J. Mob. Biol.

(1978) 119, 293328

The Low Molecular

Weight of RNAs of Adenovirus Cells MICHAEL

2-infected

B. MATHEWS

Cold Spring Harbor Laboratory P.O. Box 100 Cold Spring Harbor, New York, U.S.A. AND ULF

PETTERSSOK

Biomedical Center Uppsala, Xweden (Received

26’ July

1977, and in revised form 24 October 1977)

The cytoplasm of HeLa cells infected with adenovirus type 2 contains many species of low molecular weight RNA, including several of viral origin. In addition to a 9 S messenger RNA, the viral genome gives rise to two species of virus-associated RNA: the major species is 5.5 S RNA or virus-associated RNA,,

and the minor

species is 52 S RNA or virus-associated

RNA,,.

Virus-associated

KNA, occurs in the cytoplasm in several electrophoretically separable forms, and its sequences are also present in high molecular weight nuclear RNA but not in cytoplasmic mRNA. The structure of virus-associated RNA,, is shown to be distinct from that of the major species, and the position of its gene is mapped on the viral genome. The two virus-associated RNA genes are located on the T strand near position 30 of the adenovirus type 2 physical map, and are separated by- a spacer of about 75 base-pairs.

1. Introduction Adenoviruses code for low molecular weight RNA species of unknown function. These virus-associated RNAst possess several intriguing characteristics, the most conspicuous of which is their pre-eminence among the viral transcripts present in the cytoplasm of the infected cell, where they may outnumber viral messenger RNAs. Unlike the genes that code for mRNAs, which are transcribed by RNA polymerase II, the VAT RNAs are synthesized by polymerase III and are produced during the incubation in vitro of nuclei from infected cells (Ohe et al., 1969; Price & Penman, t The low molecular weight RNAs, both those of adenovirus-infected cells and those of uninfected cells, are known by a multiplicity of names. To facilitate comparisons and minimize confusion, we follow the terminology adopted by the authors who have published the fullest structural analysis of the species in question, with modifying suffixes where necessary. Where there are minor disparities in the placement of restriction enzyme sites on the Ad2 genome, the BanaH cleavage at position 30.0 has been taken as 8 reference point and map lengths are related to this location. $ Abbreviations used: VA RNA, virus-associated RNA; Ad2, adenovirus type 2; HnRNA, heterogeneous nuclear RNA; p.i., post-infection; bp, base-pair(s); PBS, phosphate buffered saline ; SDS, sodium dodecyl sulfate. 293

“(34

M.

13. MATHEWS

A-\SI)

1..

l’E’rTk:RSSON

1972a,b; Wallace & Kates. 1972: Weinmann et al., 1974,1975,1976: SGderlund et nl.. 1976). When made in this system, the VA RNAs carry 5’-terminal pppGp residues, the hallmark of an initiation event (Price $ Penman. 19726: Siiderlund et al., 1976). The greater part of the VA RNA present in the cytoplasm of cells infected by adenovirus 2 consists of a species called VA RNA, or 5.5 S RNA. Its nucleotide sequence was determined by Ohe & Weissman (1971), who found it to contain about 156 bases with a limited heterogeneity at its 3’ end. A number of minor species have been reported by other authors and characterized in various ways (Mathews, 1975; Siiderlund et al., 1976: Weinmann et al., 1976; Varrichio et al.. 1976). Some of these appear to be related to VA RNA, on the basis of RNA fingerprinting. while others have been claimed to be distinct species. The techniques employed are so varied, however, that it is impossible to be certain how many distinct species exist. In this paper we report the results of a systematic investigation of the low molecular weight RNAs present in the cytoplasm of Ad2-infected cells. These RNAs comprise, in addition to several host species which we partially characterize, a 9S mRNA (Pettersson & Mathews, 1977) and various forms of just two viral RNAs. One of them is the major species VA RNA,, whose map position on the viral genomc has been defined precisely (Mathews, 1975; Pett,ersson & Philipson, 1975). The other species, previously described on the basis of its electrophoretic mobility as 5.2 S RNA (SGderlund et al., 1976), is shown to be identical to VA RNA,,, a species hitherto recognized only by hybridization and RNA fingerprinting (Mathews, 1975). The sequence of this RNA is quite distinct from that of VA RNA,. The position of its gene on the viral DNA is located and t’he size of the spacer region between the genes for the two VA RNAs is measured. Because both species are initiated separately and transcribed from the same DNA strand, this short spacer region presumably contains a polymerase III initiation site. The occurrence of VA RNA, in high molecular weight nuclear RNA and cytoplasmic mRNA is also investigated. and the implications of the findings are discussed.

2. Materials (a) infection

and Methods and

labeling

of cells

HeLa cells in suspension at a density of 5 x 106/ml were infected with adenovirus 2 at a multiplicity of 50 to 100. After absorption for 1 h the cells were diluted lo-fold into fresh F-13 medium (Gibco). For labeling with tritium, r3H]adenosine (Radiochemical Centre, Amersham) was added to 10 &i/ml from 12 to 16 h p.i. For labeling with 32P, the cells were washed twice in phosphate-free F-13 medium after virus absorption, and at 2 h p.i. were resuspended in phosphate-free medium containing 2”/b horse serum and 200 to 400 &i [32P]phosphate/ml (New England Nuclear). Uninfected cultures were treated in parallel fashion. (b)

Isolation

Cells were harvested by centrifugation at phosphate buffered saline. The cell pellet Nonidet P40 (NP40), and held at 0°C for nuclei and cytoplasm by centrifugation. gradient centrifugation of the cytoplasmic RNA was extracted from the cytoplasmic Brawerman et al. (1972). Nuclear RNA was (1973). Following alcohol precipit,at,ion, RNA (pH 7.6), 1 mM EDTA).

of RNA

various times and were washed once in ice-cold was resuspended in cold PBS with 0.65% 10 min. The lysed cells were fractionated into Polysomes were obtained by sucrose density fraction as described by Lindberg et al. (1972). fraction and from the polysomes according to isolated by the method of Holmes & Bonner was dissolved in TE buffer (10 mM-Tris.HCl

SM;\LL

RNAs

OF (r)

(i) Sucrose

gradient

AI)ENOVlttUS-ISFF:(‘TEI) Fmctionation.

of KS.4

sedimentation.

RNA was sedimented for 16 h at 35,000 revs/min and 4°C in an SW41 15% to 30% sucrose gradients in 0.1 M-NaCl, 1 mM-EDTA, 10 mM-Tris.HCl 0.5% Sarkosyl. Generally, fractions containing RNA sediment,ing bet,ween ~(VYX pooled and precipitated for flnther analysis. (I;)

.\‘atitx

_“I).?

(‘ELLS

&ya,c+ylarnide

rotor through (pH 7.5), and 4 S and 10 S

gels

Gels containing 10% polyacrylamide (acrylamide: bisacrylamide, 66: l), glycerol, 0.5% (w/v) Sarkosyl and TBE buffer (89 mM-Tris base, 89 m&I-boric EDTA ; pH 8.3) were cast in slabs 25 cm x 15 cm x 0.3 cm (SGderlund et al., was generally loaded into internal slots to reduce trailing. and electrophoresis out at 4”(‘. (iii)

lIe?aat?Aring

polyacrylamide

loo/ (v/v) acid, 1 m&T1976). RNA was carried

geis

Slab gels, 25 cm x 15 cm x 0.3 cm, containing 7 M-urea were cast with 12% polyacrylamide (acrylamide : bisacrylamide, 29: 1) in TBE buffer, as described by Maniat,is et al. (1975b). Before loading, RNA was heated in 90% formamide for 2 min at 65°C. “2P-labeled infected cell RNA treated with formamide at 25°C or 100°C gave the same patter11 of bands as RNA incubat’cd with formamide at 65°C; this pattern differed very slightly from that obtained with untreated RNA. DNA fragments of up to 800 bp migrated in these gels as single strands provided that the molecules were denatured b> heating in formamide before loading, but when native DNA was loaded directly onto the grl it maintained its duplex state (Mathews and Pettersson, unpublished results; Maniatis et al., 1975b). These observations indicate that the gel system does not abolish pre-existing base-paired duplex structures but does strongly disfavor their re-formation. The possible existence of intramolecular secondary structure in RNA is not excluded, however, and t,he migration of some RNA species (particularly U-2 and 5.8 S RNAs; see Results sections (a) and (b) and Discussion section (c)) appears to depart from the linear relationship between their rlectrophoretic mobilities and the logarithm of t)heir chain lengths (Maniatis et al.. 1975h). (iv)

Two-dimensional

gels

Follo\ving electrophoresis and autoradiography to locate the regions of interest, strips \vt’r(x rxcistd from native gels. The strips were soaked fbr 30 min in 0.2 x TBE saturat’ed \vith urea. then were laid on the top of a denaturing gc.1, tamped to secur? good contact, and subjected to rlectrophoresis at right angles to the original direction. (~7) Detection

and

e&ion

of RNA

[32P]RNA was detected by autoradiography, and was eluted from the gel matrix by diffusion. Pieces of gel were broken up by extrusion through a syringe, and were shaken twice with 10 to 20 volumes of TE buffer containing 0.5% SDS. After removal of gel particltss by centrifugation and filtration, the RNA was recovered by alcohol precipitation. For fingerprinting, the RNA was further purified by chromatography on cellulose (Whatman. CFll) as devised by Franklin (1966), with slight modifications (Pettersson & Mathews, 1977). For hybridization to DNA bound to nitrocellulose membrane, the RNA was generally allowed to diffuse out of the macerated gel into thr hybridization solution during the course of the 68°C incubation (see section (e), below). (L-i)

Formamide-containing

gels

For t,he hybridization competition experiments (Results, section (f)), polyadenylated nuclear and cytoplasmic RNAs were selected by chromatography on oligo(dT)-cellulose as described by Tibbetts & Pettersson (1974), and were then fractionated through 4.2% polyacrylamide gels in 98% formamide at room temperature. The cylindrical gels werr prepared and run according t,o Pinder et al. (1974). After Gctrophoresis, the gels were I:!

29G

M.

B.

MATHEWS

AND

U.

PETTERSSON

cut into 2-mm slices by an automatic gel slicer (Gilson) and each gel slice was extracted with 0.5 ml of a buffer which contained 0.1 m-Tris.HCl (pH 9.0), 1 mM-EDTA and 0.5% SDS. Portions were assayed for radioactivity and the extracted RNA was purified by phenol extraction and chromatography on cellulose (Franklin, 1966) brfore it was used in hybridization experiments (section (e) (iii) below). (d) Preparation (i) Digestion

with

restriction

of DXA

fragments

enzymes

Ad2 DNA, isolated from virus as described by Pettersson & Sambrook (1973), was digested with restriction endonucleases in a solution containing 6 mm-Tris.HCl (pH 7.9), 6 mM-MgCl, and 6 m&r-2-mercaptoethanol. Endonuclease BamHI was isolated from Badua amyloliquefaciens H by the procedure of Wilson & Young (1975); Bali from Brevibacterium albidum by the procedure of Gelinas et al. (19776); Hind111 from Haemophik? in&enzae R, (Smith & Wilcox, 1970) as described by Lai & Nathans (1974) or as f&d from H. suis by an unpublished method of R. J. Roberts and P. A. Myers; HinfI from Haemophilus in&enzae Rr by the procedure of Middleton (1973) ; HphI from Haemophilu-9 parahaemolyticus as described by Kleid et al. ( 1976) ; and MboI from Moraxella bovis according to Gelinas et al. (1977a). (ii)

Gel electrophoresis

Large scale preparation of fragments from HinfI digests of Ad2 DNA was carried out 01, 3.5 y’ polyacrylamide gels (acrylamide : bisacrylamide, 29: 1) cast in slabs 40 cm x 20 cm x 0.3 cm and run in TBE buffer. For analytical purposes (as in Fig. 13B), gels of 0.15 cm thickness were employed. A similar gel, but composed of 5% polyacrylamide, was used for analysis of the digestion products of the HindIII-B/BamHI-D fragment (Fig. 12). All other electrophoretic separations were achieved in 1.4% agarose slab gels, measuring 20 cm x 20 cm x 0.3 cm, apart from the isolation of the BamHI-D fragment which employed 1% agarose. The agarose gels contained either 40 mM-Tris.HCl (pH 7.8), 5 mMsodium acetate and 1 mM-EDTA or, more recently, 0.5 x TBE. About I pg of DNA was loaded in each 0.5~cm slot, unless the gel was destined for screening experiments (section (e) (ii)) : in this case 20 pg DNA was applied across a l&cm well. The progress of electrophoresis was monitored by observation of xylene cyan01 FF and bromophenol blue dyes. At the end of the run, the DNA bands were located by autoradiography, or by fluorescence under ultraviolet illumination in the presence of 0.5 pg ethidium bromide/ml (Sharp et al.. 1973). (iii)

E&ion

of DNA

The gel was broken up by extrusion through a syringe and shaken 3 times for several hours each with 10 to 20 volumes of TE buffer. After filtration, the DNA was concentrated from the resulting dilute solution by passage through a small column (0.1 to 0.5 ml bed) of DEAE-cellulose (Whatman DE52). The column was washed with 2 ml of 0.15 M-NaCl in TE buffer and the DNA eluted with 3 x 1 ml of 1 M-Nacl in TE buffer. This method worked well for small DNA fragments but for large fragments such as BamHI-D it was preferable to dissolve the agarose gel in sodium perchlorate and recover the DNA by hydroxylapatite chromatography (Southern, 1975a). (iv)

Labeling

of HindlII-B/B

amHI-D

fragment

(co-ordinates

30.0

to 32.3)

This fragment was required in 2 forms: uniformly labeled along its length, and tagged specifically at its left end (the BamHI site at position 30.0). Both preparations started with the BamHI-D fragment (30.0 to 42.8). Uniform labeling was achieved by “nickeoxynucleoside triphosphates (New England translation” (Rigby et al., 1977) with [a-32P]d Nuclear) in the presence of DNA polymerase I (Boehringer) and DNase I using the conditions established by Maniatis et al. (1975a,1976). The 5’ termini were labeled with [c+~~P]ATP using T4 polynucleotide kinase in the exchange reaction described by van de Sande et aE. (1973) and Chaconas et al. (1975). In both cases, the labeled BamHI-D fragment was separated from the radioactive precursors by gel filtration through Sephadex

SMALL

RNAs

OF

ADENOVIRUS-INFECTED

CELLS

297

G50 in TE buffer, and was digested with endonuclease Hind111 yielding 4 fragments: HindIII-B/BanaHI-D (30.0 to 32.3), HindIII-I (32.3 to 381), HindIII-J (38.1 to 418). and BarnHI-D/HindIII-D (41.8 to 428). The mapping of the fmgments represents unpublished work by R. Greene, M. B. Mathews, C. Mulder, R. J. Roberts, J. Sambrook and P. Sharp. In the digestion products of the fmgment exposed to polynucleotide kinase, only the 2 terminal fragments were labeled at their BumHI-cleaved 5’ termini; whereas all 4 products were uniformly labeled in digests of nick-translated BarnHI-D. The fragments were separated by electrophoresis in 1.4O/, agarose gels and elut,ed as above. (v)

Xizing

of DNA

fragments

The estimated chain lengths given here represent the mean of several determinations made in a number of gel systems: 1.4% agarose and 3.57; and 5% polyacrylamide gels prepared as described above ; 20/ agarose gels cast in TBE buffer ; and 7 ye polyacrylamide gels run in TBM buffer according to Maniatis et al. (19756). Double-stranded phage +X174 replicative form DNA, digested with endonuclease Hind11 and kindly supplied by M. Zabrau, provided sequenced markers of known chairi length (Sanger et al., 1977). (0)

(i) Nitrocellulose

Hybridization

methods

jilters

Ad2 DNA or its fragments were denatured in 0.3 N-NaOH, neutralized and bound to nitrocelhilose circles (Schleicher and Schiill, B6) by slow filtration according to Gillespie & Spiegelman (1965). The dry filters were baked in a vacuum oven at 80°C for 4 to 10 h, and incubated with radioactive RNA for 12 to 15 h at 68°C in a solution containing 6 x SSC (SSC is 0.15 M-NaCl, 0.015 M-sodium citrate, pH 8.3), 0.5% SDS, 2 mM-EDTA and 20 pg yeast RNA/ml. Non-specifically bound RNA was removed by exhaustive washing in 2 x SSC, followed by further washes at room temperature. Protruding unhybridized tails of RNA were trimmed by incubation at 37°C for 1 h in 2 x SSC with 20 rg RNase T,/ml, followed by more washing at room temperature. For fingerprint analysis, RNA was dissociated from the filters by heating for 90 s with 3 Y 0.5 ml of TE buffer containing 50 rg of yeast tRNA. Following alcohol precipitation, insoluble matter was removed by centrifu gation . (ii) Nitrocellulose

sheets

DNA was transferred from 1.4% agarose gels to sheets of nitrocellulose (Schleicher and Schiill. B6) essentially as described by Southern (1975b). After denaturation in 0.2 MNaOH, 0.6 M-NaCl for 45 min, and neutralization in 1 ivr-Tris.HCl (pH 7.4), 0.6 M-NaCl for 45 min, the DNA was blotted onto the nitrocellulose membrane in a stream of 6 x SSC. The membrane was dried and baked as above (section (e) (i)). Screening experiments were carried out as illustrated in Figure 1. The membrane was moistened with 2 x SSC and sliced into longitudinal strips about 3 mm wide, each of which was hybridized to labeled RNA under the conditions given above. When the entire membrane was hybridized to a single RNA probe, the moistened nitrocellulose sheet was formed into a cylinder and inserted into a tube containing 3 ml of hybridization mixture. The tube was stoppered and incubated at 68°C while being rotated on its long axis to ensure even exposure to the medium. Both strips and sheets of nitrocellulose membrane were washed as above, taped to filter paper, dried and subjected to autoradiography. (iii)

Hybridization

in solution

For the competition hybridization experiment (Results, section (f)), annealing was carried out in solution at 65°C in 100 ~1 of 0.05 M-Tris.HCl (pH 7.5), 1 M-NaCl, 0.1% SDS and 1 mM-EDTA. Ad2 r-strand DNA, prepared as described by Tibbetts et aZ. (1974), was first hybridized with different preparations of 3H-labeled RNA for 5 h at 65°C. Then 5 ~1 (15,000 cts/min) of 12sI-labeled VA RNA,, prepared as described by Pettersson & Philipson (1975), was added to the hybridization mixture. After an additional 18 h at 65°C the hybridization mixture was diluted lo-fold with a buffer which contained 0.15 Jr-NaCl, 0.05 M-Tris*HCl (pH 7.5) and 1 mM-EDTA. Pancreatic RNase and RNase T,

29s

M. Ad2

DNA

Fragments

B.

MATHEWS

AND

U.

PETTERSSOK pP]cytoplasmvz

p C %LXW

I

Digestloo restriction

by eruymo

L Sucrose gradlent sedimentation

aQQ&ww&&

/

I %trophoresls

RNA

I

Polyacrylamlde electrophoresls

gel

,

Agame gel slab Fraction

‘---D

.I.-“-

one

I

,--A

Autoradlogram nrlrlrlrlrlr-

* cf hybrIdned

strips

Rephca of banding pattern on membrone

FIG.

mobilized

1. Scheme for screening restriction fragments

RNA fractions of Ad2 I>ICA.

from

gels or gradients

by hybridization

to im-

were added to final concentrations of 20 rg/ml and 40 units/ml, respectively, atid tlrc: samples were incubated at 37°C for 1 11. The reaction was stopped by addition of SDS to (%5o/o and the samples were fractionated by chromat.ography on Sephadrx Cl00 in 0.15 M-NaCl, O*O5M-‘rris.HCl (pH 7.5), 1 rn%r-EDTA. Fractions of 0.5 ml wem collected ant1 radioactivity was determined in a gammaspectrophotorneter. VA RNA, hybridized to r-strand DNA elutes in the void volume of tlrc column whereas t,hc oligonuclcotides derived from unhybridized RNA clute rrchar the t,otal volume of t,hc col~~nn~, (f) K,\TA

jingeiyrinting

Labeled RNA, together with 50 pg carrier yeast transfer RNA, was digested in 10 ~1 TE buffer with 5 pg of RNase T, or pancreatic RNase. Fingerprints were prepared by electrophoresis on cellulose acetate paper at pH 3.5 in 7 InM-llrt?a followed by a, second dimension on DEAE-cellulose paper in 7”/0 formic acid, as described by Sanger et al. (1965).

3. Results (a) Kinetics

of VA RNA

Synthesis

To achieve satisfactory resolution of low molecular weight RNAs we have used two types of polyacrylamide gels, both separately and in a two-dimensional combination. One type of gel contains glycerol and Sarkosyl, and is run at 4°C conditions which would be expected to favor the maintenance or acquisition of nucleic acid secondary structure: such gels are referred to here as native gels. The second type is called a denaturing gel. Before loading, the RNA is heated in formamide to destroy secondary structure, re-acquisition of which is discouraged by the presence of 7 Murea in the gel. A comparison of 32P-labeled cytoplasmic RNA from infected and uninfected cells is shown in Figure 2. In native gels the cytoplasm of uninfected cells yields three principa~l regions of radioactive RNA which are inva,riably prcscnt8, and a number of

SMALL

OF

~~I)ENOVlRCS-lNFEClTE1)

299

(‘ELLS

Infected

L

:: I

RNAs

B

IO

12

15

Hour 5 D.I.

>

-

-> >

‘S-

VA .VA

I II

.5*85-5s

-

rANA

-4s tRNA

0

b

c Nattve

d

e

f Dewturing

FIG. 2. Gel electrophoresis of low molecular weight cytoplasrnic RN.1 from uninfected and Ad2-infected cells. Gel A was run in native conditions (see Materials and Methods) and contained RNA from: uninfected cells labeled for 14 h (track a) ; infected cells labeled with [32P]phosphatc: from 2 h p.i. until harvesting at 8 h p.i. (track b); 10 h p.i. (track c); 12 h p.i. (track d); and 15 h p.i. (t,rack e). The amount of RNA loaded onto tracks a and e was twice the amount applied to tracks b to d. Gel B was run under denaturing conditions and contained denatured RNA from: infected cells labeled from 2 to 15 h pi. (track f); the VA RNA, band (track g) and VA RNA,, band (t,rack h) of a gel run in native conditions. In all Figs, VA1 --. \‘A Rh’4, and V.411 :~ VA RNArr.

300

M.

B.

MATHEWS

AND

U.

PETTERSSON

minor or variable bands (Fig. 2a). The major regions represent: tRNA, comprising a prominent series of bands migrating at approximately 4 S: ribosomal 5 S RNA: with a satellite band containing its slightly faster moving conformational isomer (Hindley, 1967) ; and a group of three bands, not always clearly resolved from one another, with electrophoretic mobilities corresponding to a sedimentation rate of about 7 S. Minor bands can be found in many areas of the gel : some are present in most RNA preparations, while others are less reproducible. For the purposes of this paper only two are significant: these represent the ribosomal 58 S and nuclear U-2 RNAs. both of which are more clearly seen in two-dimensional gels and will be discussed below. Additionally, at the top of the gel there is an area of intense radioactivity which contains high molecular weight or aggregated material which is unable to enter the gel. Cytoplasmic extracts prepared from infected cells exhibit the same major RNA bands, and probably share many of the minor bands too. Two new bands are prominent in autoradiograms of native gels: an intense, unusually broad band which contains the well-known viral 5.5 S RNA or VA RNA,; and a band labeled VA RNA,, which contains the viral 5.2 S RNA species described by Mathews (1975) and Siiderlund et al. (1976). Both species are apparent at eight hours post-infection, which is two hours after the onset of viral DNA synthesis under our conditions, and by 15 hours post-infection they account for a large fraction of the total label in low molecular weight cytoplasmic RNA (Fig. 2b to e). The other outstanding change that is apparent in autoradiograms of infected cell RNA is the dramatic increase of radioactivity in the 5.8 S RNA band 12 hours after infection and its decline three hours later (Fig. 2), a phenomenon which was observed in each of three experiments. When RNA extracted from infected cells is run in denaturing gels, a number of changes take place: some bands disappear, some new ones appear, and others interchange position (Fig. 2f to h). The group of tRNA bands redistribute, and an intense band of lower mobility breaks away from the cluster. The two 5 S RNA bands merge into a single band, as would be expected for conformational isomers. The disperse VA RNA, band resolves into two bands. Little alteration is visible in the regions of the 7 S RNA bands, but two new bands of lower mobility appear further up the gel. xuperimposed on a background of radioactivity which increases towards the top of the gel. It is shown elsewhere (Pett’ersson & Mathews. 1977) that the faster moving of these contains a viral 9 S messenger RNA. The region between the 5 S RNA and VA RNA, bands contains three bands. The fastest moving of these is a faint band which migrates slightly slower than 5 8 RNA in the denaturing gel, although it fails to separate from 5 S RNA in the native gel system: this species, called 5 S RNA (c), is shown clearly by two-dimensional electrophoresis (see below). Of t’he remaining two, more intense, bands the slower resembles the 5.8 S species in that it is present’ in extracts of uninfected cells as well as of infected cells, and in that’ its level increases sharply 12 hours post-infection and declines by 15 hours (data not shown). The faster migrating band of the pair is absent from uninfected cell extracts, and would therefore be expected to correspond to the minor viral RNA species. Indeed, RNA eluted from the VA RNA,, band of a native gel contains a major component which comigrates with this band in the denaturing system (Fig. 2h). Thus it would seem that the relative mobilit,ies of the 5.8 8 RNA and VA RNA,, are reversed in denaturing gels. To gain increased resolution of the RNA species and establish their relationships more firmly, we subjected the samples to elect.rophoresis in two dimensions.

SM.ZLL

RNAs

OF

AnENOVIRUS-INFECTEI)

(b) Two-dimensional

CELLS

RNA

no1

gels

For t,wo-dimensional separations, the RNA is first run in a 10:; polyacrylamide gel under native conditions. Following electrophoresis and autoradiography, the region of interest is excised as a strip which is soaked in urea. positioned on top of a 12% polyacrylamide gel containing 7 M-urea and subjected to electrophoresis at right angles to the original direction. This system affords substantially improved resolution. as can be seen from Figure 3. The individual species appear as discrete spots many of which deviate from the diagonal. Vertical streaks, or a chain of spots of lesser intensity running faster in the second dimension than a major spot, are common: presumably these result from hidden breaks, nicks in the RNA chains which are masked by the secondary structure but revealed after denaturation. In addition. horizontal streaks also become apparent after long autoradiographic exposure. These extend from major spots towards the high molecular weight region of the first dimension, and are probably attributable to the presence of aggregated forms in the native gels. The RNA species contained in such structures would run spuriously slowly in the first dimension, but migrate with their characteristic mobility in the second dimension after disruption of the aggregates by urea. Discounting derivatives which are related in these ways, more than a dozen spots are apparent in a two-dimensional gel of RNA from uninfected cells (Fig. 3A). Three 7 8 RNA species are separated: their chain lengths are estimated to be 325. 305 and 1977). The 5.8 S RNA also resolves into 300 nucleotides (Pettersson & Mathews, three spots. The two main 5 S RNA forms have the same mobility and the same chain of breakdown derivatives as one another in the second dimension. The 5 S RNA (c) snd U-2 RNA species both form distinct, spots. Gels of RNA isolated from infected cells 12 hours after infection have a more complicated appearance, but contain only three or four spots (together with their derivatives) that are absent from gels of uninfected cell RNA (Fig. 3B). Two of these new spots arise from VA RNA, which is resolved into two forms by virtue of small mobility differences in both dimensions. Calibration of the denaturing gel dimension suggests that the two forms differ in chain length by about four nucleotides. The third spot unique to infected cell extracts contains VA RNA,,: in some gels this spot was also partially resolved into two (data not shown). Relative to 58 S RNA, VA RNA,, runs slower in the native gel and faster in the denaturing gel, as predicted from the one-dimensional separations shown above (Fig. 2). All the remaining spots seem to correspond to spots present in uninfected rell extracts.

(c) Fingerprint

analysis

of RNA

species

One way in which the relatedness of RNA species can be assessed is by comparison of their RNase T, fingerprints. Following gel electrophoresis in one or two dimensions, the RNA is eluted from the gel, digested to completion with RNase T,, and resolved into its constituent oligonucleotides by two-dimensional paper electrophoresis (fingerprinting). Further information is gained by interpolating a molecular hybridization step after the gel electrophoresis. Examination of the fingerprints of RNA species selected on Ad2 DNA permits discrimination between viral and host transcripts. Figures 4, 5 and 7 present the results of such an analysis for a number of RNAs from infected and uninfected cells.

lh. 3. Two-dimensional gel separations of low molecular weight cytoplasmic RX.ls. -1: Uninfected cell RNA labeled for 10 h. R : Ad2-infected labeled 2 to 12 h p.i. The first dimension was a native gel, followed by a denaturing gel in the second dimension as described in Materials In the body of the Figure the RK’d species are indicated by coded arrowheads (for which the lwy is shown above the autoradiograms), and electrophoretic forms are designated by alphabetical suffixes.

Mock

cell RNA, and Methods. the individual

SMALL

RNAs

OF

AnENOVIRUS-TNFEC1’EI)

CELLS

:m

(i) i’ S RNAs Cytoplasmic RNA from infected cells was fractionated on a native gel. Fingerprints of the RNA eluted from two of the 7 S RNA bands are shown in Figure 4A and B. The patterns are different, indicating that the 7 8 RNA (a) and (c) bands contain distinct species of RNA, although neither RNA is pure as evidenced by the presence of trace oligonucleotides in both. The fingerprints of 7 S RNA (c) is very similar, but perhaps not identical, to that obtained by Walker et al. (1974) for a 7 S RNA species from mouse L cells. Fingerprints of the 7 S RNA (b) band (not, shown) are complex and appear to contain some oligonucleotides characteristic of the species found in both the 7 S RNA (a) and (c) bands. Only a small fraction of the radioactivity in the 7 S RNA bands from infected cell cytoplasm hybridizes to nitrocellulose filters bearing Ad2 DNA. Fingerprints of the hybridized material do not resemble those in either Figure 4A or 4B, but are very similar to fingerprints of VA RNA, (see below). These results show that the 7 S RNAs are not viral. Prolonged exposure of two-dimensional gels reveals that traces of VA RNA1 trail into the 7 S RNA region of the first gel, accounting for the hybridization findings: this is better illustrated in section (e) below by hybridization to restriction fragments. (ii) 1=4 RNA, The RN-4 contained in the \:A RNA, band of it native gel gives the fingerprint published by Ohe & Weissman (1971) andMathews (1975). The two major components resolved by electrophoresis on two-dimensiona,l gels, VA RNA, (a) and (b), present fingerprints which are virtually identical to one another and can be distinguished from that of VA RNA, fractionated in one dimension only by their freedom from contaminating background spots (Fig. 5D). The VA RNA, (c) species of Figure 3B also gives a very similar fingerprint (not shown). This species, which corn&rated with VA RNA, in the first dimension of a two-dimensional gel, but runs with a mobility similar to that of 5 S RNA in the second dimension, is clearly a breakdown product. The fingerprint of VA RNA, is not, altered by hybridization of the RNA to Ad2 RNA. (iii)

11-Z RNA

The U-2 RNA from a two-dimensional gel of cytoplasmic RNA from uninfected cell RNA gave the fingerprint shown in Figure 4D. This pattern is virtually identical to that obtained by Shibata et al. (1975) for U-2 RNA purified from the nuclei of rat hepatoma cells. We have not prepared enough of the RNA t’o attempt a hybridization selection. but its host cell origin is not in question. (iv) l’Z4 RNA,, The RNase T, fingerprint of VA RNA,, isolated by two-dimensional electrophoresis is shown in Figure 5A. This fingerprint 1, ‘c: ident’ical to that obtained by Mathews (1975) following hybridization of infected cell RNA to the BaZI-B restriction fragments of Ad2 DNA, but differs from that published by Soderlund et al. (1976). In t,he latter study the RNA was analyzed after one-dimensional separation in a native gel and was therefore probably contaminated with other species. Both the RNase T, and the pancreatic RNase fingerprints (Fig. 5B) of VA RKA,, differ markedly from the equivalent8 fingerprint~s of VA RNA,. The drt8ailed a.nalysis presented

A

B 7SRNA

(01

5.8s

RNA

C

TSRNA(c)

3 U-2

RNA

FIG. 4. RNase T1 fingerprints of some cytoplavmic RNA specw~ of host cell origin. A and R: 7 S RNA (a) and (c) species, respectively, from a native gel containing infected cell RNA. C:: 5.8 S RNA (a), from a 2-dimensional gel of infected cell RNA. D : U-2 RNA, from a Z-dimensional gel of uninfected cell RNA. Digests were fractionated by electrophoresis in the first dimension on cellulose acetate paper at pH 3.5 in 7 ~-urea from left to right,, and in the second dimension on DEAE-cellulose paper in ‘7% formic acid from bottom to top.

A

VA RNA=

VA f?NA,

T,

D

VARNAI

Pane.

(a)

2

Fig. 5. Fingerprints of viral RNA species. A and B: RKia~e of VA RNAIl purified by gel electrophoresis in 2 dimensions. of the RNase T1 oligonucleotides from VA RNArr. D: RNaw nftor 2-dimensional gel aleotrophoresis.

T, and pancreatic RNase digests C: Diagram giving the numbering T, digest of VA RN-AI (a) isolate- a more limited distribution in the neighbourhood of their gel band, where t’hey are present in greatest quantity. The 9 S mRNA is found principally in fra.ctions 2 and 3. with smaller amounts extending down the gel t’o fraction 6 or 7. The same RNA gel fractions also give rise to low levels of hybridization a-it)h a number of other DNA fragments : this is certainly attributable to penetration of the upper layers of t’he gel by viral messenger RNAs. the great majority of which are trapped at the top of the RNA gel. These rnR,RAs HI-C responsible for the intense hybridization to severa! DN=\ fragments which is seen on the strip incubated with RN’S from fraction 1. Similar results are obtained with RNA separat,ed in denaturing gels (data not, shown ). tinder these conditions VA RNA, sequences are found in the high molecular weight region of the gel in rather smaller quantity. presumably because aggregates containing this species have been disrupted. as noted above, Comparable screening experiments have been carried out, with Ad2 DNA digested by six other restriction c’rlzymc’s. RcoRl. Ba.mHT. /ip~I. HindTIT. HindTT and &ZhoI. Tn no case was

_____.

-

-

-

-

I

T

f

.+.

10

11

14

15

16

17

18

-

-

-

_ ~ .~

Alkaline UP

8a b

b

5a

4

3

2

1

Olinonucleotide number*

A

-

i

-

-

5.0

5.0

1.2

+

hydrolysis

6 + -t

-

+

-

1.1

1.2

2.2

t

+

-

product@

Analysis

+

t

+

-

1

1

1

+

1 + GP

1 + GP

GP

i- (CP)x.o

”

done)

+ (ApCp)o-s

+ (APCP),.,

(UPII.4

-!, (CP)P.B

(UP) I 4 + APGP

(UP)l.D

Wp)l.s

(Not

(CP)Z-5

(Cp)s.l

(CP)I.Z + APAPGP (APAPCP) + GP

t

+ Gp

i- GP

CP

1

[UP,

deducedd

-~PCPIGP

ApCplGp

Cp)Gp

(CPLIGP

~PAPGP

(UP,

UPGP

PGP?)

[(CP)~-S~

[(Cph,

CPAPAPGP APAPCPGP

CPAPGP A~PCPGP

L~PGP

CPCPGP

Structure

of VA RNA,,

CpGp

GP

T, digestion

productsc

by RNase

digestion

-i- GP

RNase

obtained

(CP)I 1 + SPGP (APCP),., + GP

-~PGP

(CP),

+

(CP),

+

GP

+ i

GP

Pancreatic

of oligonucleotides

TABLE

3.7

1

1

1 2.6

o-1 ‘1 (j

O-1

1

1

1 1g

1 1

4

3

3

20

Copies’ per molecule

ratio

O-6

0.5

4.6

4.1

4.3

5.1

i-9

8.8

6.0

19.6

Molar Ijucleotides” per molecule

1.0

1.2

+

1.6

2.3

2.0

2.2

3.0

7.8

+

20

23

24

25

26

27

30

31

34

35

-

2.4

4.8

3.8

2.1

1.3

0.5

-

6.1

0.7

0.8

-

3.7

2.4

-

1.2

.-

-

0.9

3.5

+

1

1

1

1

1

1

-!-

1

1

1 f

(ApCp),.,

i Gp

+ ApGp

+ (CPL.3

+- (APUP),.,

+ (Cp11.e + CP

-c GP

+ (Cp11.3

i- Gp

Wp)s.o

(UP),.,

(UP)W

(Cp)s.T

+ GP

+ (Cp)l.s

i + (AP~PL.O

-t- (APAP-~PUP),.,

(UP),., + (Cp)4.3 + GP

(UP),.,

UJp11.3

(U~12.5

(UP,,.,

(UP),., + (Cp)s.~ + Gp

(UP),.,

(APAPAPCPL.,

+ GP + ApApCp

+ Gp

2.8

UPUPGP

APAPAPUPIGP

6.2

(CPL

(CP)S-s,

UPUPUPGP

[WP)S-7,

[WP)Z,

[(UP)Z, (CPLIGP

APUPIAPAPGP

4.6

(CPMGP

3.7

9.2

10.6

6.3

4.1

CP, APUPJGP

(UP, [(UP),,

CPIGP

[(UP,,.

8.2

3.3

(UP, APCP) GP

3.2

WP, (CP)S-SIGP

ApApApCpApGp

1

1

1

1

1

1

2

1

1

1

1

a See Fig. 5C. b Base compositions are expressed as mol of nucleotide per mol of Gp. d + indicates the presence of a nucleotide; a - indicates that no significant amount of tho product was observed. c Compositions are expressed as mol of product per mol of Gp or of oligonucleotide containing Gp. Mean of 2 determinations. d Takes into account position of oligonucleotide on paper and thin-layer homochromatography fingerprints as well as composition data. e The total number of nucleotides in rach oligonucleotidc, expressed per molecule of RNA, and calculated assuming a chain length of 135 bases. Mean of 3 determinations. f The copy number for each oligonucleotide is the number of nucleotides per molecule divided by the number of bases in that oligonucleotide, and rounded to the nearest integer. @The recovery of ApApCpGp was always low. Its presence in molar yield is supported by data from pancreatic RNase digestion of VA RNA,,. In some fingerprints, spots 8a and 8b separated from one another. b Tentatively identified from its position on the fingerprints.

-

19

75

I

I

VA1 1

I 2

I 3

VAD I

I 4

55 I

I 5

4s

__--

i

i 7

6

I El’9’

I

IO

I I

,

I I I I I I I I

1-

I-

0

I

FIa. 6. Hybridization RNA from AdB-infected gel under native conditions radiogram of the gel is divided into fractions as with filters carrying 10 resistant radioactivity (-v-v--, cts/min x x 1Om6) for each fraction.

2

to Ad2 cells (at shown indicated. pg Ad2 hybridized 10e4)

3

4

I I

\ :

I

>,

5

6

7

Gel slice

number

0

8

9

IO

II

DNA of RNA fraction* from a polyacrylamide gel. C!ytoplasmic was subjected to electrophoresis through an 18% polyacrylamide room t,emperat,uro wit,h glycerol and Sarkosyl omitted). An autoat the top of the Figure, wit,h it,s origin on the left. The gel was Each fraction was crushed and the RNA within it was incubated DNA or phage X DNA (see Materials and Methods). The RNanoto Ad2 DNA (--e-a-, cts/min x 1W4) and h DNA are plotted, together with the input counts ( (_ ;s ~-~ :; --, cts/min

A

C

Frn 3

0

Fm 6

D

Frn 5

Fm IO

J?‘~G. 7. Vingorprints of RNA fractions after selectiurl by hybridization to Add DNA. The ltrl’d hybridized to Ad2 DNA in the experimmt of Fig. 6 wan cluted. digested w&h RNase T, and fingerprinted aa in Pig. 4. .I: Fraction 3 ( - 7 S). H: Fraction 5 ( 4 A.5 R). C: Frsct,irm 6 (+ 5.2 S). 13: l+action 10 (-4 S).

VAII -

5s

7s

4s I

I

I I I I I

I I I I I I 5 IO

I

I I 14

1111,111 18

I

I I 22

I

I 25

I

I

I

I 29

Bal I

-

(portiol)

-G

--M

HinfI

-1100 -580 -560

SMrZLL

RN.4s

OF

rlI)ENOVTRUS-INFECTEI>

C’ELTJS

313

hybridization detected to any fragment other than those which hybridize to the VA RNAs and the 9 S mRNA. This experiment, then, also fails t)o evidence the existence of additional species of low molecular weight viral RNA. (f) VA RNA

sequences in high molecular

weight

RNA

In the foregoing studies we consistently found VA RNA, sequences in the high molecular weight range of polyacrylamide gels, and t,heir aberrant mobility did not appear to be wholly attributable t’o aggregation with other species. These results suggest, that V9 RNA, may form part of some longer RNA species. To investigate this possibility we have carried out hybridization competjition experiments between cytoplasmic mRNA or high molecular weight nuclear RNA on the one hand, and 1251-labeled VA RNA, on the other. Poly (A)- containing mRNA and HnRNA were labeled with [3H]adenosine, isolated by standard techniques and fractionated under tlenat uring conditions by electrophoresis through polyacrylamide gels containing 98’: ;, formamide. As expected, the HnRNA contained very high molecular weight species (Fig. 9), most of which migrated as a sharp peak close t,o the top of the gel because of their large size. To ensure that this material did not trap VA RNA, the HnRNA was stored in 95% formamide at -20°C for six days. resulting in sufficient degradation or disaggregation to allow most of the nuclear RNA4 to penetrate the gel matrix further (Fig. 9(b)). Fractions containing high molecular weight HnRNA and mRh’A were pooled as indicated and used in hybridization experiments with single-stranded Ad2 DNA. The r-strand DNA was presaturated with various preparations of RNA by annealing in solution for five hours: subsequently, 1251-laheled VA RNA, was added and the mixture was incubated for a further 18 hours. Hybridization of the labeled VA RNA was measured by gel filtrat,ion following digestion of single-stranded RNA by incubation with pancreatic and T, ribonucleases : the hybrid duplex is well separated from the oligonucleotide degradation products (Fig. 10). The results, shown in Figure 10 and summarized in Table 2, show that presaturation with HnRNA from Ad2-infected cells blocks thca subsequent1 hybridization of VA RNA: on the other hand, mRNA exhibits little or no competition with the labeled probe. Nuclear RNA from uninfected cells wa,s not inhibitory: and the efficacy of tjht infected cell HnRNA was abolished by alkaline llydrolysis: these controls demonst’ratc the specificity of the competition and exclude t’he possibility that contaminating viral DNA is responsible for it. Thus \‘A RN$, sequences appear to be present in rigorously purified HnRNA from infected cells, and to he absent or present in much lower concentration in the mRNB fraction. The VA RKA, sequences found in high molecular weight cytoplasmic RNA are unlikely to derive from polyadenylabed

FIG. 8. Hybridization to restriction endonuclease fragments from Ad2 DNA of RNA fractions from a polyacrylamide gel. Low molecular weight cytoplasmic RNA labeled from 2 to 12 h p.i. was separated in a 12% polyacrylamide gel run under native conditions. An autoradiogram of the gel is shown at the top of the Figure, with its origin on the left. Fractions were taken as shown, crushed, and incubated under conditions for RNA hybridization with strips of nitrocellulose membrane carrying fragments of Ad2 DNA generated by digestion with the restriction enzymes indicated (see Fig. 1 and Materials and Methods). Photographs of the patterns of stained DNA fragments, resolved in agarose gels, before transfer to the membrane arc shown at the left. The body of the Figure consists of an autoradiogram of the nitrocellulose strips after hybridization with [s2P]RNA from the gel fractions. The fragments which hybridize to the 9 S mRNA (B&I-G, HinfI 580, HphI 900), VA RNA, (BaZI-B and -M, Hitif1 560. I HphI 590), and VA RNA,, (B&I-B, HinfI 560 and 1100, H&I 590), are named on the right,.

i IO

20

30

40

50

60

70

80

FIG. 9. Fractionation of nuclear and polysomal RNA from infected cells by gel electrophoresis. Gels were run in 9fi% formamide as described in Materials and Methods. ‘251-labeled GA RNA, was added to the [3H]adenosinc3-labeled samples as a marker. After electrophoresis, the gel vas cut into 2.mm slices which were elutetl. Portions of tho eluatc WCTP analyzed for radioactivity: -e-e-, 3H; -~ ;I-c;--, 1251, (a) Poly(A-containing (b) As (a), but (c) Poly(A)-containing

after

cytoplasmic mRNA, the VA RNA region.

nuclear RNA. storage of the RNA polysomal RKA.

for

6 days

but may be related (g) Map position

at

- 20°C’

in 9576

to large nuclear

formamitiv.

transcripts

which

include

of VA KNA,,

Previous work has shown that the gene for VA RNA, maps at position 30.0 on t’hrx Ad2 genome, where it straddles a BamHI restriction enzyme cleavage site (Mathews, 1975). The gene for VA RNA,, has not been located with equal precision: it map.3 to the right of the minor species, in the 800 bp region between the BamHI site and a Hind111 cleavage site at position 32.3 (Mathews, 1975; Sijderlund et al., 1976). Thesr results are illustrated in Figure 11A: which shows the hybridization of VA RNAs to fragments derived from Ad2 DNA by double digestion with the enzymes BamHl and HindIII. VA RNA,, hybridizes exclusively to the BamHI-D/HindlIT-B fragment (map co-ordinates 304 to 32.3). while VA RNA, hybridizes t’o this fragment, and to

l500-

1000

(b)

-

,-

Fractm

numbev

10. Fractionation of hybridized 1251-labeled VA RNA, by gel filtration. The hybridization, RNase treatment and chromatography conditions are described in Materials and Methods. VA RNA, elutes in the void volume, fractions 9 to 17, approx., whereas oligonucleotide fragments resulting from the RNase treatment elute near the total volume of the column. (a) [1a51]VA RNA, incubated without DNA. (b) [lz51]VA RNA, hybridized with 0.09 pg r-strand DNA. (c) [lz51]VA RNA, hybridized with 0.09 pg r-strand DNA after presaturation with 0.9 pg HnRNA from Ad2infected cells. (d) [?K]VA RNA, hybridized with 0.09 pg r-strand DNA after presaturation with 2.8 pg mRNA from AdZ-infected cells. FIG.

the BanzHI-B/HindIII-B fragment (co-ordinates 17.0 to 30.0). Earlier studies also suggested that the gene for VA RNA, probably lies to the right of the Bali restriction site at position 30.3, 120 bp from the Bam,HI site. This conclusion is verified by the data of Figure llA, which show that VA RNA,, h.vbridizes t,o the B&-B fragment (co-ordinates 30.3 to 50.3), whereas V.4 RSA, hvhridizes t,o t,he B&T-M fragment (co-ordinates 28.8 to 30.3).

816

hl.

13. MATHEWS

AND TABLE

IT.

PETTERSSOX

2

Hybridizations between 1251-labeled 1’8 RNA, presaturated with different preparatiom Amount of r-strand DNA (rg)

Amount

0.09 0 0.09 0.09 0.09 0.09 0.09 0.09 0.09 0.09 t The RNA 1 The RNA

of anlabelr(l (P!z)

and r-strand of unlabeled

12”I-labeled Br\‘A in void volumt~ (cts/min)

RNAt

0 0 0.3 0.9 S.6 1.8 0.6 2.1 1.4

pg pg pg pg pg pg pg 2.8 pg

was pooled mas boiled

Ad2 HnRN-4 pool (a) Ad2 HnRNA pool (a) Ad2 HnRNA pool (a) hydrolyzed Ad2 HnRNA Ad2 HnRNA pool (b) HnRNA from uninfected Ad2 mRNA pool (c) Ad2 mRN.4 pool (c) as shown in Fig. 9. for 15 min in 0.3~--NaOH,

DNA RNA

oj’ Adz,

Tnhibition (“i,)

0 100 62 93 9x 0 9:s 0 0 12

“965

11 112:3 ?I0 fi”

pool

(a):

3 1:30 208 3067

cells

3051 2601

then

n~~~tralizecl,

hrforc,

hybridization.

More precise location of the VA RNA,, gent requires a knowledge of restriction sites in the interval between the Ball cleavage at 30.3 and the Hind111 cleavage at 32.3. We have found three enzymes which make a single cut in this region, HphI, MboI and Hinfl, and have mapped their cleavage sites by digestion of the BarnHID/HindIII-B fragment (co-ordinates 30.0 to 32.3) wit’h each enzyme. In order to gather enough information to order the digestion products unambiguously, it was necessary to prepare the fragment in two forms. one labeled uniformly along its length by “nick-translation”, and the other specifically labeled at, its left end by “kinase-labeling” at the BaniHl clravagc site (see MatSerials and Methods). The results arc shown in Figures 11 and 1%. and t.he conclusions schcmat,icall,y represented in Figure 15. Digestion of Ad2 DNA with the :MOol restriction enzyme gives rise to one fragment of 610 bp which hybridizes to both VA RNAs, and a fragment of 900 bp which only hybridizes to VA RNA, (Fig. 11A). E‘rom what is already known about the relative positions of the two VA RNA genes, the 900 bp fragment must lie to the left of the 610 bp fragment. Digestion of the BamHI-D/HindIII-B fragment with MboI yields three fragments, of sizes 610, 180 and 10 bp (Fig. 12). The latter carries the end label. and its existence is consistent with the sequence of VA RNA, (Ohe & Weissman, 1971) and of the MboI cleavage site (Gelinas et al.. 1977a), but it is so small that it has run FIG. 11. Hybridization of VA RN& to Ad2 DNA fragments generated by digestion with restriction endonucleases. In A, track (a) shows a photograph of the stained DNA fragments resolved by agarose gel electrophoresis; t,racks (b) and (c) contain autoradiograms of nitrocellulose membrane strips carrying the DNA fragments transferred by blotting, after hybridization to VA RNA, and VA RNA,, RNA, respectively. In B, the portion on the left is a photograph of stained DNA fragments; the portion on the right is an autoradiogram of the corresponding nitrocellulose membrane containing the DNA fragments transferred by blotting and hybridized to (a) VA RNA,, (b) to (d) VA RNA,,. Tracks (a) and (c) contain digests of Ad2 DNA by HinfI; tracks (b) and (d) contain the Hinf 1100 and 660 bp fragments, respectively, isolated on a preparative polyacrylamide gel.

R

TEI) A

HindIII t

MboI

(‘ELLS HohI

BornHI VA1 VAII

VA1 VAl

3/D

b

0

c

(I

B

b

c

b

HmfI VA1 I-

IIDO

L

-

560

0

b

c Stamed

d

31X

MI.

B.

MATHEWS

r 2 b E :

i\NU

8~O/Hindm H/nfI

IJ.

PETTERSSON

El tro~mcnt

AtboX

HphI

UIUIJ?

---K

NT

K

NT

K

NT

K

NT

800

610 580 470

330

,180

Fro. 1% Digests of the HindIII-B/Barn-D fragment (co-ordinates 30.0 to 33.3). The fragment was uniformly labeled by nick-translation (NT); or specifically labeled at its left-hand end, the BnrnHI site at position 30.0, by the polynucleotide kinase reaction (K). The Figure shows an autoradiogram of a 5% polyacrylamide gel, in which fragments of 4X174 double-stranded DNA obtained by Hind11 dig&ion were run as markers. Tho sizes indicatcct on thr right arc the means of 3 to 6 det.erminatwns in bot.h agerose and ~~r~lyacr,ylamidr gclw.

SMAI~I,

RNAs

OF

;\T~ENOT’TK~‘S-I1;Pl~~~‘~~l~

(‘EI,I,S

DI!b

off the gel in Figure 12. These data indicate that JlhoI sites are found 10 bp and 620 bp t,o the right of t,he Bnm.HT sitcb. and 890 bp t,o its left. Thus t’hc gene for \‘A RNA,, ca,nnot lie more than 620 bp to t,he right of the BnnaHl sit,e at position 30.0. The H@I digest of Ad2 DNA contains a single DNA fragment of 590 bp which hybridizes to both VA RNAs (Fig. 11A). Th is enzyme cleaves the BarnHI-D/HindIIIB fragment into two fragments 470 and 330 bp ion,.v of which the former contains the clnd label (Fig. 12). Therefore the 580 bp fragment must, ext’end from 120 bp to the Ifaft of the BamHl site to 470 bp to it’s right, and thcl gene for the VA RNA,, must lit, \\ithin the region between the BaZI and H$l sites. 120 and 470 bp to the right’ oft he Bzn~Hl site at position 30.0. Digests of Ad2 DNA with H,irbfI contain a fragment of 560 hp which hybridizrs to both VA RNAs. and a fragment 1100 bp in length which hybridizes solely to VA RSA,, (Fig. 11B). Clearly the HinfI site is located within the gene for VA RNA,,, flanked by the 560 bp fragment on its left and the 1100 bp fragment on its right,. Hi?bfl cleavage of the BamHI-D/HindIIi-B fragment precludes two fragments: one has 220 hp and is end-labeled: while the other contains 580 bp (Fig. 12). These resultas show that’ the VA RNA,, gene spans the HiThf.fI sib:, located 220 bp from the Bnm.HI sit’o at position 30.0 (see Fig. 15). (h) Partial

pu$cation

of fragn~xnts

,frotn

Hinfr

digest

In theory it should be possible to discover t,he location of the Hinfl site within the \‘A RNA,, gene by measuring the relative amounts of radioactivity bound to the 560 I)p and 1100 bp fragments after hybridization to labeled VA RNA,,. Unfortunately, this ratio could not be determined with sufficient reproducibility to allow reliable positioning. ,4 similar difficulty has been experienced with estimates of the amounts of VA RNA, radioactivity bound to DNB fragments either side of the BamHI site the which divides its gene (Mathews, 1975). A, s in this case we have circumvented problem by measuring the complexity, rather than bhe yuantity, of the VA RNA,, sequences bound to the two H%nfI fragments. Prerequisite for this approach are t.hr purified DNA fragment’s and some knowledge of the structure of the RNA (discussed a,bove. section (d)). The 560 bp and 1100 bp fragments were isolated from a Hinfl digest of Ad2 DNA hy electrophoresis through a 3.5:; polyacrylamide gel. Examination of the purified fragments after gel electrophoresis indicated that they were free of cross-contamination although neither was pure (Fig. 11B). From the data given above, it is most unlikely that the contaminating fragments would interfere with the experiments at hand by hybridizing to VA RNA,,. The results presented in Figure 13 eliminabe this possibility entirely. The 1100 bp fragment is accompanied by a 1050 bp fragment which comigrated with it in the preparative polyacr,ylamide gel but runs substantially faster in 14O,, agarose. After transfer to nitrocellulose paper and hybridization, it is clear that onl) the larger fragment contains sequences complementary t,o VA RNB,,. As expected from the mapping data described above. this fragment is cleaved by the enzyme Hin,dIII into fragment,s 580 bp and 520 bp long. of’ \\,hich only the former hybridizes to LTA RNA (Fig. 13A). The isolated 560 bp fragment of Hin,fI-cleaved Ad2 DNA was visualized as a single somewhat’ broad band in 1.4(+(, agarose gels. but under optimal conditions it could ht. separat’ed int)o four or five fragments by electrophoresis in 3..5:0 acrylamide gels

a

+

Stairted

1100

b

a Hybridized

VA RNA,

b

.

5603

8 Hmf/AdE

c d Stoined

t Q +

I”

220

340

560

1100

t $ +

e f Stained

C Hinf/AdP

c

f e Hybridized

VA1

VAIL

f

a-

1100 bp fragment, isolated by polyacrylamide FIG. 13. Characterization of the HG&fI fragments of Ad2 DNA which h>Ibridize to VA RX.1 II, (.I) The Hi,zf of an agarose gel stained to show the fragments; on t.he right gel electrophoresis; (a) digested by HindIII, and (b) undigested. On the left 1‘J a photograph is the autoradiogram obtained after transfer of the fragments to nitrocellulose membrane and hybridization to VA RNA,,. (B) A photograph of a stained i BnmHI. The closed arrows indicate the positions of 2 of the 3 3.5% polyacrylamide gel cont,aining Ad2 DNA digested with (c) HinfI, and (d) HinfI + BarnHI, resolved by electrophoresis in an agarose fragments which contain sites of BumHI cleavage, (C) Digests of Ad2 DSd with (e) HinfI, and (f) HinfI arc sutoradiograms of nitrocellulose membranes gel. On the left is a photograph of the stained DNA fragments. In the middle and on the right, respectively, to which the fragments were transferred, following hybridization to VA RNA, and VA RN.iI,. The arrowhead indicates the position of a band which W&S too faint to be clearly visible on this reproduction of the autoradiogram.

520

580

II00 1050

A ff/nf

SMALL

RNAs

OF

ADENOVIRUS-INFECTED

CELLS

321

(Figs IIB. 13B and 13C). Results discussed in the previous section establish that the fragment of interest must contain a cleavage site for the BumHI endonuclease (the other two sites being in fragments approximately 600 bp and 1900 bp long, as indicated in the digest of Ad2 DNA with HinfI in Fig. 13B). The autoradiograms reproduced in Figure 13C show that digestion with the BamHI enzyme abolishes the hybridization of both VA RNAs to the HinfI 560 fragment region : as anticipated for the BamHIIHinfI double digest of Ad2 DNA, VA RNA, hybridizes to fragments of 340 bp and 220 bp, while VA RNA,, hybridizes to fragments 220 bp and 1100 bp in length. (The faintness of some of the bands in Figure 13C, particularly that resulting from hybridization of VA RNA,, to the 220 bp fragment, is due to poor retention of small fragments by the nitrocellulose membrane as originally noted by Southern (19753).) Thus the HanfI 560 and 1100 fragments are the only ones which hybridize to the VA RNAs, and the presence of contaminating sequences is of no consequence to the experiment set out below. (i) Length of the spacer regiola The precise location of the VA RNA,, gene, and the length of the spacer region between the two VA RNA genes, were determined by hybridization of labeled VA RNA to the 560 bp and 1100 bp fragments from a HinfI digest of Ad2 DNA. Following removal of non-specifically bound material and trimming of the overhanging singlestranded RNA tails by RNase T, treatment, the hybridized RNA was eluted, digested with RNase T, and fingerprinted. The results are illustrated in Figure 14 and tabulated in Table 3. Thirteen of the 25 oligonucleotides of VA RNA,, are certainly bound to the H&f1 1100 bp fragment and three more may be; three others are visible on the fingerprint derived from the HinfI 560 bp fragment ; the remaining six are presumably present, in the latter fingerprint but are obscured by the oligonucleotides derived from VA RNA, which also hybridizes to the 560 bp fragment. An estimate of the molar yields of those oligonucleotides bound to the 1100 bp fragment together with data on their chain length (Table 1) permits the conclusion that about, 75 bases of RNA hybridize to the right, of the HinfI site. Assuming the chain length of VA RNA,, to be 135 bases, some 60 bases should lie to the left of this site. The site is 220 bp from the BamHI cleavage at position 30.0, and 83 nucleotides of VA RNA, are coded on t’he DNA extending to the right of this point (Mathews. 1975). Therefore we conclude that, t’he spacer region between the two genes is approximately 75 bp long.

4. Discussion (a) Genes for VA Rh’A The location of the genes for the two VA RNA s is summarized in Figure 15. They are situated on the r-strand of the viral DNA, close to the 5’ terminus of a major block of ‘(late” mRNAs and to the right of a region which does not appear to encode any proteins. The two genes are separated by a spacer region of about 75 bp which includes the site for initiation of VA RNA synthesis by RNA polymerase III. The spacer must also contain sequences specifying the 3’ end of VA RNA,, although it is not yet clear whether this is generated by processing from a precursor molecule or by transcriptional termination. It is interesting to note that a similar organization exists in the 5 S rRNA repeat of Xenopus laevis. where the gene for the 5 S RNA is separated by a 73 bp spacer from a modified copy or “pseudogene” (Jacq it nl., 1977; Fedoroff &

A

Hmf

560

B

H/nf

II00

FIG. 14. Fingerprints of VA4 RNA selected on HinfI fragments. A preparation of VA RNA,,, separated in one dimension and nontaminatetl with VA RNA, scyuences, was incubated with filters carrying (A) the HinfI 560 bp and (B) the H&f1 1100 bp fragment. Following removal of unhybridized RNA tails and elution, the hybrid&xl RNA was digested with RNase T, and fingcrprinted. The orientation of the autoradiograms is the sanw as in Pig. 5.

Brown, 1977). Whether or not this is a significant feature of genes t)ranscribed I, RNA polymerasr I I r remains t’o be seen. Ln the case of the VA RNAs, present indications arc that the two genes are quite widely diverged. Ln the RNa,se T, digest, none of the oligonucleotides of more than four bases are common to the two species. and highly purified preparations exhibit little competition and cross-hybridization (data not shown). These results suggest that the degree of homology is small, although only direct comparison of the primar) sequences can decide this point with certainty. The experiments reported here afford evidence for no more than t,hree distinct viral RNA species of low molecular weight in the cytoplasm of Ad2-infected cells: the two VA RNAs and the 9 S mRNA which is discussed in detail elsewhere (Pettersson & Mathews, 1977). While it is clearly difficult to exclude the existence of further species of low molecular weight RNA, several types of experiments argue in this direction. In polyacrylamidr gels. RNA from t#h(J r.vtoplasm of infected cells is resolved into a large number of b;Lnds : all 1~16 t’hrec of t,hrstt arc%.&o stm1 in gels containing uninfected

0 0 0

16 17

18 19

t See Table 1. 1 The symbols have $ The total number C: See note f of Table number $8 The total

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f 0 0 0 0 0 + -~

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15

20 23 24 25 26 27 30 31 34 35

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the following of nucleotidos 1. of bases in

0 0 0 0

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Hybridization HkfI 560

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Oligonucleotide numbert

A naly&

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1.8

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324

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29

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PETTERSSON 100 bp H

5.2s VA RNA, 31

32

33

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FIG. 15. Map of the VA RNA gene region of Ad2 DNA. The heavy arrows indicate and direction of transcription of the RNAs. Cleavage sit,es and fragments generated wit,h certain restriction endonucleases are also shown.

the positions by digestion

cell RNA. Fingerprint analysis and hybridization to viral DNA restriction fragments show that two of the three bands contain forms of VA RNA,, while the other contains VA RNA,,. Selection by hybridization to Ad2 DNA of fractions of RNA migrating between 4 S and 7 S failed to disclose the fingerprint, of any new viral RNA. A similar conclusion was reached from the screening of RNA fractions by hybridization to restriction fragments transferred from agarose gels to nitrocellulose membrane by the Southern blotting method. No transcripts were detected from fragments other than t’hose containing the genes for the 9 S mRNA and the two VA RNAs. This type of experiment has been carried out both with enzymes such as Hind111 and Bali which cut the viral genome relatively infrequently, at &es which have been mapped, and with enzymes such as HindII, HphI and MboI, producing a large number of fragments for which no map is yet available. Although it is possible to imagine that the hybridization of a hitherto unrecognized RNA to a DNA fragment could have been obscured by the hybridization of one of the known species to the same fragment, or to one which comigrates with it, the number of different enzymes used and of different fragments produced makes this very unlikely. These arguments are reinforced by experiments in which low molecular weight cytoplasmic RNA, the 3 to 13 S pool from a sucrose gradient, is hybridized to DNA fragments spanning the VA RNA genes and neighboring regions. Fingerprints of the selected RNA are very simple and contain only the expected VA DNA oligonucleotides (Mathews and Pattersson, unpublished results). On the other hand, fingerprints of nuclear RNA hybridized to the same fragments exhibit some novel extra spots, as anticipated from the presence of VA RNA sequences in HnRNA. These data lead to the conclusion that low molecular weight viral RNA species other than those listed above either do not exist in the cytoplasm of AdB-infected cells or are present in concentrations below the limit of detectability of our methods. The experiments involving hybridization are likely to be able to pick up species at least IO2 to 103-fold less abundant than VA RNA,,. An infected cell can contain as many as lo5 to lo6 copies of this species. so we consider that any new species will probably 1.e present in fewer than 10” copies per cell. and will be difficult, or impossible to detect solely by autoradiography of a gel containing infected cell RNA. On these grounds we are disposed to think of the cytoplasmic species described by

SMALL

RNAs

OF

AT,E~OVIRUS-I

NFEC’TED

(“ELLA

32 5

Weinmann et aE. (1976) and Varrichio et al. (1976) as either cellular RNAs contaminated by viral sequences, or as forms of the VA RNAs discussed here. However, it should be emphasized that our studies relate solely bo moderately stable cytoplasmic species, which may limit, t,he scope of our conclusion. (b)

Forms

of J’A

RNA,

Although the data are consistent with the existence of just two genes for VA RNA, these species, VA RNA, in particular, exist in a surprising number of forms. The heterogeneity has three aspects. Firstly, VA RNA, gives rise to a very broad band on gel electrophoresis, and under some conditions can be resolved into two species which appear to differ in chain length by about four residues. Ohe & Weissman (1971) reported t,hat the 3’ terminus may carry one or two urid,vlate residues, but RNase T, fingerprints of the 5.5 S RNA (a) and (b) forms do not show- any significant qualitat,ive or quantitative differences in our hands. The possibility exists that the difference resides at the 5’ end of the molecule which commences wit,h a run of three guanylate residues: variations here which might not be readily apparent in fingerprints with RNasu T, have been detected by Celma et nl. (1977) and by H. VennstrGm (personal communication). Secondly, denaturation of thcb \rA RNA, generates a number of shorter products, some of which form discrete spots in the denaturing dimension of a two-dimensional gel. The most likely interpretation of this finding is that the molecule contains hidden breaks, as described above and mentioned by Ohe & Weissman (1971). It is curious that the fingerprint of a prominent 12O-base product,, VA RNA, (c), contains all the RNasa T, oligonucleotides of the full length molecule. Preliminary d&a indicate tha,t oligonucleotides from the middle of the molecule are over-represented relative to both ends, suggesting that the molecule may comprise a mixture of two forms, one lacking 30 to 40 bases from the 5’ end and the other foreshortened by a similar amount, at, the 3’ end. Thirdly, VA RNA, sequences are found by hybridization in all regions of a native polyacrylamide gel from 4 S to 7 S. In denaturing gels, t’he trail of VA RNA sequences into regions of higher molecular weight is much reduced, presumably because VA RN,4, forms aggregates or interacts with other RNB species during electrophoresis in non-denaturing conditions. Whether or not this has any physiological importance is unclear: these dissociable aggregates, and the VA RNA, sequences which persist in their slow gel mobility after denaturation. could still represent an artifact of some sort. Alternatively, they may be derived from the high molecular weight VA RNA, sequences which are found in polyadenylated nuclear RNA, possihlp by leakage of nuclear contents during fractionation of the cells. The existence of VA RNA sequences in HnRNA, for which there is also evidence from electron microscopy (Meissner et al., 1977), ma’y imply that the tsrmination or processing signals surrounding the VA RNA genes are not respected by all polymerases travelling through this region. Late in infection, nearly all of the viral r -strand is transcribed (Pettersson & Philipson, 1954; Sharp et al., 1974), and large RNA molecules corresponding in size to more than 50% of the Ad2 DNA have been detected in the nucleus (Wall et al.. 1972). Transcription of these species appears to be mediated by RNA polymerase II, judging by its sensitivity to a-amanitin (Price & Penman, 1972a; Wallace t Kates, 1972; Weinmann et al? 1974,1975), and there is evidence to suggest that’ the initiation point lies somewhere close to positions

326

M.

13. MATHEWS

ASI)

II.

1’ETTERSSON

16 to 17 (Bachenheimer & Darnell, 1975; Weber et al., 1977; Goldberg et al., 1977; Berget et aZ., 1977; Chow et al., 1977; Klessig, 1977). Thus a likely hypothesis is that RNA polymerase II, commencing transcription to the left of the VA RNA genes, continues to read through these genes and towards the right-hand end of the molecule, while RNA polymerase III, responding to initiation and termination signals in VA RNA gene region, accomplishes the discrete synthesis of these products. However, we cannot at present exclude the possibility that some of the readthrough may be due to RNA polymerase Ill, or that VA RNA, is located at the 5’ end of longer transcripts. In any case, it seems that if bhe HnRNA molecules are precursors to mRNA, the VA RNA portions are excised before transport through the nuclear membrane because VA RNA, sequences are deplet,ed in cytoplasmic mRNA. (c) C:yto$asmic

RNAs

of the host cell

In addition to tRNA and 5 S RNA, which apparently is overproduced in HeLa cells (Leibowitz et aZ., 1973): several host cell RNA species were detected in the cytoplasm of infected and uninfected cells. Most of these minor species have been identified with RNAs previously described by others, but a few remain olscure. Of the three 7 S RNA bands, only the (c) species can be accounted for with certainty. It corresponds to the 5 8 RNA which is included in RNA tumor virus particles, although it is encoded by the host’ cell, and appears to vary slightly in structure among animal species (Erikson et al.. 1973; Walker et al.. 1974). According to Walker et al. (1974), about half of the RNA is functionally associated with polysomes; however Zieve & Penman (1976). who called this RNA ScL and showed that it resides solely in the cytoplasm, found that less than lOO;, was ribosome-bound but that a large fraction was associated with cell membranes. In our hands most of the 7 S RNA in a detergent-treated cell lysat’e sediments with ribosomes (unpublished observations); whether this is an artifact of cell disruption or genuinely represents the intracellular localization is not clear. Our estimate of 300 nucleot’ides for the length of this RNA (Pettersson & Mathews, 1977) is about, 10 “$ hi g her than the sizes reported elsewhere. The identities of the other 7 S RNAs have not been est(ablished. Judging by its electrophoretic behavior, SnK (Zieve & Penman, 1976) is a likely candidate for 7 S RNA (a): this species is found in both the nucleus and cytoplasm in varying proRNA moving between SnK portions. Zieve & Penman (1976) a 1so saw a cytoplasmic and ScL, which might correspond to 7 S RNA (b). Another contender is the 8 S(28 S) species, called SnQ by Sieve & Penman (1976). which Prestayko et ccl. (1970) showed was hydrogen-bonded to 28 S ribosomal RNA in the nucleolus. The complete sequence of U-2 RNA from the nuclei of rat Novikoff hepatoma cells has been determined by Shihata et al. (1975). It consists of 196 bases of which the 5’-terminal third is heavily modified: this feature may explain t’he unexpectedly high electrophoretic mobility of U-2 RNA (Figs 2 and 3). Its occurrence in the cytoplasm is consistent with Zieve & Penman’s (1976) observation that 40’~/0 of SnC (as they term it) appears in the cytoplasm after long-term labeling of HeLa cells. but not with Goldstein & Ko’s (1974) report that in Amoeba the U-2 RNA does not “shuttle” between cytoplasm and nuclei. There are also contradictory data on the state of this RNA within the nucleus : Zieve & Penman (1976) consider that it is part of the nuclear skeleton and that its appearance in the cytoplasm may be an artifact of the fractionat’ion procedures, whereas Raj et al. (1975) found that it is present in chromatinassociated ribonucleoprotein particles.

SMALL

RNAs

Ob’

ADENOVIRUS-INFECTEIJ

(‘ELLS

327

All three forms of 5.8 S RNA give fingerprints essentially identical to those obtained previously for HeLa and other mammalian cells (Maden C%Robertson, 1974; Nazar it aZ., 1975,1976). This RNA, also known as 5.5 S, 6 S, 7 S and ScE, is 159 bases long and has been sequenced (Nazar et al., 1975,1976). The 5.8 S RNAs of various mammals differ only at their termini (Nazar et al., 1976) : it remains to be seen whether the same is true of the three forms detected here. The 5.8 S species is associated by hydrogen bonds with the 28 S RNA of rihosomes, and both are derived from the 32 S rRNA precursor in the nucleolus (Maden $ Robertson. 1974; Nazar et aZ., 1975). Its presence in the post-ribosomal supernatant of adenovirus-infected cells (Mathews, unpublished observations), and the curious rise and fall in its concentration during the infectious cycle are unexplained, but presumably reflect the disturbance of ribosome production (or other host cell functions) which occurs late in infection (Raskas et al., 1970). Several other species, such as t)he 5 S RNA (c). h ave also been detected in the cytoplasm of HeLa cells, but in quantities insufficient to permit further analysis. Their relationships, if any, to species already recognized remain to be discovered. \Vr thank Susanne Weirich and Gun-Inger work \vas supported by grants from the Medical &search Council.

Lindh National

for

skilflll

(‘anccr

technical lnstitut,e

assistance. This and the Swedish

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