Cell, Vol. 12,741-750,

November

1977, Copyright

0 1977 by MIT

The Gene and Messenger Polypeptide IX Ulf Pettersson and Michael 8. Mathews Department of Microbiology Biomedical Center Uppsala University Uppsala, Sweden and Cold Spring Harbor Laboratory Cold Spring Harbor, New York 11724

RNA for Adenovirus

The present report describes the purification and mapping of this RNA, which is shown to be the messenger RNA coding for polypeptide IX of the adenovirus capsid.

Results Isolation of a New RNA Species

Summary A small RNA species, distinct from the VA RNAs, has been identified in HeLa cells infected with adenovlrus type 2. The RNA, which has been purified using a novel screening procedure, is polyadenylated, sediments at 9S and has an estimated length of 550 nucleotides. In a cell-free translation system, the 9s RNA directs the synthesis of virion polypeptide IX, molecular weight 12,000 daltons. The location of its gene has been established by hybridization of the RNA to fragments of viral DNA produced by cleavage with restriction endonucleases: it spans position 10.0 on the r strand of the viral genome. These results unexpectedly place the gene for a “late” protein within a region of the genome which is transcribed early during infection. Introduction The adenovirus genome appears to be transcribed by RNA polymerases of the host cell. The available evidence suggests that precursors to mRNA are made by an RNA polymerase with properties similar to those of the mammalian RNA polymerase II, whereas two low molecular weight RNA species, VA RNA, and VA RNAr,, are products of RNA polymerase III (Price and Penman, 1972; Wallace and Kates, 1972; Weinmann, Raskas and Roeder, 1974; Soderlund et al., 1976). Messenger RNA from polysomes of adenovirus-infected cells has been fractionated by sedimentation through sucrose gradients and by electrophoresis in polyacrylamide gels (Lindberg, Persson and Philipson, 1972; Bhaduri, Raskas and Green, 1972; Anderson et al., 1974; Tal et al., 1974). In the late phase of the infectious cycle, adenovirus messenger RNA consists of a large number of species with prominent components sedimenting at 265 and 22S, but fractionation of viral messenger RNA has not so far permitted the isolation of pure species. We have recently made a detailed investigation of low molecular weight RNA species produced during lytic adenovirus 2 (Ad2) infection (Mathews and Pettersson, 1978). This study led to the discovery of an additional small RNA species with properties differing from those of the VA RNAs.

Screening experiments, designed to explore the repertoire of viral transcripts present in the cytoplasm of Ad2-infected cells, have been conducted with a novel variation of the Southern blotting technique (Mathews and Pettersson, 1978). Briefly, 3ZP-labeled RNA from lytically infected cells is separated into size classes, either by sedimentation through sucrose gradients or by gel electrophoresis, and each fraction is then analyzed by hybridization to Ad2 DNA fragments produced by digestion with restriction endonucleases. The fragments, resolved by electrophoresis through agarose gels, are bound to strips of nitrocellulose paper by the blotting method of Southern (1975) in such a way that each strip carries a replica of the banding pattern representing the entire viral genome. Autoradiography of the strips reveals the distribution of labeled RNA transcripts in each size class among the various fragments of the viral genome. We have used this technique to purify and characterize a novel low molecular weight RNA species. Cytoplasmic RNA was extracted from AdP-infected HeLa cells labeled with 32P-phosphate from 2-16 hr post-infection, and was fractionated through sucrose gradients. Screening of the fractions by hybridization to nitrocellulose strips carrying fragments of Ad2 DNA generated by endonuclease Sma I revealed a peak of RNA, which sedimented between the peaks of 18s and 5s RNA, hybridizing to fragment Sma I-E. The profile of the gradient and an autoradiogram of the hybridized nitrocellulose strips are illustrated in Figure 1. We pooled fractions containing RNA complementary to fragment Sma I-E and further purified the RNA by electrophoresis on 7% polyacrylamide gels in the presence of 7 M urea. A peak of RNA hybridizing to fragment Sma I-E was again identified (Figures 2A and 2C). We combined the corresponding gel slices and eluted the RNA in them for further analysis.

Properties of the New Low Molecular Weight RNA Species On centrifugation through lo-30% sucrose gradients in 50% formamide as described by Cberg et al. (1975), with ribosomal 18s and 5s RNA as markers, the peak of RNA hybridizing to fragment Sma I-E sedimented at approximately 9s (data not

Cell 742

may explain why the 9s species occupies a rather broad band after gel electrophoresis (see Figures 2A and 3), since the poly(A) tails on eucaryotic messenger RNAs are usually rather heterogeneous in length.

Map Position of the Gene for 9s RNA

5 10 15 FRACTION NUMBER

1

Figure

2

3

4

1. Screening

5

6

7

9

of Gradient

9

20

10 11 12 13 lb 15 16

Fractions

by Hybridization

Upper panel: Fractionation of 32P-labeled RNA from the cytoplasm of AdP-infected cells on a 15-300/o sucrose gradient. Sedimentation was from right to left. Lower panel: Screening of aliquots from the sucrose gradient fractions in the upper panel and by hybridization to nitrocellulose strips carrying Sma I gragments of Ad2 DNA. Hybridizations were carried out at 65°C in a total volume of 1.5 ml. The arrow indicates the position of fragment Sma I-E.

shown). To measure the length of the 9s RNA, we conducted gel electrophoresis in 7% polyacrylamide gels containing 7 M urea. Relative to suitable DNA and RNA markers, the size of 9s RNA was estimated to be approximately 550 nucleotides (Figure 3). On the same gel, the chain lengths of the three host cell 7s RNA species (Mathews and Pettersson, 1978) were about 325, 305 and 300 nucleotides. When subjected to affinity chromatography on poly(U)-Sepharose, 40-80% of the labeled 9s RNA was recovered in the poly(A)-containing fraction, and nearly all of the viral polyadenylated material hybridized to Sma I-E (results not shown). These findings suggested that the 9s RNA has a messenger function, an inference which is sustained by the data presented below. The presence of poly(A)

9s RNA, purified by sucrose gradient centrifugation and gel electrophoresis, was hybridized to nitrocellulose sheets carrying immobilized restriction fragments of Ad2 DNA. Results from hybridizations with the four Barn HI fragments of Ad2 DNA indicated that 9s RNA hybridized exclusively to fragment Barn HI-B (compare with Figure 7). Figure 4 shows the result of hybridizations with sets of fragments generated from the Barn HI-B fragment by cleavage with endonucleases Bal I, Sma I, Kpn I, Hind Ill and Bgl II. In each case, the RNA was found to hybridize to a unique fragment, and hybridization to the following fragments was manifested: Bal I-G, Sma I-E, Kpn I-B, Hind Ill-C and Bgl II-B. The map positions of these fragments are illustrated in Figure 5, and we conclude from the results that the gene for 9s RNA is contained in the region between map coordinates 9.4 and 11 .l , bounded respectively by the cleavage sites for the endonucleases Bgl II and Sma I. We have estimated the length of this interval directly, by measuring the size of the small fragment released from the Sma I-E fragment of Ad2 DNA by digestion with the Bgl II enzyme. Relative to double-stranded 4x174 DNA size standards, the region contains about 590 base pairs, or 1.7 map units (result not shown), in good agreement with the mapping data. We also performed hybridization between 9s RNA and fragments of Ad2 DNA generated by endonucleases Hph I, Mbo I and Hinf I, which all cut Ad2 DNA into a large number of fragments. In these hybridization experiments, samples of VA RNA, and VA RNA*, were included as references. With each set of fragments, the 9s RNA displayed a unique pattern of hybridization and, in each case, hybridized to a different fragment than the VA RNAs. The smallest fragment which hybridized to 9s RNA was approximately 420 nucleotides long and was contained in the endonuclease Mbo I digest (Figure 8). To determine the direction of transcription for 9s RNA, we separated the complementary strands of the fragments derived from Ad2 DNA by digestion with the endonuclease Barn HI. Strand separation was effected by gel electrophoresis, essentially as described by Hayward (1972) and by Sharp, Gallimore and Flint (1974), and the strands of the fragments were immobilized on nitrocellulose strips by the method of Southern (1975). The 9s RNA hybridized selectively to the more slowly mi-

Gene

for Adenovirus

Polypeptide

IX

743

A

B Q, 3

* Endogsnous incorporation

IS0 -

9lx 4 i?EL

12 15 18 21242730333639

. 160

-60

p nN

-60

i .

-40

OL

IO

20

30

40 60 60 Fraction Number

70

60

I 100

90

C 2

Figure

58

II

14

2. RNA Gel Fractions

172023

Scanned

2629323538

by Translation

I

4

7

IO

I3

I6

19 22

25

28

31

34

37

40

and by Hybridization

(A) Cytoplasmic RNA, isolated from about 10’ cells 16 hr post-infection and fractionated by sedimentation through sucrose gradients (see Experimental Procedures), was subjected to electrophoresis through a 7% polyacrylamide gel in 7 M urea. Slices were assayed for radioacivity (04). In addition, the RNA eluted from selected fractions was assayed for messenger activity in a wheat germ cell-free protein-synthesizing system: reactions contained amounts of RNA equivalent to 3.5% (O---U) or 10% (WA) of that present in the gel slice, as determined by recovery of radioactivity from each fraction. (B) Location of the messenger activity coding for component IX by translation. The products synthesized in vitro under the direction of RNA from selected fractions were analyzed by electrophoresis in an SDS-polyacrylamide gel. The figure contains an autoradiogram of the gel, with marker proteins from cells labeled late in the infectious cycle. (C and D) Location of the 9S mRNA by hybridization of RNA from selected fractions to nitrocellulose strips carrying fragments of Ad2 DNA generated by endonucleases Sma I and Bgl II, respectively. The figure contains an autoradiogram of the hybridized strips and a photograph of a portion of the original gel. The fragments which hybridize to 9S RNA, Sma I-E and Bgl II-B, are indicated by arrows.

Cell 744

I

800

350

1

1

2

3

4

5

6

Hybridized

7

8

9

IO

Stained

Figure 3. Measurement of the Length of 9s RNA Upper section: Autoradiogram of 7% polyacrylamide/7 M urea gel containing denatured DNA markers (upper track) and 9s RNA. partially purified by sedimentation through a sucrose gradient (lower track). The markers were made by end labeling the Barn HI-D fragment of Ad2 DNA with 32P-phosphate using T, polynucleotide kinase, followed by cleavage with Hind Ill, as described by Mathews and Pettersson (1978). The 899 and 350 base pair fragments, generated from the left and right ends, respectively, were sized by comparison

Gene

for Adenovirus

Polypeptide

IX

745

Hybridized

Stained Figure 4. Hybridization between HI-B Fragment of Ad2 DNA

Purified

9s RNA and Different

Restriction

Fragments

to 9s RNA Derived

from

the Barn

Fragment Barn HI-B is located between positions 0.0 and 30.0. The following restriction endonucleases were used: Kpn I, Hind Ill, Bal I, Bgl II and Sma I. The fragments were immobilized on a nitrocellulose membrane and used in hybridization experiments with purified 9s RNA. The left panel shows a photograph of the gel after staining with ethidium bromide. The right panel shows the corresponding autoradiogram after hybridization to 9s RNA. The map positions of the fragments are illustrated in Figure 5.

with +X174 DNA markers (not shown). VA RNA, and ribosomal 5s RNA provide additional markers, 156 and 120 bases long respectively. Lower section: Hybridization of RNA from gel fractions to fragments of Ad2 DNA derived by cleavage with endonuclease Hind II. The position of the fragment which hybridizes to 9s RNA is indicated by an arrow.

Cell 746

9s RNA D

VA RNAl

I-

I

-O

Kpn I G

Hin dliI Ball

0;

C

1

;L:$

G

-

E

Bgl II

J :

SmaI

E

E

y 1 IO

0 5. Map Position

F

of the Gene

(

A’

8’

h

8

(M’

D

I

B

Barn HI-B,

Figure

6’

1 20 for

Polypeptide

I 30 IX

The figure represents the left end of the Ad2 genome. The bottom portion illustrates the positions of the subfragments derived by restriction enzyme cleavage of the Barn HI-B fragment. Heavy lines indicate the fragments which hybridize to the 9S RNA (data from Figure 4). The map position deduced for this mRNA is shown at the top of the figure, above the transcription map which gives the locations of sequences present in cytoplasmic mRNA early (open arrows) and late (dark arrows). VA RNA, is shown stippled. Data for the restriction maps were obtained as personal communications from the following colleagues at the Cold Spring Harbor Laboratory: R. Gelinas (Bal I); R. Greene and C. Mulder (Barn HI); M. Zabeau (Bgl II); R. J. Roberts, C. Mulder, J. Sambrook and P. Sharp (Hind Ill); R. Greene and M. B. Mathews (Kpn I); H. Delius and C. Mulder (Sma I). Fragments curtailed by the Barn HI cleavage are denoted by a “prime” mark. Data for the transcription map are drawn from Pettersson et al. (1976), Flint (1977) and Chow et al. (1977).

grating strand of fragment Barn HI-B, which also hybridized to VA RNA, (Figure 7). Previous studies by Weingartner et al. (1976) have shown that this strand has its 3’ terminus at the left end of the Ad2 DNA. Furthermore, the VA RNAs are known to be transcribed from the r strand (Mathews, 1975; Pettersson and Philipson, 1975; Sdderlund et al., 1976). Thus we conclude that the gene for 9s RNA is located on the r strand of a DNA segment located between map positions 9.4 and 11 .l .

Translation

of 9s RNA

When added to a cell-free translation system from wheat germ, the 9s RNA stimulates the incorporation of YS-methionine into protein. We analyzed the product by electrophoresis through polyactylamide gels in the presence of SDS. Figure 8 demonstrates the presence of a polypeptide which has an apparent molecular weight of 12,000 daltons and co-migrates with polypeptide IX of the virus capsid. At late times, polypeptide IX is a prominent component of adenovirus-infected cells, but there is little or no suggestion of a band at this position in a gel containing proteins from cells early in the infectious cycle or from uninfected cells (Figure 8). We have obtained similar results by cell-free translation of RNA from mock-infected cells or infected cells at early times after infection. These

data demonstrate that polypeptide IX, a hexon-associated virion protein (Maizel, White and Scharff, 1968; Everitt et al., 1973), is made from 9s RNA during the late phase of infection. To ascertain that this messenger activity is precisely coincident with the 9s RNA species described here, we isolated RNA from several fractions of a 7% polyacrylamide/7 M urea gel, spanning the region from about 7-17s. The RNA from each fraction was assayed for its messenger activity in the wheat germ cell-free system, and the products were displayed on an SDS-polyacrylamide gel. Figure 2B shows that the polypeptide IX-synthesizing ability is indeed restricted to the 9s RNA and coincides with hybridization to the Sma I-E and Bgl II-B fragments (Figures 2C and 2D). The identity of the cell-free product was confirmed by tryptic peptide analysis. Authentic polypeptide IX labeled with 35S-methionine in vivo was purified by gel electrophoresis and digested to completion with trypsin. The 12,000 dalton product synthesized in vitro was similarly isolated and digested, and the resultant peptides were compared by thin-layer electrophoresis and chromatography. The peptides derived from the cell-free product co-migrated with those from the marker upon chromatography (Figure 9A) and electrophoresis at both pH 3.5 and pH 6.5 (Figures 9B and 9C). This correspondence, which was upheld during chromatography in a second solvent and’also in twodimensional fingerprints (data not shown), verifies that the 9S RNA codes for polypeptide IX of the virus capsid.

Discussion This paper describes the isolation and mapping of a small polyadenylated RNA sedimenting at 9s and having a chain length of approximately 550 nucleotides. The gene for the 9s RNA, which is shown to code for virion polypeptide IX, has been mapped on the r strand in the region between the Bgl II site at position 9.4 and the Sma I site at 11 .l (see Figure 5). This region encompasses about 590 base pairs and could accommodate the 9s RNA in its entirety. Our data do not exclude the possibility that the RNA extends a short distance across one or the other of these boundaries into the neighboring fragment, but it is clear that the major portion of the gene is included in the region surrounding position 10.0. Assuming an average amino acid composition for protein IX, about 330 bases would be required to encode its 12,000 daltons, leaving some 220 bases to be occupied by flanking sequences at the 5’ and 3’ ends. The length of the poly(A) tract has not been measured directly, but it is expected to account for 150-200 nucleotides,

Gene

for Adenovirus

Polypeptide

IX

747

VA L

Figure 6. Hybridization and a Mixture of Hind

between 9s RNA or VA RNA and Fragments Ill and Barn HI

of Ad2 DNA Generated

by Endonucleases

VA II

Bal I, Hinf

Three identical sets of fragments were separated on a 1.4% agarose gel and immobilized on a nitrocellulose membrane. From left to right, the panels of the figure contain a photograph of one section of the stained gel and autoradiograms membranes after hybridization with 9S RNA, VA RNA, and VA RNAr,.

since unfractionated adenovirus messenger RNA carries poly(A) segments in this range (Philipson et al., 1971). Our results are in good agreement with those of Anderson et al. (1974), who found that the activity coding for polypeptide IX sedimented at 9S, and of Chow et al. (1977), who observed a late transcript mapping between coordinates 9.7 and 11 .O, using RNA loop mapping techniques. Lewis, Anderson and Atkins (1977) have furthermore found that the messenger RNA for polypeptide IX can be selected by hybridization to fragment Hind Ill-C, located between positions 7.5 and 17.0. As illustrated in Figure 5, previous hybridizationsaturation studies (Pettersson, Tibbetts and Philipson, 1976) have demonstrated the existence close

I, Mbo

I, Hph I

of nitrocellulose

to position 10.3 of a “strand switch:” at this point, the 3’ end of the r strand transcript, running from approximately position l-10, adjoins the 3’ end of an I strand transcript mapping between positions 15-10 approximately. The 9S RNA belongs to the r strand block of transcripts and appears to extend to the strand switch. A similar conclusion was formed by Chow et al. (1977), who placed the strand switch at coordinate 11.2. Surprisingly, this r strand transcript from the left-hand end of the genome is synthesized in the early phase of the lytic cycle, before viral DNA synthesis begins, and also in adenovirus-transformed cells (reviewed by Flint, 1977), but polypeptide IX, a structural component of the virus particle, has not been detected in either of these conditions. This could indicate

Cell 746

123 e

AL Ar-B!Bl

IrIOOK-

p-m-- p-XII--plLFI8K-‘-

Figure 7. Hybridization between Purified 9s RNA and the Separated Strands of the Barn HI-Fragments of Ad2 DNA Ad2 DNA was cleaved with endonuclease Barn HI, and the complementary strands of each restriction fragment were separated by gel electrophoresis and transferred to a nitrocellulose membrane. 32P-labeled, denatured Ad2 DNA (I), VA RNA, (2) and 9s RNA (3) were hybridized to strips of the nitrocellulose membrane. The strands are designated according to Weingartner et al. (1976). Under our conditions, the complementary strands of fragment Barn HI-D are not well resolved.

that the sequences coding for 9s RNA are transcribed early after infection, but are present in a form which is inactive with regard to the synthesis of polypeptide IX. Alternatively, the gene for 9s RNA might be interposed between the block of early genes near the left-hand end and the site of the strand switch. Present evidence favors the

abcdaef Figure 6. Analysis SDS-Polyacrylamide

of the Products Gel

of 9s mRNA

Translation

in an

Proteins were synthesized in vitro in the absence(b) and presence (c) of 9s mRNA; marker proteins were extracted from infected cells late in the infectious cycle (a), early in the cycle (e) and from uninfected cells (f); track (d) contains a mixture of the “late” marker proteins and 9s mRNA product (tracks a and c, respectively). The a%-methionine-labeled polypeptides were detected by autoradiography. The “early” and “late” proteins are distinguished by broken and continuous lines respectively.

Gene

for Adenovirus

Polypeptide

IX

749

A

B TLC

by ethanol precipitation and layered on a 15-30% sucrose gradient in 0.5% sarcosyl, 0.1 M NaCI, IO mM Tris-HCI (pH 7.5) and 1 mM EDTA. Centrifugation was carried out at 35,000 rpm for 16 hr at 4°C in the Spinco SW41 rotor. and fractions containing RNA sedimenting at 5-12s were pooled. The RNA was recovered by ethanol precipitation and was further purified by polyactylamide gel electrophoresis.

C QH

3.5

DH 6.5

Gel Electrophoresls Electrophoresis under denaturing conditions was carried out in 7% acrylamide gels (acrylamide:bisacrylamide = 29:1) in the presence of 7 M urea as described by Maniatis, Jeffrey and van de Sande (1975). Before loading, the RNA was heated at 65°C for 2 min in 90% formamide. Double-stranded DNA markers were denatured by heating for 2 min at 90°C in 90% formamide. For hybridization, RNA was eluted from gels by shaking the crushed slices in a buffer which contained 0.01 M Tris-HCI (pH 7.9), 1 mM EDTA and 0.5% SDS.

a Figure tion

b

c

a

9. Peptide

Analysis

b

c

of the Products

ab of 9s mRNA

c Transla-

Ttyptic digests of (a) component IX purified from infected cell lysates, (c) the 12,000 dalton product of the cell-free translation of 9s RNA, and (b) a mixture of digests (a) and (c), were compared by (A) thin-layer chromatography or thin-layer electrophoresis (B) at pH 3.5 and (C) at pH 6.5. The arrowheads mark the sample origin, and the cathode is at the top of the autoradiogram

former hypothesis, since Chow et al. (1977) observed that the late RNA occupying positions 9.811 .l coincides with the 3’ end of a longer early transcript. Several precedents exist in virus systems for this state of affairs, but further studies are required to clarify the situation. To the best of our knowledge, this is the first description of an adenovirus messenger RNA which has been purified by biochemical methods. We hope that the availability of a pure species of adenovirus messenger RNA will make it possible to study the synthesis of messenger RNA precursors in the nucleus as well as the structure of the messenger RNA itself. We also anticipate that the screening procedure used here will prove useful in a variety of other situations. Expertmental

Pwsdures

Isolation of Low Milecular Weight RNA HeLa cells in suspension were infected at a multiplicity of 50-100 PFU per cell and labeled with 300-600 &i/ml of UP-phosphate from 2 hr post-infection. Cells were harvested 16 hr after infection and washed once with ice-cold phosphate-buffered saline (PBS). The cells were then lysed with 0.65% NP40 in PBS at 0°C for 10 min. Nuclei were removed by centrifugation, and the cytoplasm was extracted once with phenol at pH 9 according to Brawerman. Mendecki and Lee (1972). The extracted RNA was concentrated

RNA-DNA Hybrldizatlon Fragments of Ad2 DNA were produced by cleavage with endonucleases Barn HI, Bal I, Bgl II, Kpn I, Hph I, Hind II, Hind Ill. Hinf I. Mbo I and Sma I. Endonuclease Bgl II was associated from Bacillus globiggi by an unpublished procedure of G. A. Wilson and F. E. Young; Kpn I from Klebsiella pneumoniae OK6 by the procedure of Smith, Blattner and Davies (1976); Sma I from Serratia marcescens Sb by an unpublished procedure of R. Greene and C. Mulder; Hind II and Hind Ill from Haemopitus influenzae Rd (Smith and Wilcox, 1970) by the procedure of Lai and Nathans (1974), or from H. influenzae R, 1160 (Hind II) and H. suis (Hind Ill) by unpublished procedures of J. A. Olson, P. A. Myers and R. J. Roberts. The purification of the remaining restriction endonucleases is referenced in a separate communication (Mathews and Pettersson, 1976). Digestions with Sma I were performed at 30°C in the presence of 0.05 M Tris-HCI (pH 9.0), 15 mM KCI and 5 mM MgCI,. All other digestions were carried out at 37°C in the presence of 6 mM Tris-HCI (pH 7.9), 6 mM P-mercaptoethanol and 6 mM MgCI,. The digestion mixture was fractionated by electrophoresis on 20 x 20 cm slab gels containing 1.4% agarose. Bands were visualized during illumination with ultraviolet light in the presence of ethidium bromide (Sharp, Sugden and Sambrook, 1973), and the DNA fragments were subsequently transferred to nitrocellulose membranes by the method of Southern (1975). Hybridization to ‘*P-labeled RNA was carried out at 65°C for 16 hr in the presence of 6 x SSC (1 x SSC is 0.15 M NaCl, 0.015 M Na citrate) with 0.5% SDS, 0.01 M Tris-HCI (pH 7.9) and 1 mM EDTA. Hybridized filter strips were washed several times with 2 x SSC and incubated with fresh 2 x SSC at 65°C for 60 min. Tails of unhybridized RNA were removed by digestion with 20 pg/ml pancreatic RNAase at 37°C for 60 min. The hybridized RNA was detected by autoradiography. Poly(U)-Sepharose Chromatography Columns containing 0.5 ml packed volume of poly(U)-Sepharose (provided by Pharmacia Fine Chemicals, Uppsala. Sweden) were washed and equilibrated at 4°C with 25% formamide in 0.5 M NaCI, 2 mM EDTA, 0.01% SDS and 0.05 M Tris (pH 7.6). RNA samples were loaded in column buffer at 4”C, and the columns were rinsed with 2 X 1 ml of column buffer, followed by 2 X 1 ml of a buffer which contained 50% formamide, 0.05 M Tris (pH 7.8), 2 mM EDTA and 0.2% SDS. Poly(A)-containing RNA was then eluted with 90% formamide in 0.05 M Tris (pH 7.8) and 2 mM EDTA. Protein Synthesls RNA was extracted from crushed polyacrylamide gel fractions by shaking with 2 X IO vol of TNE [50 mM Tris-HCI (pH 7.4), 100 mM NaCI, 1 mM EDTA]. An equal volume of ethanol was added, and the RNA was bound to a column containing 0.5 ml packed volume of Whatman CFll (Franklin, 1966). Following washes with TNE:ethanol (65:35, by volume) and with ethanol, the RNA

Cell 750

was eluted with water and concentrated by ethanol precipitation. Protein synthesis was performed in extracts of wheat germ prepared as described by Roberts and Paterson (1973). Incubations (25 ~1) contained 5 ~1 wheat germ S-30 together with 16 mM HEPES-K+ (pH 7.2). 1 mM Mg acetate, 50 mM KCI, 1 mM ATP, 0.2 mM GTP, 10 mM creatine phosphate, 0.5 mM dithiothreitol, 50 PM spermine, about 1 pM gsS-methionine (Radiochemical Center, Amersham; 400-600 Cilmmole) and the remaining 19 necessary amino acids at 25 &f each. After incubation for 3 hr at 2528°C the reactions were terminated by the addition of pancreatic RNAase and EDTA to 20 rglml and 10 mM, respectively. Incubation was continued for a further 15 min at 37°C and the samples were prepared for analysis on the discontinuous SDS-polyacrylamide gel system of Laemmli (1970). The separating gel contained a linear 7-15% gradient of polyacrylamide. Following electrophoresis, the gel was fixed, stained, destained, dried and subjected to autoradiography. For peptide analysis, protein was eluted from preparative scale gels, oxidized with performic acid and digested with trypsin as described by Brownlee et al. (1972). Chromatography in 1-butanol:acetic acid:water:pyridine (15:3:12:10, by volume) and electrophoresis at pH 3.5 were performed on thin layers of cellulose (Macherey-Nagel, Cel 300).

Acknowledgments We thank R. Greene and R. Roberts for providing the restriction endonucleases, T. Maniatis for polynucleotide kinase and N. Harter for a sample of “early” proteins. The technical assistance of S. Weirich, I. Wendel and G. I. Lindh is gratefully acknowledged. This investigation was supported by a NCI Cancer Center Grant to Cold Spring Harbor Laboratory and by grants from the Swedish Medical Research Council. 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 16 U.S.C. Section 1734 solely to indicate this fact. Received

May 27, 1977;

revised

August

19, 1977

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