Proc. Nati. Acad. Scl. USA Vol. 74, No. 10, pp. 4346-4350, October 1977

Biochemistry

Use of molecular hybridization to purify and analyze albumin messenger RNA from rat liver (immunoprecipitation of polysomes/complementary DNA-cellulose/cell-free protein synthesis)

ROGER K. STRAIR, SING HIEM YAP, AND DAVID A. SHAFRITZ Departments of Medicine and Cell Biology and the Liver Research Center, Albert Einstein College of Medicine, 1300 Morris Park Avenue, Bronx, New York 10461

Communicated by Alex B. Novikoff, August 4, 1977

munoprecipitation of specific polysomes and molecular hybridization in a novel approach to prepare highly purified rat albumin mRNA. The general strategy of our procedure is to enrich for a specific mRNA, so that it represents the most abundant sequence in a given RNA population. Complementary DNA (cDNA) is transcribed from this enriched mRNA and is then used in a limited RNA-cDNA hybridization, under conditions in which only cDNA corresponding to the most abundant component of the RNA population from which it was transcribed will form hybrids. Messenger RNA is then isolated from the RNA-cDNA hybrid and is characterized for purity by molecular hybridization and cell-free protein synthesis. By these procedures we have prepared biologically active, highly purified, rat albumin mRNA and an albumin cDNA probe that can be utilized for a wide variety of molecular studies.

ABSTRACT A new procedure is described for purification of rat liver albumin m A. First a population of RNA molecules is enriched for albumin mRNA by immunoprecipitation of polysomes containing albumin nascent chains. Polyadenylylated RNA is prepared from immunoprecipitates, transcribed into complementary DNA, and shown to be enriched severalfold for a particular RNA frequency component. This enriched RNA component is then purified by molecular hybridization to a limited Rot value (product of RNA concentration and incubation time), under conditions in which only the most abundant sequence component is annealed. Potentially, this procedure can be employed for the purification of a wide variety of mRNAs present in lesser amounts in the cell. The isolated RNA appears to be a single frequency component by hybridization to complementary DNA transcribed from itself. This RNA is a 17S species and represents 5-8% of total cytoplasmic polyadenylylated RNA. In vitro translation of the purified RNA has shown that it codes for a single polypeptide that can be identified immunologically as albumin and migrates with rat serum albumin on sodium dodecyl sulfate/polyacrylamide gels. This albumin mRNA was determined to be essentially pure by comparing its kinetics of hybridization to those obtained with rabbit a + P globin mRNA and its DNA complement. The sequence complexity of purified rat albumin mRNA corresponds to 5.9 X 105 daltons. Various procedures have been employed for purification of eukaryotic mRNAs. Affinity chromatography with either oligo(dT)-cellulose or poly(U)-Sepharose has been most successful for separating mRNAs containing a 3'-poly(A) segment from the bulk of ribosomal and other RNA components (1, 2). However, this procedure cannot be used to separate specific polyadenylylated mRNAs from each other, and a portion of eukaryotic mRNA does not contain a 3'-poly(A) segment (3-7). Other commonly used methods for purification of eukaryotic mRNAs have been based either on a unique size for a specific mRNA-e.g., a and ,B globin mRNA(8), histone mRNA (3,9, 10), immunoglobin light chain mRNA (11-14), ovalbumin mRNA (15), etc.-or an unusual nucleotide composition-e.g., silk fibroin mRNA (16). For purification of mRNAs that are average sized and are present as only a small portion of total cellular mRNA, the method of immunoprecipitation of polysomes containing nascent polypeptide chains for a specific protein is the only specific procedure available at the present time. Immunoprecipitation can enrich a polysome preparation for a specific mRNA by 5- to 10-fold (18-22). However, in order to obtain intact mRNA with good biological activity, the antibody must be highly purified to remove ribonuclease activity. Furthermore, large amounts of antibody are needed to generate sufficient amounts of material for use in molecular studies. In the present study, we have combined the techniques of im-

MATERIALS AND METHODS Preparation of Total Liver Cytoplasmic Polyadenylylated RNA. Livers were excised, perfused, and homogenized as described by Taylor and Schimke (21). A postmitochondrial supernatant was prepared by centrifugation of the homogenate at 12,000 X g for 15 min and prepared for RNA extraction by addition of 1/2 volume of 0.3 M NaCl/1.5% sodium dodecyl sulfate (NaDodSO4)/15 mM EDTA/30 mM Tris-HCl (pH 8.5). After 15 min at room temperature, several successive extractions were performed with equal volumes of phenol/chloroform/ isoamyl alcohol (50:48:2 by volume). When no detectable interface remained, 2 volumes of ethanol were added. RNA was precipitated overnight and isolated by centrifugation at 12,000 X g for 30 min. For oligo(dT)-cellulose chromatography, the RNA was dissolved in 10 mM Tris-HCl (pH 7.4)/0.5% NaDodSO4 and heated to 650 for 5 min. The RNA solution was then cooled rapidly by the addition of an equal volume of 1 M NaCl (40). This material was placed over a 3-ml packed oligo(dT)-cellulose column. The column was washed with 0.1 M NaCl/10 mM Tris-HCl (pH 7.4)/0.5% NaDodSO4/10 mM Tris-HCl (pH 7.4) and then washed with 0.1 M NaCl/10 mM Tris-HCI (pH 7.4)/0.5% NaDodSO4. RNA remaining bound to the column was eluted with 10 mM Tris-HCl (pH 7.4)/0.5% NaDodSO4 and termed "cytoplasmic poly(A)+ RNA." The eluted RNA was made 0.2 M in sodium acetate buffer (pH 5.5) and precipitated with ethanol. Immunoprecipitation of Polysomes. Total rat liver polysomes were prepared as described by Taylor and Schimke (21). Polysomes containing albumin nascent chains were immunoprecipitated by incubating total liver polysomes, approximately 2200 A2W units (one A2Wo unit is the amount of material

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U. S. C. §1734 solely to indicate this fact.

Abbreviations: cDNA, DNA complementary to mRNA; NaDodSO4, sodium dodecyl sulfate; Immpt, prepared with the use of immunoprecipitation.

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having an absorbance of 1 when dissolved in 1 ml and the light path is 1 cm), with rabbit IgG specific for rat serum albumin (3.6 mg); followed by precipitation of immune complexes with goat antiserum to rabbit IgG (455 mg) as described by Palacios et al. (18). Preparation of RNase-free IgG was as described by Palacios et al. (23). The yield of RNA extracted from immunoprecipitates was generally 100-150 A2j0 units. cDNA Synthesis. DNA complementary to polyadenylylated RNA was prepared as described by Housman et al. (39). Standard preparations sedimented on sucrose gradients. at approximately 8 S. The specific activity of the cDNA was 7.5 X 106 cpm/Aug. cDNA-cellulose was synthesized as described by Levy and Aviv (31). RNA-cDNA Hybridization. Analytical RNA-cDNA hybridizations were performed at 650 in sealed 5-Mil or 10-lA capillary tubes containing 0.2 M sodium phosphate buffer (pH 6.8), 0.5% NaDodSO4, a constant amount of cDNA (indicated in the figure legends), and various amounts of RNA. After the desired Rot value was achieved (Rot is the product of RNA concentration in moles of nucleotide/liter and incubation time in seconds), the reaction mixture was processed as described by Housman et al. (39). Preparative RNA-cDNA hybridization reactions were performed under the same conditions in volumes varying from 140 Itl to 17 ml. To prepare "S, nuclease" cDNA, hybridizations were performed in approximately 10-fold RNA excess. After the desired Rot value was achieved, the reaction was processed as described by Housman et al. (39). After S, nuclease digestion, the sample was adjusted to 0.1 M NaCl, 0.5% NaDodSO4, and Escherichia coli tRNA at 10 sg/ml, and RNA-cDNA hybrids were extracted with phenol/chloroform/isoamyl alcohol as described above. After ethanol precipitation the hybrids were alkali digested (0.6 M NaOH for 60 min at 370), neutralized, and sedimented on a 5-20% exponential sucrose gradient containing 10 mM Tris-HCl (pH 7.4), 0.5% NaDodSO4. Material sedimenting faster than 4 S was pooled and used as "Si cDNA." For cDNA-cellulose hybridization, RNA was used in approximately 10-fold excess of the cDNA. Conditions for the hybridization were the same as those described above. cDNA-cellulose was kept in suspension by continuous shaking during the reaction period. After the desired Rot value was achieved the reaction was diluted into 50 ml of 10 mM Tris-HCI (pH 7.4)/0.5% NaDodSO4 at room temperature and transferred to a column. After extensive washing of the column with 10 mM Tris-HCI (pH 7.4)/0.5% NaDodSO4 at room temperature, hybridized RNA was eluted by washing with 85% (vol/vol) formamide at 37'. Eluted RNA fractions were precipitated with ethanol as described above. RESULTS Sequence Complexity Analysis of Polyadenylylated RNA Extracted from Immunoprecipitated Polysomes. The first step in the purification scheme, i.e., the creation of a population of RNA molecules in which albumin mRNA is the most frequently represented sequence, was approached by the immunoprecipitation of albumin-synthesizing polysomes. To test whether we could distinguish a very abundant class of Rna in polyadenylylated RNA prepared from immunoprecipitated polysomes [termed "Immpt poly(A)+ RNA"], we prepared 3H-labeled cDNA to Immpt poly(A)+ RNA and analyzed its kinetics of hybridization in RNA excess. This method of analysis has been used to define the frequency and sequence complexity components of total cytoplasmic (25-27), polysomal (28, 29), and nuclear (27, 30) RNA populations. As can be seen in Fig.

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FIG. 1. Hybridization analysis of liver and reticulocyte mRNA preparations. (A) Polyadenylylated RNA extracted from immunoprecipitated liver polysomes was transcribed into cDNA. This cDNA (Immpt cDNA) was then hybridized to either the RNA from which it was transcribed (0-*) or to total liver cytoplasmic polyadenylylated RNA (O---0). RNA concentrations in these experiments were varied from 0.1 to 14 jg/ml. Approximately 500 cpm of [3H]cDNA was used for each determination. (B) Rabbit a + ft globin mRNA was prepared from reticulocyte lysates by a combination of oligo(dT)-cellulose chromatography and sucrose gradient centrifugation. [3H]cDNA was prepared from this RNA and used for hybridization analysis to determine sequence complexity. Approximately 500 cpm of [3H]cDNA was used for each determination.

1A, when Immpt poly(A)+ RNA was hybridized to its cDNA (termed "Immpt cDNA"), approximately 25-30% of the cDNA annealed in the most rapidly hybridizing component. Because the hybridization of this component occurs over approximately 1.5 logs of Rot values, it presumably represents the annealing of one frequency component of RNA (25). Assuming that all polyadenylylated RNAs are transcribed into cDNA with equal efficiency, this indicates that there is a major abundancy component that accounts for 25-30% of the RNA in polyadenylylated RNA extracted from immunoprecipitated polysomes. The Rot1/2 of this component, 5.0 X 10-3 mol-sec/liter (Fig. 1A), when corrected for the percentage of RNA driving the reaction (25-S0), gives a value of 1.25-1.50 X 10-3 molsec/liter. Comparison of this value with -the Rot1/2 for hybridization of a + ,B globin mRNA (4.0 X 105 daltons) to its own cDNA, 6.6 X 10-4 mol-sec/liter (Fig. 1B), indicates that our rapidly hybridizing RNA component has a maximum sequence complexity of approximately 8.0 X 105 daltons. Therefore, we conclude that this component is composed of at most two and probably only one average-sized mRNA molecule (24). Also shown in Fig. 1A is the hybridization of Immpt cDNA to liver cytoplasmic poly(A)+ RNA. The curve is similar in shape to that obtained with Immpt poly(A)+ RNA and, as expected, is displaced towards higher Rot values. Preparation of Single Complexity Component cDNA from Immpt cDNA. To analyze the nature of these hybridizations in greater detail, we purified the most rapidly annealing component of Immpt cDNA by hybridizing Immpt cDNA to cytoplasmic poly(A)+ RNA to a Rot value of 1.75 X 10-2 mol-sec/liter (12% of cDNA hybridized). The reaction mixture was treated with SI nuclease to digest unhybridized cDNA. Hybridized cDNA was purified as described in Materials and Methods. This cDNA (termed "Si nuclease-purified cDNA") was then used in RNA excess hybridization reactions driven by either Immpt poly(A)+ RNA or total cytoplasmic poly(A)+ RNA (Fig. 2). Purified cDNA hybridized to these RNAs as one component with ROt1/2 values of 5.0 X 10-3 mol-sec/liter and 1.75 X 10-2 mol-sec/liter, respectively. These values were indistinguishable from the RotI/2 values of the rapidly hybridizing component of Immpt cDNA when hybridized to these same RNAs (compare Figs. 1 and 2). These results support our interpretation that 25-30% of the Immpt cDNA indeed represents

4348

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Proc. Natl. Acad. Sci. USA 74 (1977)

Strair et al.

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one component. If two or more components with similar

abundancies were present, then the Rot1/2 value of the most rapidly hybridizing sequences of Immpt cDNA (12%) should be reduced when the cDNA is purified and reannealed. Our results also indicate that the Immpt cDNA that reacts as the rapid component when hybridized to total liver polyadenylylated RNA represents the same sequences that react rapidly with Immpt poly(A)+ RNA. This conclusion is based on the observation that purified Immpt cDNA sequences that hybridize rapidly to cytoplasmic poly(A)+ RNA hybridize to Immpt poly(A)+ RNA with the same kinetics as the 25-30% component of unfractionated Immpt cDNA. The ratio of the Rotl/2 values of hybridizations driven by cytoplasmic poly(A)+ RNA and Immpt poly(A)+ RNA is 3.5 (Fig. 2). This value is an estimate of the fold enrichment during immunoprecipitation of this most abundant RNA component. It also indicates that the amount of this component in the original RNA preparation of total cytoplasmic poly(A)+ RNA was approximately 5-8%. Purification of RNA Complementary to Rapidly Hybridizing cDNA. Our strategy was to perform a limited hybridization to a Rot value such that only cDNA transcribed from the most abundant component of Immpt poly(A)+ RNA would anneal. Subsequent isolation of RNA-cDNA hybrids should yield RNA corresponding to that most abundant component. Because the most rapidly hybridizing component in Immpt cDNA contains the same sequences regardless of whether it is hybridized to immunoprecipitated polyadenylylated RNA or total cytoplasmic polyadenylylated RNA (Figs. 1 and 2), either one of these RNA fractions could be used. We decided to use cytoplasmic poly(A)+ RNA because it was more easily obtained and its isolation procedure subjected it to less chance of degradation. To facilitate separation of hybridized from unhybridized RNA and to allow us to reutilize our preparations of Immpt cDNA, we enzymatically linked Immpt cDNA to cellulose (17, 31). In a pilot synthesis of Immpt cDNA-cellulose, using [3H]dCTP as a substrate, the yield of cDNA was approximately 20% of the input RNA. Labeled Immpt cDNA-cellulose was also used to determine its effect on the kinetics of hybridization. When hybridizations were performed in a shaker bath (so that the cellulose remained in suspension), the rates of hybridization, as determined by SI nuclease digestion, were 1.2-1.5 times slower than the corresponding rates of solution hybridization (data not shown). Immpt poly(A)+ RNA was transcribed into approximately 10 jig of Immpt cDNA-cellulose. Total cytoplasmic poly(A)+

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RNA was then hybridized to Immpt cDNA-cellulose to an Rot value of 2.5 X 10-2 mol-sec/liter. This represents approximately 10-15% hybridization of the Immpt cDNA linked to cellulose. High concentrations of RNA (25 ,ug/ml) were used both to maintain RNA excess and to achieve the desired Rot value quickly (within 10 min). After the desired Rot value was achieved, unhybridized RNA was removed by extensive washing of the Immpt cDNA-cellulose at 230 with 0.5% NaDodSO4/10 mM Tris-HCI (pH 7.4). This procedure also removed RNA that was bound to the column as a consequence of poly(rA)-oligo(dT) annealing. Hybridized RNA was then eluted from Immpt cDNA-cellulose by washing with 85% formamide at 37°. Estimation of the Purity of RNA Eluted from cDNA-Cellulose. The purity of RNA eluted from Immpt cDNA-cellulose was determined by RNA excess hybridization to cDNA transcribed from itself. As is shown in Fig. 3, the eluted RNA appears to consist of a single frequency component with a Rot,/2 of 9.8 X 10-4 mol-sec/liter. By comparison to the Rot1/2 of 6.6 X 10-4 mol-sec/liter for purified a + f globin mRNA (4 X 105 daltons), the sequence complexity of the purified RNA is 5.9 X 105 daltons. When cDNA prepared from the purified RNA is used to titrate fractions of total cytoplasmic poly(A)+ RNA isolated from a sucrose gradient, it is shown to be complementary to RNA that sediments slightly slower than 18S ribosomal RNA (Fig. 4). The same sedimentation profile was obtained with purified RNA eluted from Immpt cDNA-cellulose. In comparison to 18S RNA (about 7.0 X 105), there is a correlation between the apparent molecular mass of the purified RNA (as determined by sucrose gradient analysis to be 17 S or about 6.0 X 105 daltons) and the sequence complexity (as determined by RNA-cDNA hybridization to be 5.9 X 105 daltons). Although neither of these methods gives an absolute determination of molecular mass, these results indicate that the purified RNA represents a single RNA species. Furthermore, the existance of only a single hybridizing component indicates that the mRNA is at least 90% pure and that any contaminants must be present at very low concentrations. Characterization of RNA Eluted from Immpt cDNACellulose. To show that the purified RNA does indeed represent albumin mRNA, we have translated this fraction in a wheat germ cell-free system and have analyzed the cell-free reaction product. Synthesis of "albumin-like" material was demonstrated by indirect immunoprecipitation with rabbit anti-rat serum albumin and goat antirabbit IgG. Ninety-two percent of the [35S]methionine-labeled polypeptides precipitated under these

Biochemistry:

Strait et al.

Proc. Natl. Acad. Sc; USA 74 (1977)

4349

Table 1. Immunoprecipitation of product synthesized under direction of exogenous RNA in the wheat germ cell-free system

cpm protein cpm utilized for precipitated Addition to wheat immunowith antibody to: % germ system precipitation Ricin Albumin "4albumin"* None 26,210 168 239 1.0 Purified rat liver albumin mRNA 10,650 127 8,367 92 Immpt poly(A)+-liver RNA 17,570 129 3,357 22 HeLa polysomal RNA 48,730 278 1,562 3.7 Rabbit reticulocyte polysomal RNA 66,630 267 763 1.3 Chemically purified rat [14C]albumin 4,170 32 3,591 100 Cell-free products synthesized in vitro were assayed for "albumin-like" material as described by Grossman et al. (32). * All figures were corrected for 85% efficiency of immunoprecipitation of [14C]albumin.

conditions (Table 1). Control experiments, using wheat germ cell-free products synthesized under direction of HeLa polysomal RNA, showed less than 4% immunoprecipitation. The cell-free product under direction of rabbit reticulocyte polysomal RNA gave a value of approximately 1% immunoprecipitation. In addition, antiserum to ricin, a plant protein, immunoprecipitated only 1% of [s5S]methionine-labeled products of the wheat germ system under the direction of exogenous albumin mRNA. Fig. 5 shows the autoradiogram of an NaDodSO4/polyacrylamide slab gel of the cell-free reaction products from the wheat germ system under the direction of cytoplasmic poly(A)+ RNA, Immpt poly(A)+ RNA, and the purified mRNA. A comparison of the products synthesized by the various RNAs shows progressive enrichment for a polypeptide that migrates with purified rat serum albumin standard (68,000 daltons). Under the direction of the purified mRNA, this product represents the only detectable high molecular weight component. Trace quantities of lower molecular weight components synthesized in the presence or absence of added

RNA presumably represent endogenous wheat germ proteins.

DISCUSSION In previous studies, controlled molecular hybridization has been used to fractionate specific DNA fractions [e.g., Davidson et al. (37) and Levy and McCarthy (27)]. Molecular hybridization has also been used to isolate specific RNA fractions for which purified complementary nucleic acids were already available [e.g., Venetianer and Leder (17) and Lewis et al. (38)]. In this study we have used controlled molecular hybridization to purify albumin mRNA. This technique should prove useful in the purification of various other mRNAs, because all that is required for this technique to be successful is that the mRNA for selection be present as the most frequent polyadenylylated RNA component. Several characteristics of the purified RNA indicate that it is indeed albumin mRNA. Its size, as determined on sucrose

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FIG. 4. Sucrose gradient analysis of the purified RNA. Cytoplasmic polyadenylylated RNA and the purified RNA eluted from Immpt cDNA-cellulose were sedimented in parallel 5-20% exponential sucrose gradients containing 10 mM Tris-HCl (pH 7.4)/0.5% NaDodSO4. Centrifugation was at 230 for 2.5 hr at 58,000 rpm in the Beckman SW 60 Ti rotor. Samples were heated to 650 in 10 mM Tris-HCl (pH 7.4)/0;5% NaDodSO4 for 5 min and rapidly cooled on ice prior to application to the gradients. The arrows indicate the positions of 28S and 18S ribosomal RNAs and duck reticulocyte 10S globin mRNA in a parallel gradient. Samples from each fraction of the gradients were assayed for sequences complementary to [3H]cDNA transcribed from the purified RNA. Hybridization conditions were as described by Housman et al. (39). Hybrid formation with fractions of total cytoplasmic polyadenylylated RNA (0-0) or purified RNA (O---O) was detected by incubation of an aliquot of each gradient fraction with [3H]cDNA (600 cpm) in 5,gl for 40 hr under the conditions described in Materials and Methods.

FIG. 5. NaDodSO4/polyacrylamide slab gel electrophoresis of cell-free reaction products. Various RNA preparations were prepared as described in Materials and Methods and translated in a wheat germ extract, prepared as described by Marcu and Dudock (33). The wheat germ was a gift of General Mills Inc., Vallejo, CA. Reaction conditions for the cell-free system were as described by Grubman et al. (34). Radioactively labeled product synthesized in vitro was analyzed electrophoretically on 1096 polyacrylamide/NaDodSO4 slab gels [Laemmli (35), Maizel (36)]. Dried slabs were exposed to x-ray film. Slot a, rat serum albumin standard (Coomassie Blue stained). Slot b, cell-free product synthesized under the direction of 2 gg of total cytoplasmic polyadenylylated RNA. Slot c, same as b with a different preparation of wheat germ extract. Slot d, cell-free product synthesized under the direction of 1 ,ug of Immpt poly(A)+ RNA. Slot e, cell-free product synthesized under the direction of 0.5 ;1g of purified RNA eluted from Immpt cDNA-cellulose. Slot f, wheat germ extract to which no exogenous RNA was added. The positions of protein standards isolated from intact vesicular stomatitis virus (G = 67,000 daltons, N = 52,000 daltons, and M = 25,000 daltons) are shown between slots d and e.

4350

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gradients, agrees with previous estimates for albumin mRNA (21, 22). This RNA is a very abundant component in cytoplasmic polyadenylylated RNA (5-8%) and represents 25-30% of polyadenylylated RNA extracted from polysomes immunoprecipitated by monospecific rabbit antiserum to rat albumin. When this RNA is placed into a wheat germ mRNAdependent cell-free translation system, 92% of the product can be identified immunologically as albumin. The single high molecular weight component produced under the direction of exogenous, purified mRNA migrates on NaDodSO4/polyacrylamide gels with essentially the same mobility as chemically purified rat serum albumin. On some occasions we have found that labeled cell-free product migrates in the trailing region slightly behind the major portion of serum albumin stained with Coomassie Blue. This cell-free product may represents a precursor to rat serum albumin as reported by Strauss et al.

(40).

Taylor and Tse (22) have reported the isolation of albumin mRNA by using other techniques. In their study, cell-free protein synthesis was used as the criterion of purity. Subsequently it has been demonstrated that the amount of albumin synthesized in an in vitro protein-synthesizing system may vary considerably depending upon the reaction conditions (41). In the present study we have used molecular hybridization as a criterion of purity. We have determined that our preparation of albumin mRNA has a maximum sequence complexity of 5.9 X 105 daltons, in close agreement with its size on a sucrose gradient. In conjunction with our cell-free protein synthesis data, these results lead us to conclude that we have isolated highly purified, intact, biologically active albumin mRNA. The authors would like to thank Dr. Arthur I. Skoultchi for advice and many helpful discussions during the course of these studies, and for his critical evaluation of this manuscript. This work was supported by National Institutes of Health Grants AM-17609, AM-17702, Cell and Molecular Biology Training Grant 5-TO1 GM 02209, the Netherlands Organization for Advancement of Pure Research (Z.W.O.) and Niels Stensen Stichting to S.H.Y., a National Institutes of Health Research Career Development Award to D.A.S. and the Irma Hirschl Charitable Trust of New York. 1. Aviv, H. & Leder, P. (1972) Proc. Nati. Acad. Sci. USA 69, 1408-1412. 2. Adesnik, M, Salditt, M., Thomas, W. & Darnell, J. E. (1972) J.

Mol. Biol. 71, 21-30. 3. Adesnik, M. & Darnell, J. E. (1972) J. Mol. Biol. 67,397-406. 4. Schochetman, G. & Perry, R. P. (1972) J. Mol. Biol. 63, 577590. 5. Greenberg, J. R. & Perry, R. P. (1972) J. Mol. Biol. 72,91-98. 6. Micarek, C., Price, R. & Penman, S. (1974) Cell 3, 1-10. 7. Nemer, M., Dubroff, L. M. & Graham, M. (1975) Cell 6, 171178. 8. Lockard, R. E. & Lingrel, J. B. (1969) Biochem. Biophys. Res. Commun. 37, 204-209. 9. Jacobs-Lorena, M., Baglioni, C. & Borun, T. W. (1972) Proc. Natl. Acad. Sci. USA 69,2095-2099.

Proc. Nati. Acad. Sci. USA 74 (1977) 10. Grunstein, J., Levy, S., Schedl, P. & Kedes, L. (1973) Cold Spring Harbor Symp. Quant. Biol. 38, 717-723. 11. Delovitch, T. L. & Baglioni, C. (1973) Proc. Natl. Acad. Sci. USA 70, 173-178. 12. Honjo, T., Packman, S., Swan, D., Nau, M. & Leder, P. (1974) Proc. Natl. Acad. Sci. USA 71,3659-3663. 13. Tonegawa, S., Bernardini, A., Weinmann, B. J. & Steinberg, C. (1974) FEBS Lett. 40, 92-96. 14. Rabbitts, T. H. & Milstein, C. (1975) Eur. J. Biochem. 52, 125-133. 15. Woo, S. L. C., Rosen, J. M., Liarakos, C. D., Robberson, D. L., Choi, Y. C., Busch, H., Means, A. R. & O'Malley, B. W. (1975)

J. Biol. Chem. 250,7027-7039. 16. Suzuki, Y. & Brown, D. D. (1972) J. Mol. Biol. 63,409-429. 17. Venetianer, P. & Leder, P. (1974) Proc. Natl. Acad. Sci. USA 71,

3892-3896. 18. Palacios, R., Sullivan, D., Summers, N. M., Kiely, M. L. & Schimke, R. T. (1973) J. Biol. Chem. 248,540-548. 19. Shapiro, D. J. & Schimke, R. T. (1975) J. Biol. Chem. 250, 1759-1764. 20. Schechter, I. (1974) Biochemistry 13, 1875-1885. 21. Taylor, J. M. & Schimke, R. T. (1973) J. Biol. Chem. 248, 7661-7668. 22. Taylor, J. M. & Tse, T. P. H. (1976) J. Biol. Chem. 251,74617467. 23. Palacios, R., Palmiter, R. D. & Schimke, R. T. (1972) J. Biol.

Chem. 247,2316-2321. 24. Keller, G. H. & Taylor, J. M. (1976) J. Biol. Chem. 251, 37683773. 25. Bishop, J. O., Morton, J. G., Rosbash, M. & Richardson, M. (1974)

Nature 250,199-204.

26. Williams, J. G. & Penman, S. (1975) Cell 6, 197-206. 27. Levy, B. & McCarthy, B. J. (1976) Biochemistry 15, 24152419. 28. Getz, M. J., Elder, P. K., Benz, E. W., Jr., Stephens, R. E. & Moses, H. L. (1976) Cell 7,255-265. 29. Axel, R., Feigelson, P. & Schutz, G. (1976) Cell 7,247-254. 30. Getz, M. J., Birnie, G. D., Young, B. D., MacPhail, E. & Paul, J. (1975) Cell 4, 121-129. 31. Levy, S. & Aviv, H. (1976) Biochemistry 15, 1844-1847. 32. Grossman, S. B., Yap, S. H. & Shafritz, D. A. (1977) J. Clin. Invest., 59,869-878. 33. Marcu, K. & Dudock, B. (1974) Nucleic Acid Res. 1, 13861394. 34. Grubman, M. J., Weinstein, J. A. & Shafritz, D. A. (1977) J. Cell Biol., 74, 43-57. 35. Laemmli, U. K. (1970) Nature 227,680-685. 36. Maizel, J. F., Jr. (1971) in Methods in Virology, eds. Maramorosch, K. & Koprowski, H. (Academic Press, New York), Vol. 5, pp. 179-212. 37. Davidson, E. H., Hough, B. R., Klein, W. H. & Britten, R. J. (1974) Cell 4, 217-238. 38. Lewis, J. B., Atkins, J. F., Anderson, C. W., Baum, P. R. & Gesteland, R. F. (1975) Proc. Natl. Acad. Sci. USA 72, 13441348. 39. Housman, D., Skoultchi, A., Forget, B. G. & Benz, E. J. (1974) Ann. N.Y. Acad. Sci. 241, 280-289. 40. Strauss, A. W., Donohue, A. M., Bennett, C. D., Rodkey, J. A. & Alberts, A. W. (1977) Proc. Natl. Acad. Sci. USA 74, 13581362. 41. Tse, T. P. H. & Taylor, J. M. (1977) J. Biol. Chem. 252, 12721278.