Preparative polyacrylamide gel electrophoresis

Vol.

THE Jouaw.~ OF B~W,XCAL CQSHI~.RY 252, No 7. Issue of Apd 10. pp. 2162-2186, Printed in U.S.A.

,977

Preparative Polyacrylamide of Human cy- and @Globin

Gel Electrophoretic Messenger RNAs*

Purification

(Received for publication,

URI NUDEL,

FRANCESCO

From the Departments College of Physicians

RAMIREZ,$

of Medicine and

Surgeons,

PAUL A. MARKS, and Human New York,

The availability of a method to prepare relatively large amounts of purified human a- and P-globin mRNA would be most valuable for the analysis of the structure of each of the normal globin mRNAs as well as abnormal globin mRNAs present in certain patients with thalassemia (l-3). In addition, purified (Y- and /3-mRNAs would be useful for the preparation of globin-specific complementary DNAs. We have recently reported the use of human globin U- and P-cDNAs prepared by other methods for measurement of the globin gene content and globin mRNA content of cells from normal and thalassemic subjects (3, 4). Although these globin cDNA probes are highly enriched

for (Y- or fi-cDNA

sequences,

the method

of purifica-

tion involves the separation of these globin-specific cDNAs from a mixture of cDNAs rather than synthesis of the individual cDNAs from purified templates. Methods for the separation of U- and P-globin mRNAs of mouse (5, 6), rabbit (5, 7-9), and human subjects (10, 11) in limited amounts have been described.

We describe

here the use of preparative

polyacryl-

* These studies were supported in part by National Institutes of Health Grants GM 14552, GM 19153, CA 03526, and CA 13696, Contract NOl-CP-6-1008, National Science Foundation Research Grant 27388, National Foundation March of Dimes Grant l-304, and the Cooley’s Anemia Foundation. $ Visiting Fellow from the Istituto di Anatomia Comparata Palermo (Italy). I Faculty Research Scholar of the American Cancer Society.

2182

BANK$

Genetics and Development, New York 10032

Columbia

University,

amide gel electrophoresis in formamide to separate n- and pmRNA from human reticulocyte RNA. The separated a- and p-mRNAs, when assayed in a wheat germ cell-free system, yield globin which is more than 90% CI or p, respectively. We have also developed a relatively rapid

assay for the separation and quantitation of the a- and pglobins synthesized in the cell-free system, using cellulose acetate

strip

electrophoresis.

The cDNAs

prepared

using

the

purified a- and P-globin mRNA fractions as templates are also highly chain-specific by hybridization studies. The methodolo&y described here permits the relatively large scale preparation of a- and /I-mRNAs. MATERIALS

AND

METHODS

Preparation of RNA-Total human reticulocyte RNA from patients with autoimmune hemolytic anemias, prepared by phenol extraction (12), was subsequently purified by oligo(dT)-cellulose chromatography (13, 14). Preparative Polyacrylamide Gel Electrophoresis - The polyacrylamide gels were prepared as described by Gould and Hamlyn (15) with the following modifications. A linear gradient gel of 4 to 6% acrylamide and 2 to 10% glycerol in 90% formamide was prepared using a two-chamber system. The outlet chamber contained 2 ml of 10% (v/v) glycerol, 5.1% acrylamide, 0.9% bisacrylamide, 0.6% (v/v) N,N,N,N’-tetramethylethylenediamine, 0.07% ammonium persulfate, 0.05 M diethylbarbituric acid, and 90% formamide, pH 9.0. The second chamber contained 2 ml of 2% (v/v) glycerol, 8% (v/v) H,G, 3.4% acrylamide, 0.6% bisacrylamide, 0.6% (v/v) N,N,N,N’-tetramcthylethylenediamine, 0.07% ammonium persulfate, 0.05 M diethylbarbituric acid, and 90% formamide, pH 9.0. The formamide

(Merck) was freshly deionized by shaking for 2 to 3 h with 4% (w/v) of a mixed bed resin (AG 501-X8, Bio-Rad); all solutions were prepared just before pouring the gels. The gels were formed in glass cylinders, 1.5 cm in diameter. Then 1 ml of 90% buffered formamide (0.05 M diethylbarbituric acid in 90% formamide, pH 9.0) was layered on the top of the gel; this solution was removed and a fresh aliquot was added just prior to the application of the RNA sample. The polymerized gel was placed m the cold room overnight. The RNA sample (50 to 150 pg) was lyophilized and dissolved in 5 to 10 ~1 of H,O; 0.1 ml of sample buffer (5% sucrose and 0.005% bromphenol blue in buffered

formamide)

was then added to the sample. The sample was applied to

the gel through the 1 ml of 90% buffered formamide overlying the gel. Reservoir buffer (Buffer A: 0.02 M NaCI, 0.001 M EDTA, pH 7.2) was added carefully to fill the remainder of the gel cylinder. Electrophoresis was performed using a Savant preparative polyacrylamide gel apparatus (16), cooled at o”, at a constant voltage of 50 V and current of approximately 5 mA. A flow of Buffer A of about 7.5 ml/h was pumped under the bottom of the gel column and used to elute the RNA from the gel chamber. The 1.5ml fractions were collected into tubes containing 0.15 ml of 1% sodium dodecyl sulfate. An aliquot of 0.025 ml of every fifth fraction was assayed for its mRNA content by hybridization to [“Hlpoly(U) (4.5 Ci/mmol, New England Nuclear) (17). Appropriate tubes containing the mRNA were pooled depending on the elution pattern, and yeast tRNA (Miles, re-extracted with

Downloaded from www.jbc.org by guest, on July 9, 2011

Previous studies have reported the use of globin chainspecific complementary I)NAs to quantitate the amount of human globin mRNA and human globin genes in normal and abnormal cells. In order to obtain larger amounts and more purified globin mRNAs as templates for these experiments, preparative polyacrylamide gel electrophoresis in formamide has been used to separate LY- and /3-globin mRNA from polyadenylate containing RNA of human reticulocytes. Fifty to one hundred-fifty micrograms of RNA can be applied to the preparative gel and the recovery of the globin mRNA is about 50%. The isolated a- and &globin mRNAs were assayed in a wheat germ cell-free system, and the (Yand /I-globin synthesized quantitated by cellulose acetate electrophoresis. The purified a- and /3-globin mRNAs direct globin synthesis which is more than 90% either a- or /3globin, respectively. The cl)NAs prepared using each of the isolated mRNAs as template are also shown to be specific for U- or /3-mRNA sequences. The gel electrophoresis technique used permits the relatively large scale isolation of a- or /3globin mRNAs from human cells.

AND ARTHUR

October 28, 1976)

Polyacrylamide

Gel Purification

(4). RESULTS

Separation of Human a- and PGlobins - Purified human aand P-globin chains were prepared by carboxymethyl-cellulose chromatography (20) and run on cellulose acetate strips. As

a- and @Globin

mRNAs

2183

shown in Fig. 1, the cu-globin migrates toward the cathode, while the p-globin migrates toward the anode. When human globin prepared from red cell hemolysates is electrophoresed, two bands of equal intensity (measured at 510 nm) are seen comigrating with (Y- and P-globin (Fig. 1). Separation of Human cx- and /%Globin mRNAs - Poly(A)containing RNA (80 pg) prepared from reticulocytes of a patient with hemolytic anemia was applied to a 4 to 6% polyacrylamide gradient gel in formamide and the electrophoresis carried out as described under “Materials and Methods.” The poly(A)-containing RNA, as assayed by hybridization to 13HIpoly(U) (see “Materials and Methods”), is eluted from the gel as a single broad peak (Fig. 2, Table I). The fractions were pooled as indicated in Fig. 2, and the poly(A)-containing RNA recovered. The RNA in each pooled fraction was dissolved in 0.05 ml of H,O, a 1.25 to 2.5 ~1 aliquot was removed and assayed for globin mRNA in a wheat germ cell-free system. Predominately a-globin is formed with RNA from pools 3 to 6, while mainly /3-globin is synthesized by RNA from pools 8 to 10 (Fig. 3 and Table I, Experiment B). The mRNA from pool 7 led to the synthesis of 39% a-globin and 61% P-globin, while pool 5 led to 96% cu-globin and pool 9, 94% /3-globin synthesis. The [35S1methionine incorporated per amount of poly(A)-containing RNA is approximately twice as great for the early eluting fractions (primarily cY-globin mRNA) as that of the late eluting fractions (predominantly p-globin mRNA) (Fig. 2). This is probably due to the fact that p-globin contains 1 methionine molecule per chain while a-globin contains 2 (25). The poly(A) content serves as a useful measure for the amount of RNA in each fraction since it has been shown previously that 01- and p-mRNAs contain similar amounts of poly(A) (14). The cell-free assays for translation of mRNA were perI

+

2 FIG. 1. Separation of human P- and p-globins by cellulose acetate electrophoresis. Unfractionated human globin was prepared from red cell hemolysates as described (24). Purified human a- and pglobin chains were separated by chromatography on carboxymethylcellulose (24). The peak fractions of the separated chains were dialyzed for 3 days against 0.5% formic acid, lyophilized, and dissolved in the electrophoresis Buffer B (see “Materials and Methods”). The 3 ~1 containing about 2 pg (p chain), 6 pg ((u chain), and about 5 Gg of unfractionated human globin were applied on the cellulose acetate strip as samples 1,2, and 3, respectively. The electrophoretic procedure is as described under “Materials and Methods.” The strips were stained with Amido black.

Downloaded from www.jbc.org by guest, on July 9, 2011

phenol) was added to a final concentration of 1 AZRO/ml. NaCl to a final concentration of 0.3 M and 2 volumes of ethanol were added, and after 18 h at -2O”, the RNA was recovered by centrifugation at 20,000 x g for 30 min. Each RNA pool was dissolved in 1 ml of H20, 1 ml of 1 M NaCl containing 0.02 M TrislHCl, pH 7.5, was added, and the RNA solution applied to a 0.4-ml oligo(dT)-cellulose column. The column was washed with 5 ml of 0.5 M NaCl, 0.01 M Tris/HCl, pH 7.5, and the poly(A)-containing mRNA eluted with four 0.5-ml aliquots of 0.01 M TrislHCl, pH 7.5. The RNA was eluted in the initial 1.5 ml under these conditions and was then concentrated to 0.15 ml by lyophilization; NaCl to a final concentration of 0.3 M and 2 volumes of ethanol were added; after 18 hat -2O”, the RNA was recovered by centrifugation at 20,000 x g for 60 min at -15”. The RNA was then dissolved in H,O and stored in liquid nitrogen. Cell-free Translation of Globin mRNA - Globin mRNA translation was measured in a wheat germ cell-free system (18) using 10 to 20 &i of P5SImethionine (Amersham, 200 to 400 Ci/mmol)/BO ~1 of assay as the labeled amino acid and 0.01 to 0.2 pg of mRNA. After 90 min of incubation at 25”, 5-~1 samples were removed for measuring hot trichloroacetic acid-precipitable counts (19), and the remaining 45 ~1 were used to determine the amount and type of globin formed. Five microliters of human red blood cell lysate containing about 100 pg of hemoglobin were added to each sample as carrier and globin was prepared by adding 1 ml of cold (-20”) 0.1 M HCl in acetone in 1.4-ml Eppendorf plastic centrifuge tubes. After standing overnight at -2O”, the samples were centrifuged in the cold for 5 min at 8000 x g. The supernatant was removed and the pellet resuspended in 1 ml of cold acetone at -20”. After standing for 1 h at -2O”, the protein was precipitated and washed again with acetone. The protein pellet was then resuspended in 150 ~1 of Hz0 and lyophilized. The protein was dissolved in 50 ~1 of Buffer B (see below). After about 2 h on ice, all of the protein is solubilized. Separation of Human a- and PGtobin Chains by Electrophoresis on Cellulose Acetate Strips - The conventional method employed to separate and quantitate human LY- and P-globins, chromatography on carboxymethyl-cellulose (20), is limited by the relatively small number of samples that can be analyzed at one time. In order to assay the globins synthesized in the cell-free system, we used a modification of the method described by Ueda and Schneider (21), in which globin chains are separated by electrophoresis on cellulose acetate strips. We found that by changing the electrophoresis buffer, a better separation between the globin chains is achieved. The electrophoresis buffer (Buffer B) contained: 4.66 M urea (freshly deionized with Bio-Rad mixed bed resin AG 501-X8D), 0.5% (v/v) pmercaptoethanol, and 0.045 M Tris/HCl. The final pH was 9.0 to 9.05. The lower urea concentration prevents precipitation of urea on the cellulose acetate strips during the 5-h run. The cellulose acetate strips (Cellogel 250, Chemotron, Milano, Italy) were equilibrated with Buffer B by slow shaking for 1 to 2 h. The extra buffer was removed from the strips by pressing them between two layers of filter paper. The origin in the middle of the strips was marked with a pencil and 3-~1 samples were applied in O&cm-long sample wells. The electrophoresis was carried out at a constant voltage of 225 V for 5 h at 4”. The strips were stained with an Amido black solution (0.5% Amido black, 45% methanol, 10% acetic acid) for 10 min, destained in 47.5% methanol, 5% acetic acid for 30 min, and then soaked for 1 min in formalin and 3 min in 7% glycerol; they were then dried for 20 min at SO”, pressed between two layers of filter paper. Quantitation of radioactive o(- and P-globin was achieved either by cutting out the bands corresponding to the carrier (Y- and P-globin bands and counting them in a liquid scintillation counter, or by scanning an autoradiogram of the strip using a densitometer. Preparation of cDNA and Hybridization to Human mRNA and DNA-Globin cDNA was prepared as described previously using reverse transcriptase from avian myeloblastosis virus obtained from Dr. Joseph W. Beard (Life Sciences Research Laboratories, St. Petersburg, Fla.) (22, 23). After incubation, the reaction mixture was passed through a 2-ml Sephadex G-25 syringe column, digested with alkali, and neutralized. The sire of the cDNA was measured by alkaline sucrose density gradient centrifugation (23). Hybridizations to globin mRNA and to cellular DNAs were as described previously

of Human

2184

Polyacrylamide

activity

of Human

885 19,610 9,048 2,500 3,433

95 22 7 15 48

5 78 93 85 52

2,327 29,024 16,383 4,941 6,858 2,651 1,470 730 33,520

449 1,040 339 699 5,350 10,794 12,064 3,289 15,294

72 93 96 78 39 11 6 10 52

28 7 4 22 61 89 94 90 48

a0

70

90

3, 66to

71to

73;5,74to

I

I 2 3 4 5

79;

7, 80 to 83; 8, 84 to 88; 9,89 to 93; lo,94 to 98. The poly(A)-containing mRNA was assayed in a wheat germ cell-free system (see “Materials and Methods”). The incorporation of [Wlmethionine was normalized as counts incorporated per 1 ~1 of each original pool. The percentage of total globin which is (Y- or @globin is calculated by recovery from cellulose acetate strips.

using a limiting amount of mRNA. The counts incorporated into a- and /3-globin are proportional to the amount of mRNA used in the range of RNA concentrations assayed (0.01 to 0.2 wg). Under these conditions, the ratio of a- to P-globin synthesized by human globin mRNA is essentially 1 after formed

I

6 7

8 9 IO II

FIG. 3. Cellulose acetate electrophoresis of cell-free products. Three-microliter samples of the cell-free product of RNA pools eluted from the acrylamide gel (Fig. 2) were analyzed by cellulose acetate electrophoresis as described under “Materials and Methods.” In this experiment, the strip was radioautographed for 24 h. Samples for electrophoresis were prepared from wheat germ cell-free reaction mixtures as described under “Materials and Methods.” Sample 1, no added mRNA; 2 t.010, cell-free products of pools 2 to 10, respectively; 11, unfractionated human globin mRNA.

100

76; 6, 77to

I i

,

NUMBER

70;4,

I

+

FIG. 2. Elution pattern of poly(A)-containing RNA separated by preparative gel electrophoresis. Eighty micrograms of poly(A)-containing human reticulocyte RNA were applied on the gel. The details of the preparation of the 4 to 6% polyacrylamide gradient gel and the electrophoresis conditions are described under “Materials and Methods.” The poly(A) content in 0.025 ml of the fractions indicated in the figure was measured by hybridization to [3Hlpoly(U) (17) (see “Materials and Methods”). The fractions were pooled as follows: pool 1, tubes51to60;2,61to65;

mRNAs

correcting for the differences in methionine content of the two chains. Recovery of Globin mRNA -The recovery of globin mRNA was calculated using both recovery of A,,, and of translational activity. The amount of globin formed in the cell-free system with the purified a- and P-globin mRNA fractions was about 50% that synthesized by the total, unfractionated RNA initially applied to the gel. The Az6,, recovered from the gel column in the a- and P-globin mRNA fractions was about 50% of the A260 of 9 to 10 S RNA applied to the gel. Globin mRNA may be lost during the two steps involving ethanol precipitation of the RNA and some mRNA might be degraded during the electrophoresis procedure. Hybridization of a- and PcDNAs to Globin mRNA and Cellular DNA - The a- and pglobin cDNAs were prepared using RNA Tom pool 3, Table I, Experiment A (a-mRNA), and pool 5, Table I, Experiment A (p-mRNA), as templates. The cDNAs were between 6 and 7 S in size and migrated as a fairly homogenous peak by alkaline sucrose gradient centrifugation. Hybridization of the a- and P-cDNAs with their homologous chain mRNAs yields C& values of 2 to 5 x 10e9 (Fig. 4). These Cot,,, values are based on the RNA concentrations recovered from the oligo(dT) purification step (after gel elution). The values are probably higher than expected because no correction is made for RNA losses during subsequent RNA precipitation. When each of the cDNAs is hybridized to its template mRNA and to the other mRNA (a-mRNA with /?cDNA or vice versa), a greater than IO-fold difference in Cot,,, is found for the a-cDNA, and 22-fold for the @DNA (Fig. 4). These data are consistent with a greater than 90% enrichment of each of the cDNAs (Table II). When a-cDNA or P-cDNA is hybridized to preparations of total polyadenylate-containing reticulocyte mRNA, the C& values of hybridization are 4.8 x 10e3 and 5.3 x 10e3, respectively. The relative Cot,,, values for a- and /3-cDNA with normal reticulocyte mRNA is approximately 1.08 (Fig. 5) indicating comparable amounts of a- and /3-mRNA in total human reticulocyte mRNA. When the acDNA is hybridized to hydrops fetalis mRNA known to lack aglobin mRNA, 15% hybridization above background is obtained (Fig. 6A). Similarly, when /3-cDNA is hybridized to mRNA from a patient homozygous for S/3 thalassemia and

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36,915 10,803 1,386 900 6,344

FRACTION

i

96 totalglobin

wm

60

EGlobin

of separated RNAs

in wheat germ cell-free systems The (Y- and P-globin bands of the cellulose acetate strips shown in Fig. 3 were cut and counted in scintillation fluid (Experiment B). Experiment A presents the product analysis data from another experiment. In the calculation of the percentage of a- and P-globin in total globin, the cr-globin counts were divided by 2 to correct for differences in methionine content of the two chains. Pool ol-Globin @Globin a-Globin @Globin Experiment A 3 4 5 6 Unfractionated mRNA Experiment B 3 4 5 6 7 8 9 10 Unfractionated mRNA

a- and

I

TABLE

Translational

Gel Purification

Polyacrylamide

Gel Purification

of Human

U- and

/3-Globin

IO “t 20 2

40

:

50

EjI .-’ 4

60 70 1 so’ 30 90 It T IOOL

mRNAs

f”

P/

L. -.---I lb

L IO-’

IO’

IO’

TABLE

fi-cDNAs to the U- and p-mRNAs. 0.16 ng to 0.2 I.rg of o-mRNA were (specific activity = 14,000 cpm/ngl u-cDNA). B, 40 pg to 8 ng of n p-mRNA were hybridized to 2500 p-cDNA; A-A, u-cDNA1.

u 4.8 x lo...’ 1.3 x 10 ’ 2.6 x 10 ,’

TABLE

B

u/p”

4 purity

5.2 x 10 :’ 5.0 x 10 :I 2.8 x 10 1

1.08 0 04 10.8

96” 92’

” Ratio of n-mRNA

to j&mRNA calculated as 8, ,,, ,/q ,,, f from Pig. as the midpoint between the initial and final hybridization plateaus. b p-mRNA/a-mRNA = 25; P-mRNA/totaI mRNA = 25/26 = 96%. ‘. u-mRNA/&mRNA = 10.6; a-mRNAltotal mRNA = 10.8/11.8 = 92%.

4. The Cd, 2 is calculated

IO

30-

5

40.

G

50-

$

60.

=

70,

O\” 90 90

0 IO

III

Hybridization of u- ad p-cDNAs to cellular DNAs Previously published values obtained using n- and [&cDNA probes isolated from total cDNA (4) are shown in last 2 columns. In these experiments, 3.3 x 10 i as much cDNA was used as cellular DNA. In a typical experiment, 150 pg of DNA (3 mglml) were mixed with 0.050 ng of U- or /3-cDNA (specific activity = 14,000 cpm/ng) and hybridized to obtain C,,r, ? values of 8.6 x lo” and I .72 x 10’ and assayed as described previously (41. Cellular

DNA

.wurce

Normal spleen, 1 Normal spleen, 2 Hydrops fetalis liver 6~ heterozygote white

,I

cell DNA

46 52 20 42

51 55 43 30

Xl-49 47 20-21 41

51-56 51 s3-*in 41

I

20. 6

!oY

-1

FIG. 5. Hybridization of U- and &cDNAs to normal reticulocyte mRNA; 20 pg to 60 ng of normal polyadenylate-containing RNA were hybridized to 2500 cpm of either a- or &cDNA for 4 h (A-A, ficDNA; A-A, a-cDNA,. known to lack 6- and ,9-mRNA, about 20% hybridization ofthe /%cDNA occurs (Fig. @I). These data provide additional evidence that the u- and p-cDNAs are highly enriched for u- and /Hike globin mRNA sequences, respectively. The u- and fi-cDNAs prepared from purified globin mRNAs were also hybridized to cellular DNAs from several different sources. Using normal cellular DNA, approximately 48% of the a-cDNA is hybridized, and 53% of the /I-cDNA is hybridized at the ratio of cDNA to cellular DNA used (Table TIT). The a-cDNA hybridizes to hydrops fetalis DNA to only 20%, while P-cDNA is hybridized to 43%. These results indicate that the a-cDNA probe can detect deletions of a gene material in cellular DNA. Similarly, when the cDNA probes are hybridized to DNA prepared from the cells of the patient heterozygous for GP-thalassemia, 42% of the WCDNA is hybridized,

while only 30% of the /rl-cDNA is hybridized. These results indicate that the purified P-cDNA probe is able to detect deletions of p-globin gene material in cellular DNA. The uand /J-cDNA probes described here arc comparable in their hybridization characteristics to these prepared by separation of (I- and P-cDNA from total cDNA (Refs. 3 and 4; Table 1111. DISCUSSION

Previous methods using polyacrylamide gel electrophoresis in formamide to separate (I- and /SmRNAs from human cells as well as those from rabbit and mnuse reticulocytes have depended on homogenization of the gel to elute the RNA (6,9, 11). Less than 20% of the RNA has been recovered using these methods and a maximum of 20 fig of RNA could be applied to each gel and permit adequate separation of the (I- and /j-globin mRNAs (6, 9, 10). The preparative polyacrylamide gel electrophoresis method described in this paper results in about 504 recovery of (I- and fi-mRNAs of high purity ( >90%) from more than 50 pg of poly(A)-containing RNA on a single gel. Our results indicate that separation of human (I- and /3-globin mRNAs can be achieved in only 90% formamide at o”, conditions which are less denaturing than those previously reported (5, 9, 10). This methodology should permit preparation of w and /S-mRNAs on a large scale for structural analysis of specific globin mRNA as well as for the analysis of abnormal messenger RNAs shown recently to be present in the thalasscmia syndromes (l-3). The rapid assay ofglobin mRNA activity using cellulose acetate electrophoresis of the cell-free product increases the feasibility of utilizing this methodology. The purity of the separated mRNAs has been demonstrated to be approximately 95% for tu-mRNA and 93% for /&mRNA

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Normal fi-mRNA n-mRNA

0.’

FIG. 6. Hybridization of U- and &cDNAs to U- or P-thalassemis mRNA. A, 10 ng to 10 pg of hydrops fetalis RNA were hybridized to 2500 cpm of U- or &cDNA for 4 h (A---a, &cDNA; A---A. UcDNA1. B. 0.42 pg to 3.3 ng of homozygous 68-thalassemia mRNA were hybridized to 2500 cpm of n- or /3-cDNA for 24 h (L-L, pcDNA; A-A, rr-cDNA).

II

of globin cDNA probes by hybridizatmn

Purity mRNA source

IO.’ CJ

Q 4. Hybridization of o- and A, 80 pg to 0.1 pg of /+mRNA and hybridized to 2500 cpm of P-cDNA for 4 h. (A-A, /ScDNA; A-A, mRNA and 0.6 ng to 0.12 fig of cpm of a-cDNA for 4 h (A-A, FIG.

2185

2186

Polyacrylamide

Gel Purification

of Human

’ U. Nude], F. Ramirez, P. A. Marks, and A. Bank, manuscript preparation.

in

BGlobin

mRNAs

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Bank, A. (1975) Proc. Null. Acad. Sci. II. S. A. 72,1550-1554 5. Morrison, M. R., Brinkley, S. A., Gorski, J., and Lingrel, .J. B. (1974) J. Biol. Chem. 249, 5290-5295 6. Orkin, S. H., Swan, D., and Leder, P. (1975)5. Binl. Chem. 2:&, 8753-8760 I. Temple, G. F., and Housman, D. (1972) Proc. Natl. Acad. Sci. U. S. A. 69, 1574-1577 8. Berns. A.. Jansen. P.. and Bloemendal. H. (1974) FE‘BS Lett. 47, 343-347, 9. Hamlyn, P. H., and Gould, H. J. (1975) J. Mol. Biol. 94, 101-109 10. Kazazian, H. H., Jr., Ginder, G. D., Synder, P. G., VanBenden, R. J., and Woodhead, A. P. (1975) Proc. Natl. Acad. Sci. U. S. A. 72, 567-57-l 11. Forget, B. G., Housman, D., Benz, E. J., Jr., and McCaffrey, R. P. (1975)Proc. Natl. Acad. Sci. 1J. S. A. 72. 984-988 12. Terada, M., Metafora, S.. Banks, J., Dow, L. W., Bank, A., and Marks, P. A. (1972) Biochem. Btophys. Res. Commun. .19,775781 13. Aviv, H., and Leder, P. (1972) Proc. Natl. Acad. Scz. U. S. A. 69, 1408-1412 14. Cann, A., Gambino, R., Banks, J., and Bank, A. (1974) J. Rio/. Chem. 249, 7536-7540 30,301-304 15. Gould, H. J., and Hamlyn, P. H. (1973)FEBSLelt. 13,3394-3400 16. Hagen, F. S., and Young, E. T. (1974)Biochemistry 17. Milcrek, C., Price, R., and Penman, S. (19741 Cell 3, I-10 18. Roberts, B. E., and Patterson, B. M. (1973)Proc. Null. Acad. Sci. U. S. A. 70, 2330-2334 19. Revel, M., Aviv, G., and Hezherg. M. (1971) Methods 1Snz.ymol. 20. 261-277 20. Cleeg. J. B.. Nauehton. M. A.. and Weatherall. D. J. (1966)J. Mol. Biol.’ 19, Sr-108 21. Uecla. S.. and Schneider. R. G. (1969) Blood 34, 230-235 22. Kacian, D. L., and Spiegelman, S. (1974) Methods Enzymol. 29, 150-173 23. Kacian, D. L., Spiegelman, S., Bank, A., Terada, M., Metafora, S., Dow, L., and Marks, P. A. (1972) Nukre New Biol. 235, 167-169 24. Dow, L. W., Terada, M., Natta, C., Metafora, S., Grosshard, E., Marks. P. A., and Bank, A. (1973) Nature New Biol. 243, 114116 25. Dayhoff, Y. O., ed. (1969) Atlas of I’rotein Sequence and Strutture Vol 4. on. D3S.D49. National Biomedical Research Foundation, Maryland 26. Pritchard. J.. Lonalev. J.. Cleaa, J. B., and Weatherall, D. J. (1976) ir. J. Haemkol. ‘2, 473-485 27. Lanyon, W. G., Ottolenghi, S., and Williamson, R. (1975) Proc. Natl. Acad. Sci. U. S. A. 72, 258-262 28. Taylor, J. M., Dozy, A., Kan, Y. W., Varmus. 1~. E., Lie-Injo, I,. E., Ganesan, J., and Todd, D. (1974) ‘Vatwe 251, 392-393 29. Venetianer, P., and Leder, P. (1974) Proc. Natl. Acad. Sci. 11. S. A. 71, 3892-3895 30. Levy, S., and Aviv, H. (1976) Biochemistry 15, 1844-1847 31. Woo, S. L. C., Smith, R. G., Means, A. R., and O’Mallcy, B. W. (1976) J. Biol. Chem. 251, 3868-3874 vu,

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when assayed in a wheat germ cell-free system for globin synthesis (Table I). The possibility exists that differences in the translational capacity of u- uersus ,!+mRNA could affect the relative amounts of (Y- and j3-globin synthesis in this system. This is unlikely, however, since the relative amounts of u- and @globin mRNA in human reticulocyte 10 S RNA are comparable as measured by analytical gels (10, 11) and by hybridization (Fig. 5). At the same time, the data ofothers (26) as well as those reported here indicate that using total human reticulocyte mRNA c~- and @globin are translated in a ratio close to 1 in the wheat germ cell-free system. These results indicate that the relative synthesis of a- and ,!&globin at limiting mRNA concentrations is a faithful reflection of the content of a- and P-globin mRNA, respectively. The cDNAs prepared from the separated globin mRNAs also show high specificity for a- and fi-mRNA sequences. Hybridization of the a- and /3-cDNAs to the separated (x- and j+ mRNAs indicate 96% purity of the p-cDNA and 92% purity of the a-cDNA (Table II). It should be noted that contamination of the separated mRNAs with the other species (i.e. (Y- with /3mRNA) will lead to an underestimation of the purity of the cDNA probes in these experiments. In addition, the ru-cDNA hybridizes to 15% above background to hydrops fetalis mRNA lacking a-mRNA sequences, while the p-cDNA hybridizes to 20%, with Gfi-thalassemia mRNA lacking 6 and /3 sequences. The higher levels of hybridization of the separated cDNAs to these cellular RNAs may reflect the partial homology of (Y-and P-cDNA with non-a- or p-mRSAs present in these RNAs. The LY-and p-cDNAs synthesized using the purified globin mRNAs appear to be at least comparable in their purity to those described previously using other methods (4, 27, 28). There are several advantages to having the template mRNAs available. First, it permits evaluation of the relative purity of the two probes by cell-free assay of translational activity and by hybridization of the separated cDNAs to the purified RNAs. In addition, the purified RNAs will be useful both to analyze the structure of normal and abnormal human u- and p-globin mRNAs and to prepare specific globin cDNA columns linked to oligo(dT)-cellulose column supports (29, 30) which may permit quantitation of newly synthesized globin mRNA and its precursors. The purified cDNAs prepared by other methods (3,27) cannot easily be covalently linked to cellulose. The purified mRNAs can also be linked to cellulose and used to specifically bind a- or P-globin gene sequences in cellular DNA in a fashion analogous to that recently reported using ovalbumin mRNA (31). We have recently used the methodology described in this paper to separate a- and p-globin mRNAs from rabbit and mouse poly(A)-containing reticulocyte RNA.’ The degree of purity of the separated mRNAs and recovery from the gel column are similar to those of human globin mRNA.

a- and