J. Mol. Biol. (1977) 116, 525-548

Quantitative Electron Microscopy of Early Adenovirus RNA HEINER

WESTPHAL

AND SING-PING LAI

Laboratory of Molecular Genetics National Institute of Child Health and Human Development National Institutes of Health, Bethesda, Md 20014, U.S.A. (Received 24 May 1977)

We present methods of electron microscopy which allow simultaneous mapping and quantitation of individual populations of RNA8 which are complementary to distinct regions of one template DNA. Adenovirus transcription, which has been thoroughly studied in the past, was chosen as a test system for our procedures. The cytoplasm of human cells harvested at early stages of productive infection with adenovirus type 2 contains RNA which hybridizes to four well defined regions of the viral genome, two on each DNA strand. We annealed the RNA to the separated strands of virion DNA. Hybrids (RD molecules) were visualized either directly or after duplexing with the complementary DNA strand (HD molecules). The four early regions were mapped between positions 1.1 to 10.6, 61.6 to 68.1, 76.7 to 83.7, and 91.5 to 96.9. At six and at eight hours after infection, RNAs were observed in these four locations at relative abundances of 1: 1.62: 2.60: l-35, and 1: 1.54: 2.46 : l-27, respectively. At least five distinct populations of RNA have been discerned, two in region 1, and one in each of the other regions, Special features detected in the electron microscope include a loop of singlestranded DNA bridged by RNA at position 773/79*0 end a tiny denaturation bubble within HD molecules near position 29. Both structures may have interesting biological implications. All map positions represent mean values, with standard deviations of -60 nucleotides in the best and of -600 nucleotides in the worst cases, on a map comprising -35,000 nucleotides. Individual transcripts contained in nanogram amounts of crude preparations may be assayed on single or double-stranded templates by our procedures. The experiments are more precise and less time consuming than comparable biochemical studies. They should be amenable to a variety of biological systems dealing with templates that are of, or can be restricted to, a size suitable for electron

microscopy.

1. Introduction Electron microscopy has become an important tool for the identification and mapping of individual transcripts complementary to specific regions of a complex genome. Map positions of ribosomal RNA and messenger RNA have been obtained by direct visualization of RNA-DNA hybrids. A long stretch of single-stranded DNA carrying an RNA annealed to the complementary region within that DNA, when spread by the standard cytochrome c method, appears as a thin filament with a heavy contour in the area of the RNA-DNA hybrid. Accurate mapping depends on the recognition of the junction between single and double-stranded regions of the molecule. Initial measurements have been difficult, due to insufficient contrast between singlestranded DNA and RNA-DNA hybrid (Hyman, 1971; Hyman & Summers, 1972; 525

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Deonier et al., 1974; Ohtsubo et al., 1974; Forsheit et al., 1974). However, minimal alterations in the spreading technique have since increased the contrast and thereby the fidelity of measurements (Dawid & Wellauer, 1976; Klukas t Dawid, 1976; Wellauer & Dawid, 1977). An elegant way to enhance the visualization of RNA-DNA duplex regions within single-stranded DNA was immediately realized. If the DNA strand that carries the RNA is hybridized to its antiparallel complement, the RNA generates a loop within the hornduplex DNA. One branch of this loop is double-stranded and represents the RNA-DNA hybrid, the other branch is single-stranded and represents the displaced region of DNA that is homologous to the RNA (Hyman $ Summers, 1972; Ohtsubo et al., 1974; Forsheit et al., 1974). In analogy to D loops (Robberson et al., 1972) which have a similar appearance in the electron microscope, these structures were later called R loops (White & Hogness, 1977). An accidental observation by Bick et al. (1972) originated the R loop technique of White & Hogness (1977) which is now widely used for the mapping of RNA in double-stranded DNA. These authors noted that RNA incubated with doublestranded DNA in the presence of formamide is able to insert itself into the duplex thereby generating an R loop which marks the region of homology. An analysis of the kinetics of this reaction (Thomas et al., 1976) revealed that the rate of R loop formation is maximal near the temperature at which the strands of DNA separate within the region of homology; in all likelihood, during partial melting of the DNA in formamide, local instabilities of the DNA duplex within this region offer nucleation events for the generation of a thermodynamically more stable RNA-DNA hybrid. The aclenovirus system is uniquely suited to compare and further refine the various techniques of RNA visualization and mapping. Thorough biochemical studies (for review, see Flint, 1977) have defined the units of transcription both in the nucleus and in the cytoplasm of cultured cells harvested at various stages of lytic infection with Atit. We used the technique of White $ Hogness (1977) to determine the relative abundances and the map positions of R loops generated by nuclear (Meissner et al., 1977) and by cytoplasmic (Westphal et al., 1976; Meyer et al., 1977; Neuwdd et al., 1977) viral RNA in the double-stranded Ad2 DNA. Our studies supported the idea that the various viral mRNAs are derived from large nuclear precursors, and established accurate map positions for several early and late cytoplasmic Ad2 transcripts. During the course of our experiments we realized that the rate of R loop formation varied among individual Ad2 transcripts. It appeared that loops formed more readily in the (A+T) rich regions of the double-stranded DNA than in the (G+C) rich regions. This fact, predicted by the kinetic studies of Thomas et al. (1976), revealed an unfortunate limitation of the White & Hogness (1977) technique for projects such as ours. The method does not allow quantitative estimates of individual transcripts since the rate of R loop formation is not only dependent on the amount of individual transcripts present in the assay but also on the primary structure of DNA at each site of hybridization. We therefore decided to return to the original t Abbreviations used: Ad2, sdenovirus type 2; Tricine, N-(Tris(hydroxymethyl)methyl) glycine; RD, hybrids of early RNA and either the rightward or the leftward strand of Ad2 DNA; HD, duplex DNA, formed by annealing an RD molecule with the complementary strand of either Ad2 or Ad2 +ND4 DNA; map locations and map units, points and lengths, in peroent of Ad2 DNA.

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527

procedures outlined at the beginning of this introduction, namely, to hybridize Ad2 RNA to single-stranded DNA in order to obtain a direct estimate of the abundance of individual transcripts by electron microscopy. We chose a preparation of early viral RNA for our analysis which anneals to four accurately mapped regions of the DNA (Neuwald et aZ., 1977). The limited complexity of this probe would seem to make it a well suited test substrate for quantitative electron microscopy.

2. Materials and Methods (a) Nucleic acids Ad2 was propagated in cultured KB cells as described by Meyer et al. (1977). A spinner culture of 6 x lo8 cells was infected with 1011 virion particles; at 6 h after infection, 125 PCi [methyZ-14C]thymidine (spec. act. 45 mCi/mmol; SchwarzfMann, Inc.) was added, and cells were harvested at 42 h after infection. Virions were puriiled by extraction with freon (Meyer et al., 1977), and 8~ Lola particles were disrupted and complexed with poly(U,G) (lot no. 7001; Sohwarz/Mann, Inc.) aa described by Tibbetts et al. (1974). Following CsCl buoyant density oentrifugation (Fig. l), the separated rightward and leftward strands of Ad2 DNA (as defined by Sharp et al., 1974) were dialyzed at 4°C for 5 h werau9 1% (v/v) formamide, 0.2 M-NaCl, 3 mivr-EDTA, 50 m&f-Tris-HCl (pH 7.5); precipitated with 2.5 vol. of 95% ethanol overnight at - 20°C ; sedimented in polyallomer tubes by 60-min centrifugation at -5% at 48,000 revs/min in the Spinco SW50 rotor; taken up at a concentration of 50 pg/ml in 18 mu-EDTA, 0.18 M-Tricine*NaOH (pH 8-O), and stored in polyethylene vials at 4°C. In accordance with observations by Patch et al. (1974), there was no need to remove traces of poly(U,G) from the preparations of separated Ad2 DNA strands. The integrity of DNA strands was examined by electron microscopy. When spread under conditions which allow annealing of the inverted terminal repetition of the DNA ends (Garon et al., 1972; Wolfson & Dressler, 1972) nearly all of the molecules formed circles. Cross-contamination of the preparations of separated strands was negligible. Under the hybridization conditions of this study, maximally 6% and 3% homoduplex formation occurred in the preparations of rightward and leftward strands, respectively. Two preparations of poly(A)-containing RNA, extracted by Neuwald et al. (1977) at 6 or 8 h after infection from the cytoplasm of cells incubated in the presence of cycloheximide, were employed in this study. (b) Hybridization Hybridization assays (100 4) contained 66% (v/v) repurified formamide (Pinder et al., 1974), 3 M-urea (Ultrapure; Schwarz/Mann, Inc.), 20 mu-EDTA, O-5 pg of the rightward or leftward Ad2 DNA strands, l-5 pg RNA (different concentrations of nucleic acids were used in a single case: see the legend to Fig. 7), 0.2 M-Trioine*NaOH (pH 8-O). The assays were incubated at 37’C in tightly capped polyethylene vials for the times indioated in the text; diluted into 2 ml of Cs,S04 in 50 maa-EDTA, 0.1 M-Tris*HCl (pH 7.5) (density = 1.783 g/cm3); overlaid with 2-l ml CsaS04 buffer of lighter density (I.197 g/cm3); and centrifuged at 15°C for 16 to 20 h at 37,000 revs/mm in a Spinco SW66 polyallomer tube. An example of this rapid buoyant density centrifugation (Brunk & Leick, 1969) is given in Fig. 2. Pools of RNA-DNA hybrids = RD molecules (see the legend to Fig. 2) were dialyzed, precipitated, and stored aa described above. Portions of the preparation of RD molecules were duplexed either with Ad2 DNA or with Ad2 +ND4 DNA to obtain HD molecules carrying R loops. Assays (10 ~1) contained 5 ng RD molecules ; 5 ng either of the complementary Ad2 DNA strand or of unfraotionated Ad2+ND, DNA that had been denatured for 20 min at 25°C in 0.1 M-NaOH, 62% (v/v) repurified formamide, 2.8 M-urea, 36 mu-EDTA, 0.36 r+Tricine*NaOH (pH 8-O). The reaction mixture was sealed in a disposable 20 4 pipet, heated for 1 min at 66”C, and incubated for 5 h at 41°C.

536

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(c) Electron microscopy Spreading conditions have been described in previous articles (Westphal et aE., 1976; contained in a hyperphase of 87% (v/v) repurified Meyer et al., 1977). RD molecules, formamide, 3.9 M-urea, 7 mu-EDTA, 70 m&r-TricinemNaOH (pH 8.0), were heated for 1 min at 40°C prior to spreading; HD molecules were spread from a hyperphase of 76% (v/v) repurified formamide, 3.4 M-urea, 18 m&r-EDTA, 0.18 M-TricineaNaOH (pH 8.0). Cytochrome c was added to 100 pg/ml immediately before spreading. Concentrations of DNA in the hyperphases ranged from 0.1 to 0.5 pg/ml. All spreadings were performed at room temperature on a hypophase of water (Dawid & Wellauer, 1976). The tracing of molecules from electron micrographs and the method of data processing have been described (Meyer et al., 1977). In order to ensure that the evaluation was representative for all molecules within a given sample, only those spreadings were examined in which at least 80% of the molecules could be traced. In HD molecules, tracings followed the double-stranded contour throughout, assuming the linear density of RNAduplex (Meyer et al., 1977). When tracing DNA hybrids to be close to that of a DNA-DNA RD molecules, however, corrections had to be made for the observed difference in linear density between single and double-stranded nucleic acid. Using mixtures of single and double-stranded Ad2 or simian virus 40 DNA we determined that under our RD spreading conditions, the contour length of single-stranded DNA is, on average, 1.16 times that of duplex DNA.

3. Results (a) Viswlization

of early adenovirus separated

strands

type 2 RNA DNA

hybri&ed

to

of viral

We separated the rightward and leftward strands of Ad2 DNA (Fig. 1) and annealed each strand with early RNA. The resulting hybrids, which we will call RD molecules, were purified by cesium sulfate buoyant density centrifugation (Fig. 2), and mounted for electron microscopy by the cytochrome c spreading technique. Figure 3 depicts the rightward strand (top) and the leftward strand (bottom), each carrying RNA at two distinct locations. The regions of RNA-DNA hybridization were recognized by their heavy contour, and could be oriented with respect to the conventional Ad2 DNA map on account of previously published information (Sharp et al., 1974; Pettersson et al., 1976; Neuwald et al., 1977). After proper alignment, the molecules were traced, and measurements were plotted by a computer as shown beneath the line drawings in Figure 3. Note that a tail is protruding from the end of one of the hybridized RNAs in the bottom micrograph of Figure 3, and that a small loop is seen within one of the RNA-DNA hybrid regions of the rightward DNA strand (Fig. 3, top micrograph). These particular features are discussed below. In order to assess the qualitative and quantitative accuracy of our measurements of RNA-DNA hybrid regions within single-stranded DNA, we annealed RD molecules of the type shown in Figure 3 with naked DNA of opposite polarity. In the resulting duplex, or HD, molecules, RNA-DNA regions are marked by R loops. The heavy branch of these loops consists of the RNA-DNA duplex, the light branch of displaced DNA sequences within the opposite DNA strand. In Figure 4 we see two R loops generated by RNA which hybridized to the rightward strand of DNA. The rest of the molecule represents a continuous homoduplex of Ad2 DNA. One sees in the background of this electron micrograph three molecules of intact single-stranded DNA which formed circles due to hybridization of their terminal inverted repeat sequences (Garon et al., 1972; Wolfson & Dressler, 1972). A similar HD molecule is depicted in Figure 5. Here, the rightward DNA strand

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Ad2

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629

Fractions

FICA 1. &per&ion of the strands of Ad2 DNA. Complexes of Ad2 [‘*C]DNA and poly(U,G) in C&l (see Materials and Methods) were adjusted to a volume of 8 ml and a density of 1-766 g/ems, and centrifuged in a polyallomer tube at 16% for 3 days st 35,000 revs/min. Fractions were collected from the bottom of the tube, and 10-d portions were diluted with 0.7 ml water and 7 ml Aquasol (New England Nuclear Corp.) for scintillation counting. Fractions containing the leftward (L) and the rightward (R) DNA strands were pooled as indicated.

carries RNA at three locations, two at the left end, one near the right end of the molecule. Note the small loop structure within the RNA-DNA duplex branch of the rightmost R loop. This structure, which is similar to the one we observed in the RD molecule of Figure 3 (top), will be discussed separately. The arrow points to a tiny bubble within the DNA duplex. This bubble is not generated by RNA but represents a local melt of DNA, since it forms in the absence of RNA as well, yet only at the elevated concentrations of formamide and urea which we chose in order to improve the spreading of HD molecules. In the experiment of Figure 5, we observed this bubble in 33 out of 100 HD molecules. Its use as a visual orientation marker is suggested by the fact that its midpoint appears quite reproducibly near position 29 (mean = 28.6, S.D. = 1.4, n = 20). In the light of recent work by Seeburg et al. (1977) on RNA polymerase-promoter interaction it may be interesting to note that the DNA bubble appears to be located within the immediate vicinity of the promoter of VA RNA, a small virus-coded transcript (Price & Penman, 1972). Small local melts of HD molecules were also observed, though less frequently, at various other map positions.

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)-

l-

)-

c IO

15 Fractions

FIQ. 2. Equilibrium isopycnic centrifugcttion of RNA-DNA hybrids hybridization aesay (see Materials and Methods) containing the rightward (-m-e-), and [‘)H]RNA (-- l -- l --) isolated 8 h after infection, and centrifuged in CsaSOl for 20 h. Fractions were collected from the portions were removed for ecintilletion counting (see the legend to Fig. 1). of individual fractions was determined by weighing portions in calibrated hybrids (fractions 16 + 10) were processed 8s indicated in the text.

in a CsaS04 gradient. A strands of Ad2 [14C]DNA was inoubated for 18 h, bottom of the tube, and The density (-AA-) 60 ~1 pipets. RNA-DNA

The molecule shown in Figure 6 represents a heteroduplex between the rightward strand of Ad2+ND, DNA and the leftward strand of Ad2 DNA carrying RNA at two locations within the right half of the molecule. The deletion/substitution bubble which characterizes the region of heterology between Ad2 and Ad2+ND, (see Meyer et al., 1977), clearly identified by its two single-stranded branches, lies between the two R loops. Note the tails of varying lengths that protrude from the forks of the R loops. The two-step hybridization, first the annealing of RNA to single-stranded DNA (RD), and second its inclusion into homo- or heteroduplex DNA (HD), served several purposes. (1) It monitored the accuracy of the determination of the ends of RNADNA regions within single-stranded DNA. In HD molecules, these ends correspond to the forks of R loops which can easily be identified. (2) The experiment examined the stability of RNA-DNA hybrids. In the hybridization shown in Figure 6, Ad2 RD molecules were incubated in the presence of denatured Ad2+ND, DNA. Since both strands of Ad2+ND, homoduplex DNA were present in the reaction mixture, part

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531

of this DNA reannealed to form Ad2+ND, homoduplex DNA. If during the incubation, RNA had melted from RD molecules and reannealed to either single or doublestranded DNA, we would have expected to see at least a few R loops in Ad2+ND, homoduplex DNA. The fact that none (O/100) was observed underscores the stability of RD molecules during prolonged incubations under our conditions of homo- or heteroduplex formation. (3) HD molecules served as a visual probe for the integrity of DNA. Duplex molecules with single-stranded ends, indicative of partial degradation of one or the other of the reannealed strands of DNA, could be spotted at a glance during electron microscopy, and thus be eliminated from photography and data processing. Figure 7 demonstrates that early cytoplasmic Ad2 RNA can be mapped with comparable accuracy either in RD or in HD molecules. Saturating amounts of RNA were annealed to the leftward strand of Ad2 DNA. Tracings of a random sample of the resulting RD molecules are shown in (a). As a control, part of the preparation of purified RD molecules were annealed with denatured Ad2 +ND, DNA to generate HD molecules of the type shown in Figure 6. Figure 7(b) shows the position of R loops (light lines) and of deletion/substitution loops in the region of heterology distinguishing Ad2 and Ad2+ND, DNA (heavy lines). A comparison of the RD and the HD tracings reveals a good correspondence of RNA measurements by either technique. The positions and the sizes of RNA match well, and are in good agreement with our published data (Neuwald et al., 1977). The outlay of the experiment, saturation of DNA with RNA and small sample size, prevented any conclusion with respect to the relative abundance and the exact map positions of the RNAs shown in Figure 7. These questions will be dealt with below. (b) Tails at the ends of RNA hybdized

to DNA

In some RD and HD molecules, tails protruded from the 3’ or 5’ ends of RNA hybridized to DNA. These tails varied in length and configuration. Most of them appeared as bush or rod-like structures of RNA partially folded back onto itself. While Figures 3 to 6 were selected to show examples of these tails, Table 1 lists their abundances. Tails appeared on average in about 5% of all RNA ends, with the exception of the 5’ end of t,he leftmost RNA. There was no obvious accumulation of tails at any particular map position. Ohtsubo et al. (1974), who observed tails at the forks of R loops, were able to relate their presence to partial displacement of RNA by reannealing DNA. Our spreading conditions guard against such displacement (Meyer et al., 1977), and the fact that Table 1 lists comparable numbers of tails in RD and in HD molecules, would seem to indicate that displacement of RNA by DNA is not a major contributing cause for the RNA tails seen in this study. Since tails were rare, were not confmed to any particular location, and were not easily traceable due to folding, we decided not to include them in our measurements. Part of the tails at 3’ ends of RNA may actuahy reflect poly(A) moieties (Philipson et al., 1971) rather than genome transcripts. Less than 10% of all taiIs appeared as long as

FIG. 3. Electron micrographs of RD molecules of the rightward (top) and the leftward (bottom) Ad2 DNA strand. Each molecule contains 2 RNA-DNA hybrid regions, which are recognized by their heavy contour. The map positions of these hybrid regions are indicated below the line drawings of each RD molecule. In this, as well as in the following electron micrographs, a bar designates 1 pm. (a) Electron micrographs; (b) line drawings.

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n

I

I

I

1

I

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I

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I

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FIG. 3.(b)

1

I

carrying Fra. 4. An Ad2 homoduples (HD) UN-4 molecule micrograph. be discerned in the background of the electron

2 RN4s

hybridized

to the rightward

strand.

Three

single-stranded

Ad2

DNA

circles

may

I

I

FIG. 6. Ad2 homoduplex (HD) DNA containing RNAs bound to the rightward DNA small DNA bubble is pointed out by an arrow. The map position of each of these features

Ln n

I

1

I

I strand. Two R loops appear at the left, one at the right is indicated below tho line drawing of the molecule.

P

side.

A

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I

I

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I

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1

1

FIG. 6. A het,eroduplex (HD) DNA molecule composed of the leftward strand of Ad2 DNA and the rightward strand of Ad2+ND4 DNA (see Fig. 7). The molecule carries 2 R loops, and a substitution/deletion loop marking the ND, region. The map positions of the R loops (white boxes) zmd of the ND, region (black box) are indicated below the line drawing.

c

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’

I

’

OF I-’

(a)

5 2E 15_ ”

-

- --

--

(b)

--

0 --

-

-

-

I 20

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I

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----------

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RNA

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unils

Fm. 7. Tracings of RNA hybridized to the leftward strand of Ad2 DNA. RD molecules were formed, as described in Materials and Methods, by incubating 1 Fg DNA with 2.6 pg 8-h early RNA for 18 h. Purified RD molecules were observed in the electron microscopy, and measured from enlarged negatives with a gmphics calculator. DNA molecules were normalized by dividing each length by itself. The positions of RNAs are shown on a DNA map divided into 100 equal units. (a) Contains measurements of 16 RD molecules selected at random from the population. Part of the preparation of RD molecules was duplexed with Ad2+ND* DNA (see Meterials and Methods), and the known (Meyer et al., 1977) map position of the deletion/substitution loop generated within the HD molecules facilitated the orientation of the DNAs with respect to the conventional map. Tracings of 16 molecules selected at random from the HD preparation are shown in (b). Light lines mark the positions of R loops, heavy lines those of deletion/substitution loops. Note that the positions and lengths of RNA coincide well in both diagrams.

TABLET Abundances of tails observed in HD and RD molecules at the ends of hybridized RNA Map region

Polarity

HDt

R”t

6’ 3’

0.00 0.07

0.00

2

5’ 3’

0.03 0.06

0.03 0.05

3

5’ 3’

0.03 O-06

0.05 0.08

4

5 3’

0.06 0.04

0.03 0.05

1

t The fraction of RNAs terminating in each of the 8 positions listed.

in tails is derived

0.05

from the observation

of at least 100 ends

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the one demonstrated in the leftmost R loop of Figure 6. RNAs carrying tails of that length were excluded from measurement, (c)

A DNA loop bridged by RNA at map position

774179.0

When discussing Figures 3 (top) and 5, we pointed out a small loop within an RNA-DNA hybrid region near position 80. Figure 8 depicts some enlargements of this area within rightward strand RD molecules which had been treated with RNase. We concluded that the loop consisted of single-stranded DNA bridged at its stem

430f

60

...

... 1500+ zoo

76.6+1.1

77,s + I .o / 79.o-fo.9

strand of Ad2 DNA. A preparation of Fm. 8. A DNA loop bridged by RNA in the rightward RD molecules (76 ng) was exposed for 6 min at 37°C to 30 ng pancreatic RNase I RAF and O-1 ng RNase T1 (Worthington Biochemical Co.) in a 3-~1 assay volume containing 30 ng vesicular stomatitis virion (VSV) RNA, 18 mba-EDTA, 0.18 M-Trioine.NaOH (pH 8.0). Prior to DNA spreading in standard formamide/urea hyperphase, ribonucleases were inactivated by adding 1% (v/v) diethyl pyrocarbonate (Fedorosak t Ehrenberg, 1966) in order to prevent enzymatic attack of hybridized RNA which has been reported to occur in solutions containing formamide (Forsheit et al., 1974). Controls lacking RNase (not shown) displayed a dense background of single-stranded VSV RNA (Weber et al., 1974), whereas no free RNA could be discerned in the spreadings of enzyme-treated assays which served as the souroe of the RD molecuIes examined for this Figure. The 3 electron micrographs depict the specific features of a small single-stranded loop (light contour) protruding from an RNA-DNA hybrid region (heavy contour). A schematic drawing of this region, based on the measurement of 21 RD molecules lists the map positions as well as the length in nuoleotides of the loop and of the RNA segments to the left and to the right of the loop. Error limits indicate one standard deviation.

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by the hybridized RNA. Exact measurements were obtained from 21 RD molecules, selected at random, which carried the loop. The data, listed in the schematic drawing of Figure 8, place the loop at map position 77*8/79.0. Intact Ad2 DNA consists of about 35,000 nucleotide pairs (Green et al., 1967). Based on this number, the loop measured 430 nucleotides (1 RD. = 60), and its distances from the 5’ and 3’ end of the RNA were 420 (1 S.D. = 120) and 1500 (1 S.D. = 200) nucleotides, respectively. Roughly 50% of the RD molecules carrying an RNA in this region of the DNA displayed the loop. In a control group of 21 RD molecules lacking the loop each RNA was located to the right of position 80. The loop looks remarkably similar to one of the smaller insertions observed by White t Hogness (1977), Wellauer BEDawid (1977), and Pellegriui et al. (1977) in rDNA-rRNA hybrids of Drosophila melanogaster. In our case, it may represent a DNA substitution not present in the DNA which served as template for early RNA synthesis inside the cell. If so, that template appears not to be packaged into virions. For, both the virion DNA and the RNA were derived from cells infected with the same virus stock. In addition, no substitution loops (O/100) were detected in homoduplex DNA that was generated from the purified strands of two different DNA preparations separated by a time interval of several months. The loop is not a feature of single-stranded DNA by itself since it is not observed in naked DNA spread under equal conditions. Since the distance from the 5’ end of RNA to the stem of the loop is very similar to the length of the loop, the loop may represent a tandem repeat of the preceding DNA sequence. If so, we would have expected, but did not see, some of the DNA lacking the loop but carrying RNA in its place. Attractive models that would account well for the observed facts have been offered by Wellauer & Dawid (1977). There may be a ligation of two transcripts, separated by the sequence of DNA in the loop, or RNA polymerase might transcribe across the stem of the loop. Experiments need to be devised to test these novel ideas. Whatever the nature of the DNA loop at position 77+3/79*0 may turn out to be, it was gratifying for the further course of our experiments to note that the tracings of as few as 21 randomly selected RD molecules provided suf%cient accuracy to measure their specific features within very narrow error limits, down to one standard deviation of 60 nucleotides. (d) The relative

abundances of early adenovirus type 2 RNAs

With the knowledge that RNA could be recognized with equal fidelity both in RD and in HD molecules, we now proceeded to quantitative electron microscopy of early Ad2 RNA. In order to prevent competition of two or more RNA molecules for the same hybridization site, annealing conditions were chosen such that maximally 27% of all DNA molecules carried an RNA molecule at any given position. Since each viral DNA strand contains only two early regions, the fraction of DNA molecules carrying more than one RNA was expectedly small. Orientation of the RNA within the rightward or leftward DNA strand presented no problem because, in either case (see Figs 3 to 6) the two early regions are located asymmetrically, one close to one end, the other more toward the middle of the molecule. Therefore, observation of RD or HD molecules in the electron microscope was sufficient to allocate each RNA to one of the four regions of the genome. 35

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In a pretest to our quantitative analysis of Table 3, we determined that observations of RD versus HD molecules produced overlapping results, and that nearly identical figures were obtained by two observers scoring independently. The actual experiment involved two RNA preparations, one extracted six hours, the other eight hours after infection. Eaeh RNA was incubated in separate assays with either the rightward or the leftward strands of DNA, and the amount of nucleic acids was equal in each of the four assays. Although DNA was present in excess, conditions were not set to exhaust RNA during the time allowed for hybridization. Under these circumstances, relative abundances of individual RNA populations could only be established if all RNAs hybridized with comparable rates. Rate differences observed in our earlier studies had been attributed to the particular shortcomings of the White & Hogness technique (Neuwald et al., 1977). In our present hybridizations we predicted the rates to bc quite similar because RNAs of comparable complexity were annealed with single rather than double-stranded DNA. Our expectations were confirmed by the experiment of Table 2. We annealed RNA with the rightward strand of Ad2 DNA containing the early region 3 which displays very slow kinetics of R loop formation by the White & Hogness technique (Neuwald et al., 1977). The results show that the number ratio of RNAs observed in regions 1 and 3 was quite similar at 6.8 hours and at 26 hours of incubation, while the absolute amount of hybridized RNA increased with time. This control enabled us to proceed to the actual quantitation, based on the fact that the kinetics of hybridization of DNA to RNA are subject to the same variables as the well studied DNA renaturation (Hutton & Wetmur, 1973). TABLE 2 Hybridization

Time of incubation (h) 6.8 26.0

kinetics of six-hour early RNA and the rightward adenovirus type 2 DNA

Total number of DNA molecules observed 760 600

Fraction

of DNA molecules carrying RNA Near middle Both Near end 0.101 0.263

0.033 0.093

0,011 0.013

strand of

Number ratio of RNAs (middle/end) 2.65 2.60

Table 3 summarizes the data collected from the observation of 3200 DNA molecules. The relative abundances of individual populations of six or eight hour Ad2 RNA are listed in two ways. The upper line reflects the actual measurements in terms of the fraction of all DNA molecules carrying RNA at any of the four early regions of the genome. The lower line indicates the relative abundances of the four RNA populations. The RNA with the lowest abundance was given the value 1. Very similar distributions were obtained with both RNA preparations, with individual populations appearing, in the order of increasing relative abundances, in regions 1, 4, 2 and 3. From the fact that much longer time of hybridization was needed for six hour RNA than for eight hour RNA to generate comparable numbers of hybrids we conclude that the amount of viral RNA per cell had increased considerably during that period after infection.

QUANTITATIVE

MICROSCOPY TABLE

OF

Ad2

541

RNA

3

The relative abundances of i~ividual populations of early cytoplasmic adenovirus type 2 RNA Map region Fraction of DNA molecules carrying RNA Relative abundances of individual RNA populations

6-h 8-h 6-h 8-h

RNA RNA RNA RNA

1

2

3

4

0.106 0.113 1 1

0.172 0.174 1.62 1.54

0.276 0.278 2.60 2.46

0.143 09144 1.35 1.27

Hybridization assays containing RNA extracted 6 h or 8 h after infection were incubated for 26 and 3 h, respectively. Each RNA was hybridized in separate assays with either rightward or leftward Ad2 RNA strands. Part of the purified RD molecules were annealed with the complimentary Ad2 DNA strand to generate HD molecules. A total of 1500 and of 1700 DNA molecules (mostly from the HD batches) were scored for 6-h and for 8-h RNA, respectively. DNA molecules carrying more than one RNA were rare (1 to 3%). Abundances have been calculated relative to the number of RNAs (normalized to the value 1) appearing at the left end of the DNA.

(e) Map positions of early RNA The preparation of cytoplasmic RNA extracted eight hours after infection was subjected to a detailed analysis of map positions. As in the experiment of Table 3, RNA was incubated with excess quantities of rightward or leftward DNA, and HD molecules were selected at random for photography. The measurements are contained in Figure 9. The upper part of the Figure shows tracings of individual RNA molecules. Within each of the four separate early regions, RNAs were arbitrarily lined up in order of increasing distance from the left end of DNA. The direction of transcription and the positions and frequencies of 5’ and 3’ ends of RNA are listed in the center diagrams. The histograms at the bottom of the Figure indicate the fraction of DNA molecules that carried RNA at any given 0.2 map unit interval. Vertical lines separate the four domains of early transcription. A crude inspection of the data indicated that each of the three regions in the right half of the map contained a single population of RNA, whereas the leftmost region appeared to be shared by more than one RNA population. A seemingly homogeneous population of smaller RNAs was observed in the left of this region. On the right side, a smaller number of RNAs appeared which were more heterogeneous in size and location although some of them appeared to bridge most of this particular area. We decided to treat all RNAs in region 1 as belonging to one or the other of two populations separated at position 5 by a point of minimum overlap. In all areas of the map, except at the left end of region 1, RNA termini were quite scattered, suggesting RNA breakdown either before or after cell harvest. Predictably, therefore, a calculation of the map limits of each RNA population from the mean of all 3’ and 5’ ends underestimated the size of mature mRNA. Fortunately, we could refer to the RNA population centering near map position 65 (region 2) to correct this underestimation. The polarity of transcription changes at the beginning and at the end of this particular early region (Sharp et a,l., 1974; Pettersson et al., 1976). We have previously mapped the strand switches near positions 61 and 68, respectively (Meyer et al., 1977). From the data of Figure 9, we calculated the mean positions of 5’ ends and 3’ ends within region 2. The values (see Table 4) are 66.5 (1 SD. = leg), and 63.1 (1 S.D. = 1.5), respectively. If we extend

542

H.

WESTPHAL

AND

S.-P.

LA1

L.-

XI=.---

-

L

--

2

_ ..--

I

-

3I-

535_1 jI)-

'5

80

85 90

95

I

Map units

Fro. 9. Map positions of early Ad2 RNAs. Leftward or rightward DNA strands were hybridized with early RNA extracted 8 h after infection, Purified RD molecules were annealed with the complementary Ad2 DNA strand, and the resulting HD molecules were selected at random for photography and tracing. Vertical lines separate the 4 major regions of early transcription on the Ad2 DNA map. Most of the RNA in the left column had been hybridized for 18 h. At that time. 17% of all DNA molecules carried RNA between map units 1 and 12. The RNAs to the right of map unit 69 were derived from the experiment of Table 3. The top part of each of the 4 columns shows tracings of individual RNA molecules. The quantity of each set of RNAs wa+z arbitrarily chosen, and does not reflect their relative abundance. All molecules were lined up in the order of increasing distance from the left end of DNA. In the center of the Figure, needles pointing upward or downward reflect the abundances of 5’ or 3’ ends of RNA per 0.2 map unit. The histograms at the bottom of each column list the fraction of the HD molecules within eaoh group that carried RNA at any given 0.2 map unit interval.

QUANTITATIVE

MICROSCOPY TABLE

Map positions of individual

OF

Ad2

543

RNA

4

populations of early cytoplusmic

adenovirus type 2

RNA

_

~ ~~

RNA population Number of molecules 5’ end Mean S.D. 3’ end Mean S.D.

Map limits

2

1

Map region 1 69 1.9 0.8 4.0 0.8 1.1-4.8

2 51 6.0 1.9 8.7 1.9 4.1-10.6

3 52 66.5 1.6 63.1 1.5 61.6-68.1

3 4 101 7R.2 1.5 81.9 1.8 76.7-83.7

4 5 50 95.7 1.2 92.4 0.9 91.5-96.9

the distance between these mean values by one standard deviation to either side, we obtain map positions (61.6 and 68-l) which coincide well with the independently corroborated data. On the basis of this observation we felt justified in treating the rest of the data accordingly. The slight overlap of map limits at the junction of RNA populations 1 and 2 within region 1 is explained by the arbitrary selection of position 5 as the point separating the two populations. In most instances there was about as much scatter among 3’ ends as there was among 5’ ends. Since the RNA had been selected for poly(A) content on oligo(dT) columns, one may presume that the in vivo addition of poly(A) to 3’ ends of Ad2 RNA (Philipson et al., 1971) does not require one particular terminus, and/or that the observed scatter is due to breakdown of RNA after purification. Some molecules in region 3 extend beyond the 3’ end of that RNA population well into late sequences of DNA encoding for the fiber protein (Meyer et al., 1977). It seems attractive to regard them as intermediates in a,processing pathway leading to mature Ad2 mRNA.

4. Discussion We annealed leftward or rightward strands of Ad2 DNA with two crude preparations of RNA containing several distinct populations of early viral RNA, and visualized the hybrids as RD or as HD molecules in the electron microscope. Our measurements confirm and further refine the map positions of early cytoplasmic transcripts which we had determined earlier by the R loop technique of White & Hogness (Neuwald et al., 1977), and add information concerning the relative abundances of individual populations of early Ad2 RNA. (a) Qualitative analysis Ad2 mRNAs giving rise to proteins in cell-free translation assays fall within rather sharp size boundaries (Anderson et al., 1974; Westphal et al., 1974; oberg et al., 1975). The considerable size variation among the RNAs visualized in this study indicates that, apart from functional mRNA, our preparations contain also precursors and degradation products of early cytoplasmic viral RNA. Therefore, we avoid the term mRNA when referring to the RNAs which we visualized. We are unable to make any statements concerning the map boundaries of mature mRNAs except in region 2 where the size of the RNA matches its coding requirements.

544

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LA1

Several laboratories have allocated early Ad2 RNAs, as well as the gene products they encode, to defined restriction fragments of Ad2 DNA. This procedure provided a general orientation of RNAs and proteins on the Ad2 DNA map. Sharp et al. (1974), Flint et al. (1975), and Pettersson et al, (1976) separated the strands of restriction fragments, and hybridized them to excess early RNA. From the amount of DNA participating in hybrid formation, as well as from its location within the DNA map, these authors were able to assign crude map positions to four separate regions of early transcription, located asymmetrically, two on each strand. Estimates of the length of transcripts within each region were based on the assumption that RNA sequences were contiguous within each early region. Sizes of individual populations of RNA were determined more directly by gel electrophoresis of the preparations either prior to or after hybridization to DNA restriction fragments (Tal et al., 1974; Craig et al., 1975a,b; Biittner et al., 1976). Polypeptides specified by mRNA molecules within the various populations of early Ad2 RNA were identified by translation i7~ vitro of RNAs eluted from DNA restriction fragments (Lewis et al., 1976). According to our results (Table 4), the four regions of early transcription are flanked by the following map positions. Region 1 (rightward strand), 1.1 to 10.6 ; region 2 (leftward strand), 61.6 to 68.1; region 3 (rightward strand), 76.7 to 83.7; region 4 (leftward strand), 915 to 96.9. This is in good agreement with our R loop data (Neuwald et al., 1977), and confirms our contention that the White & Hogness (1977) technique is able to provide qualitatively accurate measurements. Within region 1, on the extreme left of the map, we see a short RNA to the left, and a longer one to the right, of map position 5. The longest of these RNA molecules measures approximately six map units. The entire region comprises 9.5 map units. This compares well to values of ~8.7 units (Flint et al., 1975) and -9.7 units (Pettersson et al., 1976), which were determined under conditions of exhaustive hybridization. Thus, it seems unlikely that our analysis missed any rare RNA sequences extending beyond the boundaries which we assigned to region I. Craig et al. (1975b) a,nd Biittner et al. (1976) located a short RNA (3.3 to 3.6 map units) to the left of position 44, and they observed RNAs of similar size adjacent to the right. Their results are in reasonable agreement with our data. A long RNA (11 map units) which would correspond to the entire region 1, has so far only been observed by Biittner et al. (1976) who do not exclude the possibility that it represents a precursor to the shorter RNAs of t.his region. Graham et al. (1974) reported that the leftmost part of Ad2 DNA is sufficient to transform cell cultures, and all AdS-transformed cell lines studied by Gallimore et al. (1974) contain the leftmost 14% of the genome. Ad2 RNAs found in transformed cells have not yet been mapped by electron microscopy, but results of Flint et al. (1975) suggest that they constitute a subset of transcripts synthesized during early lytic infection. Tumors have been induced in animals, using cells that contain only the leftmost part of adenovirus DNA. Antibodies contained in the sera of these animals were used to selectively precipitate virus-specific proteins contained in extracts of cells harvested at early stages of lytic infection. Levinson $ Levine (1977) observed an adenovirus type B-specific, 58,000 n/r, polypeptide, Gilead et al. (1976) saw two Ad2-specific components of 53,000 and 15,000 Mr, in the immune precipitates. Although not proven by their results, it is quite possible that the leftmost part of adenovirus DNA is encoding for these polypeptides, for experiments of Lewis et al. (1976) have shown that Ad2 RNA from the leftmost early region gives

QUANTITATIVE

MICROSCOPY

OF

Ad2

RNA

545

rise to polypeptides of comparable size in cell-free translation assays. A 44,000 M, polypeptide was placed to the left of position 4.1, and a 15,000 M, polypeptide somewhere between map positions 4.1 and 167. Although we observed many RNA molecules in region 1 which were long enough to accommodate coding sequences of polypeptides in the range of 15,000 to 53,000 M,, none of the RNAs observed to the left of position 4.1 was of a size required to encode for a 44,000 M, polypeptide. Thus, the map position of this polypeptide may have to be reassessed. Each of the other three early regions of the Ad2 genome appears to encode for one homogeneous RNA population. We find region 2 located on the leftward strand between positions 61.6 and 68.1. This agrees well with our previous observations of strand switch points which flank this region near positions 61 and 68 (Meyer et al., 1977). The RNA of region 2 specifies a 72,000 M, polypeptide in cell-free translation (Lewis et al., 1976), which, in its native configuration, represents the DNA binding protein (Levine et al., 1974) implicated in adenovirus DNA replication (van der Vliet et al., 1975). An RNA sequence of 6.3% genome length, required to encode for this polypeptide, is well accommodated within the limits we determined for region 2. Tal et al. (1974), Craig et al. (1975a), Pettersson et aE. (1976) and Biittner et al. (1976) have all reported a similar size for this RNA whereas the values of Sharp et al. (1974) and of Flint et al. (1975) are closer to four map units, and appear therefore to be underestimated. For region 3, which we have positioned between map limits 76.7 and 83.7, most laboratories agree on an RNA size of about seven units. The exception is an estimate of about ten map units by Pettersson et al. (1976). This is interesting, because we see some long RNA molecules which extend to the right into a late region containing the fiber gene (Meyer et al., 1977), and which may represent unprocessed precursors of mRNA. The only mRNA so far located in region 3 specifies a 15,500 M, polypeptide of unknown function in cell-free translation (Lewis et al., 1976). A portion of Ad2 DNS which includes the right half of region 3 (Kelly & Lewis, 1973; Mulder et al., 1974) can be deleted without adverse effect on viral DNA replication in cultured cells (Lewis et al., 1969). Thus, although an RNA spanning seven map units could encode for polypeptides of a combined size of >80,000 M,, it remains unclear which, if any, additional gene products are located here. Region 4 appears between positions 91.5 and 96.9 on the leftward DNA strand. A strand switch point near position 91 (Meyer et al., 1977) coincides well with our positioning of the left end of region 4. Our size estimate for the RNA, 5.4 map units, agrees well with previous estimates of Sharp et al. (1974) and Flint et al. (1975). Larger figures have been reported by Pettersson et al. (1976) (“7 map units) and by Biittner et al. (1976) (~8 map units), yet none of the molecules we visualized (O/50) were of that size. Two early polypeptides of 19,000 and 11,000 M, are specified by mRNA located within region 4 (Lewis et al., 1976). This leaves a considerable proportion of the RNA sequences in this region without known function. (b) Quantitative analysis The main objective of this study was the quantitative estimate of the relative proportions of individual Ad2 RNA populations contained in our crude preparations of early cytoplasmic RNA. The remarkable coincidence of relative abundances of viral RNAs measured at two different times after infection (Table 3) indicates a high degree of accuracy of quantitative electron microscopy within this system.

546

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The meaning of the relative abundances of early RNAs remains to be elucidated as more facts become known concerning the nature, function and quantity of individual early viral gene products. At present, we can only state a few observations. Individual populations of early RNA are present in quite similar relative abundances six and eight hours after infection, whereas their absolute concentrations appear to increase substantially during this period, as inferred from the observed kinetics of hybridization. The least abundant RNA populations are located in regions 1 and 4. It is puzzling to see that, within region 3, containing the most abundant RNA population, a large proportion of the RNA hybridizes to DNA sequences that appear dispensable with respect to virus growth in cell culture. In region 2, we had observed very slow kinetics of hybridization by the White C%Hogness technique (Neuwald et al., 1977). Under the hybridization conditions of our present study, the RNA of this region anneals readily with the DNA and is second in relative abundance. In light of the fact that the same RNA preparations were used in both types of experiment, this comparison underscores the validity of our change in hybridization procedures for the purpose of quantitative electron microscopy of Ad2 RNA. Flint & Sharp (1976) have published an indirect quantitation of Ad2 RNA sequences based on the observation of hybridization kinetics in assays containing separated strands of radioactive DNA restriction fragments and excess crude unlabeled RNA. They find up to twofold variance between individual measurements. This may suffice to explain the discrepancies between their figures and ours. On the basis of an estimate of the total amount of RNA present in the cell, these authors expressed their numbers in terms of copies of RNA complementary to probe per cell. In our opinion, there is no accurate measure of the absolute quantity of RNA per cell, and therefore we have chosen to determine relative abundances only. The methods described in this paper appear suitable to provide accurate qualitative and quantitative measurements of transcripts, not only in the adenovirus field, but generally, in all systems that deal with single or double-stranded templates which are of, or can be restricted to, a size suitable for electron microscopy. Individual populations of transcripts can be assayed direct,ly from nanogram amounts of crude RNA preparations, and the experiments are less time consuming and considerably more precise than comparable biochemical studies. We thank Dr J. V. Maizel, Jr for the use of his electron microscope facilities and for many helpful comments; Dr P. Howley for advice concerning the statistical evaluation of our data; Dr C. Patch for a sample of poly(U,G) ; Dr R. Lazzarini for vesicular stomatitis virus RNA; Mrs M. Sullivan and Mr S. Quill for expert technical assistance; and Mrs C. Harvey for editorial help. When this manuscript was completed, a preprint of a paper by Chow et al. (1977) reached us which deals with R loop mapping of Ad2 RNA, using the White & Hogness technique. Their work coincides well with our published reports, and with the data presented in Fig. 9 of our present study. REFERENCES Anderson, C. W., Lewis, J. B., Atkins, J. F. & Gesteland, R. F. (1974). PTOC. Nat. Acad. Sci., U.S.A. 71,2756-2760. Rick, M. D., Lee, C. S. & Thomas, C. A. Jr (1972). J. Mol. Bid. 71, 1-9. Brunk, C. F. & Leick, V. (1969). &o&m. Biophys. Acta, 179, 136-144. Biittner, W., Veres-Molnar, Z. & Green, M. (1970). J. MoZ. Biol. 107, 93-114. Chow, L. T., Roberts, J. M., Lewis, J. B. & Broker, T. R. (1977). Cell, 11, 819-836. Craig, E. A., Zimmer, S. & Raskas, H. J. (197.5~). J. V’irol. 15, 1202-1213. Craig, E. A., McGrogan, M., Mulder, C. & Raskas, H. J. (19753). J. Viral. 16, 905-912.

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Dawid, I. B. & Wellauer, P. K. (1976). Cell, 8, 443-448. Deonier, R. C., Ohtsubo, E., Lee, H. J. & Davidson, N. (1974). J. Mol. Biol. 89, 619-629. Fedorcsak, I. & Ehrenberg, L. (1966). Acta Chem. &and. 20, 107-112. Flint, S. J. (1977). Cell, 10, 153-166. Flint, S. J. & Sharp, P. A. (1976). J. Mol. BioZ. 106, 749-771. Flint, S. J., Gallimore, P. H. & Sharp, P. A. (1975). J. Mol. Biol. 96, 47-68. Forsheit, A. B., Davidson, N. & Brown, D. D. (1974). J. Mol. BioZ. 90, 301-314. Gallimore, P. H., Sharp, P. A. & Sambrook, J. (1974). J. Mol. Biol. 89, 49-72. Garon, C. F., Berry, K. W. & Rose, J. A. (1972). Proc. Nat. Acad. Sci., U.S.A. 69, 23912395. Gilead, Z., Jeng, Y.-H., Wold, W. S. M., Sugawara, K., Rho, H. M., Harter, M. L. & Green, M. (1976). Nature (London), 264, 263-266. Graham, F. L., Abrahams, P. J., Mulder, C., Heijneker, H. L., Warnaar, S. O., de Vries, Harbor Symp. Quant. BioZ. F. A. J., Fiers, W. & van der Eb, A. J. (1974). Cold Spring 39, 637-650. Green, M., Pica, M., Kimes, R., Wensink, P. C., MaeHattie, L. A. & Thomas, C. A., Jr (1967). Proc. Nat. Acad. Sci., U.S.A. 57, 1302-1309. Hutton, J. R. & Wetmur, J. G. (1973). J. Mol. BioZ. 77, 495-500. Hyman, R. W. (1971). J. Mol. BioZ. 61, 369-376. Hyman, R. W. & Summers, W. C. (1972). J. Mol. BioZ. 71, 573-582. Kelly, T. 5. Jr & Lewis, A. M., Jr (1973). J. P&+oZ. 12, 6433652. Klukas, C. K. & Dawid, I. (1976). Cell, 9, 615-625. Levine, A. J., van der Vliet, P. C., Rosenwirth, B., Rabek, J., Frenkel, G., & Ensinger, M. (1974). Cold Spring Harbor Symp. Quant. BioZ. 39, 559-566. Levinson, A. & Levine, A. J. (1977). PiroZogy, 76, l-11. Lewis, A. M., Jr, Levin, M. J., Wiese, W. H., Crumpacker, C. S. & Henry, P. H. (1969). Proc. Nat. Acad. Sci., U.S.A. 63, 1128-1135. Lewis, J. B., Atkins, J. F., Baum, P. R., Solem, R., Gesteland, R. F. & Anderson, C. W. (1976). CeZZ, 7, 141-151. Meissner, H. C., Meyer, J., Maizel, J. V., Jr & Westphal, H. (1977). Cell, 10, 225-235. Meyer, J. Neuwald, P. D., Lai, S. P., Maizel, J. V. Jr, & Westphal, H. (1977). J. PiroZ. 21, 1010-1018. Mulder, C., Arrand, J. R., Delius, H., Keller, W., Pettersson, U., Roberts, R. J. & Sharp, P. A. (1974). Cold Spring Harbor Symp. Quant. BioZ. 39, 397-400. Neuwald, P. D., Meyer, J. Maizel, J. V., Jr & Westphal, H. (1977). J. Viral. 21, 1019-1030. Cberg, B., Saborio, J., Persson, T., Everitt, E. & Philipson, L. (1975). J. Viral. 15, 199-207. Ohtsubo, E., Soll, L., Deonier, R. C., Lee, H. J. & Davidson, N. (1974). J. Mol. BioZ. 89, 631-646. Patch, C. T., Lewis, A. M., Jr & Levine, A. S. (1974). J. Viral. 13, 677-689. Pellegrini, M., Manning, J. & Davidson, N. (1977). Cell, 10, 213-224. Pettersson, U., Tibbetts, C. & Philipson, L. (1976). J. Mol. BioZ. 101,479-501. Philipson, L., Wall, R., Glickman, G. & Darnell, J. E. (1971). Proc. Nut. Acad. Sci., U.S.A. 68, 2806-2809. Pinder, J. C., Staynov, D. Z. & Cratzer, W. B. (1974). Biochemistry, 13, 5373-5378. Price, R. & Penman, S. (1972). J. Mol. BioZ. 70, 435-450. J. (1972). Proc. Nat. Acad. Sk., U.S.A. 69, Robberson, D. L., Kasamatsu, H. & Vinograd, 737-741. Seeburg, P. H., Niisslein, C. & Schaller, H. (1977). Eur.J. Biochem. 74, 107-113. Sharp, P. A., Gallimore, P. H. & Flint, S. J. (1974). Cold Spring Harbor Symp. Quant. BioZ. 39,457-474. Tal, J., Craig, E. A., Zimmer, S. & Raskas, H. J. (1974). Proc. Nat. Acad. Sci., U.S.A. 71, 4057-4061. Thomas, M., White, R. L. & Davis, R. W. (1976). Proc. Nat. Acad. Sci., U.S.A. 73, 22942298. Tibbetts, C., Pettersson, U., Johansson, K. & Philipson, L. (1974). J. Viral. 13, 370-377. van der Vliet, P. C., Levine, A. J., Ensinger, M. J. & Ginsberg, H. S. (1975). J. Viral. 15, 348-354.

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Weber, G. H., Dahlberg, J. E., Cottler-Fox, M. & Heine, U. (1974). Vhwlogy, 62, 284-287. Wellauer, P. K. & Dawid, I. B. (1977). Cell, 10, 193-212. Westphal, H., Eron, L., Ferdinand, F.-J., Callahan, R. & Lai, S. P. (1974). Cold Spring Harbor Symp. Quant. BioZ. 39, 575-579. Westphal, H., Meyer, J. BE Maizel, J. V. Jr (1976). Proc. Nat. Acad. Sci., U.S.A. 73, 2069-2071. White, R. L. & Hogness, D. S. (1977). CeZZ,10, 177-192. Wolfson, J. & Dressler, D. (1972). Proc. Nat. Acad. Sci., U.S.A. 69, 3054-3057.