadex

178

150

The results shown are from one representative experiment . 4 .4 X 107 cells were cultured for two days in the presence of 8 µg PHA/ml and were subjected to the enzyme solubilization procedure. The extract was chromatographed on a 1 ml DEAESephadex column, and 0 .086 ml fractions were collected as described in Experimental Procedures . Assays of fractions F1 A, F1 B, and F2 were performed at 0 .05 M ammonium sulfate, and the chromatographic fractions were assayed as described in Figure 1 . The units of I + III in the crude fractions indicate activity resistant to 0 .5 µg a-amanitin/ml, while units of II represent activity sensitive to 0 .5 µg a-amanitin/ ml. For the DEAE-Sephadex fraction the units of activity of each enzyme were calculated from the individual peaks eluted from the column (see Figure 2) . All values represent activity with calf thymus DNA as template .

10

20

30

Fraction No. Figure 2 . DEAE-Sephadex Chromatography of RNA Polymerases from Human Peripheral Lymphocytes Lymphocytes were isolated and RNA polymerase activity was solubilized and chromatographed on DEAE-Sephadex as described in Experimental Procedures . (A) Fraction F2 enzyme derived from 4 x 108 resting peripheral lymphocytes (day 0 control) was chromatographed on a 3 .5 ml DEAE-Sephadex column, and 0 .17 ml fractions were collected . (B) Fraction F2 enzyme derived from 4 .6 x 107 lymphocytes which had been cultured in 8 µg PHA/ml for 4 days was chromatographed on a 1 .5 ml DEAE-Sephadex column, and 0 .086 ml fractions were collected . Activity from the fractions was measured at the salt concentration resulting from dilution of 10 µI of each column fraction to a final volume of 25 µi . Assays were performed with calf thymus in the absence (0-0) or presence (o---o) of 0 .5 µg a-amanitin/ml or with poly[d(AT)] in the presence of 0 .5 µg a-amanitin/ml (A--- A) . (-) ammonium sulfate concentration .



Cell 54

fractions are assayed in the presence of 0 .5 µg aamanitin per ml to selectively inhibit RNA polymerase II, two additional peaks of activity are observed in the upper region of the salt gradient with either native calf thymus DNA or poly[d(A-T)] as template . These correspond to the Class III RNA polymerases reported in other systems (Schwartz et al ., 1974b) on the basis of their elution positions, their insensitivities to low a-amanitin concentrations, and their increased activities on poly[d(A-T)] relative to DNA . In addition, both of these enzymes, designated here as III A and III B , exhibit the same sensitivity (data not shown) to high concentrations of a-amanitin (50% inhibition at 20 pg/ml), as do RNA polymerases IIIA and IIIB from mouse plasmacytoma cells (Schwartz et al ., 1974b) . RNA Polymerase Levels during PHA Induced Blast Transformation It is evident from Figure 2 that both resting lymphocytes and transformed lymphocytes (cultured for four days in the presence of PHA) contain all three classes of RNA polymerases . However, the absolute levels of the enzymes per cell are markedly higher in the transformed lymphocytes . This is more readily apparent in Table 2, which summarizes the analyses in Figure 2 in addition to similar analyses performed on lymphocytes cultured in the presence or absence of PHA for varying periods of time between 0 and 4 days . The data are expressed as enzyme activities per cell to facilitate direct comparisons . Table 2 also presents for comparison the cellular

Table 2 . RNA Polymerase Levels in PHA Stimulated Lymphocytes Days in Culture

enzyme levels in two metabolically active mammalian tissues (mouse liver and malignant mouse plasmacytoma) . The cellular activity levels of RNA polymerases I, II, and III are much lower in the resting lymphocytes than in the liver cells . During the course of lymphocyte transformation and proliferation in response to PHA, the levels of all enzymes increase greatly . After four days in culture the RNA polymerase II level approximates that found in the liver cell, while the RNA polymerase I and III levels exceed by several fold those seen in this comparison tissue . In no case, however, do the enzyme levels in stimulated lymphocytes reach those found in the malignant, rapidly proliferating plasmacytoma cells . The basal enzyme levels remain low (or decrease slightly) in the lymphocytes cultured in the absence of PHA for four days . The temporal increase in the levels of the enzymes in PHA stimulated lymphocytes are more readily visualized in Figure 3, in which the data from Table 2 have been replotted as relative increases in activity over the day-0-levels . The enzymes all appear to increase at approximately the same time insofar as is possible to judge from these data . However, the levels of both RNA polymerases I and III appear to increase to a greater extent over the five day period (about 17 fold in each case) than does the level of RNA polymerase II (about 8 fold) . As a result, RNA polymerases I and III comprise larger fractions of the total activity in transformed lymphocytes (53% and 10%, respectively) than in resting lymphocytes (39% and 6%, respectively) . Conversely, RNA polymerase II accounts for a smaller fraction of the total activity in stimulated lymphocytes (36%) than in resting lymphocytes (55%) .

Units/108 Cells PHA

1

II

20

III T

0

135

191

23

0 .5

+

151

213

23

1

+

433

477

67

2

+

1013

663

148

3

+

1768

953

350

4

+

2241

1502

406

77

141

12

764

1350

186

5500

2600

1100

4 Mouse Liver Mouse Plasmacytoma

Enzyme levels were calculated from DEAE-Sephadex chromatographic analyses of solubilized enzyme preparations similar to those shown in Figure 2 . The enzyme activity is expressed as units/ 108 lymphocytes . RNA polymerase III activity is the sum of the peaks of III A and 111 8 measured with calf thymus DNA as template and in the presence of 0 .5 µg a-amanitin/ml . Values represent the average of from 3-8 experiments . Values for mouse liver and plasmacytoma RNA polymerase levels are from Schwartz et al . (1974b) .

16 Q C a) 0 a) U C a) 0

12

8

4

0 0

1

2

3

4

Days in Culture Figure 3 . Relative Increases in Cellular RNA Polymerase Levels in PHA Stimulated Lymphocytes The levels of enzyme activity in lymphocytes cultured for varying periods of time in the presence (closed symbols) or absence (open symbols) of PHA are plotted relative to the enzyme levels present in resting peripheral lymphocytes at day 0 . The data is calculated from that presented in Table 2 .

Levels of Lymphocyte RNA Polymerase 55

Discussion The present data confirm previous reports of dramatic increases in the apparent rates of RNA and DNA synthesis and the initiation of cellular proliferation during the response of cultured peripheral lymphocytes to PHA (Cooper, 1970 ; Loeb et al ., 1970) . However, the magnitude of the increase in the apparent rate of RNA synthesis is much greater than previously reported, possibly reflecting the increased cell viability in the present system . Both resting and stimulated lymphocytes contain Class I, II, and III RNA polymerases which synthesize, respectively, rRNA, hnRNA, and tRNA and 5S RNA (see Introduction for references) . Both cell types also contain two chromatographically distinct forms of RNA polymerase III analogous to those described in mouse tissues (Schwartz et al ., 1974b) . Although the catalytic properties and subunit structures of III A and Ills appear quite similar (Schwartz et al ., 1974b; Sklar, Schwartz, and Roeder, 1975), it remains possible that the two enzyme forms have discrete functions . The cellular levels of all enzymes (I, II, and III) change dramatically in response to PHA, increasing from very low levels to levels which exceed those found in other normal adult mammalian tissues (but not those found in malignant, rapidly proliferating tissues) . This suggests that the enzyme levels in resting lymphocytes are insufficient for the increased RNA synthesis which is observed in stimulated lymphocytes . The increase in total RNA polymerase activity (about 9 fold by day 3) is not as great as the apparent increase in the rate of RNA synthesis (100 fold by day 3) . This could reflect an excess of the RNA polymerases in resting lymphocytes, modulation of RNA polymerase activity in vivo by other factors (see below), or discrepancies between rates of labeled uridine incorporation and actual rates of RNA synthesis due to variations in precursor pool specific activities or rates of RNA turnover between resting and stimulated cells (Abelson et al ., 1974) . These latter possibilities might also explain the apparent discrepancy between the increasing levels of RNA polymerase activity in the stimulated lymphocytes between 3 and 5 days in culture and the decreasing rate of uridine incorporation during the same time period . Although cellular levels of all classes of RNA polymerase increase in stimulated lymphocytes, enzymes I and III increase to a greater extent (17 fold) than does enzyme II (8 fold) . These findings agree well with previous conclusions that while the rates of synthesis of all classes of RNA increase in stimulated lymphocytes, the rates increase to a greater extent for rRNA and for tRNA than for hnRNA (see

Introduction) . Other studies also suggest correlations between levels of solubilized RNA polymerase activities and rates of synthesis of specific classes of RNA . Thus Mauck and Green (1973, 1974) found that the cellular rates of rRNA and tRNA synthesis in cultured fibroblasts increased during serum stimulated growth, whereas the cellular rate of hnRNA synthesis remained constant . Although solubilized RNA polymerase levels were not examined in this system, an analysis of various mouse tissues has revealed that the cellular levels of RNA polymerases I and III are greater in tissues with apparently greater rates of cellular growth and proliferation, whereas the levels of RNA polymerase II are relatively constant (Schwartz et al ., 1974b) . In other cell types various growth promoting stimuli (for example, hormones) may effect increases of RNA polymerase I activity with little or no change in RNA polymerase II activity (RNA polymerase III has not been examined) (references in Schwartz et al ., 1974b), which is in agreement with the preferential stimulation of rRNA synthesis in these systems . The observed change in RNA polymerase II activity in the PHA-stimulated lymphocytes contrasts with these observations and with those of Mauck and Green (1973), but may simply reflect the greatly reduced rates of synthesis of all classes of RNA, including the hnRNAs, in these metabolically inactive lymphocytes . The results observed in all these systems are consistent with the idea that the activities of specific genes or classes of genes may be regulated in part by specific enzyme levels . The present studies do not permit conclusions regarding the mechanisms responsible for variations in RNA polymerase levels which could be due to increased enzyme concentrations or to modulations of the activity of a constant cellular population of enzyme molecules . In mouse plasmacytoma cells the increased level of RNA polymerase I activity per cell (relative to that in a mouse liver cell) is due to an increased concentration of enzyme molecules (Schwartz and Roeder, 1974) . In addition, it appears that RNA synthesis by solubilized RNA polymerases on heterogeneous templates is largely nonspecific and may measure RNA polymerase catalytic activity independent of any regulatory modification of the enzyme which may have occurred in vivo (see, for example, Schwartz et al ., 1974a ; Weinmann, Raskas, and Roeder, 1974 ; Yu and Feigelson, 1972) . It thus is probable that increased levels of solubilized RNA polymerases in the present system reflect primarily increased enzyme concentrations . There is also evidence that other factors may be involved in the regulation of enzyme activity in vivo . During viral infection of animal cells (Weinmann et al ., 1974 ; Schwartz et al ., 1974a) or during

Cell 56

inhibition of protein synthesis (Yu and Feigelson, 1972 ; Mauck and Green, 1974 ; Gross and Pogo, 1974), specific RNA polymerase activities in intact cells or in isolated nuclei may be inhibited or stimulated even though the solubilized levels of enzyme are unaffected . In addition, some effects on RNA synthesis may be too rapid to be accounted for by synthesis of new RNA polymerase molecules (Mauck and Green, 1973) . Whether these modulations of RNA polymerase activity reflect alterations of the chromosomal template(s), or effects directly upon the enzyme(s), is not known . Experimental Procedures Cell Isolation and Culture Venous blood from normal human donors was drawn into 15 ml heparinized glass tubes which were subjected to centrifugation for 10 min at 360 x g . The upper half of the packed cells was removed and mixed with 5 vol of 2 .5% Dextran (Sigma) in a modified Eagle's minimal essential medium (a-MEM, Flow Lab .) adjusted to pH 7 .3 with NaOH . The red cells were allowed to settle for 15-30 min, and the supernatant was layered over 9 ml of 6 .4% Ficoll (Sigma), 10% Hypaque (Winthrop) in a 50 ml glass centrifuge tube . The isopycnic gradient was subjected to centrifugation for 12 min at 1600 x g . The cells at the interface were removed and washed 3 times by resuspension and centrifugation with culture medium (a-MEM supplemented with 10% fetal calf serum and 100 units penicillin and 100 µg streptomycin/ml) . All centrifugation steps were at room temperature. The resulting cell preparation contains 95-100% small lymphocytes with essentially no red cell contamination . The cells were cultured in a-MEM as described (Stewart et al ., 1975) with 50 ml of culture medium per 75 cm2 culture flasks (Falcon) and 2 x 105 cells/ml . Phytohemagglutinin (PHA P batch #584,000, Difco), when present, was used at concentrations of 4-8 µg/ml . The flasks were incubated at 37°C in a9% CO2 humidified atmosphere . Counting and viability measurements were done by the method of Stewart et al . (1975), using pronase-treated cells lysed in a cetrimide solution to count nuclei with a Nuclear Chicago electronic particle counter . At appropriate times cells were harvested by combining the contents of similar flasks and collecting cells by centrifugation for 10 min at 620 x g at 4°C . Cells were resuspended at 108 cells/ml in Buffer A (0 .05 M Tris-HCI (pH 7 .9 at 23°C), 25% (v/v) glycerol, 0.1 mM EDTA, and 0 .5 mM dithioerythritol) . Suspensions were quick frozen in a dry ice acetone bath and were stored at -70°C . Thymidine and Uridine Incorporation and DNA Measurements One half ml volumes of 3H-thymidine or 3 H-uridine (specific activity 6 .0 Ci/mmole) solutions diluted to 4 µCi/ml in culture medium were added to replicate 1 ml aliquots of cells. The cells were incubated for 1 hr as described above . Cultures were chilled and centrifuged at 1000 x g for 10 min and were washed once by centrifugation at 1000 x g for 10 min with normal saline . Cells were precipitated with cold 10% trichloroacetic acid and were collected on Millipore filters (0 .22 µ) which were washed with 10 ml cold 10% trichloroacetic acid . Filters in glass vials were incubated for 2 hr at 70°C with 0 .5 ml of 5% trichloroacetic acid . After cooling, 10 ml of liquid scintillation fluid (Kinard, 1957) was added to each vial and radioactivity determined after dispersing the aqueous solution . DNA was measured according to Burton (1956) . RNA Polymerase Solubilization The method used for enzyme solubilization was a modification of the procedure of Roeder (1974) . Cell suspensions in Buffer A (above) were thawed and bovine serum albumin (Pentex) was

added to a final concentration of 1 mg/ml . Buffers used in all subsequent stages, including chromatography, contained the same concentration of bovine serum albumin . The suspensions were lysed by the addition of 4 M ammonium sulfate (pH 7 .9) to a final concentration of 0 .3 M . The viscous solution was sonicated 5-7 times for 10 sec with a Branson S-75 sonifier (ultramicro probe, setting 1) at 0-4°C . The suspension (F1 A) was subjected to centrifugation for 70 min at 45,000 rpm . The supernatant (17113) was diluted to 0 .1 M ammonium sulfate with Buffer A and was again subjected to centrifugation for 50 min at 50,000 rpm . The supernatant (F2) was diluted to 0 .05 M ammonium sulfate with Buffer A in preparation for ion exchange chromatography . All centrifugations were performed at 0-4°C with a Spinco type 65 rotor. Chromatography DEAE-Sephadex (A-25) columns were prepared as described (Roeder, 1974) and equilibrated with Buffer A containing 0 .05 M ammonium sulfate. The sample was applied at <_ 2 mg of protein (excluding the carrier bovine serum albumin) per ml of bed volume . The enzymes were eluted with a 3 column vol linear gradient from 0 .05-0.5 M ammonium sulfate in Buffer A . RNA Polymerase Assay The assay conditions were as described (Roeder, 1974) except for the following modifications : 10 µl of enzyme solution was assayed in a final volume of 25 µl ; calf thymus DNA (Sigma, Type I) or poly [d(AT)] (Miles Lab.) were used as template at a final concentration of 100 or 50 µg/ml, respectively . 3 H-UTP was present at 1 µCi/25 µl . Reactions were incubated at 37°C for 20 min and 3H-UMP-labeled RNA was measured as described . One unit of activity represents incorporation of 1 picomole of UMP into RNA in 20 min under standard assay conditions . Acknowledgment We thank Barbara Wagner and Barbara Nardacci for advice and assistance . This work was supported by grants from the National Institutes of Health and the National Cancer Institute. Received September 9, 1974 References Abelson, H . T ., Johnson, L . F., Penman, S ., and Green, H . (1974) . Cell 1, 161-165 . Burton, K . (1956). Biochem . J . 62, 315-323 . Cooke, A ., and Brown, M . (1973) . Biochem . Biophys . Res . Commun . 51, 1042-1047 . Cooke, A ., and Kay, J . E . (1973) . Exp . Cell Res . 79, 179-185 . Cooper, H. L . (1969) . In Biochemistry of Cell Division, R . Baserga, ed . (Springfield, III . : C. C . Thomas), pp. 91-111 . Cooper, H . L . (1970) . In Proceedings of the Fifth Leukocyte Culture Conference, J . E . Harris, ed . (New York : Academic Press), pp . 15-27 . Gross, K . J ., and Pogo, A . 0 . (1974) . J . Biol . Chem . 249, 568-576 . Kay, J . E ., and Cooper, H . L . (1969) . Biochim . Biophys . Acta 186, 62-84 . Kedinger, C ., Gissinger, F ., and Chambon, P . (1974) . Eur. J . Biochem . 44, 421-436 . Kinard, F . E . (1957) . Rev . Sci . Instr. 28, 293-294 . Loeb, L . A., Ewald, J . L., and Agarwal, S. S . (1970) . Cancer Res . 30,2514-2520 . Mauck, J . C ., and Green, H . (1973) . Proc . Nat . Acad . Sci. USA 70, 2819-2822 . Mauck, J . C ., and Green, H . (1974) . Cell 3, 171-177 . Nowell, P . C . (1960) . Cancer Res . 20, 462-466 . Pogo, B . G . T. (1972) . J . Cell Biol. 53, 635-641 .

Levels of Lymphocyte RNA Polymerase 57

Roeder, R . G . (1974) . J . Biol . Chem . 249, 241-248 . Roeder, R . G ., and Rutter, W . J . (1969) . Nature 224, 234-237 . Roeder, R . G ., Chou, S . M ., Jaehning, J . A ., Schwartz, L . B ., Sklar, V . E . F ., and Weinmann, R . (1974) . Annals New York Acad . Sci ., in press . Schwartz, L . B ., and Roeder, R . G . (1974) . J. Biol . Chem . 249, 5898-5906 . Schwartz, L . B ., Lawrence, C ., Thach, R . E ., and Roeder, R . G . (1974a) . J . Virol . 14, 611-619 . Schwartz, L . B ., Sklar, V . E . F ., Jaehning, J . A., Weinmann, R ., and Roeder, R . G . (1974b) . J . Biol . Chem . 249, 5889-5897 . Schwartz, L. B ., and Roeder, R . G . (1975) . J . Biol . Chem . 250, in press . Sklar, V . E . F ., Schwartz, L . B ., and Roeder, R . G . (1975) . Proc . Nat . Acad . Sci . USA, in press . Stewart, C . S ., Cramer, S . F ., and Steward, P . G . (1975) . Cell, Immunol ., in press . Weinmann, R ., and Roeder, R . G . (1974) . Proc . Nat . Acad . Sci . USA 71, 1790-1794 . Weinmann, R ., Raskas, H ., and Roeder, R . G . (1974) . Proc. Nat . Acad . Sci . USA 71, 3426-3430 . Yu, Fu-Li, and Feigelson, P . L . (1972) . Proc . Nat . Acad . Sci . USA 69,2833-2837 .