Physical and chemical characterization of purified ovalbumin messenger RNA.

Preparative agarose gel electrophoresis under denaturing conditions has been successfully employed to purify large quantities of ovalbumin mRNA from hen oviducts. The mRNA thus prepared is physically homogeneous based on its migration as a single component on electrophoresis in both analytical acid-urea agarose gels and formamide-containing, neutral polyacrylaminde gels; it also sediments as a single peak in sucrose gradients containing 70% formamide. The mRNA is chemically free of ribosomal RNA contamination since its oligonucleotide fingerprint map after complete T1 ribonuclease digestion contains no detectable specific large oligonucleotide markers of ribosomal RNAs. It is also not contaminated by other biologically active messenger RNAs because, when it is added to the cell-free wheat germ translation system, the only protein product synthesized is ovalbumin as analyzed by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate and specific immunoprecipitation. Ovalbumin mRNA has a nucleotide composition of 32.3% A, 21.0% G, 25.7% U, and 20.7% C [(A+U)/(G+C) equal 1.41]. The mRNA contains a heterogeneous poly(A) tract ranging from 20 to 140 residues with a number average chain length of 62 adenylate residues. The molecular weight of the sodium salt of the purified mRNA is approximately 650,000 +/- 63,000, corresponding to a chain length of 1890 +/- 180 nucleotides, as determined by electron microscopy under completely denaturing conditions. This value is in close agreement with the values obtained from: (a) sucrose gradient centrifugation in the presence of 70% formamide; (b) evaluation of poly(A) content in the mRNA and the number average chain length of its poly(A) tract; and (c) sedimentation velocity studies in the presence of 3% formaldehyde. When 125I-labeled ovalbumin mRNA is allowed to hybridize with a large excess of chick DNA, the observed kinetics of hybridization reveal no appreciable reaction between the mRNA and the repeated sequences of the chick DNA, although the mRNA appears to be approximately 600 nucleotides longer than necessary to code for ovalbumin. It thus appears that the entire ovalbumin mRNA is primarily transcribed from a unique sequence in the chick genome.


Preparative
agarose gel electrophoresis under denaturing conditions has been successfully employed to purify large quantities of ovalbumin mRNA from hen oviducts. The mRNA thus prepared is physically homogeneous based on its migration as a single component on electrophoresis in both analytical acid-urea agarose gels and formamide-containing, neutral polyacrylamide gels; it also sediments as a single peak in sucrose gradients containing 70% formamide. The mRNA is chemically free of ribosomal RNA contamination since its oligonucleotide fingerprint map after complete T, ribonuclease digestion contains no detectable specific large oligonucleotide markers of ribosomal RNAs. It is also not contaminated by other biologically active messenger RNAs because, when it is added to the cell-free wheat germ translation system, the only protein product synthesized is ovalbumin as analyzed by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate and specific immunoprecipitation.
The mRNA contains a heterogeneous poly(A) tract ranging from 20 to 140 residues with a number average chain length of 62 adenylate residues. The molecular weight of the sodium salt of the purified mRNA is approximately 650,000 f 63,000, corresponding to a chain length of 1890 f 180 nucleotides, as determined by electron microscopy under completely denaturing conditions. This value is in close agreement with the values obtained from: (a) sucrose gradient centrifugation in the presence of 70% formamide; (b) evaluation of poly(A) content in the mRNA and the number average chain length of its poly(A) tract; and (c) sedimentation velocity studies in the presence of 3% formaldehyde.
When 12SI-labeled ovalbumin mRNA is allowed to hybridize with a large excess of chick DNA, the observed kinetics of hybridization reveal no appreciable reaction between the mRNA and the repeated sequences of the chick DNA, although the mRNA appears to be approximately 600 nucleotides longer than necessary to code for ovalbumin.
It thus appears that the entire ovalbumin mRNA is primarily transcribed from a unique sequence in the chick genome.
Our previous work has shown that the estrogen-inducible RNAs in total tissue nucleic acid extracts (3). Preparative ovalbumin mRNA can be substantially purified from total agarose gel electrophoresis under conditions of acidic pH and nucleic acid extracts of hen oviducts (1,2 A piece of Nytex screen was secured at the bottom of the adapter with the aid of a snap ring. The adapter was fitted into the gel column, and the gel was cast inside the adapter. The in uitro translation assay using wheat germ S-30 was carried out essentially as previously described (5 T, were added, and the solution was incubated at 37" for 1 hour. The digest was subjected to electrophoresis on 12% polyacrylamide gels as described (13). The gels were sliced into l-mm slices after electrophoresis.
Each gel slice was soaked in 0.1 ml of 0.5 M NaCl and homogenized with a Teflon pestle. The homogenate was centrifuged at 12,000 x g for , 15  The values obtained were corrected for a control value obtained by determining the ribonuclease resistance of the ""I-mRNA in the reaction mixture in which the DNA was not denatured.
The remaining 0.5.ml portions of the assay mixture were used to determine the extent of DNA-DNA reassociation by hydroxylapatite chromatography as described previously (2).
Analysis of Nucleotide Composition of Ovalbumin mRNA Nucleotide composition analysis of ovalbumin mRNA was accomplished using the tritium derivative method (19). Ovalbumin mRNA was digested for 18 to 24 hours at 37' with a mixture of pancreatic RNase A, alkaline phosphatase, and snake venom phosphodiesterase. The resulting nucleosides were oxidized to 2',3'-nucleoside dialdehydes with NaIO, and subsequently reduced to Wnucleoside trialcohols with KB3H, (10 Ci/mmol).
In each determination, an enzyme blank (no RNA) and a buffer blank (no RNA and no enzymes) were carried out simultaneously with the RNA sample. Individual nucleoside trialcohols were resolved by two dimensional chromatography on 0.10.mm cellulose thin layers (20 x 20 cm, EM Laboratories, Inc., Germany).
The 'H-nucleosides were visualized by autoradiography as described (20) and eluted with 2 N NH,OH for counting in an Omnifluor (New England Nuclear) scintillator. The mean of five 2-min cycles (3 to 8 x lo5 cpm) was used to calculate the mole percentage of each 'H-nucleoside trialcohol.

RESULTS
The purification of ovalbumin mRNA from total hen oviduct nucleic acid extract is shown in  of each sample were assayed in the wheat germ translation system, and the values obtained above represent initial activities. Each picomole of valine is equivalent to 457 cpm.
* Per cent of total protein synthesized in the wheat germ translation system represented by ovalbumin. Since only 85 to 90% of a "C-labeled ovalbumin is precipitable with the antibody in this assay, a correction factor of 10% had been added to these values. In this experiment ribosomes were not removed prior. to immunoprecipitation. Absorbance at 260 nm (0-O) was also determined for each fraction.
Thus, the purification of ovalbumin mRNA by this procedure was 146-fold, with an over-all activity yield of approximately 6.7%. From 1300 mg of total extract, 0.6 mg of purified ovalbumin mRNA was obtained. The yield generally ranged from 0.5 to 1.0 mg of ovalbumin mRNA for different preparations.
Preparative agarose gel electrophoresis is capable of separating RNA species that are very similar in molecular size. This technique was employed at the last step in the purification procedure to remove quantitatively the residual contaminating 18 S rRNA from the ovalbumin mRNA. It consistently resolved RNA preparations from the second nitrocellulose filtration step into two major RNA peaks (Fig. 1). The faster migrating peak was subsequently identified as the 18 S rRNA, and when assayed in the wheat germ translation system, all ovalbumin mRNA activity was associated with the slower migrating RNA peak. Total mRNA activity in the fractions also was assayed in the wheat germ translation system by measuring the incorporation of labeled amino acid into trichloroacetic acid-precipitable material.
The major peak of activity was associated with the peak of ovalbumin synthesizing activity.
The presence of two additional small peaks of activity in fraction eluted prior to and coincident with the 18 S rRNA indicates the separation of some other mRNA species from ovalbumin mRNA. The content of nucleic acids at each stage of purification was analyzed by analytical agarose gel electrophoresis (Fig. 2). Total nucleic acid extract contains many minor RNA bands in addition to the major stable cellular RNA species and DNA ( Fig. 2, Gel A). Consequent to nitrocellulose filtration, there appears a preferential enrichment in a single RNA species migrating at 21 S (Fig. 2, Gel B). After Sepharose 4B column chromatography and a second nitrocellulose filtration step, the only remaining major species of nucleic acid are the 21 S RNA and the 18 S rRNA (Fig. 2, Gel C). DNA 28 S rRNA, and most of the minor RNA species have been quantitatively removed. Finally, the only RNA species present after preparative agarose gel electrophoresis is the 21 S RNA (Fig. 2 3. Sodium dodecyl sulfate polyacrylamide gel electrophoresis of total protein products synthesized in the wheat germ translation assay using purified ovalburnin mRNA. The entire postribosomal supernatant was applied and electrophoresis was carried out as described (7). The radioactivity contents in each 2 mm gel slice were determined by liquid scintillation counting after solubilizing the gel in 30% H,Oz. A-A, background radioactivity in the gel slices where ovalbumin mRNA was not added to the translation system; CL--Cl, total proteins synthesized in response to purified ovalbumin mRNA. The ovalbumin marker represents the migration of immunoprecipitated radioactive ovalbumin synthesized in the same translation system. BPB, bromphenol blue. detectable contamination by other RNAs (10). When examined by sucrose gradient centrifugation in 70% formamide, it sediments as a single peak at 16 S (Fig. 9). This property of ovalbumin mRNA to migrate at 21 S in gels but sediment at 16 S in sucrose gradients provides another means for the removal of any potential rRNA fragments that may also be 21 S-migrating during gel electrophoresis.
The wheat germ translation system, which has an intrinsically low endogenous protein synthesizing activity, was next employed to assess the biological purity of ovalbumin mRNA. The ratio of the ovalbumin antibody-precipitable radioactivity to total trichloroacetic acid-precipitable radioactivity in the postribosomal supernatant is a measure of the fraction of total biologically active messenger RNAs represented by ovalbumin mRNA.
When this analysis was carried out for the RNA samples at each stage of purification, the per cent ovalbumin mRNA increased from 45% in the total nucleic acid extract to over 90% in the purified ovalbumin mRNA preparations (Table  I). This is at best a minimum estimation because incomplete ovalbumin molecules due to premature release from the ribosomes are not recognizable by the antibody (6) but will be precipitated by trichloroacetic acid. The total protein products synthesized in the wheat germ translation system in response to purified ovalbumin mRNA were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis.
The entire postribosomal supernatant fraction was applied to the gel and the radioactivity distribution in the gel is shown in Fig. 3. Only one peak of radioactivity is present and its electrophoretic mobility is identical with the ovalbumin marker.
The broadness of the peak is probably not due to the presence of different peptide products, but rather to a distribution of various lengths of ovalbumin synthesized in uitro. This interpretation is supported by the observation that an identical radioactivity profile was obtained when immunoprecipitated product was analyzed. The absence of radioactivity peaks in other areas of the gel suggests that proteins of other sizes are not being synthesized.
These experiments thus indicate that purified ovalbumin mRNA is not demonstrably contaminated by other biologically active oviduct messenger RNAs. The nucleotide fingerprint patterns of the complete pancreatic RNase A digestion products of "'I-labeled ovalbumin mRNA, 18 S rRNA, and 28 S rRNA are shown in Fig. 4. The nucleotide fingerprint maps from the complete T, RNase digest of 'Y-ovalhumin mRNA, '25I-18 S rRNA, and a mixture of these two RNA species are shown in Fig. 5 while those of a similar experiment using I*?-ovalbumin mRNA and 'Y-28 S rRNA are shown in Fig. 6. The reproducibility of each of these nucleotide fingerprint patterns was established by mapping several preparations of each of the Y-labeled RNA species. Each RNA species gave the same characteristic and qualitatively distinct pattern of large oligonucleotide spots from preparation to preparation. In each mixture experiment, the RNAs were labeled separately with '25I and mixed prior to enzyme digestion.
Each large oligonucleotide spot is assigned to messenger RNA (unshaded) or ribosomal RNA (cross-hatched) on the basis of its position relative to the other oligonucleotide spots by comparison with the maps of the individual RNA species shown above and below the mixture map. The pattern of large oligonucleotides of the mixed RNA species appears to be largely additive, with possible overlaps occurring only between 18 S rRNA spots 7 and 10, and mRNA spots 19 and 21, respectively (Fig. 5b, arrows). Furthermore, each mixture map exhibits localized patterns of oligonucleotides characteristic of the individual RNA species. For example, Areas A and B (Fig.  5b, schematic) represent oligonucleotide patterns characteristic of 18 S rRNA (Fig. 5c, schematic) while Area C (Fig. 6b,
The absence of ribosomal RNA-specific patterns of large oligonucleotides from the T1 RNase fingerprint map of ovalbumin mRNA (Fig. 5a or 6~) indicates that the messenger RNA contains no detectable contamination with 18 S or 28 S ribosomal RNAs or with their degradation products which contain the large oligonucleotide marker fragments.
All of these data provide strong evidence that the ovalbumin mRNA has been purified to apparent homogeneity. Therefore, it seems suitable to further characterize this molecule with respect to various physical and chemical parameters. The first such experiment undertaken was to determine the nucleotide composition of ovalbumin mRNA. This analysis was performed using the tritium derivative method (19) and the nucleotide composition of ovalbumin messenger RNA is presented in Fig. 7 (inset tablti). These data represent the average of four determinations on two different RNA preparations: A + U = 58.0% and G + C = 41.4%. An autoradiograph of the chromatographic separation of the four nucleoside trialcohols (A,U,G,C) and the tritiated background spots (Bl, B2, B3) produced in both the buffer and enzyme blank reactions is also shown in Fig. 7. The spots labeled Xl, X2, and X3 appear in all of the nucleotide composition determinations and represent less than 1% of the total radioactivity (A+U+G+C+X) eluted from the plate. These X spots have not been identified as yet but could represent modified nucleotides occurring in the ovalbumin mRNA molecule. The molecular weight of ovalbumin mRNA was estimated by several independent methods. Initially, we utilized gel electrophoresis, assuming that the molecular weights of Escherichia coli 16 S and 23 S rRNAs are 560,000 and 1,080,000, and hen oviduct 18 S and 28 S rRNAs are 700,000 and 1,580,000, respectively (13). The molecular weight of ovalbumin mRNA was estimated to be 900,000 * 90,000 from both acid-urea agarose gel electrophoresis (Fig. 8, Panel A) and neutral formamide polyacrylamide gel electrophoresis (Fig. 8, Panel  B). Since the average residue molecular weight (sodium salt) is 343.5 in ovalbumin mRNA according to its nuclebtide composition (Fig. 7) was analyzed in the same manner, a sharp RNA peak sedimenting at approximately 16 S was noted, which was essentially 'superimposable with the peak of ovalbumin mRNA activity when assayed in the wheat germ translation system (Fig: 9,, instif). Compared to the stable cellular RNAs, the molecular weight of ovalbumin mRNA can be estimated by this technique to be '550,000 h 30,000 (Fig. 9), which coriesponds'to a polynucleotide chain length of 1600 * 90 residues. It is apparent that this molecular weight estimation-is considerably 'different from the value obtained by the gel electrophoretie techniques'.
Consequently, other procedureswere required to determine the true figure. ;  of less than 2%. The s20,w value obtained from three independent experiments was 9.12 + 0.10, which converts to a molecular weight of 450,000 =t 12,000. This value, however, must be corrected by a factor of 1.2, since there appears to be one chain breakage per 5 molecules (see Fig. 12). Hence the corrected value should be 540,000 * 14,000, corresponding to a chain length of 1572 f 42 nucleotide residues. The final approach to determine accurately the molecular weight of purified ovalbumin mRNA is to measure its length directly by electron microscopy under denaturing conditions. When spread in 4 M urea dissolved in formamide as previously described (ll), ovalbumin mRNA molecules appear to be completely denatured as evidenced by the uniform contour throughout their length (Fig. 11). Length measurements on two independent ovalbumin mRNA preparations provide length distributions that are representative of a homogenous species of RNA (Fig. 12). Both the number average length and the weight average length for the two preparations are 0.5 f 0.05 pm (Fig. 12). Since RNA molecules have an average residue spacing of 2.65 Alnucleotide in this spreading procedure, FIG. 7. Nucleotide composrtron analysis of ovalbumin mRNA determined from cellulose, thin layer chromatograms. OR = origin; Bl, B2, and B3 are tritiated background spots also produced in the buffer and enzyme blank reactions.
A, U, G, and C are the [3H]trialcohol derivatives of adenosine, urrdme, guanidine, and cytosine, respectively, which were eluted and counted to determine the nucleotide composltion (insert Hen oviduct 4 S, 18 S, and 28 S RNAs were used as standards. The log of molecular weight was plotted versus distance of migration through the gradient. Inset, the gradient containing purified ovalbumin mRNA was monitored at A,,, (-) during fractionation; RNA in each fraction was precipitated separately from alcohol and redissolved in water, and ovalbumin mRNA activity (O----O) was determined by the wheat germ translation system. [3H]cDNA probe used in these studies, however, was not a complete copy of the mRNA and could only reveal the sequence complexity of the 3'-terminal portion of the mRNA molecule. We have extended this study by performing hybridization experiments using "9-ovalbumin mRNA and a large excess of chick DNA to examine whether the entire ovalbumin mRNA molecule is transcribed from the unique sequence portion of the oviduct genome. A typical DNA reassociation curve is shown in Fig. 13 The purified mRNA migrates as a single component in both acid-urea agarose gels and formamide-containing neutral polyacrylamide gels (Fig. 2, Gel D, and Ref. 10). It also sediments as a single symmetrical peak in formamide-containing neutral sucrose gradients (Fig. 9, inset). When examined by electron microscopy under completely denaturing conditions, length distributions for two ovalbumin mRNA preparat,ions appear homogeneous (Fig. 12). Furthermore, prolonged radioautography of agarose gels of lZ51ovalbumin mRNA preparations failed to develop other radioactive RNA bands. These combined results indicate a high degree of size homogeneity of the purified ovalbumin mRNA.
When purified ovalbumin mRNA was assayed in the wheat germ translation system, greater than 90% of the protein synthesized was immunoprecipitable with a monospecific antibody against ovalbumin. Furthermore, when analyzed by electrophoresis in sodium dodecyl sulfate-polyacrylamide gels, the in vitro synthesized proteins present in the post-ribosomal supernatant fraction migrate as ovalbumin. These data suggest that the ovalbumin mRNA preparations are essentially free of other functionally active oviduct mRNAs. Comparison of the T, RNase resistant oligonucleotides from the different RNA species by routine nucleotide composition analysis is not possible since lZ51 is incorporated largely into cytidine, and to a much lesser extent into uracil (16). Therefore, the TImresistant oligonucleotides of '251-ovalbumin mRNA were compared with those from '""I-labeled 18 S or 28 S rRNA by mapping a mixture of equal amounts of mRNA and 18 S or 28 S rRNA. T,-resistant oligonucleotides that are identical with respect to nucleotide composition and chain length (although not necessarily nucleotide sequence) will cochromatograph.
Since additive maps were obtained, these mixture experiments demonstrate that the large, T,-resistant oligonucleotides of ovalbumin mRNA and 18 S or 28 S rRNA chromatograph independently of each other in a specific pattern characteristic of the parent RNA species. The absence of patterns of large, T,-resistant oligonucleotides specific for 18 S or 28 S rRNA from the T1 RNase map of ovalbumin mKNA indicates that the mRNA is not contaminated with either of these rRNAs or with any of their degradation products which contain the rRNA-specific, large oligonucleotides. This, however, does not rule out the remote possibility of contamination of mRNA with an rRNA degradation product longer than 1500 nueleotides and completely devoid of the rRNA-specific, large oligonucleotides.
Furthermore, since the mixture experiments were carried out with approximately equal amounts of mRNA and rRNA (both in terms of mass and radioactivity), it is also possible that small amounts of rRNA might exist in purified ovalbumin mRNA preparations that were undetected by autoradiography for 1 day. However, this appears unlikely since prolonged (4 to 7 days) autoradioagraphy of the mRNA thin layer plates failed to develop any additional rRNA-specific, large oligonucleotide spots. Thus, the contamination of ovalbumin mRNA with either 18 S or 28 S chick rRNA or their degradation products appears to be highly unlikely. The preparative nature of the purification method described (greater than 0.5 mg of purified material/preparation) has permitted detailed physical and chemical characterization of the mRNA. In this regard, the molecular weight of ovalbumin 7037 mRNA has previously been determined in our laboratory as well as others (10,24). However, the results were inconclusive since a large discrepancy existed between the values obtained by gel electrophoresis and by sucrose gradient centrifugation. The mRNA migrates as a 21 S species during electrophoresis in both acid-urea agarose gels and formamide-containing polyacrylamide gels, corresponding to a molecular weight of about 900,000 * 90,000 or a chain length of 2620 * 262 nucleotides; however, it sediments at approximately 16 S in sucrose gradients containing 70% formamide, corresponding to a molecular weight of about 550,000 * 30,000 or a chain length of about 1,600 f 90 nucleotides.
Therefore, we employed three additional independent methods to estimate the molecular weight of ovalbumin mRNA, and the results are summarized in Table II. The molecular weight estimates from poly(A) analysis, sedimentation velocity in 3% formaldehyde and electron microscopy are in reasonable agreement with the value obtained from sucrose gradient centrifugatiqn, but considerably less than the value obtained from the gel electrophoresis methods. Among the methods employed, only electron microscopy has the capability of measuring the linear lengths of RNA molecules independent of secondary structure and nucleotide composition (11). The absence of secondary structure within ovalbumin mRNA molecules under conditions employed is demonstrated by the uniform contour (Fig. 11). The only remaining variable in this method of molecular weight determination for RNAs is the residue spacing which ranges only between 2.6 to 2.7 A/residue for a variety of RNAs tested (11). Therefore, the value of 650,000 * 63,000 obtained by electron microscopy should be a more reliable estimate of the molecular weight of ovalbumin mRNA. The discrepancy in RNA molecular weight determination between the methods of gel electrophoresis and sucrose gradient sedimentation is by no means unique to ovalbumin mRNA. Molecular weights of the two HeLa cell mitochondrial ribosomal RNAs were estimated to be 720,000 and 420,000 based on their electrophoretic mobilities (25,26) and 540,000 and 350,000 by sucrose gradient sedimentation analysis (27), respectively. More recently, a similar observation has also been made in several messenger RNAs. Hemoglobin and myosin mRNAs sedimented at 9 S and 26 S in sucrose gradients, while migrated at 12 S and greater than 28 S in gels, respectively (28,29). Gel electrophoresis at two different temperatures yielded a