VIROLOGY

72,443-455 (1976)

Adenovirus III. Mapping

of Viral RNA Sequences

Transcription in Cells Productively

Infected

by Adenovirus

Type 5

S. J. FLINT,l

S. M. BERGET, AND PHILLIP

Center for Cancer Research, Massachusetts

Institute of Technology, Massachusetts 02139

A. SHARP

77 Massachusetts

Avenue,

Cambridge,

Accepted March 16, 1976

Separated strands of restriction endonuclease Eco Rl and Hpa 1 fragments of 32Plabeled Adenovirus 5 DNA have been used in saturation hybridization experiments with cytoplasmic RNA extracted from human cells infected with Adenovirus type 5. The results of such experiments have allowed the construction of maps of the regions of the viral genome complementary to both “early” and “late” Adenovirus 5 RNA. At early times during the lytic cycle, about 25% of the viral genome is expressed as mRNA. Adenovirus 5 DNA sequences complementary to “early” mRNA comprise four discrete regions, two on each strand, of the viral genome: except in one instance, these correspond to the regions of the Adenovirus 2 genome complementary to “early” Adenovirus 2 mRNA. In the exceptional case, “early” Adenovirus 5 mRNA appears to contain not only sequences corresponding, at least in position, to those expressed in Adenovirus 2infected cells, but also some additional, adjacent sequences, reminiscent of the situation in human cells infected with the Adenovirus S/Simian virus 40 hybrid viruses, AdB+ND, and Ad2+ND, (Flint et al., 19’75a). All, or almost all, the information encoded by the Adenovirus 5 genome is expressed as mRNA during the lytic cycle and most of the exclusively “late” mRNA is complementary to the r strand of Adenovirus 5 DNA. The separated strands of fragments of 3*Plabeled Adenovirus 2 DNA generated by the restriction endonuclease Barn Hl have been used to improve the resolution of maps of both Adenovirus 2 and Adenovirus 5 “late” mRNA, which are described here. INTRODUCTION

Bacterial restriction endonucleases cleave double-stranded Adenovirus DNA into sets of overlapping fragments (Nathans and Smith, 1975), powerful tools with which to locate viral functions on the genome. Accordingly, many studies to map Adenovirus 2 (Ad2) have been performed in the last few years. The regions of the Ad2 genome coding for “early” and “late” viral mRNA have been mapped from the results of saturation hybridization experiments in which the separated strands of restriction endonuclease fragments of Ad2 DNA were used as probes: 1 Author to whom reprint requests should be addressed. Copyright 0 1976by Academic hess, Inc. All rights of reproduction in any form reserved.

DNA sequences complementary to “early” viral mRNA, reported to comprise about 20% (Sharp et al., 1974) or about 40% (Philipson et al., 1974) of the genome, are located at four sites along the DNA, two on each strand (Sharp et al., 1974;Flint et al., 197513).The sequences of “late” Ad2 mRNA account for nearly all the information encoded by Ad2 DNA and are transcribed predominantly from the r strand (Philipson et al., 1974; Sharp et al., 1974; Tibbetts et al., 1974). A similar methodology has been used to investigate the relationship between “early” and “late” Ad2 mRNA (Sharp et al., 1974); to define the viral sequences expressed as mRNA in several lines of rat cells transformed by Ad2 and its relation to “early” Ad2 RNA (Flint et 443

444

FLINT,

BERGET AND SHARP

al., 1975b); and to demonstrate the presence of many additional viral RNA sequences, complementary to both strands of the genome, in the nuclei of productively infected cells at both early and late times (Flint and Sharp, 1974; Pettersson and Philipson, 1974; Sharp et al., 1974). Restriction endonuclease fragments of Ad2 DNA have also been used to define various size classes of both “early” and “late” mRNA (Tal et al., 1974; Craig et al., 1975). Genes coding for several “late” Ad2 polypeptides (Lewis et al., 1975), the termini of replication @chilling et al., 1975; Tolun and Pettersson, 1975), and the sites of temperature-sensitive mutations (Grodzicker et al., 1974; Sambrook et al., 1975) have also been mapped. The studies outlined above (see Sharp and Flint, 1976, for a review) have contributed greatly to our understanding of the topography of the Ad2 genome. Our present picture, however, is rather static and we still lack understanding about the underlying dynamics, that is, the mechanisms by which the expression of the Ad2 genome is achieved and regulated. It therefore seemed that a study of RNA synthesis by Adenovirus mutants might prove useful in this respect. Until very recently, however (Begin and Weber, 1975; Weber et al., 1975), no mutants of Ad2 had been described. On the other hand, the genetics of Adenovirus 5 (Ad@, a closely related group C Adenovirus, have been studied in some detail: a large number of mutants have been isolated and partially characterized (Williams et al., 1971; Ensinger and Ginsberg, 1972). When a complementation index of 10 is used, 80 randomly isolated, temperature-sensitive mutants of Ad5 fall into 17 complementation groups (Williams and Ustacelebi, 1971; Williams et al., 1974), two of which represent early functions whose expression is required for viral DNA syntheses (Russell et al., 1972; Wilkie et al., 1973; Russell et al., 1974). As we wished to characterize a selection of these mutants with respect to viral RNA synthesis (Berget et al., 1976), it was necessary to define the viral mRNA sequences synthesized during productive infection of human cells by wild-type Ad5 Accord-

ingly, the strands of the three Eco Rl fragments and five of the seven Hpa 1 fragments of 32P-labeled Ad5 DNA (see Fig. 1) were separated by electrophoresis of denatured DNA and used as probes in saturation hybridization experiments with unlabeled RNA extracted at both early and late times from cells productively infected by Ad5. This exercise has also allowed us to compare the coding regions of the DNA genomes of Ad2 and Ad5: only one major difference, in one of the four regions complementary to “early” mRNA, has been observed. The separated strands of the four fragments of Ad2 DNA generated by cleavage with endonuclease Barn HI have been used to confirm and to improve the resolution of the maps of Ad2 and Ad5 “late” mRNA. A parallel study on the expression of Ad5 ‘mRNA in AdBtransformed rodent cell lines is described in the accompanying report (Flint et al., 1976). MATERIALS

AND METHODS

(a) Cells and virus. HeLa cells were grown in suspension cultures in Eagle’s minimal essential medium (MEM) supplemented with 5% horse serum. Plaque assays were performed as described by Grodzicker et al. (1974) using HeLa cells, originally obtained from Dr. J. Williams, Insti0 0 20 30

so 60 70 80 90 loo

40

Adenovirus 5 lw 840 ‘c’

A 4.0 I E’

C

242265 II %

548 A

8

B 864 904 1 I ‘F’ D

&cJ Ri

?!!?I

FIG. 1. Restriction endonuclease cleavage maps of Ad5 and Ad2 DNA. The solid horizontal line represents the genome of either virus. Vertical lines and numerical coordinates above the line indicate sites of cleavage, while letters below the line give the nomenclature of each cleavage pattern. These data are taken from Mulder et al. (19741and Roberts et aE., manuscript in preparation.

ADENOVIRUS

5 LYTIC

tute of Virology, Glasgow, cultivated on plastic dishes (NUNC, Denmark) in Dulbecco’s modification of Eagle’s medium (DME, Dulbecco and Friedman, 1959) supplemented with 10% calf serum. Ad5 and Ad2 were propagated in suspension cultures of HeLa cells (Pettersson et al., 1967) at an input multiplicity of 10 PFU/cell. Purification of virus and preparation of 32P-labeled Ad5 and Ad2 virus are described in the accompanying report (Flint et aZ., 1976). (b) DNA. Ad5 and Ad2 DNA were extracted from purified virions as described by Pettersson and Sambrook (1973). (c) Restriction endonucleases. Endonucleases Eco Rl and Hpa 1 were prepared as described in the accompanying report. Bum Hl was the generous gift of R. J. Roberts. (d) Preparation of specific fragments of Adenovirus DNA. Eco Rl and Hpa 1 fragments of Ad5 DNA were prepared as described previously for the Ad2 fragments (Gallimore et al., 1974). Digestion of Ad2 DNA by Barn Hl was performed as described by Sambrook et al. (1975) and the resulting 4 fragments were separated by electrophoresis on 0.7 x 15-cm cylindrical, 1.4% agarose gels at 2 V/cm for 8-10 hr. (e) Separation of the strands of restriction endonuclease fragments of Ad5 and Ad2 DNA. Restriction endonuclease frag-

ments of Ad2 and Ad5 DNA were recovered from agarose gel sites by electrophoresis (Pettersson et al., 1973) and purified by phenol extraction. The DNA of each fragment was concentrated by ethanol precipitation and dissolved in 10 ~1 of 0.02 M Tris-HCl, pH 8.5, containing 0.002 M EDTA and denatured in 0.2 M NaOH at 37” for 10 min. Three microliters of solution of 60% sucrose and 0.5% bromphenol blue were added to each sample, which was then layered onto an 0.7 x 15cm cylindrical 0.7% agarose gel in 0.036 M Tris, 0.03 M NaH,PO,, pH 7.7, containing 0.001 M EDTA (Hayward, 1972). Electrophoresis at 2 V/cm for 8 hr was in this buffer. In some cases, complete Eco Rl digests of Ad5 DNA were denatured and subjected to electrophoresis in this manner. Each gel was stained by immersion in

RNA

SEQUENCES

445

electrophoresis buffer containing 0.5 /.@l ml of ethidium bromide and the fluorescence of the bound dye observed during excitation with ultraviolet light (Sharp et al., 1973). Each agarose slice cut from such gels was placed in 0.5 ml of 0.1 M phosphate buffer, pH 6.8 (Kohne and Britten, 1968) containing 1.0 M NaCl and 0.5% SDS and boiled for 10 min. Each solution was then incubated at 68” for 10 min in 0.3 M NaOH, neutralized, and further incubated at 68 for a time equivalent to lo-20 X P/2, calculated from the complexity of the DNA fragment and its concentration to allow any contaminating complementary strand sequences to reanneal with the strand in excess. Any double-stranded DNA thus formed was removed by chromatography on hydroxylapatite. (f) RNA. HeLa cells, at a concentration of 3-4 x 106/ml, were infected with lo-20 PFU/cell of Ad5. After adsorption of the virus at 37” for 30 min, the cells were diluted lo-fold and incubated at 37” for 8 hr in the presence of 20 pg/ml of cytosine arabinoside (“early”) or for 18 hr in the absence of drugs (“late”). Cells were harvested at the appropriate time, and nuclear and cytoplasmic fractions prepared by two brief t,reatments with 0.65% NP40 in 0.01 M Tris-HCl, pH 7.9, containing 0.001 M EDTA and 0.15 M NaCl (Kumar and Lindberg, 1972). The cytoplasmic fraction was diluted with an equal volume of 0.02 M Tris-HCl, pH 7.9, containing 7 M urea, 0.35 M NaCl, 0.02 M EDTA, and 1% SDS (Holmes and Bonner, 1972), and deproteinized by phenol:chloroform extraction. RNA preparations were purified fur-, ther by treatment with deoxyribonuclease I and chromatography on G-75 sephadex, as described previously (Sharp et al., 1974) and finally dissolved at concentrations of 5-10 mg/ml in 0.01 M Tris-HCl, pH 7.9, containing 0.001 M EDTA and 0.15 M NaCl. (g> Hybridization conditions. RNA:DNA hybridization reactions contained, in a final volume of 0.1-0.2 ml, 0.10 it4 sodium phosphate, pH 6.8, 1.0 M NaCl, 0.5% SDS, about 250 cpm of 32P-labeled singlestranded DNA (0.2-0.5 x 10e3 pg) and

446

FLINT,

BERGET AND SHARP

various concentrations of unlabeled, infected cell RNA. Incubation was at 68”, generally for 24 hr. Reactions were then diluted with 1.0 ml of 0.125 M sodium phosphate, pH 6.8, containing 0.4% SDS, and stored at 4” until analyzed by hydroxylapatite chromatography (Sambrook et al., 1972; Flint et al., 1975b).

native

Eco Rl A

RESULTS

Strand Separation Single-stranded DNA probes representing discrete regions of the viral genome are a prerequisite to the mapping studies we wished to perform. Gel electrophoresis of denatured DNA separates the strands of T7 and A DNAs (Hayward, 1972) and can also be applied to fragments of Ad2 and SV40 DNA generated by cleavage with restriction endonucleases (Sharp et al., 1974; Flint et al., 1975a). This method will also separate the strands of the three Eco Rl fragments of Ad5 DNA, as illustrated in Fig. 2, which shows the electrophoresis of a denatured Eco Rl digest of Ad5 DNA on 0.7% agarose. As the three DNA fragments generated are very different in size, 77, 16, and 7% of the Ad5 genome, it is possible to separate the strands of all three fragments on one gel: as Fig. 2 shows, the three doublets corresponding to denatured Eco Rl fragments A, B, and C DNA are well resolved from one another and are also clearly distinguishable from doublestrand DNA of the three fragments. The two DNA bands of each doublet stain with equal intensity, suggesting that they do represent complementary strands. This has been confirmed by hybridizing 32P-labeled DNA of each fast and slow band with excess, unlabeled DNA of the two strands of Ad2 DNA. These unlabeled strands were prepared by equilibrium sedimentation in CsCl gradients after binding poly(U, G) to denatured Ad2 DNA (Land graf-Leurs and Green, 1971): we have previously shown that the strand with the lesser affinity for poly(U, G) and therefore of lower bouyant density in CsCl, is transcribed to the right and have therefore designated it the r strand, and its complement the 1 strand (Sharp et al., 1974). The purified DNA of each fast and slow band of

denatured

-Eco Rl A

native

-Eco Rl B

native

-Eco Rl C

denatured

-Eco Rl B

denatured

-Eco Rl C

FIG. 2. Electrophoresis of denatured DNA of the three Eco Rl fragments of Ad5 DNA. Two micrograms of Ad5 DNA were digested with Eco Rl as described in Materials and Methods. The total digest was denatured in 0.2 M NaOH and layered onto an 0.7 x 15-cm gel of 7% agarose, as described in Materials and Methods. Electrophoresis was for 8 hr at 2 V/cm. The gel was stained in 0.5 pg/ml ethidium bromide in electrophoresis buffer and the fluorescence of the bound dye photographed during excitation by ultraviolet light (Sharp et al., 1973). Some native DNA of each of these fragments is seen on this gel, as some renaturation takes place during loading of the denatured digest onto the gel.

ADENOVIRUS TABLE

5 LYTIC RNA SEQUENCES

1

AWGNMENT OF THE SEPARATED STRANDI? OF THE THREE Eco RI FRAGMENTS OF Ad5 AND THE FOUR Barn HI FRAGMENTS OF Ad2 DNA TO THE r AND 1 STRANDS OF THE VIRAL GENOME~

32P-labeled fragment strand

(a) Eco Rl Ad5 DNA fragments AF AS BF BS CF cs (b) Bum HI Ad2 DNA fragments BF BS CF cs DF DS

Percenta e 32P- La&i labeled Dk A in assignHybrid ment r 1

75.2 4.3 4.1 84.2 5.5

3.2 54.5 82.5 4.3 84.6

78.9

1.9

95.5 0 0 62.3 88.8

0

0 68.4 85.2 0 7.6 67.6

1 r r 1 r 1 1 r r 1 r 1

n In the experiments shown in parts (a) and (b), about 250 cpm (0.25-0.5 x lo+ pg) of 3ZP-labeled DNA of each fast or slow band were hybridized with about lo-* pg of unlabeled r and 1 strand Ad2 DNA in 0.10 M phosphate buffer, pH 6.8, containing 1.0 M NaCl and 0.5% SDS at 68” for 16 hr. In experiment (a), the strands of the whole Ad2 genome, separated by equilibrium sedimentation in CsCl after binding of poly(U, G) to denatured DNA (Sharp et al., 1974), were used, while in experiment (b) the unlabeled r and 1 strands were those of Eco Rl fragment A of unlabeled Ad2 DNA, separated by gel electrophoresis. Hybridization was analyzed by chromatography on hydroxylapatite.

the three Eco Rl fragments of Ad5 DNA annealed with only one of the two strands of whole Ad2 DNA, as Table 1 shows. Thus, the bands of each DNA doublet observed after electrophoresis of denatured Eco Rl fragments of Ad5 DNA do indeed represent separated strands. It is also clear from this experiment that the parameter(s) responsible for the observed separation, presumably some sequence difference between the two strands, is asymmetrically distributed along the Ad5 genome: fast strand DNA of Eco Rl fragment A is complementary to the r strand of Ad2 DNA and thus comprises 1 strand sequences, while 32P-labeled fast strand

447

DNA of fragments B and C hybridizes exclusively with the 1 strand of Ad2 DNA and therefore represents the r strand of these fragments. Table 1 also shows the results of a similar experiment in which purified fast and slow band DNA of three of the four fragments of Ad2 DNA generated by cleavage with endonuclease Bum HI were hybridized to unlabeled r and 1 strand Ad2 DNA. The fast strands of those fragments comprising the left-hand 40% of the genome, Bum HI B and D, are homologous to 1 strand DNA, while the 1 strands of Bum HI fragments A and C migrate as the slow band. Hybridization of 32P-labeled fast or slow DNA to its homologous unlabeled strand is low in all cases, indicating that purification by one cycle of self-annealing followed by hydroxylapatite chromatography removes contaminating complementary strand sequences. Electrophoresis of denatured DNA also separates the strands of five of the seven Hpa 1 fragments of Ad5 DNA, but denatured DNA of Hpa 1 fragments A and G migrates as one band under these conditions and we have not achieved separation of their strands. As hybridization reaction between unlabeled, infected cell RNA and 32P-labeled, single-strand viral DNA have been analyzed by chromatography on hydroxylapatite, the 32P-labeled DNA must be suitably degraded: if the DNA segments are too short, less than about 50 nucleotides in length, DNA:RNA hybrids may not bind to hydroxylapatite (Wilson and Thomas, 1973). On the other hand, hydroxylapatite chromatography does not distinguish between DNA:RNA hybrids that are completely double-strand and those that have single-stranded DNA “tails”: it is thus possible to overestimate the fraction of 32Plabeled DNA probe that is complementary to a given RNA if that probe is not sufficiently fragmented. The suitability of each preparation of separated strands of restriction endonuclease fragments of 32P-labeled viral DNA is therefore tested routinely. The purified and fragmented DNA separated strands of a restriction endonuclease fragment of 32P-labeled viral DNA are hybridized with unlabeled strands of the DNA of a second fragment that overlaps

448

FLINT,

BERGET AND SHARP

TABLE 2 !hsr

FOR THE SUITABILITY

OF 32P-~~~~

SINGLE-

STRANDED PROBES WHEN HYBRIDIZATION AMAYED BY CHROMATOGRAPHY ON HYDROXYLAPATITE”

IS

3ZP-labeled fragment DNA strand

Unlabeled DNA

Percentage 32P-labeled DNA in hybrid

Eco Rl Br

None HpalDr Hpu 1 Dl None Hpa 1 Dr HpalDl

1.7 2.3 60.6 3.6 64.8 1.9

Eco Rl Bl

a The =P-labeled, separated strands of Eco Rl fragment B of Ad5 DNA were annealed with unlabeled r and 1 strand DNA of Hpu 1 fragment D of Ad5, as described in the legend to Table 1. Formation of DNA:DNA hybrid was assayed by chromatography on hydroxylapatite.

arabinoside, as described in Materials and Methods. Increasing amounts of such RNA were hybridized with the separated strands of the three Eco Rl fragments of 32P-labeled Ad5 DNA, with the result shown in Fig. 3. Ad5 “early” RNA saturates 7-8% of both the r and 1 strands of Eco Rl fragment A, 20 and 30% of the r and 1 strands, respectively, of Eco Rl fragment B, and 80% of the r strand of Eco Rl fragment C. However, Eco Rl fragment A of Ad5 comprises nearly 80% of the viral genome (see Fig. 1). Similar hybridizations between Ad5 “early” cytoplasmic RNA and the 32P-labeled strands of five of the seven Hpa 1 fragments of Ad5 DNA 100

(a) Ad5 “Early” Cytoplasmic &Q RlA

&Q RlB

RNA &g RlC

60

the first only partially. In the example 60 shown in Table 2, the 32P-labeled strands e 40 of Eco Rl fragment B of Ad5 DNA were ii2 ,F--‘--A hybridized to the separated strands of Hpa 20 t’ .--. .s / 1 fragment D, which is 60% homologous to ag 0 L-L 0.5 IO 0 / 05 IO r 0.--- *--*05 ---. IO . . Eco Rl fragment B (see Fig. 1). In this case, 58.9 and 61.2% of the fast and slow w z (b) Ad5 “Late” Cytoplasmic RNA strands, respectively, of Eco Rl fragment g 100 &o RlA & I?18 4 B formed hybrid with the complementary /strand of Hpa 1 fragment D. These 32Plabeled strands were fragmented by boiling for 10 min at neutral pH, followed by 40incubation for a further 10 min at 68” in 0.3 M NaOH, as described in Materials and Methods, section (e). We have found that I 1 this method, rather than boiling in alkali, 1.0 reproducibly degrades 32P-labeled, singleRNA Concentration, mg/ml strand viral DNA to fragments of approFIG. 3. Hybridization of Ad5 “early” and “late” priate length for use in hybridization reac- Ad5 cytoplasmic RNA to 3ZP-labeled, separated tions to be analysed by hydroxylapatite strands of the three Eco Rl fragments of Ad5 DNA. chromatography. RNA was purified, as described in Materials and Mapping of Ad5 RNA Sequences

‘%arly”

Cytoplasnic

Previous studies on AdB-infected cell RNA have shown that polysomal RNA, poly[A)-containing RNA and total cytoplasmic RNA contain the same viral RNA sequences (Flint and Sharp, 1974). Ad5 “early” RNA was therefore purified from the total cytoplasmic fraction of HeLa cells infected for 8 hr with lo-20 PFU/cell of Ad5 in the presence of 20 pg/ml of cytosine

Methods, from the cytoplasm of HeLa cells infected with lo-20 PFU/cell of Ad5 either for 8 hr in the presence of 20 pg/ml of cytosine arabinoside (“early”) or for 18 hr (“late”) at 37”. Increasing concentrations of “early” or “late” cytoplasmic RNA were annealed with 250 cpm (-2.5-5 x 10e4 pg) of 32P-labeled, single-stranded DNA in 0.10 M phosphate buffer, pH 6.8, containing 1.0 M NaCl and 0.5% SDS for 24 hr at 68”. The fraction of DNA entering hybid was determined by chromatography on hydroxylapatite. (O-0-0) and (A- - -A- - -A) represent annealing to the r and 1 strands, respectively, of each fragment.

ADENOVIRUS

5 LYTIC

Hpa 1C

-Hpo 16

_

/.-.Li 20 ,* --=---.w -/ d5 ,loa”-,; 8 o(/ ,

,*--A---‘A P 1'

.L.

-/I ,b {.-*--; RNA

Hpa 1F

-Hpa 1E

Hpa 1D

l y.-.-

449

SEQUENCES

is accounted for by hybridization to the r strands of five Hpa 1 fragments used here. Thus, Ad5 “early” RNA is not complementary to the r strands of Hpa 1 fragments A and G. There is, however, nearly a 2% difference in the values of the 1 strand complementary to “early” RNA computed from the two sets of data. However, when the separated strands of Barn HI fragments B, C, and D of Ad2 DNA, which comprise the left-hand 60% of the viral genome (see Fig. 1) and therefore include the region homologous to Hpa 1 fragments A and G, were hybridized with Ad5 “early” RNA, no hybridization to the 1 strand of any fragment was observed (data not

were therefore performed to achieve some resolution of the left-hand end of the genome. The results of one such set of hybridizations are shown in Fig. 4, while the saturation values observed with both the Eco Rl and Hpa 1 fragments are summarized in Table 3: the fraction of the total Ad5 genome complementary to “early” Ad5 RNA, calculated assuming one strand equivalent is informational, is also given. The values of Ad5 “early” RNA complementary to the r strand of Ad5 calculated from the two sets of data are identical within experimental error, indicating that all hybridization observed to the r strands of the three Eco Rl fragments of Ad5 DNA 0 100 b r” c so4 5 60n i 402

RNA

,.‘5~-..---~;o

Concentration,

.-•--. 7 ~-“~~;l---,~

mg/ml

FIG. 4. Annealing of Ad5 “early” cytoplasmic RNA to the separated strands of Hpa 1 fragments B-F of Ad5 DNA. RNA was extracted and hybridization reactions were performed as described in the legend to Fig. 3. (O-0-0) and A- -A- -A) represent the hybridization of Ad5 “early” cytoplasmic RNA to the r and 1 strands, respectively, of each fragment. TABLE Ad5 DNA

SEQUENCES

Percentage

Eco Rl fragment

Hpa 1 fragment

3’0

B

Percentage total gemone expressed

C

1

r

1

r

1

r

1

7

8

20

30

80

0

14.4

11.1

r 14.9

1 9.3

C 1 15

mRNA”

r

B

Strand

TO Ad5 “EARLY”

32P-labeled DNA in hybrid

A

Strand

3

COMPLEMENTARY

2:

D 1 0

r 0

E 1 50

2:

F 1 0

2;

1 10

a The fraction of 3ZP-labeled DNA entering hybrid with excess Ad5 “early” cytoplasmic RNA is given for each Eco Rl fragment and strand combination and for the strands of Hpa 1 fragments B-F. These saturation values are taken from the data shown in Fig. 3 and 4 for the Eco Rl and Hpa 1 fragments, respectively. The percentage of the total genome expressed as cytoplasmic RNA was calculated as the product of the fraction of a fragment strand annealing to RNA and the fractional length of that fragment (see Fig. 1). The fractional lengths of Eco Rl fragments A, B, and C of Ad5 DNA are 0.77, 0.16, and 0.07, respectively, while Hpa 1 fragments, B, C, D, E, and F comprise 0.28, 0.20, 0.10, 0.04, and 0.04 fractional length of the Ad5 genome, respectively.

450

FLINT,

BERGET AND SHARP

shown). The difference shown in Table 3, then, probably reflects difficulties in measuring the low levels of hybridization to Eco Rl fragment A where an error of 1% in the saturation value taken results in error of 0.8% in the fraction of the total genome calculated to be complementary to “early” mRNA. Thus, Ad5 “early” mRNA is transcribed from about 25% of the viral genome. These two sets of overlapping saturation hybridization values allow a map of the Ad5 DNA sequences complementary to “early” mRNA to be constructed. Consider, for example, the hybridization of Hpa 1 fragments C and E: “early” Ad5 mRNA saturates 20 and 25%, respectively, of the r strands of Hpa 1 fragments E and C, but is not complementary to 1 strand DNA of these fragments (Fig. 4). This hybridization corresponds to a total of 5.9 units of the genome and thus accounts for the 7% of the r strand of Eco Rl fragment A, 5.4 units, complementary to “early” mRNA. In constructing our map, we have assumed that when RNA sequences are complementary to the same strand of adjacent restriction endonuclease fragments, they form one chain. Thus, we map the “early” mRNA transcribed from the r strands of Hpa 1 fragments E and C from a position corresponding to 20% of fragment E, 0.8 units, to the left of the Hpa 1 cleavage site at position 4 (see Fig. 1) to a position 25% of fragment C, 5.1 units, to its right, that is, between 3.2 and 9.1, when the genome is represented as 100 units. Similarly, Ad5 “early” mRNA complementary to the r strand of adjacent Eco Rl fragments B and C would map from position 78.2 to position 87.4, 9.2 units in total, if these formed one RNA chain. The hybridization observed to the r strands of overlapping Hpa 1 fragments D and F, a total of 8.9 units, would confirm this arrangement. “Early” Ad5 RNA is also complementary to 30-35% of the 1 strand of Eco Rl fragment B: within this region, 10 and 50% of the 1 strands of adjacent Hpa 1 fragments F and D, respectively (see Fig. 11, form hybrid with Ad5 “early” RNA. This message can therefore be mapped from position 90.1 to position 95.5 on the 1

strand. The final region of the Ad5 genome coding for “early” mRNA, complementary to about 8% of the 1 strand of Eco Rl fragment A and to 15% of the 1 strand of Hpa 1 fragment B must lie within the region of overlap of these two fragments, that is, somewhat between positions 58.8 and 76.7, but cannot be located further from this data. The map based on these considerations of the data shown in Table 3 is given in Figure 5, which shows the regions of the Ad5 genome complementary to Ad5 “early,” mRNA. Mapping

of AdS “Late” mBNA

Similar experiments to those described for Ad5 “early” cytoplasmic RNA have been performed with RNA extracted from cytoplasm of HeLa cells 18 hr after infection with Ad5. Such RNA was first hybridized with 32P-labeled, separated strands of the three Eco Rl fragments of Ad5 DNA, with the result shown in Fig. 3. Ad5 “late,” cytoplasmic RNA contains sequences complementary to 75, 50, and 92% of the r strands of Eco Rl fragments A, B, and C, respectively, and to 20 and 40%, respectively, of the 1 strands of Eco Rl fragments A and B. These sequences comprise a total of 72 and 22% of the r and 1 strands, respectively (Table 4), indicating that almost all the information encoded by the viral genome is expressed at this time. The predominance of RNA sequences complementary to the r strand of late times has also been observed with Ad2 “late” RNA (Green et al., 1970; Sharp et al., 1974; Tib-

.

Hpa -

1

-+-

A

&o Rl E,

c

I

A

F,

8

*C II 0

,c,

* B

I

f&D1

6 10

1 20

1 30

1 40

1 x)

1 60

1 70

1 80

1 90

I 100

FIG. 5. The regions of the Ad5 genome coding for

“early” and “late” mRNA. The Ad5 genome is represented by the two solid horizontal lines, which also indicate the Eco Rl and Hpu 1 cleavage fragments. The thin lines above and below the genome indicate these regions of the genome complementary to Ad5 “early” mRNA, while those seqvences expressed only during the late phase of infection are represented by thick lines. Arrows show the direction of transcription (Sharp et al., 1974).

ADENOVIRUS

451

5 LYTIC RNA SEQUENCES TABLE 4

Ad5 DNA

SEQUENCES

TO Ad5 “LATE” 32P-labelecl DNA in hybrid

mRNA”

COMPLEMENTARY

Percentage

Percentage total

A

Eco Rl fragment r 75

Strand

1 20

B

Hpa 1 fragment r 70

Strand

1 25

1 22

6:

r 55

Strand

5’s

3:

1 55

8:

r 72.2

1. 21.7

(3~3

1 (17.7)

F 20 1

7:

10 1

D

C 1 25

1 0 E

3’0

B 1 30

r 92

1 40 D

C

A

Barn Hl fragment

r 50

r 85

1 0

1 0

6if.8

1 19.8

” The data is expressed as in Table 3. The saturation values for the separated strands of the three Eco Rl fragments and Hpa 1 fragments B-F of Ad5 DNA and Barn HI fragments B-D of Ad2 DNA are taken from Fig. 3,6, and 7, respectively. The Barn HI fragments A, B, C, and D of Ad5 DNA are 0.41,0.28,0.18, and 0.13 fractional lengths of the intact genome. 0” 0

IOO-

r” .c

go-

-Hpa 16

-

-Hpa 1C

-

-Hpa ID

i

-Hpa 1E

-

-Hpa 1F .,*--r-1

RNA

Concentration,

mg/ml

FIG. 6. Hybridization of Ad5 “late,” cytoplasmic RNA to the separated strands of Hpa 1 fragments B-F of Ad5 DNA. Hybridization reactions were performed and analyzed as described in the legend to Fig. 3. (O-O-0) and (A- -A- -A) represent the hybridization of Ad5 “late,” cytoplasmic RNA to the r and 1 strands, respectively, of each fragment.

betts and Pettersson, 1974). “Late,” cytoplasmic RNA was then hybridized to the separated strands of Hpa 1 fragments B-F, as shown in Fig. 6. These saturation values and the fraction of the total genome they represent are summarized in Table 4. Clearly, these sequences do not correspond to all the hybridization observed with the separated strands of the Eco Rl fragments of Ad5 DNA. As we could not separate the strands of the other two Hpa 1 fragments of Ad5 DNA, A and G, it was necessary to study the region of the genome these fragments encompass, positions 26-59, using another set of fragments, the Barn HI frag-

ments of Ad2 DNA, which span this region conveniently and are at least S-88% homologous to Ad5 DNA, as measured by chromatography on hydroxylapatite (data not shown). The results of hybridizing the separated strands of Barn HI fragments B, C, and D of 32P-labeled Ad2 DNA to both Ad5 and Ad2 “late,” cytoplasmic RNA are shown in Fig. 7. The saturation values observed with Ad5 RNA are summarized in Table 4. The saturation values observed when the 32P-labeled separated strands of these three sets of restriction endonuclease fragments of Ad5 or Ad2 DNA are hybridized

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may lead to under or overestimation of the fraction of a probe complementary to a given RNA, as discussed above. Figure 5 shows a map of the regions of the Ad5 genome from which Ad5 “early” and “late” mRNA sequences are transcribed. This map was derived from saturation hybridization experiments between “early” and “late” cytoplasmic RNA extracted from Ad5-infected human cells and the separated strands of the three Eco Rl FIG. 7. Annealing of Ad5 and Ad2 “late” cytofragments and Hpa 1 fragments B-F of plasmic RNA to the separated strands of Bum HI 32P-labeled Ad5 DNA. Ad5 and Ad2 “late” fragments B-D of Ad2 DNA. Ad2 “late,” cytoplasmic RNA was purified from HeLa cells infected for 18 hr mRNA sequences were also analyzed with at 37” with lo-20 PFU/cell Ad2 as described for Ad5 the separated strands of the four frag“late” cytoplasmic RNA. ‘Hybridization reactions ments of Ad2 DNA generated by cleavage were performed and analyzed as described in the with Bum HI. The arrangements of “early” legend to Fig. 3. (O-0-0) and (A- -A- -A) show the and “late” coding regions shown in Fig. 5 annealing of Ad5 “late” cytoplasmic RNA to the r were deduced from the saturation values and 1 strand, respectively, of each fragment, while summarized in Tables 3 and 4, respec(O--O--O) and (A-A-A) show hybridization of tively, assuming that: Ad2 “late,” cytoplasmic RNA to the r and 1 strands, (1) One strand equivalent of the Ad5 respectively. genome is informational, or, to put it anto Ad5 “late” cytoplasmic RNA can be used other way, mRNA sequences are not comto construct a map of the regions of the plementary to one another. (2) When RNA sequences are compleviral genome complementary to Ad5 “late” mRNA. This map, which is shown in Fig. mentary to the same strand of adjacent 5, was derived from the saturation values restriction endonuclease fragments, they given in Table 4 using the principles and form one chain. Thus the arrangements shown in Fig. 5 assumptions outlined in detail above for are the most simple that are consistent Ad5 “early” mRNA. with the data reported here. DISCUSSION The map of Ad5 “early” mRNA seThe complementary strands of restric- quences described here is identical, as far tion endonuclease fragments of Ad5 and as we can tell, to that of Ad2 “early” Ad2 DNA can be separated by electropho- mRNA previously described except in one resis of denatured fragment DNA. When region, approximately position 78 to posisuch 32P-labeled, single-stranded probes tion 87. Within the region, Ad2 “early” are used in saturation hybridization exper- mRNA was originally mapped from posiiments with unlabeled RNA extracted tion 80 to position 86 on the r strand from Ad5-infected cells, the fraction of (Sharp et al., 1974; Flint et al., 1975b). This arrangement was confirmed by studies on DNA from different regions of the viral genome complementary to given RNA can the “early” mRNA sequences expressed in be determined. The saturation values ob- human cells infected by two Ad2lSV40 served are reproducible to within 5% when nondefective hybrid viruses, Ad2+ND, and assayed by hydroxylapatite chromatogra- Ad2+ND3, in which this region of the gephy (and similar values are observed when nome is deleted (Flint et al., 1975a). The hybridization reactions are analyzed by only mRNA complementary to the region digestion with S1 nuclease (Flint and of the Ad2 genome between positions 78 Sharp, manuscript in preparation)), pro- and 87 synthesized in Ad2+ND, and vided that the degree of fragmentation of Ad2+ND3 infected cells, some 1000bases in 32P-labeled, single-stranded DNA probes is all, appears to map from about position 77 controlled: ignorance of this parameter to position 80. Thus, Ad5 “early” mRNA

ADENOVIRUS

5 LYTIC

complementary to the r strand from position 78 to position 87 appears to correspond, in position at least, to Ad2 “early” mRNA described above plus the cytoplasmic RNA sequences characteristic of Ad2+ND, and Ad2+ND, infection. Unfortunately, attempts to compare directly these Ad5, Ad2 and Ad2+ND, or Ad2+ND, “early” RNA sequences were not successful, as Ad2 and Ad5 are not sufficiently homologous at this location (Garon et al., 1973). The nature of the polypeptide(s) encoded by this region of the viral genome is not known, but it does not seem to be essential for the growth of the virus in tissue culture (Flint et al., 1975a). Indeed, the observed variation in transcription of mRNA from this region of the r strand, positions 78 to 87, may perhaps reflect on the nonessential nature of this gene product and is clearly of interest. The Ad5 “early” mRNA sequences described here account for a total of 25% of the viral genome. Our previous studies on human cells productively infected by Ad2 indicated that 22.5% of the viral genome is expressed at early times (Sharp et al., 1974). As discussed above, this difference reflects the expression of some additional DNA sequences as “early” mRNA in Ad5infected cells. Others have reported a rather higher value, some 40%, for the fraction of the Ad2 genome complementary to “early” mRNA (Philipson et al., 1975). The reason for this difference is not clear, but could reflect a number of experimental factors. Figure 5 also shows our current map of Ad5 DNA sequences complementary to “late” mRNA: the thick lines represent those regions expressed at late times only, but all “early” mRNA sequences can also be detected at this time. This map corresponds, within experimental error, to similar maps of Ad2 “late” mRNA. Although the three sets of restriction endonuclease fragments have allowed a reasonable delinition of both “early” and “late” coding regions as described here, some uncertainties remain. These include the detailed arrangement of the “early” RNA sequences complementary to the r strand of Hpa 1 C and E, the exact location of the 1 strand

RNA

SEQUENCES

453

sequences between positions 59 and 71 which are complementary to “early” mRNA and the precise position of “late” 1 strand sequences within Bum HI fragment B. The use of the separated strands of smaller fragments of Ad2 DNA, such as those generated by cleavage with endonuclease Hind III, and measurement of the size of mRNA species, should resolve these ambiguities. Recently, polypeptides of various structural proteins of Adenovirus 2 and 5 have been located on the genome by several methods, including physical mapping of sites of temperature-sensitive mutation (Grodjicker et al., 1974; Sambrook et al., 1975), analysis of the antigenic determinants of structural proteins of recombinants of Ad2+ND, and Ad5 (Mautner et al., 1975), and in vitro translation of viral RNAs selected by hybridization to specific fragments of the Ad2 genome (Lewis et al., 1975). These studies agree in placing the genes for polypeptides II and IV, constituents of the hexon and fiber within positions 40-60 and 85-100, respectively, but give no information about the strand from which the appropriate mRNA is transcribed. Inspection of Fig. 5, however, reveals that the mRNAs for both hexon and fiber polypeptides must be transcribed from the r strand, as no exclusively “late” mRNA is complementary to the 1 strand within this region. Clearly, these powerful techniques in combination will soon provide detailed structural maps of the adenovirus genome. ACKNOWLEDGMENTS We thank Jan Haverty for technical assistance and R. J. Roberts for a generous gift of endonuclease Barn HI. S. J. Flint was supported by a fellowship from the Science Research Council of Great Britain, and S. M. Berget by a fellowship from N.I.H. P. A. Sharp is supported by an American Cancer Society faculty grant. This work was funded by grants from the National Cancer Institute (No. CA 13106-03) and the American Cancer Society (No. VC-151). REFERENCES B&GIN, M., and WEBER, J. (1975). Genetic analysis of Adenovirus type 2. I. Isolation and genetic

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characterization of temperature-sensitive mutants. J. Viral. 15, l-7. BERGET, S. M., FLINT, S. J., WILLIAMS, J. F., and SHARP, P. A. (1976). Adenovirus transcription. IV. Synthesis of viral-specific RNA in human cells infected with temperature-sensitive mutants of Adenovirus 5. J. Viral., in press. CRAIG, E. A., ZIMMER, S., and RASKAS,H. J. (1975). Analysis of early Adenovirus 2 RNA using Eco R.Rl viral DNA fragments. J. Viral. 15, 12021213.

DULBECCO,R., and FRIEDMAN, G. (1959). Plaque production by the polyoma virus. Virology 8,396397. ENSINGER,M. J., and GINSBERG,H. S. (1972). Selection and preliminary characterization of temperature-sensitive mutants of type 5 Adenovirus. J. Viral. 10, 328-339. FLINT, S. J., and SHARP, P. A. (1974). Mapping of viral-specific RNA in the cytoplasm and nucleus of adenovirus a-infected human cells. Brookhaven Symp. Biol. 26, 333-358. FLINT, S. J., WEWERKA-LUTZ, Y., LEVINE, A. S., SAMBROOK, J., and SHARP, P. A. (1975a). RNA sequences complementary to SV40 and Adenovirus 2 DNA in Ad2+ND, and Ad2+ND, infected cells. J. Viral. 16, 662-673. FLINT, S. J., GALLIMORE, P. H., and SHARP, P. A. (1975b). Comparison of viral RNA sequences in Adenovirus a-transformed and lytically-infected cells. J. Mol. Biol. 96, 47-68. FLINT, S. J., SAMBROOK,J., WILLIAMS, J. F., and SHARP, P. A. (1976). Viral nucleic acid sequences in transformed cells. IV. A study of the sequences of Adenovirus 5 DNA and RNA in four lines of Adenovirus 5 transformed rodent cells using specific fragments of the viral genome. Virology 72, 456-470. GALLIMORE, P. H., SHARP,P. A., and SAMBROOK, J. (1974). Viral DNA in transformed cell lines. II. A study of the sequences of Adenovirus 2 DNA in nine lines of transformed rat cells using specific fragments of the viral genome. J. Mol. Biol. 89, 49-72. GARON, C. F., BERRY, K. W., HIERHOLZLR, J. C., and ROSE, J. A. (1973). Mapping of base sequence heterologies between genomes from different adenovirus serotypes. Virology 54, 414-426. GREEN, M., PAREONS, J. T., PiNA, M., FUJINAGA, K., CAFFIER, H., and LANDGRAF-LEURS, M. (1970). Transcription of Adenovirus genes in productively-infected and in transformed cells. Cold Spring Harbor Symp. &aunt. Biol. 35,803-818. GRODZICKER, T., WILLIAMS, J. F., SHARP, P. A., and SAMBROOK, J. (1974). Physical mapping of temperature-sensitive mutations of adenoviruses. Cold Spring Harbor Symp. Quant. Biol. 39,439-446. HAYWARD, G. (1972). Gel electrophoresis separation

of the complementary strands of bacteriophage DNA. Virology 49, 342-344. HOLMES,D. S., and BONNER, J. (1973). Preparation, molecular weight, base composition and secondary structure of giant, nuclear ribonucleic acid. Biochemistry 12, 2330-2338. KOHNE, D. E., and BRITTEN, R. J. (1971). Hydroxylapatite techniques for nucleic acid reassociation, In “Procedures in Nucleic Acid Research” (G. L. Cantoni and D. R. Davies, eds.), Vol. 2, 500-512. Harper and Row, New York. KUMAR, A., and LINDBERG, U. (1972). Characterization of messenger ribonucleoprotein and messenger RNA from KB cells. Proc. Nat. Acad. Sci. USA 69, 681-685. LANDGRAF-LEURS, M., and GREEN, M. (1971). Adenovirus DNA. III. Separation of the complementary strands of Adenovirus 2,7, and 12 DNA molecules. J. Mol. Biol. 60, 185-202. LEWIS, J. B., ATKINS, J. F., ANDERSON, C. W., BAUM, P. R., and GESTELAND, R. F. (1975). Mapping of late adenovirus genes by cell-free translation of RNA selected by hybridization to specific DNA fragments. Proc. Nat. Acad. Sci. USA 72, 1344-1348. MAUTNER, V., WILLIAMS, J., SAMBROOK, J., SHARP, P. A., and GRODZICKER, T. (1975). The location of genes coding for hexon and flbre proteins in adenovirus DNA. Cell 5, 93-99. MULDER, C., ARRAND, J. R., KELLER, W., PETTER& SON, U., ROBERTS, R. J., and SHARP, P. A. (1974). Cleavage maps of DNA from adenovirus types 2 and 5 by restriction endonucleases Eco Rl and Hpa 1. Cold Spring Harbor Symp. Quant. Biol. 35, 397-400. NATHANS, D., and SMITH, H. 0. (1975). Restriction endonucleases in the analysis and restructuring of DNA molecules. Ann. Rev. Biochem. 44, 273-293. PETTER~SON, U., PHILIPSON, L., and HOGLUND, S. (1967). Structural proteins of Adenovirus. I. Purification and characterization of the hexon antigen. Virology 33, 575-590. J. (1973). The PETTEREISON, U., and SAMBROOK, amount of viral DNA in the genome of cells transformed by adenovirus type 2. J. Mol. Biol. 73,125130. PETTERSSON, U., MULDER, C., DELIUS, H., and SHARP, P. A. (1973). Cleavage of adenovirus type 2 DNA into six unique fragments by endonuclease R.Rl. Proc. Nat. Acad. Sci. USA 70, 200-204. PETTER&SON, U., and PHILIPSON, L. (1974). Synthesis of complementary RNA sequences during productive adenovirus infection. Proc. Nat. Acad. Sci. USA 71, 4887-4891. PHILIPSON, L., PETTERSSON, U., LINDBERG, U., TIE+ BETTS, C., VENNSTROM, B., and PERSSON, T. (1974). RNA synthesis and processing in adenovirus-infected cells. Cold Spring Harbor Symp.

ADENOVIRUS

5 LYTIC RNA SEQUENCES

Quant. Biol. 39, 447-456. RUSSELL, W. C., and WILLIAMS, J. F. (1972). Charac-

terization of temperature-sensitive mutants of Adenovirus type 5-serology. J. Gen. Viral. 17, 265-279. RUSSELL, W. C., SKEHEL, J. J., and WILLIAMS, J. F.

(1974). Characterization of temperature-sensitive mutants of Adenovirus type 5 -Synthesis of polypeptides in infected cells. J. Gen. Viral. 24, 247259. SAMBROOK, J., SHARP, P. A., and KELLER, W. (1972).

Transcription of simian virus 40. I. Separation of the strands of SV40 DNA and hybridization of the separated strands to RNA extracted from lytitally-infected and transformed cells. J. Mol. Biol. 70, 57-71. SAMBROOK, J., WILLIAMS, J. F., SHARP, P. A., and GRODZICKER, T. (1975). Physical mapping of temperature-sensitive mutations of Adenoviruses. J. Mol. Biol. 97, 369-390. SCHILLING, R., WEING~~RTNER, B., and WINHACKER,

E. L. (1975). Adenovirus type 2 DNA replication. II. Termini of DNA replication. J. Virol. 16, 767774. SHARP, P. A., SUGDEN, B., and SAMBROOK, J. (1973).

Detection of two restriction endonuclease activities in Hemophilus paminfluenzae using analytical agarose ethidium bromide electrophoresis. Biochemistry 12, 3055-3063. SHARP, P. A., GALLIMORE, P. H., and FLINT, S. J.

(1974). Mapping of Adenovirus 2 RNA sequences in lytically infected cells and transformed cell lines. Cold Spring Harbor Symp. Quant. Biol. 39, 457-474. SHARP, P. A., and FLINT, S. J. (1976). Adenovirus transcription. In “Current Topics in Microbiology

and Immunology” (P. Vogt, ed.). Springer-Verlag,

455

Berlin, in press. TAL, J., CRAIG, E. A., ZIMMER, S., and RASKAS, H.

J.

(1974). Localization of Adenovirus 2 messenger RNA to segments of the viral genome defined by endonuclease Eco R.Rl. Proc. Nat. Acad. Sci. USA 71, 4057-4061. TIBBETTS, C., and PETTERSSON, U. (1974). Comple-

mentary strand-specific sequences from unique fragments of adenovirus type 2 DNA for hybridization-mapping experiments. J. Mol. Biol. 88, 767-784. TOLUN, A., and PETTER~~ON, U. (1975). Termination sites for Adenovirus type 2 DNA replication. J. Viral. 16, 759-766. WEBER, J., BEGIN, M., and KHITOI, G. (1975). Ge-

netic analysis of adenovirus type 2. II. Preliminary phenotypic characterization of temperaturesensitive mutants. J. Virol. 15, 1049-1056. WILKIE, N. M., USTACELEBI, S., and WILLIAMS, J. F. (1973). Characterization of temperature-sensitive mutants of adenovirus type 5: Nucleic acid synthesis. Virology 51, 499-503. WILLIAMS, J. F., and USTACELEBI, S. (1971). Complementation and recombination with temperaturesensitive mutants of adenovirus type 5. J. Gen. Virol. 13, 345-348. WILLIAMS, J. F., GHARPURE, M., USTACELEBI, S., and MCDONALD, S. (1971). Isolation of temperature-sensitive mutants of adenovirus type 5. J. Gen. Virol. 11, 95-102. WILLIAMS, J. F., YOUNG, C. S. H., and AUSTIN, P. E.

(1974). Genetic analysis of human adenovirus type 5 in permissive and non-permissive cells. Cold Spring Harbor Symp. Quant. Biol. 39,427-437. WILSON, D., and THOMAS, C. A. (1973). Hydroxylap-

atite chromatography of short double-helical DNA. Biochim. Biophys. Acta 331, 333-340.