nucleotide polymerases in the developing avian …the journal of b~log~cal chemi~ry vol. 252. no. 1....

THE JOURNAL OF B~LOG~CAL CHEMI~RY Vol. 252. No. 1. Issue of January 10, pp. 273-283, 1977

Printed in U.S.A.

Nucleotide Polymerases in the Developing Avian Erythrocyte*

(Received for publication, April 7, 1976)

SHIRLEY S. LONGACRES AND WILLIAM J. RUTTER

From the Department of Biochemistry and Biophysics, University of California, San Francisco, San Francisco, California 94143

Avian erythroid cells were separated into five developmental stages by sedimentation on discontinuous isotonic albumin gradients. Solubilized enzyme activities from whole cells were partially purified and characterized by ion exchange and ion filtration chromatography and velocity sedimentation analysis. Three nucleotide polymerase types were investigated: (a) DNA-dependent RNA polymerases; (b) RNA-dependent terminal ribonucleotidyltransferases, and (c) DNA-dependent DNA polymerases.

The two characteristic forms of eucaryotic DNA-dependent RNA polymerases, polymerase I (nucleolar) and polymerase II (nucleoplasmic), were identified. Polymerase III was only marginally detectable even in the earliest developmental populations. At least two species of RNA-dependent terminal ribosyltransferases were present. One apparently was the po1ytA) polymerase observed in other systems. The other terminal transferase was present in two chromatographic forms, required an RNA primer, and used UTP and/or CTP as particularly efficient substrates. Three DNA polymerase activities were resolved, two of which were characteristic of the (Y and /3 DNA polymerases described in other eucaryotic systems. The third polymerase was not the y polymerase but a separate entity. Poly(dC)-dependent RNA polymerase activity, associated with the (Y polymerase, was relatively enriched in the third DNA polymerase species.

The activity levels of the nucleotide polymerases were monitored as a function of red cell maturation. Characteristic declining patterns of activity were obtained for each enzyme which correlate well with the synthetic rates of their in uioo products where these are known. These results are consistent with the postulate that the genera1 transcriptive and replicative control processes operating during development may involve changes in the level of the requisite polymerases.

The avian erythrocyte goes from a state active in macromolecular synthesis (DNA, RNA, protein) in erythroblasts to a relatively quiescent but stable state in the mature erythrocyte (l-5). Nuclei are retained in the mature forms of avian erythrocytes, unlike their mammalian counterparts. It is axiomatic that the activation of macromolecular synthesis must involve specific selective mechanisms, e.g. enzyme synthesis and modification of operator-promoter interactions with a regulatory protein. However, for a genera1 deactivation of cellular processes, it is easier to invoke a nonspecific process such as general template inactivation or enzyme degradation. The unique histone V (f&j, associated with the nucleated erythrocyte, might be an obvious candidate for mediating the genera1 repression of gene expression via the template (6-8). In the work presented here the forms and activity levels of nucleotide polymerizing enzymes have been examined in the developing chicken erythrocyte in an effort to assess their possible role in controlling the gross inactivation of gene activity evident in

* This work was supported by Grant BMS72-02222 from the Na- tional Science Foundation and by Grant HD04617 from the National Institutes of Health.

$ Present address, Department of Pathology, 1211 Geneva 4, Switzerland.

these cells. Three classes of nucleotide polymerizing enzymes in avian erythrocytes have been defined: (a) DNA-dependent RNA polymerases, (b) DNA polymerases, and (c) RNA-dependent terminal ribonucleotidyl transferases. The three forms of DNA-dependent RNA polymerase (I, II, and III) generally present in eucaryotic organisms (9) were identified. Polymerases I and II have been implicated in the synthesis of ribosomal RNA and heterogeneous nuclear RNA, respectively. Recently, Weinmann and Roeder (10) have demonstrated the role of polymerase III in the transcription of the tRNA and 5 S RNA genes.

Two forms of DNA polymerase, the LY and p polymerases, generally present in eucaryotes (review Ref. 11) were identified. In addition, a third chromatographic form of DNA polymerase is present which does not correspond to any obvious DNA polymerase in the literature. An anomalous poly(dC)- dependent RNA polymerizing activity may be associated with (Y DNA polymerase and the third DNA polymerase form.

At least two RNA-dependent ribonucleotidyltransferases have been defined. One is characteristic of the terminal riboadenyltransferase (poly(A) polymerase) described by Tsia- palis et al. (12). The other is present in two chromatographic forms which are otherwise indistinguishable. It is primer-

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274 Nucleotide Polymerases in Erythrocyte Development

dependent rather than template-dependent and appears to be

distinct from the DNA-dependent RNA polymerases. Similar activities have been described in the literature (13-16).

The activity levels of these various enzymes have been monitored in the course of erythroid maturation. The patterns of declination of enzyme activity vary for the different enzymes and may be correlated with the loss of nuclear functions.

EXPERIMENTAL PROCEDURES

Materials

13HlUTP (40 Cilmmol), 13H1ATP (30 Ci/mmol), I”HlCTP (20 Gil mm&, 13HIGTP (10 Cilmmol), and PHITTP (50 Cilmmol) were obtained from New England Nuclear, Boston, Mass. Nucleotides were from P-L Biochemicals; calf thymus DNA was from Sigma; poly[d(A-T)] was from Miles; dithiothreitol was from Calbiochem; Q- amanitin was obtained from Henley Co., N. Y. Bidistilled glycerol and ammonium sulfate were enzyme grade reagents. DEAE-Sepha- dex (A-25) was from Pharmacia Fine Chemicals, Sweden; phosphocellulose (Whatman Pll) was from Reeve Angel, N. Y. Poly(dC), poly(dT), and rA(pA), were the generous gifts of Dr. Fred J. Bollum of the University of Kentucky.

Erythroid Cell Preparation

Six-month-old white leghorn hens were made anemic with daily injections of 25% phenylhydrazine/HCl (Sigma) in ethanol:water (l:l, v:v) neutralized with NaOH. Dosages varied between 125 and 175 mg/day with the lowest amounts given in the middle of a 5 to 8 day injection program. Cells were collected on the day following the last injection. The extent of anemia on the day of collection was monitored using microdiscontinuous gradients of isotonic bovine serum albumin in capillary tubes (5). Blood was collected only when the majority of cells banded at the interface of bovine serum albumin of densities 1.086 g/cm?l.lll g/cm3 and 1.086 g/cm?1.074 g/m+, corresponding to reticulocyte and polychromatophilic stages, respectively (see below). Celh from the peripheral circulation of both normal and anemic animals were collected by heart puncture into Buffer NKM (140 rn~ NaCl, 3 rn~ KCl, 3 IUM MgCI,, and 3 mM potassium phosphate, pH 7.9) containing 0.1% heparin (Sigma). Bone marrow cells were obtained by scraping and aspirating the contents of the cracked leg bones from anemic birds into Eagle’s minimal medium and straining through three layers of cheesecloth to remove fatty tissue and other debris. The collected cells from all sources were washed three times in Buffer NKM and pelleted after each wash by centrifugation for 10 min at 600 x g. After each wash the buffy coat (containing mostly lymphocytes) was removed from normal and anemic peripheral blood populations with a Pasteur pipette.

Fractionation of Cells by Isopycnic Centrifugation

Cells obtained as described above were fractionated on discontinuous gradients of isotonic bovine serum albumin, hereafter referred to as albumin (17). This procedure is based on the observed correla- tion between density and cell maturity in the erythroid developmental process (2).

The preparation of isotonic albumin was based on the methods of Leif and Vinograd (18) and Shortman (19). Plasma fraction V powder (Sigma) was dissolved in water at 50 g/100 ml and dialyzed exten- sively against at least five changes of glass-distilled water over a period of 3 days. The dialyzed albumin was lyophilized and resuspended in an isotonic salt solution (Buffer NKM). Albumin solutions of varying density were obtained by further diluting this stock with Buffer NKM. Densities were calculated from refractive index meas- urements using the relation p = 1.0540 + 1.543 (refractive index = 1.3670) (18). In this manner solutions of the following densities were obtained: 1.111 g/c&, refractive index = 1.4036; 1.086 g/c&, refractive index = 1.3880; 1.074 g/cm3, refractive index = 1.3800; 1.059 g/ cm3, refractive index = 1.3700. All of the following operations were carried out at O-4” unless otherwise specified.

After the final wash, the pelleted cells were resuspended in 4 to 5 volumes of Buffer NKM and 1.111 g/cm3 of albumin solution (l:l, v:v). Up to 10 ml were then layered on top of a discontinuous gradient in a 30-ml Corex tube containing 5 ml each of albumin of densities 1.111 g/cm3, 1.086 g/cm3, and 1.074 g/cm3 for cells from the peripheral

circulation of anemic animals or 5 ml each of albumin of densities 1.074 g/cm3 and 1.059 g/cm” for cells from the bone marrow of anemic animals. Centrifugation at 4000 x g was for 1 hr. Cells banding at a given interface were recovered with a hooked Pasteur pipette and washed three times in 10 volumes of Buffer NKM. Aliquots of each fractionated population were suspended in Buffer NKM and smears were stained with Wright’s stain, new methyl blue reticulocyte stain, or benzidine as described by Lucas and Jamroz (20). Wright’s stain consists of a basic dye (methyl blue) which stains acid groups blue (nucleic acids) and an acid dye (cosine) which stains basic groups pinkish orange (hemoglobin). New methyl blue reveals the cytoplasmic basophilic granules (residual ribosomes) characteristic of reticulocytes which may otherwise look like mature erythrocytes. Staining with benzidine is diagnostic for the presence of hemoglobin. This was necessary to identify early stages as definitively erythroid because of the morphological similarity in the early forms of erythroid, lymphoid, and granulocytic cell lines. Cells were classified according to Lucas and Jamroz (20).

Solubilitation of Enzyme Activity

After fractionation the washed and pelleted cells were resuspended in 2 to 3 volumes of Buffer TGMED (50 rn~ Tris/HCl, pH 7.9, 25% v/v glycerol, 5 rn~ MgCl,, 1 IIIM EDTA, and 0.5 rn~ dithiothreitol). At this point an aliquot was taken for determination of DNA content by the Burton diphenylamine procedure (211, using calf thymus DNA as a standard. This was used as a normalization factor for cell number in comparing enzyme activity levels from different developmental populations. Solid ammonium sulfate was added to a final concentration of 0.3 M and the cells were homogenized for 1.5 to 2 min at full speed using the small probe in a Dispex Polytron homogenizer; sonication under varying conditions of salt concentration and intensity at this point dramatically reduced enzyme activity. This preparation, before or after homogenization, could be kept frozen in liquid nitrogen at least 2 months with no substantial loss in activity.

The high salt homogenate was diluted with Buffer TGMED to 0.1 M ammonium sulfate and centrifuged at 105,000 x g for 45 min to pellet chromatin. All enzyme activities discussed in this paper were recovered quantitatively in the supernatant.

Ion Exchange Chromatography

DEAE-Sephadex (A-25) was prepared by suspension in 0.5 M HCL, 0.5 M NaOH, and 0.5 M ammonium sulfate, pH 7.9, for 20 min each and thorough washing with deionized water after each suspension. It was stored in 2 x Buffer TGMED at 4”. For DEAE-resolution chromatography a column bed volume of 5 ml (0.8 x 10 cm) was equilibrated with 0.05 M ammonium sulfate (RNA polymerase chromatography) or 0.05 M KC1 (DNA polymerase chromatography) in Buffer TGMED. The 105,000 x g supernatant was either diluted to 0.05 M ammonium sulfate with Buffer TGMED or dialyzed against 0.05 M KC1 in Buffer TGMED and 1 to 8 ml were applied to the column. The column was washed with 3 volumes of equilibration buffer and eluted with a gradient of 4 column volumes from 0.05 to 0.45 M ammonium sulfate (RNA polymerase) or from 0.05 to 0.65 M KC1 (DNA polymerase) in Buffer TGMED. Enzymatic assays were performed immediately.

Alternatively, ion filtration chromatography (22) on DEAE-Seph- adex (A-25) was performed. The 105,000 x g supernatant was brought to 0.4 M ammonium sulfate and a sample of less than 7% of the column volume was applied to a column previously equilibrated with 0.1 M ammonium sulfate in Buffer TGMED. The column was eluted with 0.4 M ammonium sulfate in Buffer TGMED. Collected fractions were assayed immediately, quick frozen in a dry ice/ ethanol bath, and stored at -70” without loss of activity for at least 2 weeks. Ion exchange gradient fractions were assayed at the ambient salt concentration. Since the enzymes elute at the same chromatographic position regardless of the developmental stage, the relative activities at different stages could be directly compared.

For each developmental stage the fractions containing relevant enzyme activities were pooled, dialyzed to reduce the salt concentration, and reassayed at optimal salt concentration. In all cases the pooled activities exhibited linear kinetics for the time period of the standard assays.

Phosphocellulose (Whatman Pll) was prepared by suspension in 0.5 M HCl, 0.5 M NaOH, and 0.5 M KC1 for 20 min each and washing thoroughly with deionized water after each suspension. It was stored in 2x Buffer TGMED at 4”. A bed volume of 5 ml (0.8 x 10 cm) was


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Nucleotide Polymerases in Erythrocyte Development 275

poured and equilibrated with 0.05 M KC1 in Buffer TGED (no magnesium). The DEAE ion filtration fractions containing DNA polymer- aee activity were pooled and dialyzed against 0.05 M KC1 in Buffer TGMED for 2 h before loading onto the column. After loading, the column was washed with 2 column volumes of 0.05 M KC1 in Buffer TGED and eluted with a gradient of 4 column volumes from 0.05 to 0.65 M KC1 in Buffer TGED. The O.&ml fractions were collected in tubes containing 5 ~1 of 0.5 M MgCl, (5 mM in O.&ml fraction). This was done because magnesium ion interferes with phospbocellulose chromatography but is necessary for enzyme stability.

Salt Concentration Measurement

The concentration of ammonium sulfate or KC1 in enzyme samples and cbromatographic fractions was determined by measuring the conductivity of a lo-/~1 sample in 1 ml of water at 23” with a Radiometer, type CDM2e conductivity meter. Salt concentrations were determined from standard curves generated with known concentrations in Buffer TGMED.

Velocity Sedimentation

The high salt homogenate was diluted with 2 volumes of Buffer TMED (no glycerol) before centrifugation at 105,000 X g. A linear 15- ml gradient of 10 to 30% glycerol in 0.05 M Tris/HCl, pH 7.9, and 0.1 M ammonium sulfate was prepared in SW40 cellulose nitrate tubes and 0.7 ml of 105,000 x g supernatant was layered on top. The sample was centrifuged for 25.5 h at 40,000 rpm in the Spinco SW40 rotor (200,000 x g). Chicken hemoglobin and beef liver catalase were included as internal markers and monitored by absorbance at 407 nm in a Zeiss spectrophotometer. Sedimentation coefficients were determined according to the method of Martin and Ames (23).

Assays

RNA Polymerase - RNA polymerase was assayed as described by Roeder and Rutter (24). The reaction was begun by adding 20 yl of enzyme sample to 30 ~1 of assay mix. The assay contained (including contributions from Buffer TGMED) 75 rnM TrislHCl, pH 7.9, 1.6 mM MnCl,, 2 mM MgCl,, 3 mM P-mercaptoethanol, 6.3 mM NaF, 0.064 mM EDTA, 0.32 mM dithiothreitol, 0.6 mM GTP, CTP, ATP, 0.01 mw 13HlUTP (1000 cpmlpmol), 320 rg/ml of native calf thymus DNA (Sigma, type I). a-Amanitin was used at 1.3 pg/ml unless otherwise specified. When used as an alternative template to calf thymus DNA, polyld(A-T)] (Miles) was present at 50 @g/ml. After incuba- tion at 30” for 10 min, reactions were terminated by spotting 40 ~1 of reaction mix onto DEAE-cellulose discs (Whatman DE81) which had been presoaked in 5% sodium phosphate and 1% sodium pyrophos- phate, followed by immersion in the same solution. The filters were washed six times for 5 min each in 5% sodium phosphate, twice in distilled H,O, twice in 95% ethanol, once in ether, and air dried. The discs were counted in toluene containing Omnifluor (New England Nuclear) (15.2 g/gallon), a mixture of water (16.5 ml/gallon), and NCS solubilizer (100 ml/gallon) (Nuclear Chicago).

Poly(dC) and Poly(dT)-dependent RNA Polymerase Activity - Ac- tivity on poly(dC) was measured in a reaction mixture containing 50 mM Tris/HCl, pH 7.9, 6.3 mM NaF, 0.63 mM [3HlGTP (28 cpmlpmol), 3 mM p-mercaptoethanol, 3 mglml of crystallized bovine serum albumin (Pentex), 1.66 mM MnCl*, and 153 PM poly(dC). The reaction was begun by adding 10 ~1 of enzyme sample to 20 ~1 of assay mixture. Reactions were carried out at 30” for 10 min and terminated as for RNA polymerase by spotting 25 ~1 of reaction mixture onto DEAE-filters. The subsequent steps were as for RNA polymerase except that filters were washed 10 times in 5% sodium phosphate since GTP binds more tightly to DEAE-filters than UTP and high backgrounds were otherwise obtained. Activity on poly(dT) was measured under identical conditions except that poly(dT) and FHIATP replaced poly(dC) and FHIGTP, respectively.

Terminal Riboadenyltransferase -Terminal riboadenyltransferase was assayed as described by Bollum et al. (25). The final incuba- tion mixture contained 0.01 mM rA(pA),, 0.5 mM [3H]ATP (6 cpml pmol), 0.5 mM MnCl,, 4.2 mM P-mercaptoethanol, 100 mM Tris/HCl, pH 8.3, and 0.2 mg/ml of bovine serum albumin. Reactions were started by adding 5 /ill of enzyme samples to 25 /Al of assay mixture and incubated at 35” for 30 min. They were terminated by spotting 25 ~1 onto DEAE-filters and further processed as described above for activity on poly(dC).

Ribonucleotidyltransferuse - Conditions of the assay are the same as for RNA polymerase above except that the DNA template is replaced by 10 to 25 pg/ml of 9 S chicken erythrocyte mRNA or yeast

polysomal RNA. Reaction mixtures were incubated for 30 min at 35” and processed as above. Unlabeled triphosphates were deleted where noted under “Results.”

DNA Polymerase-DNA polymerase activity was assayed as described by Bollum et al. (25). The assay contained 20 mM potassium phosphate buffer, pH 7.0, 0.1 mM EDTA, 1.5 mM p-mercaptoethanol, 8.35 mM MgCl,, 0.1 mg/ml albumin, 0.1 mM dATP, dCTP, 13HldTTP (10 cpm/pmol), and 250 pg/ml of calf thymus DNA “activated” by exposure to 0.05 kg/ml of pancreatic DNase for 15 min at 37” (26). The 5 ~1 of enzyme sample were incubated with 25 ~1 of assay mixture at 35” for 30 min and 25 ~1 were spotted on DEAE-filters and processed as above. Where noted N-ethylmaleimide was present at a concentration of 3 mg/ml.

RESULTS

Fractionation of Erythroid Developmental Cell Popula- tions -Five populations of hematopoietic cells were resolved by discontinuous albumin gradients as shown in Fig. 1. Avian erythroid development is a continuous process so precise clas- sification of developmental populations at intermediate stages is not always rigorous. Mature erythrocytes are contaminated with less than 1% reticulocytes as revealed by new methyl blue staining (Fig. lA, Stage V). The reticulocyte fractions (Fig. lB, Stage IV) contained a few midpolychromatic cells and no cells exhibiting more immature morphology. The late and midpolychromatophilic erythrocyte stages (Fig. lC, Stage III) contain about 5% early polychromatophilic cells and occa- sional late blast cells. Stage II erythroblast populations (Fig. 1 D) have greater proportions of late small blast cells and early polychromatophilic cells while Stage I populations (Fig. 1E) contain primarily the large early blast cells. Early erythroblasts have a large nuclear to cytoplasmic volume ratio. Greater than 95% of these cells were judged erythroid by benzidine-positive staining criteria.

DNA-dependent RNA Polymerases -At each developmental stage, RNA polymerase activities were solubilized from total cell homogenates (see “Experimental Procedures”) to obviate considerations of cellular localization or of enzyme leakage (or both) from isolated nuclei (predominantly a- amanitin-insensitive activity). DEAE-Sephadex chromatography was used to distinguish the various RNA polymerase forms.

DEAE-Sephadex resolution chromatography of RNA polymerase from erythroblasts gave the activity profile shown in Fig. 2. Two major peaks were present which correspond to polymerase I (or A) and polymerase II (or B) in their elution position on DEAE-Sephadex and in their a-amanitin sensitivity. The ionic strength optima of 0.05 and 0.10 M ammonium sulfate, respectively, for polymerase I and II activities were also characteristic. Polymerase I eluted as a single peak of activity whereas form II was resolved into a minor and a major component. A very low activity eluted at the position of polymerase BIB when a-amanitin (1.3 pg/ml) was used to inhibit polymerase II (27-29). Only a marginal stimulation by poly[d(A-T)] of the putative polymerase III activity was observed (27). The polymerase I activity was higher with the poly[d(A-T)] template (Fig. 2) than with the usual calfthymus DNA template.

The enzyme activities from different developmental populations were monitored by DEAE-Sephadex ion filtration chromatography. This procedure is rapid and mild, yet effectively removes endogenous template and substrates while partially fractionating enzyme activities. Greater than 90% of the RNA polymerase activity was routinely recovered, compared to 50% or less recovery from DEAE-Sephadex resolution chromatography. To test for the presence of endogenous inhibitors (espe-


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FIG. 1. Fractionation of erythroid cell populations. Cells were fractionated on discontinuous gradients of isotonic albumin as described under “Experimental Procedures.” Smears were stained with Wright’s stain. A, mature erythrocytes (Stage V): cells from normal blood centrifuged through a layer of albumin of density 1.111 g/cm3. B, reticulocytes (Stage IV): cells from anemic blood sedimenting at the interface of albumin of densities 1.086 g/cm3 and 1.111 g/cm3. C, mid- and late polychromatic erythrocytes (Stage III): cells from

FRACTION NUMBER

FIG. 2. Resolution of multiple forms of RNA polymerase on DEAE-Sephadex. RNA polymerase from a fractionated early erythroblast population (Fig. 1E) was solubilized as described under “Experimental Procedures” and diluted to 0.05 M ammonium sulfate with Buffer TGMED. Then 8 ml were chromatographed on a column (0.8 x 10 cm) (5 ml of resin). The hemoglobin in the sample does not bind to DEAE-Sephadex. Fractions of 0.5 ml were collected. Activity was measured at the ambient salt concentration resulting from a dilution of 20 ~1 of each column fraction to a final volume of 50 ~1. RNA polymerase activity was measured with calf thymus DNA in the absence (0-O) and in the presence (0-O) of 1.3 @g/ml of cu-amanitin; and with poly[d(A-T)] in the presence of 1.3 fig/ml of 01- amanitin (A-A). -, ammonium sulfate concentration.

RNA polymerases I and II in the major peak were distinguished by their differential sensitivity to low a-amanitin concentration (1.3 pg/ml). Most of the activity insensitive to low cr-amanitin concentrations from erythroblasts was apparently polymerase I since only the trailing edge was sensitive to high concentrations of a-amanitin (250 pg/ml) (30). In addition, only 5% of the activity observed with poly[d(A-T)] and low a-amanitin in the crude extract (105,000 x g supernatant) is sensitive to high a-amanitin concentrations. This was judged to be polymerase III according to the criterion of Schwartz et al. (27). Thus we have not quantitatively estimated changes in polymerase III activity in these studies.

The response of the RNA polymerase to the homopolymer templates, poly(d0 and poly(dT) was also examined (30). Polymerase I as compared to polymerase II showed a decided preference for either poly(dC) or poly(dT) relative to the re- spective activities on calf thymus DNA. This correlates quali- tatively with previously reported observations from this labo- ratory (311, and might suggest a particular affinity of polymerase I for a pyrimidine-rich sequence.

cially in mature cell populations), partially purified rat liver RNA-dependent Ribonucleotidyltransferases -Two appar- polymerase (I and II) was added to the red blood cell extracts. ently distinct species of DNA-dependent ribonucleotidyltrans- Over 90% of this exogenous activity was recovered after ion ferases can be identified in this system. One requires an RNA filtration. primer and is capable of utilizing ribotriphosphates other than

anemic blood sedimenting at the interface of albumin of densities 1.086 g/cm3 and 1.074 g/cm3. D, early polychromatophilic erythrocytes and late erythroblasts (Stage II): cells from anemic bone marrow sedimenting at the interface of albumin of densities 1.074 g/cm3 and 1.059 g/cm3. E, early erythroblasts (Stage I): cells from anemic bone marrow sedimenting on top of the albumin layer of density 1.059 g/cm3.

Two peaks of RNA polymerase activity were resolved by DEAE-ion filtration chromatography (30). The front minor peak corresponded to the low unbound activity observed in Fig. 2 and is discussed in more detail below. The major peak was dependent on exogenous template and all but a minor fraction was sensitive to actinomycin D and dependent on the four nucleoside triphosphates. This anomalous minor activity was very low relative to polymerases I and II, and was disre- garded in the determination of the developmental profiles for these enzymes.


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ATP. The other appears to be the terminal riboadenyltransferase which presumably mediates the synthesis of poly(A) associated with mRNA.

The first RNA-dependent ribonucleotidyltransferase activity was originally identified as the minor peak of nucleotide polymerizing activity that did not bind to DEAE-Sephadex at 0.05 M ammonium sulfate (Fig. 2, see above). This was not an artifact of column overload since it appeared regardless of the protein load. It was insensitive to actinomycin D (48 Kg/ml), to cr-amanitin (1.3 pg/ml), and the activity was severalfold greater in the absence of the other three nucleotides. The activity probably depends on the presence of contaminating RNA in commercial calf thymus DNA since no activity was seen when the DNA was deleted (Fig. 3) or after prior base or ribonuclease treatment of the DNA.

A small portion of the RNA polymerase activity bound to DEAE-Sephadex was also actinomycin D-insensitive and independent of the other three nucleotides (see above). It was subsequently shown to be dependent on RNA (see below).

The standard assay conditions for these RNA-dependent ribonucleotidyltransferases were identical with those for RNA polymerase, with the substitution of chicken red cell 9 S RNA or yeast polysomal RNA for calf thymus DNA. Yeast RNA was somewhat more active than the red cell RNA on a microgram basis. Using this assay, two peaks of activity were resolved by DEAE-Sephadex ion filtration (Fig. 3) or resolution (Fig. 4) chromatography and were coincident with the triphosphate independent, actinomycin D, and cu-amanitin-insensitive RNA polymerase activities described above (Fig. 3). To diatin- guish between a template and a primer function for the polyri- bonucleotide, the activity of this enzyme was examined using substrates and polyribonucleotides where no extensive base pairing is expected. Tritiated CMP incorporation was observed with either yeast polysomal RNA or poly(rA) (data not shown) and the incorporation was exclusively seen or greatly en- hanced in the absence of the three complementary triphosphates (Fig. 4). These data suggest a primer rather than a template function. This activity is distinct from either the

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FRACTION NUMBER

FIG. 3. Properties of DEAE-Sephadex ion filtration ribonucleotidyltransferase activity. RNA polymerases from an early chromatophilic erythrocyte-late erythroblast population (Fig. lD) was solubilized as described under “Experimental Procedures.” Then 1 ml of the 105,006 x g supernatant was brought to 0.4 M ammonium sulfate and chromatographed on a 15-ml column (1.3 x 10.5 cm). Fractions of 0.45 ml were collected and assayed at the ambient salt concentrations. Ribonucleotidyltransferase was assayed in the absence of ATP, GTP, and CTP with 25 pg/ml of 9 S chicken erythrocyte mRNA in the absence (O-O) and in the presence (0-O) of 1.3 pg/ml of a-amanitin. Activity in the absence of ATP, GTP, and CTP was assayed with calf thymus DNA (A-A) and in the absence of any added polynucleotide (A-A). -, ammonium sulfate concentration

riboadenyltransferase or the DNA-dependent RNA polymerases. A comparable ribonucleotidyltransferase activity was observed in every system examined including yeast, rat liver, sea urchin, and Chinese hamster ovary cells.

Terminal riboadenyltransferase activity was assayed using a rA(pA), primer which reportedly gives optimal activity under these assay conditions (12). The activity recovered in the 105,000 x g supernatant did not bind to DEAE-Sephadex (data not shown). Its molecular weight as estimated by sucrose gradient centrifugation of the crude soluble protein (Fig. 5) is

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/ :

FRACTfON NUMBER

FIG. 4. Resolution of ribonucleotidyltransferase activities on DEAE-Sephadex. Chromatography conditions are as detailed for Fig. 2. Reactions contained 25 @g/ml of yeast polysomal RNA in the standard assay mixture. Activity with 0.1 rnM L3H]UTP (500 cpml pmol) was measured in the presence (0-O) and in the absence (0-O) of 0.6 rnM each ATP, GTP, and CTP. Activity with 0.1 mM k3HlCTP (500 cpm/pmol) was measured in the presence (A-A) and in the absence (A-A) of 0.6 mM ATP, GTP, and UTP. -, ammonium sulfate concentration; - - -, DNA-dependent RNA polymerase activity profile

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FRACTION NUMBER

FIG. 5. Velocity sedimentation of terminal riboadenyltransferase and DNA polymerases from a crude extract. Sedimentation was performed on a 10 to 30% glycerol gradient as described under “Experimental Procedures.” Fractions of 0.6 ml were collected. Bo- vine serum catalase (11.3 S) and chicken hemoglobin (4.32 S) were run as internal standards and monitored by absorbance at 407 nm. A-A, terminal riboadenyltransferase activity; O-O, DNA polymerase activity. Sedimentation is from right to left.


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about 70,000, analogous to the calf thymus enzyme (12). The activity required manganous ion and ATP, GTP, CTP,

UTP, and dTTP were incorporated into polynucleotides at less than 2% of the ATP level using the rA(pA), primer. The low activity apparent with UTP but not ATP using calf thymus DNA (contaminated with RNA) is attributed to the RNA- dependent RNA polymerase activity described above.

DNA Polymerases -The DNA polymerase activity from avian erythroblasts was resolved into three components by DEAE-Sephadex or phosphocellulose ion exchange chromatography and by velocity sedimentation. Two of these polymerases have properties characteristic of the a! and /3 DNA polymerases which have been described in other eucaryotic systems (11). The third does not correspond to any DNA polymerase activity previously reported. FRACTION NUMBER

The nuclear DNA polymerase /3 (32, 33) is evident as the first peak in DEAE-Sephadex chromatography (Fig. 6) (331, the third peak on phosphocellulose chromatography (Fig. 71 (32-34), and as the slowly sedimenting (5.2 S) activity (Fig. 5). This activity did not bind to DEAE-Sephadex and was only partially inhibited by a thiol reagent, N-ethylmaleimide (11, 35). The first peak on DEAE-Sephadex ion filtration was associated with rA:oligo(dT)-dependent activity which is characteristic of purified p but not a: polymerases (36, 37). The S value for this activity is somewhat higher than has been observed for the fi polymerase in other systems; this may be due to enzyme aggregation or the presence of bound nucleic acid.

FIG. 6. Resolution of multiple forms of DNA polymerases on DEAE-Sephadex. Chromatography of a late erythroblast-early polychromatophilic erythrocyte population was performed as described under “Experimental Procedures.” After dialysis versus 0.05 M KCl, 1.4 ml of the crude extract were applied to the column. Fractions of 0.45 ml were collected. DNA polymerase activity was measured in the absence (0-O) and in the presence (0-O) of 3 mg/ml of N- ethylmaleimide. -, KC1 concentration.

I a

The major DNA polymerase component in avian erythroblasts evident as the second component in Figs. 6 and 7 and as the 10.2 S sedimenting component in Fig. 5 was analogous to the large cytoplasmic DNA polymerase a observed by others (11, 32, 35, 38, 39). Its activity was completely inhibited by N- ethylmaleimide (Fig. 6). The S value for this activity is also somewhat higher than is usually observed for DNA polymerase cy.

-.

30 -

.

The third DNA polymerase component was detected by ion exchange chromatography (Fig. 6, third peak; Fig. 7, first peak) and by velocity sedimentation (18.2 S) (Fig. 5). This component was N-ethylmaleimide-sensitive like the (Y polymerase (Fig. 6). FRACTION NUMBER

This rapidly sedimenting activity is not DNA polymerase y (11) since no rA:oligo(dT)-dependent activity eluted late in DEAE-Sephadex ion filtration chromatography. It is apparently not mitochondrial polymerase (39) since the enzyme is resistant to thiol reagents (401, and persists at developmental stages which have no mitochondria.

FIG. 7. Resolution of multiple forms of DNA polymerase on phosphocellulose. The DEAE-ion filtration fractions with DNA polymerase (and no RNA polymerase activity) were pooled, dialyzed against 0.05 M KC1 in Buffer TGMED, and chromatographed on phosphocellulose as described under “Experimental Procedures.” DNA polymerase activity, O-O; RNA polymerase activity on poly(dC), 0-O; -, KC1 concentration.

As with RNA polymerase, DEAE-Sephadex ion filtration chromatography increased recovery but decreased resolution of DNA polymerase activity. Nevertheless three poorly resolved peaks were still distinguishable (Fig. 121. N-Ethylmal- eimide resistance tended to be associated with early eluting activity (30).

Poly(dC)-dependent RNA Polymerase Activities -RNA polymerase activity was assayed using poly(dC) as a primer since this template has been shown to detect forms of RNA polymerase which are inactive on double-stranded DNA templates. I

This activity was associated with DNA polymerase activity on both DEAE-Sephadex and phosphocellulose chromatography (Fig. 7 and data not shown). No typical RNA polymerase

activity was present in these fractions. Agarose 1.5m gel filtration chromatography of DEAE-Sephadex ion filtration DNA polymerase activity which had been pooled and concen- trated failed to separate poly(dC) and DNA polymerase activities. In addition the poly(dCl-dependent RNA polymerase activity was sensitive to N-ethylmaleimide. However, this poly(dC) activity was not proportionate to the DNA synthetic activity on activated calf thymus DNA. In relative terms poly(dC) activity was lowest on p, intermediate on cr, and highest on the third component (Fig. 7 and data not shown). The poly(dC) activity of the latter component was not exactly coincident with activity on activated calf thymus DNA, suggesting the possibility of a dissociable component. No activity was seen in the presence of poly(dT) and ATP.

* M. Goldberg, J. C. Perriard, and W. J. Rutter, submitted for Developmental Activity Profiles -The activity profiles of the publication. various polymerases resolved by ion filtration chromatogra-


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Nucleotide Polymerases in Erythrocyte Deuelopment 279

21% 15% 0%

18 2 6 10 14 6 10 14 18 4 8 12 16 4 8 12 16

FRACTION NUMBER

phy from fractionated erythroid cells are displayed in Figs. 8 to 12.

Profiles of DNA-dependent RNA polymerases I and II are displayed in Figs. 8 and 9, respectively. RNA polymerase II activity was taken as the Lu-amanitin sensitive activity. Po- lymerases I and III were not distinguished in the a-amanitin insensitive peak, but polymerase III activity was considered negligible in these experiments. The percentages given in the figure are representative of the integrated areas under each peak.

All the RNA polymerase activities declined progressively but not coincidentally with maturation. Polymerase I activity decreased very quickly after the erythroblast stage and disap- peared entirely in the mature erythrocyte. By comparison, polymerase II activity declined much more slowly with development and was still present at maturity although at reduced levels (15%). The low polymerase III activity like the polymerase I activity must disappear with maturation since no (Y- amanitin insensitive activity is evident in the mature erythrocyte.

The developmental profile for the riboadenyltransferase is shown in Fig. 10. There was a large decrease in activity early in development. Thereafter activity declined more slowly to the low level of the mature cell.

In contrast to the other activities discussed above, there were no large decreases in the activity of the ribonucleotidyltransferase during red cell development through the reticulocyte stage (Fig. 11). In the mature stage, however, the activity dropped to about 10% of the erythroblast level.

The DNA polymerase activity profile is shown in Fig. 12. The three peaks of activity were discernible at all stages, although the third peak was present primarily as a shoulder. The first peak (p polymerase) remained at relatively constant levels and the decline of enzyme activity associated with erythroid development occurred mainly in the second (o( polymerase) and third peaks. In the final process of maturation, all activities disappear.

DISCUSSION

This survey of nucleotide polymerizing enzymes in the developing avian erythrocyte is carried out during a period in which there are dramatic qualitative and quantitative

1

FIG. 8. Developmental profile of RNA polymerase I activity. DEAE- Sephadex ion elution chromatography was performed on each cell population shown in Fig. 1. Conditions were as for Fig. 3. The eluted activities were nor- malized to the DNA content in the cell suspension before homogenization in 0.3 M ammonium sulfate as described under “Experimental Procedures.” The profiles are RNA polymerase activity in the presence of 1.3 pglml of a-amanitin. The percentages represent the integrated area under the large peak in each case relative to the early erythroblast stage.

II

79%

IL IO 14 18

m

.9%

nz

47% ix, 16 20 16 20

P

15%

A, 16 20

FRACTION NUMBER

FIG. 9. Developmental profile of RNA polymerase II activity. Same as for Fig. 8 except the profiles represent the differential between total activity and activity in the presence of 1.3 pglml of IX- amanitin.

changes in gene expression leading to a stable quiescent state. Several of the enzyme activities detected are analogous to previously characterized eucaryotic enzymes, two other unu- sual activities of particular interest were also found. Charac- teristic patterns of decline for each enzyme activity were observed during erythrocyte maturation.

Four characteristic DNA-dependent RNA polymerase activities were resolved by DEAE-Sephadex ion exchange chromatography (9). The two major peaks are polymerases I and II as judged by their chromatographic behavior, cy-amanitin sensitivity, and ammonium sulfate activity profiles. No heteroge- neity in polymerase I as reported in some other systems (29, 41) is seen. However, two RNA polymerase II peaks were detected, the first elutes analogously to form IIA described for Xenopus Zaeuis cytoplasm (28). The polymerase III activity is very low in avian erythroblasts even when assayed under optimal conditions (27). Form IIIB seems to be the predomi- nant if not the only species present. Polymerase III activity levels have been analyzed from a number of tissues (24,27-29, 31). Generally the levels of this enzyme relative to the other RNA polymerase species are low in slowly growing differen- tiated tissues (such as calf thymus, mouse liver, spleen, cul-


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Q 5 500 0 z \ n 400 E 2 0 & 300

FIG. 10. Developmental profile of 0 terminal riboadenyltransferase activ- g ity. Details are the same as for Fig. 8. -

4 2oo Q

ii? d 100

5 L

A 8 12 16 2 6 10 A 8 12 2 6 10 14 4 8 12 16 20

FRACTION NUMBER

5 9 13 17 2 6 10 14 18 4 8 12 16 20 A 8 12 16 20 A 8 12 16 20

FRACTION NUMBER

FIG. 11. Developmental profile of ribonucleotidyltransferase. Chromatography details are as in Fig. 8. Assays at all stages were performed with 25 @g/ml of yeast polysomal RNA in the absence of ATP, GTP, and CTP. The percentages given represent the total integrated activitv from both peaks at each staee relative to that of the ervthroblast population. The numbers above each peak are indicative of their activity levels relative to one another.

tured Xenopus kidney cells) and are high in rapidly growing tissues (such as Xenopus and sea urchin embryos and yeast).

The two distinct RNA-dependent ribonucleotidyltransferases were observed in these cells. One is typical of terminal riboadenyltransferases (poly(A) polymerases) described in other systems (12, 15) with regard to its Mn*+ dependence, its size, its chromatographic behavior on DEAE-Sephadex, its use of rA(pA), as a primer, and its exclusive use of ATP as a substrate. The observed enzyme presumably mediates the post-transcriptional addition of poly(A) to messenger RNA. Since globin mRNA is well known to carry a terminal poly(A) sequence, the existence of polyadenylate polymerase in the erythroid system is logically expected although not previously demonstrated. We have not detected the Mgz+-dependent polymerase activity reported by Edmonds and Abrams (42).

A second RNA-dependent ribonucleotidyltransferase is distinguished from DNA-dependent RNA polymerases and terminal riboadenyltransferases. The two peaks of this RNA-de-

pendent activity co-elute on DEAE-Sephadex ion filtration with the two peaks of actinomycin D-insensitive and complementary triphosphate-independent activity first measured with calf thymus DNA. These activities were shown to be dependent on RNA contaminants present in the commercial DNA preparation. In DEAE-Sephadex chromatography, the RNA-dependent activities elute in the flow through and between DNA-dependent forms I and II. The second form co- elutes with the minor DNA-dependent RNA polymerase (IIA) activity. The partial sensitivity to cr-amanitin of this peak may be due to the presence of both activities.

The RNA-dependent ribonucleotidyltransferase is defined by the following observations. Its activity is dependent on exogenous RNA. The activity is increased in the absence of the three complementary triphosphates, suggesting that the four bases are related competitively as for a priming reaction in- stead of cooperatively as for a ternplating reaction. The various bases are not equally competitive since UMP is incorpo-


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Nude&de Polymerases in Erythrocyte Development

6 10 14 18 22 3 7 11 15 19 4 8 12 16 20 4 8 12 16 20 6 10 14 18

FRACTION NUMBER

281

I 2

FIG. 12. Developmental profile of DNA polymerase activity. Chromatography details are as for Fig. 8. The percentages given represent the total integrated activity from all peaks at each stage relative to that of the erythroblast population. The numbers above Peak I and above Peaks II and III are indicative of their activity levels relative to one another.

rated far more readily in the presence of a large excess of guanosine, cytidine, and adenosine than is CMP in the presence of a corresponding excess of guanosine, uridine, and adenosine. A priming function for the RNA is indicated from the incorporation of CMP alone in the presence of various RNA molecules.

We have found similar ribonucleotidyltransferase activities in yeast, sea urchin, Chinese hamster ovary cells, and rat liver. Analogous activities have been described by others from both procaryotic and eucaryotic sources (13-15, 43, 44) including avian erythrocytes (16, 45). Thus this activity seems to be widespread. We believe the RNA-dependent RNA polymerase activity described by Downey et al. (46) is due to this enzyme.

Contrary to the proposal of these authors, however, hemoglobin mRNA is neither a template, nor a preferred substrate for this activity. These ribonucleotidyltransferases appear to be located in the cytoplasm associated with the microsome fraction (13, 16, 43) or with ribonucleoprotein particles containing HnRNA (15). Thus, the RNA-dependent ribonucleotidyltransferase(s) may play some role in the metabolism of cytoplasmic poly(A) containing mRNA. Although no cogent suggestions regarding a function for this curious activity have been presented, modulation of translational efficiency or message turn- over are obvious possibilities. A specific model might involve primer-dependent ribonucleotide addition on poly(A) message termini. The partial specificity of ribonucleotide addition suggests a limited ternplating function; thus partially double- stranded structures may result at the termini of these molecules. Such modifications might then mediate a stimulation or inhibition of message translation or a modification in message stability. This activity may be related to the translational control RNA postulated by Bester et al. (47) which is associated with mRNA in ribonucleoprotein particles or polysomes.

Three peaks of DNA polymerase activity are resolved by DEAE-Sephadex and phosphocellulose chromatography. Sev- eral criteria including elution behavior, size, inhibition by

thiol reagents, and activity on an rA:oligo(dT) template suggest that two of these peaks are analogous, respectively, to the DNA polymerase p and DNA polymerase (Y observed in other eukaryotic (particularly mammalian) systems (11, 38). The third peak of activity is a novel activity; it is much higher in molecular weight, is N-ethylmaleimide-sensitive like (Y DNA polymerase, and is associated with disproportionately high poly(dC)-dependent RNA polymerase activity.

Poly(dC)-dependent RNA polymerase (riboG incorporation) activities are coincident as expected with RNA polymerase activities but in addition, and unexpectedly, with DNA polymerase activities, suggesting that this activity may be a subsidi- ary activity of the DNA polymerase. Although such an activity has not been previously reported for the eucaryotic enzymes, several considerations support this possibility. First, van de Sande et al. (48) and Berg et al. (49) have shown that Escherichia coli DNA polymerase I in the presence of Mn*+ incorporates CMP and GMP at rates comparable to their deoxy analogs while AMP and UMP are incorporated either slowly or not at all. Similarly, we observe CMP but not AMP incorporation in response to the complementary homopolymers with the avian enzyme(s). Secondly, single chain homopolymers are generally inactive templates for DNA synthesis since they lack the required initiator. The only exception is poly(dC) which forms folded structures at neutral pH (50). Finally, purified calf thymus DNA polymerase LY, but not p, exhibits RNA polymerizing activity (GMP incorporation) on a poly(dC) template.2 The skewed poly(dC) RNA polymerase activity relative to the third, rapidly migrating DNA polymerase activity might reflect dissociation from a complex. The propensity of the third enzyme to polymerize ribonucleotides might suggest an initiator function. As such it could represent merely a modified form of the (Y polymerase. The nature and function of this third heavy activity clearly deserves further study.

2 F. Bollum, personal communication.


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Developmental Changes in Enzyme Activities -The relatively high activities of RNA polymerases I and II at early developmental stages are consistent with previous reports demonstrating higher rates of RNA synthesis in less mature populations of erythroid cells (l-3, 52). The preferential cessation of ribosomal RNA synthesis relative to heterogeneous nuclear RNA synthesis in the erythroid maturation process (3, 53, 54) correlates well with the preferential decline in form I relative to form II activity. It has been generally observed that polymerase I is the most labile species both in vivo and in vitro (review, Refs. 9 and 55 to 59).

The failure of van der Westhuyzen et al. (60) to observe polymerase I activity in anemic erythrocyte populations is probably due to the mild conditions employed to induce anemia and to the nuclear isolation and sonication procedures which result, respectively, in preferential leakage and inactivation of polymerase I as reported here. The polymerase II in mature erythrocytes (found also by Schechter (61)) is presumably responsible for the residual levels of RNA synthesis observed in the mature avian erythrocyte (52, 54).

The parallel decrease in RNA polymerase II and riboadenyltransferase with development of the erythroid cell can be correlated with their related roles in synthesis and post-transcriptional modification of heterogeneous nuclear RNA. Re- tention of residual riboadenyltransferase activity in mature erythrocytes suggests that this enzyme may be required for effective utilization of the products of the RNA polymerase II activity which remains in these cells.

The RNA-dependent ribonucleotidyltransferase activity levels are not significantly altered in the course of erythroid maturation until the final stage when a precipitous decline is observed. Its presence at normal levels when most other nucleotide polymerizing activities have declined suggest that it is very stable and/or may have a basic continuing function in cellular metabolism.

The developmental profiles of DNA polymerase show the same progressive decline with maturation observed with the other enzymes investigated here. Cell division in avian erythropoiesis generally ceases after the erythroblast stage (21). Williams (5) has reported that erythroblasts are the last cells in the developmental cohort to synthesize DNA while Attardi et al. (52) suggest that the polychromatic erythrocytes from anemic blood also synthesize DNA. We have observed that significant DNA-polymerizing activity is present as late as the reticulocyte stage. Whether this residual activity serves a function or not is unknown.

The developmental profiles indicate that the attrition of DNA polymerase activity during erythrocyte maturation oc- curs primarily at the expense of the DNA polymerase (Y (second peak) and the unidentified third peak. This corresponds to the pattern observed in other eucaryotic systems where changes in the activity of the a: polymerase species are correlated with replication (review Fkfs. 38, 62, and 63). The (Y polymerase and the third peak are most likely the species which mediate DNA replication.

By correlative inference these data are consistent with the postulate that nucleotide polymerase activity levels play a regulatory role in the gross attenuation of gene activity in avian erythropoiesis. These changes in activity may be related to the functional alterations in the maturing erythrocyte nu- cleus. The loss of gene activity may be mediated by alterations in the template and/or in the enzymes involved in gene expression, particularly nucleotide polymerizing enzymes. Histone V

tar of general nuclear repression. For example, Appels et al. (64) have shown that reactivation of chick erythrocyte nuclei in chick/HeLa heterokaryons is associated with the loss of histone V. However, more recent studies (65) have demonstrated that erythroblasts have histone V levels nearly comparable to those found in later stages, so histone V cannot be the sole regulatory component involved. The studies presented here suggest that the cessation of specific cellular functions may be caused by the loss of particular polymerizing activities.

Acknowledgment-We would like to thank Dr. Fred Bol- lum particularly for his helpful suggestions and discussions and for his generous gifts of poly(dT), poly(dC), and (rA,),.

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S S Longacre and W J RutterNucleotide polymerases in the developing avian erythrocyte.

1977, 252:273-283.J. Biol. Chem.

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