Synthesis of messenger-like RNA in avian erythrocyte nuclei

ARCHIVES OF BIOCHEMISTRYAND BIOPHYSICS Vol. 242, No. 1, October, pp. 90-103, 1985

Synthesis of Messenger-Like PAUL

RNA in Avian Erythrocyte

A. WIERSMA’

AND G. STANLEY

Nuclei

COX’

Department of Biochemistry, University of Nebraska Medical Center, .42nd and Dewey Avenue, Omaha, Nebraska 68105 Received December 26,1984, and in revised form May 21,1985

Cell ghosts have been prepared from mature chicken erythrocytes using 0.05% saponin. Such preparations are capable of incorporating label from rHJUTP and provide a system, where the nucleus is permeable to nucleotides and macromolecules, for studying the low-level RNA synthesis characteristic of these cells. RNase A (50 pg/ml) eliminated all radioactivity binding to DE-81 filters, indicating that the product was RNA; and DNase (10 pg/ml) and actinomycin D (10 pg/ml) each inhibited UMP incorporation by 70%) suggesting that the synthesis was DNA-dependent. Polymerization was inhibited 90% by 0.1 pg/ml cy-amanitin, and maximum synthesis occurred in the presence of high salt (0.175 M KCl) and Mn2+ (0.5 mM). Polyacrylamide gel electrophoresis indicated that the newly synthesized RNA was heterogeneous in size, having a distribution from 5 to 60 S with a significant fraction migrating as 8-12 S. Approximately 15% of the total RNA was bound by an oligo(dT)-cellulose column, suggesting that some RNA processing was occurring, although attempts to detect the incorporation of label from [a-32P]GTP into a 5’-cap structure were unsuccessful. In comparison to RNA synthesis in reticulocyte nuclei, both the rate and extent of transcription in erythrocyte nuclei were much reduced. Moreover, about 25-30% of the reticulocyte nascent RNA was released from the nuclei during a 60-min incubation, while no release was observed for the erythrocyte nuclei. Hybridization of radiolabeled RNA to excess chicken DNA indicated that the majority (80%) of the in vitro transcripts were complementary to unique sequence DNA (C&/z = 4.5 X 103). When RNA synthesized by either erythrocyte or reticulocyte nuclei was hybridized to cDNA complementary to reticulocyte polysomal mRNA, about 8% of the reticulocyte nuclear RNA but less than 1% of the erythrocyte nuclear RNA were resistant to RNase A digestion. Taken together, these data suggest that nuclei prepared by saponin lysis of chicken erythrocytes synthesize messenger-like RNA via endogenous polymerase II activity. A fraction of this RNA is polyadenylated but contains few, if any, globin sequences or other transcripts found on reticulocyte polysomes. o 19%AcademicPWS, I~C.

Various forms of cell differentiation are characterized by a progressive cessation of RNA synthesis. In the case of mature avian erythrocytes which retain their nuclei, there is a general decline in transcriptional activity and replication is arrested as the

cells mature (l-6). This shut down in nuclear functions is probably the consequence of a combination of events, including loss of nonhistone chromosomal proteins (7), alterations in histone composition (7, 8), degeneration of nucleoli (9), and overall chromatin condensation and nuclear contraction (9). These, however, are not irreversible changes since transcription and replication can be restored by fusion of erythrocytes with mammalian cells in culture (5, 10). It is thought that the reacti-

‘Present address: Department of Medical Biochemistry, Health Science Center, University of Calgary, Calgary, Alberta, Canada. a To whom reprint requests and correspondence should be addressed. 0003-9861185 $3.00 Copyright All rights

0 1985 by Academic Press, Inc. of reproduction in any form reserved.

90

MESSENGER-LIKE

RNA

SYNTHESIS

vation process results from an influx of macromolecules into the erythrocyte nucleus from mammalian constituents in the heterokaryons (H-14), though the exact mechanism is not understood. Thus, the nucleated avian erythrocyte provides a system for examining gene regulation under conditions of both repression and derepression. It has generally been held that mature erythrocytes were incompetent for RNA synthesis (1, 2). However, Zentgraf et al. (15) and Madgwick et al. (16) have demonstrated that whole cells in culture, in contrast to those in Gvo, can incorporate [3H]uridine into RNA. As part of a larger study in the development of an in vitro system to examine various aspects of erythroid differentiation and reactivation, we have isolated nuclei from mature chicken erythrocytes and examined their capacity for transcription. The following report characterizes the transcripts produced by these preparations. In general, the isolated ghosts exhibit a synthetic capacity which is quite similar to that of intact erythrocytes as reported by Zentgraf et al. (15). MATERIALS

AND

METHODS

Mutetic&. The following were obtained from Sigma Biochemicals: saponin, a-amanitin, actinomycin D, Escherichia coli RNA polymerase (Type l), RNase A, nuclease Pl, protease (Type XIV, Pronase E), and E. coli alkaline phosphatase (Type III). P & L Biochemieals was the source for oligo(dT)1z+ G(5’)ppp(5’)G, and ATP, CTP, GTP, and UTP. Chicken erythrocyte DNA was obtained from Calbiochem and DNase I was from Worthington Biochemicals. Collaborative Research provided oligo(dT)-cellulose (type 1). Whatman, Inc. supplied DE-81 paper. [$H]UTP (35-42 Ci/mmol) and [a-“P]GTP (>300 Ci/mmol) were purchased from New England Nuclear. DNA-grade hydroxylapatite was obtained from Bio-Rad Laboratories. Avian myeloblastosis virus (AMV) reverse transcriptase, deoxynucleoside triphosphates, and calf thymus primers for cDNA synthesis were generously provided by Dr. Duane P. Grandgenett. Preparation ofnuclei. Erythrocytes were collected into a heparinized vessel from adult white Leghorns by cardiac or wing vein puncture. All subsequent procedures were carried out at 0-4°C. Cells were washed several times with sterile 0.9% (w/v) NaCI. After each centrifugation, the upper quarter of packed cells was removed to minimize white cell and immature red cell contamination, and the preparations were routinely

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91

examined for retieulocytes by staining with phenyl cresyl blue. The cells from 20 ml of blood were lysed by gentle shaking on ice in 400 ml of nuclei buffer (0.25 M sucrose, 25 mhf KCI, 50 mM Tris-Cl, pH 7.4, 5 mM MgClz) containing 0.05% (w/v) saponin. The cell ghosts were collected by centrifugation at 7OOg for 5 min. The pellet was resuspended by shaking gently into 400 ml of the nuclei buffer and again centrifuging. The pellet was washed twice more and finally resuspended at 10’ nuclei/ml in nuclei buffer and frozen at -80°C until use. Reticulocyte nuclei were prepared similarly, but only the buffy layer of the packed cells was discarded, and lysis with saponin required somewhat longer incubation times. Microscopic examination suggested that lysis with dilute saponin released all hemoglobin and presumably all cytoplasm from the erythrocytes without affecting the nucleus. As long as the saponin treatment was for less than 2 min and centrifugation forces were kept to a minimum, the nuclei could be thawed and resuspended without clumping. Transcription activity of nuclei stored at -80°C remained constant over several months. Nuclei prepared at different times and from different birds varied in their transcriptional activity although the results remained qualitatively consistent. The terms “nuclei” and “erythrocyte ghosts” both refer to materials prepared as described in this section and will be used interchangeably in this paper. Transcription assay. Frozen nuclei were thawed rapidly and dispersed by gentle vortexing. The standard transcription assay contained 10s nuclei in a final volume of 0.2 ml and, unless otherwise noted, the mixture also contained 0.3 M sucrose; 25 mM Tris-Cl (pH 7.4); 2.5 mM MgClz; 0.5 mM MnClz; 175 mM KCl; 2 mM dithiothreitol (DTT)a; 0.1 mM each ATP, GTP, and CTP; and 5 &i of [‘HIIJTP. Incubations were carried out at 26”C, and were stopped by the addition on ice of unlabeled UTP to 0.1 mM. Each sample was sonicated in 0.3% sodium deoxycholate, and 100 ~1 of the mixture was applied to each of two Whatman DE-81 filters. The tube was rinsed with 100 pl of water which was spotted onto a third filter. The filters were soaked 10 times for 15 min each in 0.5 M NazHPOa; rinsed twice with water, twice with 95% ethanol, and once with ether; dried; and counted by liquid scintillation in a toluene cocktail. Isolation of nascent RNA. All solutions were autoclaved, and all glassware was baked overnight. Nuclei were incubated with 25 &i/ml of [3H]UTP in a lotimes scaled up reaction mixture for gel electrophoresis and oligo(dT)-cellulose chromatography and with 125 &i/ml of [a-“P]GTP in a fivefold mix for cap structure and hybridization analysis. The samples

a Abbreviations used: DTT, dithiothreitol; dium dodecyl sulfate; HAP, hydroxylapatite; avian myeloblastosis virus.

SDS, soAMV,

92

WIERSMA

were diluted into 15-20 ml of buffer containing 50 rnM Na acetate (pH 5.1) and 10 rnbf EDTA. Sodium dodecyl sulfate (SDS) was added to a final concentration of 0.5% (w/v), and the RNA was extracted with hot phenol as described previously (17). RNA in the aqueous phase was precipitated with 0.1 vol of 2 M NaCl and 2 vol of 95% ethanol at -20°C. HeLa rRNA (100 pg) was added as carrier. The precipitate was resuspended in 50 mM Tris-Cl (pH 7.4) containing 2 mM MgClz and 100 pg/ml of RNase-free DNase I, and was incubated for 30 min at 37°C. EDTA was added to 10 mM and SDS to 0.5%, and the solution was extracted with phenol:choloroform:isoamyl alcohol (25:24:1). The RNA was again precipitated with ethanol and the precipitate was brought up in a small volume of sterile water. Capping analysis. Assay for 5’-terminal cap structures was carried out essentially as described by Mizumoto and Lipmann (18). RNA labeled with [a“P]GTP was incubated at 37°C in 8 mM Na acetate (pH 6.0) with 1 mg/ml of RNase A and 1.5 mg/ml of nuclease Pl. After 60 min, the pH was raised to 8.0 with the addition of Tris-Cl to 70 mM, and bacterial alkaline phosphatase was added to 250 pg/ml. The mixture was incubated for a further 75 min with the addition of aliquots of the phosphatase at 15-min intervals. Samples were applied to 26 X 20-cm Whatman DE-81 paper, dried, and electrophoresed at 900 V for 4 h at 10°C in 5% (v/v) acetic acid, 0.5% (v/v) pyridine buffer (pH 3.5). The paper was washed with 70% (v/ v) ethanol and dried between blotters. Unlabeled samples were visualized by uv irradiation and radioactivity was observed by autoradiography. DNA-RNA hybridization analysis. Total chicken DNA was obtained commercially and further purified as follows. DNA (2 mg/ml) was digested for 3 h at 37°C with 0.35 mg/ml of protease in 1.8% (w/v) SDS, 75 mM NaCl, and 35 mM EDTA. After this mixture was extracted several times with phenokcholoroform: isoamyl alcohol (25:24:1), the DNA was precipitated with ethanol and resuspended at a final concentration of 0.4 mg/ml in 0.1 M NaCl, 10 mM Tris-Cl (pH 7.4). DNA was sheared by three passes through a French press at 18-29kpsi, precipitated with ethanol, collected by centrifugation, and resuspended in 2 ml of water. Before use, the DNA was dialyzed extensively against deionized HzO. Absorption at 260 nm was determined after boiling of a sample diluted in 0.18 M NaCl. Hybridizations were carried out in 27-~1 volumes sealed in capillaries containing 150 Am units/ml DNA. The various Cot values were obtained by using four concentrations of potassium phosphate buffer (pH 6.9). Samples containing 0.55 M phosphate were incubated at 66”C, and those containing 0.2, 0.06, and 0.04 M phosphate were incubated at 6O”C, correcting to a standard rate as previously described (23, 30). Each sample contained approximately 5000 epm of “P-labeled RNA (estimated specific activity of 13 cpm/ pmol) with a DNA:RNA ratio of at least 2000. DNA-DNA hybrids were analyzed by chromatog-

AND

COX

raphy on hydroxylapatite (HAP). Capillary samples were diluted to 1.5 ml with water and applied at room temperature to 0.5 g of HAP which had been pretreated by boiling 15 min in 0.1 M potassium phosphate buffer (pH 6.9). Hot potassium phosphate buffer (50 mM) was pumped through the column while the water jacket was simultaneously brought to 60°C. When all single-stranded material had been eluted, doublestranded DNA was eluted with 0.2 M potassium phosphate buffer (pH 6.9). Each sample was dialyzed against distilled water and boiled for 10 min, and the absorbance at 260 nm was determined. DNA-RNA hybrids were analyzed by RNase A digestion. The 27-~1 capillary sample was added to 206 pl of phosphate buffer so that the final concentration was 0.24 M. A lOO-~1 aliquot was assayed directly on a DE-81 filter while another was incubated at 37’C for 30 min with 50 pg/ml of RNase A and then assayed. The amount of RNA in hybrid formation was determined from the fraction of radioactivity resistant to digestion. Hybridization parameters were analyzed by least-squares and the data were computer-fit (smooth curve) as described by Davidson (28). DNA reassociation was modeled by C/C, =&hybrid&d + c&/(1 + k,COt), and RNA hybridization in DNA exc”,ss

was

modeled

by

U/U,

=.fu.hrbridired

+ z

fn

exp[hm(l - (1 + k,,,C&)‘-“/k,(l - n)]; the value for n equals 0.43. Preparation of reticulocyte polyscnnd mRNA. Preparation of polysomes and mRNA was by slight modification of published procedures (19-21). All solutions and equipment were autoclaved or treated with 0.1% diethyl pyrocarbonate. White Leghorn roosters were made anemic by daily injections (0.22 ml/kg) of 2.5% (w/v) phenylhydrazine for 6 days, and were bled by cardiac puncture on the eighth day. The cells were washed three times with sterile 0.15 M NaCl, removing the buffy coat. One volume of packed cells was resuspended in 3 vol of lysis buffer containing 10 mM ethanolamine, 2 mM MgClz, 20 mM KCl, and 5 mM @-mercaptoethanol, pH 7.4, and allowed to sit on ice for 10 min. Several strokes with a loose-fitting dounce homogenizer ensured lysis. The nuclei and other large debris were removed by centrifugation at 30,OOOg for 15 min. The supernatant fluid was layered over a 12ml pad of 25% (w/v) sucrose in 20 mM Tris-Cl (pH 7.4), 50 mM KCl, 5 mM MgClz, and centrifuged for 3 h in a Spinco 45 Ti rotor at 35,000 rpm. The supernatant fluid was removed, and the pellets were carefully rinsed with several milliliters of polysome buffer (20 mM Tris-Cl, pH 7.4, 10 mM KCl, 5 mM MgClz, 0.5 mM EDTA). The pellets were resuspended in polysome buffer and gently homogenized with a Teflon-glass homogenizer. The polysome solution was adjusted to 0.1 M TrisCl (pH 9.0), 0.1 M NaCI, 10 mM EDTA, 1% (w/v) SDS, and extracted twice with an equal volume of phenol: choloroform:isoamyl alcohol (25241). After centrifugation, the aqueous layer was precipitated with

MESSENGER-LIKE

RNA

SYNTHESIS

ethanol and resuspended in a small volume of 0.5 M KCl, 10 mM Tris-Cl (pH 7.4). Chromatography of the RNA on oligo(dT)-cellulose was carried out as described by Aviv and Leder (22). Material which bound to the column was eluted with 10 mM Tris-Cl (pH ‘7.4), and the pooled fractions were precipitated with 2 vol of absolute ethanol (-2O’C) in the presence of 0.2% (w/v) potassium acetate (pH 5.6). The precipitate was collected by centrifugation and resuspended in sterile water. Preparation and characterization of cDNA. Reverse transcription was carried out with AMV RNA-directed DNA polymerase as described (24) with the following components in a total volume of 300 ~1: 10 mM Tris-Cl (pH 8.3); 10 mM DTT; 5 mM MgClz; 30 pg/ ml actinomycin D; 0.3 mM dATP, dCTP, dGTP, and dTTP (with [3H]TTP included at 160 &i/ml); approximately 100 pg/ml of unfractionated oligo(dT)-bound polysomal RNA; 400 pg/ml oligo(dT)iz-1s; 30 ~1 of enzyme buffer containing 50 mM Tris-Cl (pH 7.5), 3 mM DTT, 1.0 M NaCl, 17.5% (w/v) glycerol, 0.1 mM EDTA, 0.002% (w/v) Nonidet P-40 (NP-40); and 3.3 units of AMV RNA-directed DNA polymerase. The reaction was run at 42°C for 2 h. The product was purified as previously described (24) and had a specific activity of 160 cpm/pmol. Synthesis of cDNA was dependent on the addition of oligo(dT) primers, suggesting that only poly(A)-containing molecules were involved as templates. When the mRNA preparation used for reverse transcription was analyzed by sedimentation through linear sucrose gradients, a major component was observed sedimenting at 9 S with additional peaks at 18 S and 28 S, presumably rRNA (Fig. 1A). Recentrifugation of the pooled 9 S material produced no further separation (Fig. 1B). The 9 S RNA was collected by ethanol precipitation and chromatographed on oligo(dT)-cellulose as shown in Fig. 1C. The cDNA was hybridized in excess to either total unfractionated polysomal poly(A)+ RNA (used for reverse transcription) or the purified 9 S poly(A)+ component. Figure 2 demonstrates that the cDNA hybridized to 9 S RNA with a R&z of 5.5 X 10m3 and to total polysomal RNA with a R&z of 1.4 X 10-l. Thus, the 9 S material was enriched in sequences hybridizing to the cDNA preparation. Both preparations protected about 30% of the labeled cDNA from digestion by Sl nuclease. Hybridization of in vitro transcripts to reticulocyte cDNA. %P-Labeled RNA from erythrocyte and reticulocyte nuclei were hybridized in DNA excess to the cDNA complementary to reticulocyte polysomal mRNA. Approximate specific activities of the isolated RNA were determined as indicated in the text. These were 10 and 20 cpm/pmol for erythrocyte and reticulocyte RNA, respectively. The total reaction volume was 3 ~1 and contained DNA at a concentration of 20 pM in 0.55 M phosphate buffer (pH 6.8). Samples containing either 5000 cpm (reticulocyte) or 2500 cpm (erythrocyte) of RNA were sealed in capillaries, boiled for 10 min, and incubated at 66°C for 22.5 h to a C,,t

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93

NUCLEI

A

5

16

26

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+ FRACTION

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FIG. 1. Purification of 9 S mRNA from the poly(A)+ RNA of chicken reticulocyte polysomes. Poly(A)+ RNA was prepared as described under Materials and Methods. (A) Six Am units of total poly(A)-containing RNA was applied to a linear 15-30% sucrose gradient made up in gradient buffer (10 mM Tris-HCl, pH 7.4, 20 mM NaCl, and 1 mM EDTA) and centrifuged for 14.5 h at 40,000 rpm in a Spinco SW40Ti rotor at 4°C. Fractions of approximately 0.5 ml were collected, and the absorbance was determined for each at 260 nm. The three peak tubes of material sedimenting at 9 S were pooled, precipitated with ethanol, and resuspended in 200 ~1 of gradient buffer. (B) This sample (fractions 21-23) was reanalyzed by centrifugation as in (A). Material in the entire peak was precipitated with ethanol and resuspended in 400 ~1 of gradient buffer. (C) The RNA was diluted with an equal volume of 1 M KC1 and applied to a 0.5-ml column of oligo(dT)cellulose. The column was washed with 0.5 M KC1 in 10 mM Tris-HCl, pH 7.4, and eluted at the arrow with 10 mM Tris-HCl, pH 7.4. of around 10. Equivalent tubes were frozen immediately after boiling as controls, and all were analyzed by RNase digestion as described under DNA-RNA hybridization.

94

WIERSMA

AND

COX

on DE-81 filters (rather than precipitation with CC13 COOH), the low-level transcriptional activity in the erythrocyte preparations could be easily monitored. The saponin-isolated nuclei exhibited linear incorporation of label over a wide range of nuclear concentrations (Fig. 3A). The data presented in Fig. 3B show that incorporation was linear for 40-60 min, though some preparations ceased incorporation after 15-20 min. The reason for this variability is not presently understood. The effects of varying cation concentrations are shown in Figs. 3C and D. Maximum synthesis occurred at 175 mM KCl, where incorporation was fivefold greater I I I I I I I I 40 -4 -?I -2 -I 0 than that with no salt. Maximum tranLOG ERoT scription occurred at the lowest total diFIG. 2. Hybridization of reticulocyte polysomal valent ion concentration tested (2 mM) and cDNA to excess total polysomal and purified 9S at a Mg2+/Mn2+ ratio of 0.7510.25 (i.e., 1.5 mRNA. ‘H-Labeled cDNA, prepared from total polymM Mg2+ and 0.5 mM Mn2+). somal poly(A)+ RNA as described under Materials and Incubating reaction mixtures at 3’7°C Methods, was incubated with total polysomal poly(A)+ with RNase A (50 pg/ml, 20 min) or KOH RNA (0) or purified 9 S RNA from the poly(A)+ ma(0.3 M, 20 h) after synthesis eliminated all terial of Fig. 1 (0). Equivalent R& values were obradioactivity binding to the filters, while tained using 0.5 or 10 PM RNA, incubation times from little change from control values was ob2 min to 12 h, and corrections for salt concentrations served when DNase I (100 pg/ml, 20 min) as previously described (23). Hybridization conditions and analysis by Sl digestion (130 Sigma units/ml) or protease (100 pg/ml, 20 min) were used were according to Longacre and Rutter (48). (data not presented). These results demonstrate that the radioactive product was RNA. The template properties of the sysRESULTS tem were probed by preincubating nuclei Transcription in erythrocyte nuclei. Pre- for 15 min at 26°C with DNase I (10 pg/ vious reports have suggested that the ml) or actinomycin D (10 pg/ml). Subsequent incorporation of [3H]UMP was intranscriptional activity of avian erythrocytes was very low (15,16,25). Hence, con- hibited about 70% relative to nuclei which ditions were sought for developing a stan- had been preincubated with no additions dard cell-free assay with maximum sen- (data not presented). a-Amanitin had a of label sitivity. In the initial phases of this study striking effect on the incorporation it was found that erythrocyte nuclei pre- by the erythrocyte nuclei as depicted in Fig. pared by the method of acid extraction (26) 4. At concentrations between 0.1 and 1 pg/ were unable to incorporate label from ml about 90% of the synthesis was inhibited, while a further 3-5% was inhibited at rH]UTP (55). Consequently, other methods were examined for isolating nuclei, and 200 Kg/ml. It should be mentioned that mithose obtained following cell lysis with sa- tochondrial RNA synthesis is not inhibited ponin incorporated low (but measurable) by Lu-amanitin (54). Transcription systems derived from eulevels of radioactivity. When RNA synthesis in such a preparation was compared to caryotic cells have often been supplethat of HeLa nuclei, it was noted that the mented with E. coli RNA polymerase in efrate of [3H]UMP incorporation was about forts to increase their synthetic capacity. 30-fold higher in the tumor cell nuclei (data Since transcription from endogenous polymerases in the erythrocyte nuclei was exnot presented). Nevertheless, by using tremely low compared to HeLa nuclei (see high-specific-activity isotopes and assaying

MESSENGER-LIKE

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NUCLEI

SYNTHESIS

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FIG. 3. Parameters affecting the incorporation of rH]UMP by isolated chicken erythrocyte nuclei. (A) Increasing numbers of nuclei were incubated for 60 min at 30°C in standard reaction mixtures containing 125 mM KCI. (B, C) Nuclei equivalent to 100 pg of DNA were incubated in standard reaction mixtures at 26°C for the times indicated in (B) and at the KC1 concentrations indicated in (C). (D) Nuclei were pelleted and resuspended in Mg-free buffer and then incubated in reaction mixtures containing a total divalent metal ion concentration of 2 mM (O), 5 mM (A), or 10 mM (A), and the Mgzf/MnZ+ ratios as indicated in the figure. The KC1 concentration was 125 mM.

above), experiments were carried out to determine whether the bacterial enzyme could be used to stimulate RNA synthesis in these preparations. The addition of 5 units/ml E. coli RNA polymerase produced only a 20% increase in the incorporation of label from [3H]UTP (data not presented). Moreover, when the endogenous activity was eliminated by a-amanitin (10 pg/ml), the rH]UMP incorporation was equal to the increase observed in the absence of the drug. This suggests that the increase in incorporation was the product of the bacterial enzyme since it was resistant to cyamanitin. Because the increase in synthesis was small, and the effects of using bacterial polymerase to model eucaryotic synthesis

are in question, it was decided to use only the endogenous polymerases for further study. Characterization scripts. Routinely,

of the nascent

tran-

70-8070 of the radioactivity incorporated by the erythrocyte nuclei could be recovered by extraction with hot phenol-SDS. 3H-Labeled RNA isolated in this way was analyzed by polyacrylamide gel electrophoresis. As shown in Fig. 5, the population of radiolabeled RNA was heterogeneous in size with no distinct peaks. Sizes ranged from greater than 50 S down to 5 S as judged by the migration of wheat germ ribosomal RNA markers. It was of interest to determine whether the erythrocyte nuclei could further pro-

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V. ”

-1

-----.. -1

0.01 0.1 o(-AMANITIN

1.0 IO 1 pg /ml)

100

FIG. 4. Effect of cY-amanitin on RNA synthesis by endogenous polymerases in erythrocyte nuclei. Incubations were at 26°C for 30 min in reaction mixtures containing 200 mM KCl, 5 mbf MgC12, 1 mM MnC&, nuclei equivalent to 95 pg of DNA, and the amounts of a-amanitin as indicated in the figure.

cess the newly transcribed RNA. Figure 6 illustrates that 15-20s of the nuclear RNA was retained by oligo(dT)-cellulose at high ionic strength and was subsequently eluted in low-salt buffer, suggesting that a fraction of the transcripts contain a segment of poly(A). 5’Capping activity was also monitored by examining the incorporation of label from [w~~P]GTP into G(5’)ppp(5’)N or its methylated derivatives which are resistant to RNase A, nuclease Pl, and bacterial alkaline phosphatase. However, under the conditions employed, capping at the 5’ end could not be detected (data not presented). Hybridization anal&s. Maclean and Madgwick (27) observed the presence of distinct rRNA species in the newly synthesized RNA of intact erythrocytes. Because other studies (4, 15), including this one, show little if any rRNA synthesis in

AND

COX

erythrocyte nuclei as judged by size distribution of the RNA products, cation requirements for transcription, and aamanitin sensitivities, this point was examined further. Hybridization kinetics in DNA excess was used to identify the class of DNA from which the RNA was transcribed, and the results are shown in Fig. ‘7. Reannealing of total chicken DNA was analyzed on HAP, and the experimental points were fit by nonlinear least-squares as described previously (28). About 20% of the DNA appeared either not to dissociate or to reanneal very rapidly. This has been alternatively treated in the literature as background (29) or as an individual component of the kinetic analysis (30). Since even the samples denatured without added salt and frozen immediately after boiling showed this initial binding, the values were subtracted as background as in the former of the two treatments (29). The remainder of the DNA reassociated as two kinetic components with rate constants of 0.165 and 0.0021 liter mol-’ s-l, accounting for 10 and ‘70% of the sequences, respectively. These data are similar to results previously reported (29-31). Similarly, 32P-labeled RNA transcribed in erythrocyte nuclei was hybridized to excess chicken DNA, where the fraction of RNA remaining single stranded was calculated as 1 - (RNase-resistant cpm/total cpm). Each point is the average of duplicate samples, and the radioactivity that was resistant to digestion with RNase A immediately after thermal denaturation (18%) has been subtracted. The kinetic parameters were obtained by fitting the data to the appropriate equation (28) by leastsquares analysis. About 7% of the in vitro transcripts hybridized with a rate constant of 0.094 liter mol-’ s-l and 36% was driven into hybrid with a rate constant of 0.00022 liter mol-l s-l. About 50% of the labeled RNA did not hybridize at the highest Cot tested, 2.5 X lo4 mol s-l liter-‘. The reasons for this are not evident, but similar observations have been reported previously (32, 33). Self-annealing of the labeled nuclear RNA during hybridization was minimal, suggesting limited symmetric transcrip-

MESSENGER-LIKE

RNA

SYNTHESIS

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3

4

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,

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FIG. 5. Size distribution of 3H-labeled RNA synthesized in erythrocyte nuclei. Reaction mixtures contained about lo9 nuclei and 25 &i of [aH]UTP (40 Ci/mmol) in a final volume of 1 ml. Incubation was at 26°C for 30 min, after which RNA was isolated (17) and analyzed by electrophoresis on 2.4% polyacrylamide gels (50). Samples were heated for 5 min at 60°C before electrophoresis, and wheat germ rRNA was run on a parallel gel for molecular weight markers (arrows).

tion (data not presented). The data indicate that, of the sequences driven into hybrid, the majority were derived from unique sequence DNA, while a small, but significant, fraction were products of moderately repetitive sequences. In contrast to erythroblasts and reticulocytes, mature erythrocytes reportedly have little or no cytoplasmic RNA nor carry out protein synthesis (3, 34). Since the erythrocyte nuclei appeared to synthesize messenger-like RNA, it was of interest to determine whether reticulocyte cytoplasmic sequences were represented among the nascent transcripts of erythrocyte nuclei. To investigate this, it was necessary to hybridize 32P-labeled erythrocyte nuclear RNA to an unlabeled DNA probe complementary to reticulocyte polysomal poly(A)-containing mRNA. The reticulocyte products were 99% sensitive to RNase A and appeared to be predominantly synthesized from unique sequences as judged by hybridization kinetics (data not pre-

sented), so in these ways the reticulocyte and erythrocyte transcripts were similar. As shown in Table I, the cDNA protected only about 1% of the erythrocyte nuclear RNA from digestion by RNase A. The data indicate that this is probably not a statistically significant change from zero-time values. In contrast, the cDNA rendered about 8% of reticulocyte nuclear transcripts resistant to RNase A run in parallel, and this value was statistically significant. Because the quantity of RNA synthesized was extremely small, and the material isolated contained unlabeled erythrocyte RNA, it was not possible to determine directly the concentration of labeled RNA in the hybridization reactions. Rough calculations based on the RNA content of erythrocyte nuclei (15) suggest that the ratio of cDNA to RNA was no less than 10. The DNA does not appear to be limiting, however, because samples containing half the amount of RNA gave hybridization values not significantly different from those

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FIG. 6. Chromatography of 3H-labeled erythrocyte nuclear RNA on oligo(dT)-cellulose. Transcription was carried out and RNA was isolated as described under Materials and Methods and in Fig. 5. The sample in 0.2 ml was applied to a column (l-ml bed volume) of oligo(dT)-cellulose at room temperature in high-salt buffer (20 IIIM Tris-Cl, pH 7.4, 0.5 M KCl) and eluted (arrow) with low-salt buffer (20 mM Tris-Cl, pH 7.4) essentially as described by Aviv and Leder (22). The radioactivity represents material precipitated from the l-ml fractions by 2 ml of ice-cold 10% (w/v) CClaCOOH. The precipitates were collected on nitrocellulose filters (Millipore, 0.45 pm), washed with cold 5% (w/v) CCl&OOH, dried, and counted in a toluenebased scintillation fluid.

shown. The actual values for RNA hybridized may be slightly in error (underestimates) due to these concentration effects, but the difference between erythrocyte and reticulocyte should remain significant. Nuclear release of nascent RNA. Isolated nuclei from rat liver, HeLa, or KB cells have been shown to release mRNA and rRNA upon incubation with ATP and cytosol (35-39). The experiments were usually performed by prelabeling RNA in intact cells and then monitoring release from isolated nuclei. In the last reference above, isolated nuclei were shown to be active for RNA synthesis under the conditions of processing and release as worked out previously. In a similar way, the conditions for transcription in erythrocyte nuclei were

AND COX

used to determine whether newly synthesized RNA was being released. Reticulocyte nuclei could be isolated with saponin under somewhat more rigorous conditions of lysis than those used for preparing erythrocyte nuclei. The total amount of RNA synthesized by each of these nuclei under conditions maximized for erythrocytes is shown in Fig. 8. In addition, that fraction of the RNA which was released was readily determined by pelleting the nuclei after incubation and removing the supernatant to be counted separately. The reticulocyte nuclei released RNA linearly with time. The erythrocytes, on the other hand, released no material during the 60-min incubation, although total RNA synthesis in these nuclei was about 40% of that observed in the reticulocyte preparation. This release did not appear to be due to a preferential breakdown of reticulocyte nuclei, as the concentration of DNA found in the supernatant after intact nuclei were pelleted out remained constant with time for both types of nuclei. There are several observations which suggest that the release of RNA in reticulocytes was at least partially independent of its synthesis: first, synthesis occurred in erythrocytes without release; second, as shown in Fig. 8, RNA synthesis reached a plateau after 30 min while release continued in a linear fashion for at least 60 min; and third, the release of RNA and total transcription responded differently to a change in pH as illustrated in Fig. 9. Whereas increasing the pH from 7.4 to 8.4 significantly increased transcription, it effected a slight decline in RNA release. DISCUSSION

The results reported here suggest that nuclei prepared from saponin lysates of mature chicken erythrocytes are active for transcription from endogenous chromatinRNA polymerase complexes and that the predominant product is messenger-like RNA. These conclusions are based on the following observations: (i) over 90% of [3H]UMP incorporation was inhibited by low concentrations of a-amanitin (0.1 pg/ ml); (ii) maximal incorporation occurred in high salt and in the presence of Mn’+; (iii) the majority of the RNA product was com-

MESSENGER-LIKE

RNA

SYNTHESIS

IN

AVIAN

ERYTHROCYTE

99

NUCLEI

00 c q -

so

2 a E z

6o

;I” ti W

SO -I

0

I

3

LOG FIG. 7. sociation centrations described corrected erythrocyte sociation, tion. The corrected

4

3

E&T

Hybridization of erythrocyte nuclear transcripts to excess total chicken DNA. (0) Reasof sheared (average length of 450 nucleotide pairs) total chicken DNA at 66°C. Salt conbetween 0.04 and 0.55 M were used, and the equivalent Cot values were calculated as (23). Duplicate samples were analyzed by HAP chromatography, and values have been for 21% double-stranded material observed at zero time. (0) Hybridization of 32P-labeled nuclear RNA to chicken DNA. In samples identical to those analyzed for DNA reasthe hybridization of tracer RNA (>300-fold DNA excess) was analyzed by RNase A digesratio of RNase-sensitive RNA to total RNA was determined for duplicate samples and for 18% RNase-resistant material present at zero-time.

plementary to unique sequence DNA; (iv) about 15-20% of the labeled RNA was selectively retained by oligo(dT)-cellulose; and (v) the transcripts were heterogeneous in size, ranging from 4 S to 50 S. Thus, the conditions for maximal synthesis are characteristic of those for RNA polymerase II, and the in vitro product has several properties expected of nuclear pre-mRNA. It should be noted, however, that this RNA shared little sequence homology with reticulocyte cDNA. The results described above for transcription in isolated nuclei are generally in accord with recent studies which examined RNA synthesis in intact chicken erythrocytes (15,16,27,40). In several reports describing the DNA-dependent RNA polymerases associated with these cells, it was concluded that the only significant activity remaining at maturation is derived from polymerase II (25,41-44). This enzyme exhibits maximal activity in reaction mix-

tures containing high salt and Mn2+ ions, is inhibited by low concentrations of cyamanitin (Ki = 0.03 pg/ml), and synthesizes pre-mRNA sequences (51). About 90% of the RNA transcribed in nuclei prepared by the saponin lysis technique was derived from polymerase II-like activity as indicated by the data in Fig. 4. About 70% of the isolated RNA which hybridized to chicken DNA did so with a rate constant of 0.00022 (Cotl,z = 4.5 X 103), indicative of single-copy sequences (see Fig. 7). The remaining 30% of the RNA hybridized with a rate constant of 0.094 (C&/z = 10.6). This might represent transcription of hnRNA containing middle repetitive sequences, or transcription of 5 S rRNA or tRNA species by RNA polymerase III. The inhibition of residual UMP incorporation by a-amanitin at concentrations greater than 100 fig/ml (Fig. 4) could indicate a minor polymerase III activity in these preparations and would be consistent with the latter alternative.

100

WIERSMA TABLE

I

HYBRIDIZATION OF NUCLEAR TRANSCRIPTS TO DNA COMPLEMENTARY TO RETKULOCYTE POLYSOMAL PoLY(A)-CONTAINING RNA Fraction Source of =P-labeled RNA Reticulocyte Erythrocyte

of label by RNase

Oh 0.846 + 0.001 0.840 + 0.025

digested A 22.5 h

0.767 + 0.025* 0.830 + 0.010**

Note. RNA synthesis was carried out in standard reaction mixtures (1 ml) containing 5 X 10’ nuclei from erythrocytes or reticulocytes and 125 pCi of [a“PIGTP. Incubation was for 60 min at 26°C. Total nuclear RNA was isolated and hybridized to cDNA prepared from reticulocyte unfractionated polysomal poly(A)-containing RNA as described under Materials and Methods. Hybridization was carried out for the indicated times at 66°C in 0.55 M sodium phosphate buffer (pH 6.8) with attainment of an equivalent Cot of 10. Protection of the =P-labeled RNA by unlabeled cDNA was determined by digestion with RNase A (Materials and Methods). * P < 0.005. ** P > 0.5.

Zentgraf et al. (15) have reported that 18-30s of the RNA synthesized by erythrocytes in culture contains poly(A) tracts as judged by poly(U)-Sepharose chromatography. By a similar criterion, about 15% of the RNA transcribed in isolated nuclei contained poly(A) (Fig. 6), demonstrating that the cell-free system reflects the in vitro situation. Other experiments were unable to detect a 5’-cap structure on the nascent transcripts. Taken together, these results are consistent with the completion and 3’-end processing of RNA molecules initiated and capped in vivo. Polyacrylamide gel electrophoresis of 3H-labeled RNA indicated that the erythrocyte nuclear RNA was heterogeneous in size, ranging from 5 S to 50 S with a large proportion centered around 14 S (Fig. 5). These results are similar to those of Zentgraf et a!. (15), who found that RNA produced by chicken erythrocytes in culture is also heterogeneous in size and contains high-molecular-weight species. Quite different results were obtained by Gregory et al. (45) in a similar analysis of in vitro

AND

COX

transcripts from X&opus erythrocyte nuclei. Most of the amphibian nuclear RNA migrated during electrophoresis to the 4 S region of the gel. These differences may reflect species specificity or may be due to the fact that the frog nuclei were supplemented with E. coli RNA polymerase, whereas the transcription observed in the present study resulted totally from endogenous polymerases. The gel electrophoresis patterns (Fig. 5) did not suggest the presence of discrete rRNA species such as had been previously observed by Maclean and Madgwick (27). The fact that over 95% of the nuclear RNA synthesis could be inhibited by high concentrations of cu-amanitin (200 pg/ml) suggests that little or no polymerase I activity was present in these preparations. The data presented in Table I suggest

1 FIG. 8. Transcription and RNA release from reticulocyte and erythrocyte nuclei. Nuclei (10’) were incubated for the times indicated in a standard transcription mixture. The reaction tubes were immediately placed on ice and centrifuged for 5 min at 9OOg and 2°C. The solution was carefully removed from the nuclear pellet and applied to a DE-81 filter. The pellets were resuspended with 100 pl of 0.3% (w/v) sodium deoxycholate in water, sonicated, and assayed on DE81 filters along with the supernates in a standard assay. Symbols: (0, 0) erthryocyte nuclei; (m, 0) reticulocyte nuclei; (0, q ) filter-bound material in the supernatant fraction; (0, n) total of nuclear material and supernatant.

MESSENGER-LIKE

RNA SYNTHESIS

IN AVIAN

ERYTHROCYTE

NUCLEI

101

quences that were each present at very low concentrations. Gariglio et al. (52) have detected sequences complementary to adult P-globin in erythrocyte nuclear transcripts by hybridizing 32P-labeled RNA to ,&globin recombinant plasmids immobilized on filters. When the elongation rate was kept low, the nuclear RNA hybridized preferentially with a 5’-globin probe; whereas, under conditions more favorable for chain elongation, there was a trend for the labeled RNA to hybridize with both 5’ and 3’ moieties of the globin gene. In contrast, RNA synthePH sized in reticulocyte nuclei under both conditions hybridized equally well to the 5’FIG. 9. Effect of pH on RNA synthesis and release in reticulocyte nuclei. Nuclei (10s) were incubated for and 3’-globin fragments. These results 60 min with 50 mM Tris buffer adjusted to the given were interpreted to mean that molecules pH and all other reaction and assay conditions as de- of RNA polymerase II are clustered in the scribed in Fig. 8. (O), total RNA synthesis; (0), RNA 5’ portion of the globin gene in mature in postnuclear supernatant. erythrocytes but are more evenly distributed over the gene in globin-synthesizing reticulocytes, suggesting that chain elonthat the RNA transcribed in vitro from gation may be important in the developof globin synthesis. In reticulocyte nuclei contained sequences mental regulation complementary to reticulocyte polysomal this context, the small amount of 32P-lacDNA while the RNA synthesized by beled erythrocyte nuclear RNA which hytotal polysomal erythrocyte nuclei did not. About 8% of the bridized to reticulocyte 32P-labeled RNA from reticulocyte nuclei cDNA (Table I and text) may contain gloand about 1% of that from erythrocyte nu- bin sequences. However, if they represent clei were rendered RNase-resistant by hy- 3’-truncated transcripts, their detection in bridization to excess cDNA for 22 h. An the present study with cDNA may have estimate of globin sequences in erythroid been limited since the cDNA hybridization hnRNA was calculated to be about 0.05% probe would likely have been enriched in by Williamson and Tobin (46), and more 3’-coding sequences. recently Landes and Martinson (47) deterZentgraf et al. (15) have shown that mined that about 0.02 to 0.05% of the tran- rH]uridine incorporated into RNA by scripts synthesized in erythroid nuclei chicken erythrocytes in culture is confined from 5-day and 12-day embryos is globinto the nucleus with little or no radioactivity specific. It seems possible, therefore, that reaching the cytoplasm. The results dethe more extensive hybridization observed scribed in Fig. 8 show that the isolated nuwith the reticulocyte cDNA in the present clei retain this characteristic since none of study may reflect the presence of nonglobin the newly transcribed RNA was released as well as globin sequences in the reticuinto the incubation mixture over the time locyte transcripts. Purification of the re- period examined. In contrast, as much as ticulocyte 9 S mRNA increased the concen- 25% of the RNA synthesized in isolated retration of those sequences which hybridticulocyte nuclei was released under idenized to the cDNA, suggesting that they tical conditions. No evidence could be found were of high concentration in the original for preferential lysis of the reticulocyte mRNA, and presumably globin. It is un- nuclei. clear why only 30% of the cDNA hybridBecause the characteristics of tranized, but suggests the possibility that the scription in nuclei prepared from saponin remaining fraction may have been complelysates of mature chicken erythrocytes are mentary to a large number of RNA se- remarkably similar to those of intact cells

102

WIERSMA

in culture, it is felt that this system may be utilized for examining further those changes which lead to the severe restriction of nuclear activity during erythropoiesis and to nuclear reactivation in heterokaryons. It will be of particular interest to determine whether cytoplasmic or nuclear extracts from more active cells can enhance transcriptional activity in the erythrocyte ghosts, particularly with respect to chain initiation.

AND

15. ZENTGRAF, H., SCHEER, Il., AND FRANKE, W. W. (1975) Exp. Cell Res. 96,81-95. 16. MADGWICK, W. J., MACLEAN, N., AND BAYNES, Y. A. (1972) Nature New Biol 238.137-139. 17. GIRARD, M. (1967) in Methods in Enzymology (Grossman, L., and Moldave, K., eds.), Vol. 12, pp. 581-588, Academic Press, New York. 18. MIZUMOTO, K., AND LIPMANN, F. (1979) Proc. Natl. Acod Sci. USA 76.4961-4965. 19. STEWERT, A. G., GANDER, E. S., MOREL, C., LUPPIS, B., AND SCHERRER, K. (1973) Eur. J. Biochem. 34,205-212. 20.

ACKNOWLEDGMENTS We thank Dr. D. Grangenett for his kind gift of AMV reverse transcriptase, deoxynucleoside triphosphates, oligo(dT)i2i8, and calf thymus primers, as well as for invaluable direction for the synthesis of cDNA. We also thank Les Williams and the Iowa State University Poultry Science Center for willing help with procurement of chickens and blood. Sincere thanks go to Barb Roberts for her skillful preparation of this manuscript. REFERENCES 1. CAMERON, T. L., AND PRESCO~, D. M. (1963) Exp. Cell Res. 30,609-612. 2. SCHERRER, K., MARCAUD, L., ZAJDELA, F., LONMIN, I. M., AND GROSS, F. (1966) Proc. Natl. Acad. Sti USA 56,1571-1578. 3. KABAT, D., AND ATTARDI, G. (1967) Biochim. Bie phys. Acta 138,382-399. 4. ATTARDI, G., PARNAS, H., ANDA~ARDI, B. (1970) Exp. Cell Res. 62, 11-31. 5. HARRIS, H. (1970) Cell Fusion, Oxford Univ. Press (Clarendon), London. 6. RINGERTZ, N. R., AND BOLUND, L. (1974) in The Ceil Nucleus (Busch, H., ed.), Vol. 3, pp. 417446, Academic Press, New York. 7. RUIZ-CARRILLO, A., WANGH, L., LIT~AU, V., AND ALLFREY, V. (1974) J. Biol. &em. 249, 73587368.

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