J. Mel

Biol. (1976)

101, 479-501

Hybridization Maps of Early and Late Messenger RNA Sequences on the Adenovirus Type 2 Genome ULF PETTERSSON? CLLARK TIBBETTS~ AND LENNART

PHILIPSON

Department of Microbiology The Wallenberg Laboratory UppsaEcc University Uppaala,

Sweden

(Received 30 June 1975, and in revised form

30 October 1975)

Specific fragments of adenovirus type 2 DNA, generated by cleavage with rastriction endonucleases endoR.EcoRI, endoR.HpaI and endoR.HindIII were The complementary strands of used in hybridization-mapping experiments. individual cleavage fragments were separated by the method of Tibbetts & Pettersson (1974). Liquid hybridizations were performed wit,h 32P-labeled separw.ted strands of cleavage fragments and messenger RNA extracted from cells early and late after adenovirus infection. The fraction of each fragment strand which was represented in “early” and “1at)e” messerlger RNA was determined by chromatography on hydroxylapatite. Early messenger RNA was found to be derived from four widely separated regions, two on the l- and two on the hstrand (h- and I- refer to the strand with heavy and light buoyant density in C’sCl when complexed with poly(U, G)). Messenger RNA, present exclusively late after infection, is derived from several locnt,ions, predominantly from the l-strand with a major block of continuous sequences extending between positions 0.25 and 0.65 on the unit map of the adelIovirua t.ype 2 genome.

1. Introduction Regions from both complementary strands of adenovirus 2 DNA are transcribed during productive infection, both before (early) and after (late) the onset of viral DNA synthesis (Green et al., 1970; Tibbetts et al., 1974; Sharp et al., 1974a). Sequences of messenger RNA synthesized early after infection persist in the cytoplasm late after infection, together with the late-specific mRNA sequences (Thomas & Green, 1969; Green et al., 1970; Fujinaga & Green, 1970; Tibbetts et al., 1974; Sharp et al., 1974u). At least some early mRNA sequences also appear to be synthesized late after infection (Lucas & Ginsberg, 1971; Fujinaga & Green, 1970; Craig & Raskas, 1974). Most of the mRNA sequences derived from h-strand$ appear to be synthesized at early and intermediate times after infection, with little or no synthesis late in the infectious cycle (Pettersson & Philipson, 1974). The portion of the Ad2 genome expressed in a line of viral-transformed rat cells (8617) was a subset of the mRNA t Present address: Department of &Microbiology, University of Connecticut, School of Medicine, Health Center, Farmington, Conn. 06032, U.S.A. $ Abbreviations used: h- and l- strands, strands with heavy and light buoyant density in CsCl when complexed w&h poly(U, G); At1 L’, adenovirus L’.

479

480

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PHILIPSON

sequences synthesized early in productivity infected cells and also represented both complementary strands of Ad2 DNA (Fujinaga & Green, 1970; Green et al., 1970; Landgraf-Leurs & Green, 1973). Digestion of viral DNA with bacterial restriction endonucleases, methods for complementary strand separat’ion of the isolated cleavage fragments and liquid phase RNA : DNA hybridization analysis have provided a powerful approach for mapping different classes of viral RNA on the genomes of papova- and adenoviruses (Khoury et al., 1973; Sambrook et al., 1973; Tibbetts & Pettersson, 1974; Philipson et al., 1974; Sharp et al.. 1974a; Tal et al.. 1974). Restriction endonucleases endoR.EcoRI (from Escherichia toll:), endoR.HpaI (from Hemophilus parainjhenzae) and endoR. Hind111 (from Hemophilus injiuelzzae) have been shown to generat’e 6, 7 and 12 unique fragments of Ad2 DNA, respectively (Pettersson et al.? 1973; Mulder et al., 1974; Roberts, personal communication). The respective order of the fragments in the linear, non-permuted Ad2 genome has been established (Gallimore et al., 1974; Mulder et al., 1974; Roberts et al., personal communication) and is shown in Figure 3. A low resolution map of the regions of the Ad2 genome corresponding Do stable mRNA late after productive infection was est’ablished by hybridization of late mRNA to complement-specific labeled DNA from each of the six endoR.EcoRI cleavage fragments (Tibbetts & Pettersson, 1974). In that map the localization of sequences complementary to mRNA within individual DNA-cleavage fragments was to some degree ambiguous. This n-as particularly true for the largest of the six endoR.EcoRI oleavage fragments which comprised nearly FOq/, of the entire Ad2 genome. By utilizand ing the additional cleavage fragments of Ad2 DNA generated by endoR.HpaI endoR.HindIII together with the general procedure for isolation of complementary &and-specific sequences of Ad2 DNA (Tibbetts & Pettersson, 1974) we have been able to construct a more detailed map to show bhe regions of the Ad2 genome which correspond to classes of stable mRNA appearing early and late in productive infection.

2. Materials and Methods (a)

T’irus

and

cell cultures

Propagation of adenovirus type 2 in suspension cultures of HeLa S3 cells has been described (Tibbetts et al., 1974). Extraction and purification of virus and viral DNA have also been described elsewhere (Tibbetts et al., 1973). 32P-labeled Ad2 DNA was prepared as described by Pettersson & Sambrook (1973). The initial spec. act. of 32P-labeled viral DNA was 1 to 4 x lo6 cts (C?erenkov)/min per pg and each preparation was used over a period of several weeks. (b)

Unlabeled

complementary strands of Ad2 DNA

Strand separation of unlabeled viral DNA with poly(U, G) was performed as previously described (Tibbetts et al., 1974; Tibbetts & Pettersson, 1974). Purified intact h- and l-strand DNA was used for preparation of complementary strand-specific sequences of a2P-labeled restriction endonuclease cleavage fragments of Ad2 DNA as described by Tibbetts & Petterson (1974). (c) Restriction

endonucleases

and cleavage of Ad2 DNA

(i) EndoR.EcoRI The enzyme was prepared as described 32P-labeled Ad2 DNA and the isolation cribed by Pettersson et al. (1973).

by Pettersson & Philipson (1974). Cleavage of of the resulting cleavage fragments were des-

HYHRIDIZATION

MAPS

OF

mRNA

OF

ADENOVIRUS

481

(ii) EndoR.HpaZ This enzyme was prepared according to the procedure of Sharp et al. (1973) and was kindly provided by Dr Robert Kamen, ICRF Laboratory, London. Incubations with Ad2 DNA were performed at 37°C in 6 m;M-Tris*HCl (pH 7.5), 6 m&f-MgCl,, 6 m&f-2mcrcaptoethanol and 100 mM-KC1 with sufficient~ enzyme and time of incubat,ion to ensurn complete digestion of the DNA. (iii) E’,rdoR.HindZZZ An enzyme preparation which was used for the initial part of this study was kindly slIpplied by Dr Ernst Winnacker, University of Cologne, West Germany. Later preparations lvere purified in our laboratory by the method of Smith (1976). Incubations were porforrnc*d at 37°C in 10 mM-Tris.HCl (pH 7.5), 60 mM-NaCl and 7 mM-MgCl, with sufficient rnzj-lncl and time of incubation to ensure complete digestion of the DNA. 32P-labeled Ad2 DNA rloaved with endoR.HindIII yielded a mixture of 12 fragments which were separated by crlectrophoresis on 2.2% acrylamide-0.7°/o agarose gels (Fig. I(a)). In order t,o acthieve satisfactory separation between fragments HindIII-D and -E or HindIII-F it was necessary to elute these fragments from the gels and perform a second and 4. ek&rophoresis on 15-cm gels. After 48 h of electrophoresis it was possible to obtain fragments D, E, F and G with about 90% purity. Complementary strands of 10 of tho 12 endoR.HindIII cleavage fragments (excepting HindIII-B and -L) were separated by the rnetjhod described by Tibbetts & Pettersson (1974). The strands of fragment HindIII-B pro\.cd difficult to separate completely by this method since they were found to contain sufficiont~ intrastrand complemontarity to make a significant fraction of even degraded, denatured DNA adsorb to hydroxylapatite at 0.13 M-phosphate (U. Pattersson, unpublist& observations). Fragment HindIII-L was not used for strand separations because its small size would require very large quantities of mRNA to achieve saturation of tlrtjc?c>tahle probe DNA and its location (Fig. 6) would not lend itself to generate clarifying data for mapping purposes. (d) Mapping

of endoR.

cleavage

fragments

of Ad2

DNA

Location of cleavage sites for restriction endonucleases required determination of the sizes and relative order of cleavage fragments in Ad2 DNA. The sizes of the six endoR. Il=colil claavage fragments of Ad2 DNA were determined earliar by Pettersson et al. (1973). The respective data for the seven fragments made with endoR.HpaI were made available by Sharp et al. (1973) and Gallimore et al. (1974) as well as via personal communications. The 12 endoR.HindIII fragments of Ad2 DNA had been studied at the Cold Spring Harl)or Laboratory prior to this study. The sizes of individual fragments were estimated from t~ho distribution of radioactivity in different segments after cleavage of 32P-labeled DNA (R. Roberts, personal communication) and are given in Table 3. T11c: ordering of the cleavage fragments of Ad2 DNA along its linear genome was establishcad for each of the 3 restriction endonucleases used in this study by the collective efforts of several individuals at, the Cold Spring Harbor Laboratory, as shown with our mRNA mapping data in Fig. ‘7 (Mulder et al., 1974; Roberts, Sharp, Mulder & Sambrook, personal communication). It should be noted that in our maps the labeling of ondoR.HindIIL fragments was done with regard to t,heir migration rates in agarose gels. Fragments H&dlII-D and -E, as u-011 as HindIII-F and -G, have been observed to migrate in reverse order on composite 0.7% agaroso-2.20/b acrylamide (R. Roberts, personal communication). lb was recently shown that electrophoretic mobility in agarose gels better correlates wit11 molecular weight than mobility in composite gels (Thomas & Davis, 1975). Therefore, wr) have chosen to designate fragments according to their mobility in agarose gels rather than in composite gels although the latter were routinely used for fragment isolation. (fs)

Preparation

of viral

mHAV’d

;2denovirus-irrfected cells were collected at 6 h (early) or 18 h HeLa cells with a multiplicity of 2000 particles per cell. Cytosine at 20 pg/ml2 h after infection for preparations of early mRNA. The by brc,aking the cells in NP40 (Shill) in isotonic buffer as described

(late) after infection of arabinoside was added cytoplasm was obtained previously by Lindborg

I

-&a1

A

-fco

RI-C

-fco

RI-D

~CO

fco G‘Hlte

RI-F RI-E

I npa

I-E

HpaI-F

ECO

RI-A

f Hpe

11-a

H/WI-G t-

ECO

RI-E

1-P

(b)

PIO. l.(a) Separation of fragments obtained by cleavage of Ad2 DNA with endoR.HindIlI. 2.2% acrylamidc~O~7~0 agaroae gel at 4 V/cm for Electrophoresis was performed on a composite 18 h. The anode is towards the bottom. Eleven fragment,s are detectable. The smallest fragment, HindIII-L, has migrated through the entire gel. Fragments D and E as well as fragments F and G have been observed to migrate in the reverse order in agaroae gels (Roberts, personal communication). The gels were stained with ethidium bromide (Sharp et al., 1973) and the bands were visualized by excitation with ultraviolet light. (b) Separation of fragments of Ad2 DNd generated by cleavage with cndoR.HpnI (left), endoR.EcoRT (middle) and a mixture of both enzymes (right). Eloctrophorasis wati performed on composite gals with l,lo/O acrylamide-0.7% agarose at 4 V/cm for 5 h.

HYBRIDIZATION

MAPS

OF

mRS.4

OF

L~DENOVIRU8

4x3

et al. (1972). RNA from cytoplasm was ext)racted as previously described by Tibbotts et a/. (1974) and purified by chromatography on oligo-(dT)-cellulose (grade T2, Collaborative Research Inc., Cambridge, Mass.) as described by Tibbetts & Pettersson (1971). All RNA preparations were treated with electrophor&ically purified DNAase (Wort,hington Inc.. N.,J.). Such RNA preparations were screened to exclude small amounts of viral DNA contamination by hybridization with complementary strand-specific 32P-labeled viral DNA probes, following alkaline hydrolysis of the RNA (0.3 M-NaOH, 37’C, 18 h). RNA sampltis wclr(L finally dissolved in 0.01 bI-Tris.HCl (pH 7.5), 0.001 PI-Na,EDTA at a concentration of 2 to 5 mg per ml. (f ) Hybridization

and ana1ysi.r

DNA samples were first degraded in 0.3 x-NaOH for 20 min at 100°C to an average chain length of about 350 nuoleotides (Tibbetts et al., 1974). More than 98% of the input raclioactivity was recovered as DNA fragments for subsequent incubations. Hybridization conditions were: 65°C in 1 M-NaCl with from 0.05 to 0.10 M-phosphate at pH 6.8 as oquimolar NaH,PO, and Na,HPO,. Samples were analyzed by hydroxylapatitc (BioRad as described by Sambrook et al. (1972). Some samples were also HTP) chromatography analyzed by digestion with the single-strand specific endonuclaase S, (Ando, 1966; Khoury et al., 1973). Endonucleasr S, was purified by chromatography on DEAE-crllulosr and Sephadex GIOO as described by Vogt, (1973). Samples were digested as described k)y Kl~otlry et al. (1973) except, that 1 rnnl-ZnSO, was llsc:d in the incubation mixture. (g) Analysis

of radioartivity

32P-labeled samples were analyzed by measurement of C”erenkov radiation in 0.14 nrto 0.40 nf-phosphate, 0.4% sodium lauryl sulfate at room temperature. Background and efficiency were independent of the phosphate buffer caoncentrat,ion in the range used.

3. Results Early and late mRNA from AdB-infected cells has now been studied by hybridization-mapping using separated strand probe DNA from 24 restriction enzyme cleavage fragments of Ad2 DNA. The results of saturation hybridizations of early cytoplasmic RNA with h- and l- strand probe DNA derived from the six endoR.EcoRI cleavage fragments of Ad2 DNA are shown in Table 1, along with data for late mRNA as previously reported by Tibbetts & Pettersson (1974) and Pettersson & Philipson (1974). Probe DNAs representing separated strands of fragments HpaI-A, -C, -E, and -F were also hybridized with early and late mRKA, giving saturation values, and the results are presented in Table 2. In the case of fragment HpaI-F which only represents about 800 base-pairs it was of particular importance to ascertain that the hybridization between late mRNA and the h-&and probe was significant. Therefore, the fract,ion of probe DNA in hybrid was also determined after digestion with endonuclease S, as shown in Table 2. Results of hybridization of early and late mRNI-1 with complement-specific sequences from 10 of the 12 endoR.HindIII cleavage fragments are summarized in Tables 3 and 5. Three additional fragments were isolated for hybridization of mRNA by sequential digestion of Ad2 DNA with two or more restriction endonucleases. A small fragment. designated EcoRI-A/HpaI-a, which represents about 500 base-pairs at the right-hand end of fragment EcoRI-A, was isolated after cleavage of fragment EcoRI-A with illustrating the isolation of this fragment endonuclease HpaI. An electrophoretogram is shown in Figure 2(a). Another small fragment designated HindIII-K/HpaI-a was isolated in the following way: a fragment EcoRI-C/HpaI-a was isolated after simultaneous digestion of Ad2 DNA with both endonucleases EcoRI and HpaI (Fig. l(b)).

1 h 1 h 1 h 1 h 1 h 1 h

DNA

21 7 3 37 7 53 73 1 40 4 23 11

Expt

1

cleavage

19 8 8 38 12 62 73 5 44 5 26 13

with

amounts

of adenovirus

Percentage as hybrid early mRNAt Expt, 2

fragments

DNA:

24 12 11 50 8 65 70 2 35 4 30 15

Expt

of early

2

TABLE

of 3’P-labeled

1

3

1 and

71 15 52 40 31 50 82 5 82 12 84 11 2.

with

0.125 0.054 0.009 0.061 0.009 0.061 0.053 0.002 0.024 0.002 0.011 0.006

0.424 0~090 0.063 0.049 O-032 0.051 0.061 WOO4 0.048 0.007 0.039 0.005

as hybricl h- or l-strand) Late, rnKXA$

saturatirly

Probe DNA (fraction of Ad2 Early mRNA

which hybridizes

Percentage as hybrid with late mRNA$

probe DNA RNA

was used in experiments 3. (1974).

21 9 7 42 9 60 72 3 40 4 23 13

Sverage early

and lute cytoplasmic

fraction

tCytoplasmic RNA which was selected by oligo(dT)-cellulose chromatography Total cytoplssmic RNA which was precipit,atod by LiCl was used in experiment $Data taken from Tibbrtts fir Pottersson (1974) and Pettersson & Philipson Average of three experiments.

EcoRI-A EcoRI-A EcoRI-B EcoRI-B EcoRI-C EcoRI-C EcoRI-D EcoRI-D EcoRI-E EcoRI-E EcoRI-F EcoRI-F

Probe

EndoR.EcoRI

1 h 1 h 1 h 1 h

tValues

HpaI-A HpaI-A HpnI-C HpaI-C HpaI-E HpaI-E HpaI-F HpaI-F

Probe DNA

7 3 ‘5 7 64 2 7 5

8 5 ‘5 6 72 4 3 3

Expt

2

Early

fragments

were determined

1

cbeavayr

Expt

iI1 parentheses

EndoR.HpaI

2

by analysis

12 5 38 8 73 2 5 8

Expt

mRNA

with

1

9 4 26 7 70 3

A4verage

endonucleese

90 % 67 32 81 6 69 24

Expt, 1

.l’rrcentag . o as hybrid with

of hybrids

3

of adenovirus

Y1.

93 4 6” 39 80 8 57 “3

Expt

2 94 4 70 34 79 4 51 26

Expt

Late mRNA

2 DNA: fraction of “P-labeled probe DNA amouds of early and late cytoplasmic messenger RNA

TABLE

3

which

9% 3 66 35 HO 6 69(54) i “4(19)

=\vrrage

hybridizes

saturating

0.001

04JO1

0.028 0.01% 0.052 0.014 O-O%9 WOO1

0.283 0.009 0.133 0.070 0.033 0.002 0.014 0.006

I’lobu DNA as hybrid (fract.ions of Ad2 h- or I-strand) Late Early

with

l*.

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EndoR.HindIII cleavage fragments of udenovirus 2 DNA: fraction of ““P-labeled probe DNA which hybkdizen with natu,rating amounts of early and late cytoplasm,ic messenger RNA Percentage Fraction of Ad2 DXA

Probe DNA Hind111 Hind111 Hind111 Hind111 Hind111 Hind111 Hind111 Hind111 Hind111 HindIII Hind111 Hind111 Hind111 Hind111 Hind111 Hind111 Hind111 Hind111

-Al -Ah -Cl -Ch -1~1 -D h -El -Eh -Fl -Ph -Gl -Gh -Hl -Hh -11 -I h -J 1 -J h

0.246 0.090

Early

as hybrid

mRNA

Late mRNA 74 25 47 46 94 6 !I” I3 41 61 83 15 90 5 93 8 94 5

8 25 35 4

0.088 0.086

58

0.076 0.076 0~063

with

12 77 63

0.057 0.035

Probe DNA as hybrid (fraction of 8d2 h- or l-strand) Early mRNA Lat’e mRNh 0.020 0.06% 0.03% 0.004

0.050 0.055 0.059 0.040

-.

0.182 0.062 0.04” 0.041 0.083 0.005 0.079 0.011 0.031 0.046 0.063 0.011 0.057 0.003 0463 0.005 0.033 0.00%

TABLE 4 of late messenger RNA with fragments of adenovirus 2 DNA, Hybridization generated by sequential cleavage with different restriction enzymes Probe DNA EcoRI-A/H&+ EcoRI-A/H@-ah HindIII-B/H@-alf HindIII-B/H@-mh

Percentage

probe as hybrid

with late mRNA

89 4 66 30

tThis fragment was obtained by cleaving fragment EcoRI.i\ with HpnI (Fig. 2(a)). It is derived from the right-hand end of fragment EcoRI-A. fThis fragment was obtained by cleaving fragment HindIII-B with endoR.Hpal (Fig. 2(c)). Fragment HindIII-B/HpaI-a is derived from the left-hand part of fragment HindIII-B, between positions 0.17 and 0.24.

HYBRIDIZATIOS

MAPS

OF

TIRLF,

mRS.4

OF

6

Hybridization of early and late messenger RNA cleavage fragments n,ear the right-hand terncinus

ujith! separated

strands

of the adewovirus

l’tvccntage DNA Early rnRSA

“2P-labeledt probe DNA

487

ADESOVIRUS

from

2 ge,nomP

in hybrid with Late mRN;I

Hpnl-G

1 H&-G h HindIII-K 1 H6,~dI11-K h Hi>ldIII-K/H@-r Hi,tdIII-K/H@-a

1: h

tThe DNA probes were not deliberately degraded before use in hybridization. why late mRN4 saturates more than 100°/c of some probes. IFragment HiradIII-K/H@-a is derived from fragment EcoRI-C by sequential between endoR.H@ and endoR.HindIII (Fig. 2(b)). The fragment is located and 0.986 on the unit map of the Ad2 genome.

This

explains

cleavage positions

with 0.972

Fragment EcoRI-C/HpaI-cc is located from 0398 t’o 0.986 on the unit map of Ad2 DNA. Fragment HindIII-K/HpaI-cc was then isolated by cleavage of fragment EcoRI-C/HpaI-a with endonuclease Hind111 (Fig. 2(b)). It is located from 0.972 to 0.986 on the unit map. A third fragment HindIII-B/HpaI-a was isolated by cleavage of fragment HindIII-B with endonuclease Hpal. The cleavage results in t’hree fragments as shown in Figure 2(c), the largest of which is HindIII-B/HP&-a. This fragment represents a segment, of DNA from 0.166 to 0.242 on the unit map. The results of hybridizations of complement-specific probe DNA from these fragments with mRNA from Ad2-infected HeLa cells are summarized in Tables 4 and 5.

4. Discussion We have previously reported on a general m&hod for hybridization-mapping experiments with unique fragments of adenovirus 2 DNA (Tibbetts & Pettersson, 1974). The results of these experiments using only one set of cleavage fragments. generated with restriction endonuclease endoR.EcoRI and comprising the entire Ad2 genome, were, however, not sufficient to permit construction of definitive hybridization maps of the Ad2 genome. In two cases the levels of probe DNA hybridization with late cytoplasmic RNA were low, relat’ive to background values without, RNA, and therefore of questionable experimental significance (h-strand sequences of fragments EcoRI-E and -F). Thus it was apparent that data from hybridization experiments involving overlapping sets of cleavage fragments were required to localize unambiguously Ad2 DNA sequences represent’ed in different classes of viral mRNA. In this st,udy 24 Ad2 DNA fragments, generated with three different restriction endonucleases, were used to obtain sufficient hybridization data for detailed localization of the sequences of the viral genome which are represented in viral mRNA. Cytoplasmic RNA was isolated from AdS-infected HeLa cells early and late after infection for hybridization with complementary st,rand-specific, 32P-labeled probe DNA. This RNA was purified by oligo(dT)-cellulose chromatography, to select for

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3ZP-labelsd Ad2 DNA was cleaved with FIG. Z.(a) Isolation of fragment EcoRI-A/HpaI-r. endoR.EcoRI and the resulting fragments were separated as described in the legend to Fig. l(b). Fragment EcoRI-A was eluted, extracted with phenol, precipitated with ethanol and dige&ed with endoRB@. Electrophoresis was at 4 V/cm on composite 1.1% aorylamide-0~7~/0 agarose gels for 6 h. The anode is towards the right,. The capital letters refer to fragments which are obtained by cleavage of Ad2 DNA with endoR.HpnI. The small fragment, designated “cc” is derived from the right-hand end of fragment EcoRI-A. (b) Isolation of fragment HindIII-K/H@-u. Fragment EcoRI-C/H@-a was first isolated by cleavage of 32P-lebeled Ad2 DNA with a mixture of endoR.EcoRI and endoR.HpaI (see Fig. l(b)). This fragment was eluted from the gel, extracted with phenol, precipitated with ethanol and digested with endoR.HindIII. The resulting fragments were separated by electrophoresis on composite 2.2% acrylamide-0.7°/o agarose gels at 4 V/cm for 6 h. The Figure shows fragment EcoRI-C/HpaI-a before (----O--Oo~~-~) and after (--- l ---•-) digestion with

HYBRIDIZATION

M.4P8

OF

mRN.4

OF

.1DENOVIRUS

4x9

mRXA species, following the gentle disruption of the infect,ed cells and removal of nuclei. Low levels of nuclear RNA contaminat’ion cannot be excluded. The extensive transcription of Ad2 DNA sequences which are complementary to various viral mRNA species (Pettersson & Philipson, 1974; Sharp et aZ., 1974a) may in fact account, for occasional low levels of hybridization (< 100;, of probe DNA) that appear with certain probes. In general, the background levels of hybridization obtained aftct incubation of probe DNA without RNA were less t’han 4O,, of Ohe probe. Complementary strand-specific, 32P-labeled Bd2 DNA probes were prepared from Ad2 DNA cleavage fragments a,s described by Tibbetts 8: Petbrrsson (1974). lt, should be emphasized that recoveries of probe DNA sequences t,hrough the isolation snd Non-random losses of probe DN=l sequence were hybridizations were quantitat’ive. minimized by avoiding adsorption losses of denatured DN&4. Fragment’ HdndlIl- N was not used in this study because the significant self-complementarit,y of it’s denatured strands leads to selective adsorption of some sequences to hydroxylapatitr, interfering with the preparation of complement-specific probe DNA representative of’ the entire fragment. Thrh levels of hybridizat)ion of various probe DSAs with early or late cptoplasmic Rn’A are presented in Tables 1. 2, and 3, as the percentage of probe DN4 in hybrid. The fract#ion of the Ad2 DNA strand which the hybridized sequeuces represent, defined as the product of the fraction of probe in hybrid and the ratio of the probe sequence length to that of an entire Ad2 strand. is also given in the Tables and is referred to as unit G. in the following discussion. The error associated with thch determination of the fraction of probe in hybrid is about :+5”;, of t’he probe. The error in determination of the size of cleavage fragments relat,ive t,o ,4d2 DNA is smaller and thus the error associated with values expressed in units of G are probably between 5 and I OY;, of the given value. (n) Sequences

of adenovirur

2

DNA represented

itL. rarly

cytophmic

RNA

Tables 1. 2, 3. and 5 include data from hybridization analysis of early cytoplasmic RNA with complement-specific probes representing 19 cleavage fragments of Bd2 DNA. Significant saturation levels of probe DNA hybridizeDion. corroborated by experiments using different fragments having common sequences, led to the identification of four major blocks of Ad2 DNA sequences represented in early cytoplasmica RNB. These four blocks appear widely separated on the viral genome, two on each complementary strand (Fig. 3). From the left end of the Ad2 genome map the first early gene block is near the 3’ terminus of the l-strand. In t’his region the extems of hybridization with probes representing HJ?aT-E and -C (0.08 G) on the one hand and HindlII-G and -C (CO.09G) on the other were in good agreement. A second block of early genes transcribed from the l-strand was discovered in t’hc rtgion of fra,gments EcoRI-F. -D. and -E, from about 0.76 to 0% on t’hc unit genornc~

(c) C’loarage of fragment HindHI-B with entloR.H~ul. 3ZP-labeled fragment HindIIl-B wax isolated by electrophoresis as described in the legend to Fig. l(a), extracted with phenol, precipit,ated with ethanol and digested with endoR.HpnI. The resulting fragments were separated on a composite 1.1 o/0 acrylamide-0.7Cy0 agarose gel at 4 V/cm for 5 h. Three fragments are obtained: and Hpcrl-F identified a~ 2, /3 and HJX/I-F, mspectivrl~ HindIII-B/HJxxI-~, HindIII-B/H~rrT-/3 in the Figure

490

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C I 1

C. TIBBETTS

F I I

AND

A

L.

B

PHILIPSON

, I

I

DGJ ,

HpoI

3’ 1 A

5’ h

000

I 025

I 075

I 050 Fraction

of Ad2

I.00

genome length

Fm. 3. Distribution of early mRNA sequences on the 2 complementary strands of the Ad2 genome. The map was deduced from data shown in Tables 1, 2, 3 and 6. The cleavage sites for 3 different restriction ondonuoleases are also shown. The size and order of the EcoRI- and HpaIfragments are taken from Gallimore et al. (1974). The size and the order of the HindIIIfragments were determined by R. Roberts at the Cold Spring Harbor Laborat,ory (personal communication).

map. The total extent of hybridization between early cytoplasmic RNA and l-strand probe DNA from these three fragments was equivalent to 0.09 G, in good agreement with the parallel results obtained using l-strand DNA from fragments HindIII-H and -E (0.10 G including HindIII-L). No experiments were performed with HindIII-L, but this fragment (between HindIII-H and -E) is too small (0*7:/, of Ad2 DNA) to alter significantly the early map as shown in Figure 3. It is likely that only l-strand DNA from this fragment would hybridize with early or late cytoplasmic RNA. The h-strand of Ad2 DNA also has two clusters of early genes. Within EcoRI-C there was hybridization with early cytoplasmic RNA equivalent to 0.06 G. This block extends quite close to the 3’ terminus of the h-strand since a fraction of t’he h-strand probe representing HpaI-G hybridized with early cytoplasmic RNA (Table 5, see also below). Fragments HindIII-F and -K contain almost the entire length of fragment EcoRI-C and the sum of h-strand hybridization with early cytoplasmic RNA was 0.07 G, in good agreement with the result for h-strand EcoRI-C DNA above. The second h-strand block of early genes appears at the region of junction of fragments EcoRI-B and -F, representing about 0.06 G. The low (10 to 15%) level of hybridization with EcoRI-F h-strand probe DNA has been consistently observed with all preparations of probe DNA and with both early and late cytoplasmic RNA (Table 1). An equivalent level of hybridization (0.06 G) in the EcoRI-B region was seen in experiments with HindIII-A h-strand probe and early cgtoplasmic RNA (Table 4). The four blocks of early genes described above are shown in Figure 3 along with cleavage maps for the three restriction endonucleaxes. Although in some cases the data which have been presented would be consistent with a more complex distribution of sequences represented in early mRNA, this simplest version requires the fewest) arbitrary assumptions, is corroborated by independent experiments with different cleavage fragments showing common sequences, and is also consistent with the mapping results obtained from hybridizations with late cytoplasmic RNA. The pairs of early gene clusters on the l- and h-strands of Ad2 DNA are equivalent to totals of about 0.19 G and 0.13 G, respectively. These values are lower than the levels of

HY I3RTI)TZATJON

MAPi

01’

mRN.\

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hybridization observed with a2P-labeled Bd2 l- and h-strand probe DNA (30’:,, and 157,” of I- and h-strands, respectively; Tibbetts & Pettersson, 1974). Hybridization experiments with early nuclear RNA have shown that the nucleus early after infection contains excessive sequences which never enter the cytoplasm (Sharp et al., 1974a: and our unpublished data). It is conceivable that sequences due to leakage of nuclear RNX during sample preparation provide a general background. Therefore, hybridizat’ion levels which amount, to less than 100; of a given fragment st’rand were disregarded \+hen the map shown in Figure 3 was constructed. This accounts for most of the discrepancy between sequences scored as early with probes representjing complete strands of Ad 2 DNA (Tibbetts & Pettersson. 1974) and sequences assigned as early in the map in Figure 3. The four early gene clusters range from about O-06 to 0.10 G in size. On the basis of molecular weight analysis of early mRNA isolated from adenovirus-infected cells (Parsons & Green, 1971; Lindberg et ab.. 1972; Bhaduri et al., 1972) it thus appears that each of the four clusters of early genes shown in Figure 3 corresponds to from one t,o perhaps three or four discrete early mRKA species. (I)) 8equences of ade?hovirus 2 DNA represented in lute cytoplasmic RNA The results of hybridizations of late cytoplasmic RNA with probes representing 24 different cleavage fragmenm of Ad2 DNA are presented in Tables 1, 2, 3, 4 and ii. It should be recalled that the levels of hybridization obtained with late cytoplasmic RNA include hybridization with early RNA sequences that persist in the cytoplasm late after infection (Tibbetts et al., 1974). Comparison of maps obtained with late cytoplasmic RNA with the early RNA map shown in Figure 3 reveals the location of late-specific genes on the viral DNA. (i) illupping

within EcoRI-A

The results of Tibbetts $ Pettersson (1974) indicated significant levels of hybridization of h- and l-strand sequences of EcoRI-A DNA with late cytoplasmic RNA. The large size of this fragment, around 60% of the genome, and the lack of additional data at that time precluded definitive localization, within EcoRI-A, of sequences which hybridized with late cytoplasmic RNA. With this source of ambiguity, the distribution requiring the fewest arbitrary assumptions, and compatible with data for the five remaining EcoRI cleavage fragments, was presented (Fig. 4(a)). EndoR.HpaI was used first to subdivide EcoRI-A in order to provide more details for mRNA mapping on the left half of the Ad2 genome. Hybridization-mapping with late cytoplasmic RNA was performed using complementary strand-specific sequences of 32P-labeled DNA from five cleavage fragments of EcoRI-A: H&-E, -C, -F, -A. and the small fragment between the right end of HpaI-A and the right end of EcoRI-A. This latter fragment, designated (EcoRI-A/HpaI-n), was obtained as described in Results (Fig. 2(a)). The hybridization results (Tables 2 and 4) suggested a more complex distribution of sequences represented in late cytoplasmic RNA than was predicted with simplicity assumptions applied to EcoRI-A data alone. The more refined map which results is shown in Figure 4(b). It appears that. there are two blocks of sequences which hybridize with cytoplasmic RNA at each end of the l-strand of EcoRI-A, separated by one (or more) blocks of h-strand sequences complementary to late cytoplasmic RNA. The left end block of l-strand sequences in EcoRI-A includes

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Frc. 4.(a) Possible distribution of late mRN.4 sequences within fragment E’coRl-A. I)at)a from Tibbetts & Pet,tersson (1974). (b) Distribution of late mRNA sequences as deduced from dat,a in Tables 1 and 2. (c) Possible distribution of mRNA sequences according to data presented in Tables 1, 2, 3 and 4. The map is besed on the Ending that late mRNA saturates about 60% of the h-strand of fragment HindIII-C, 30% of the h-strand of fragment HindIII-B/Hpd-a and 24% of t)hra h-strand of fragment HpaI-F.

the block of early genes described in the previous section. The h-strand sequences which hybridize with the mRNA appear to be localized within the contiguous fragments HpaI-C and -F. The sums of the extents of hybridization of I- and h-strand probes of the five fragments comprising EcoRI-A (0.48 G and 0.09 G, respectively) are in reasonable agreement with the results of hybridization using l- and h-strand EcoRI-A probe DNA (0.42 G and 0.09 G, respectively). Because of the small size of fragment HpaI-F (800 base-pairs) and the implications for mapping derived from significant levels of hybridization on each strand of that fragment, hybridization reactions were also analyzed by digestions with the single-strand specific S, endonuclease. The results of hydroxylapatite chromatography (Table 2) were confirmed by this alternative method of assay. Further experiments, employing Ad2 DNA fragments generated by endoR.HindIII, provided additional data for mapping within the EcoRI-A fragment, and led to the map shown in Figure 4(c). Hybridization of l-strand sequences at each end of EcoRI-A was corroborated by the results obtained using fragments HindIII-G (left end) and HindIII-I, -J, -D, and -A (right end). The low level hybridization of the HindIII-G h-strand probe (about 150/& Table 4) probably reflects a contamination of the probe with sequences from fragment HindIII-F (see Results and Table 4). Fra.gments HindIII-F and -G are difficult to separate completely and the former fragment gave mostly h-strand-specific hybridization with early and late cytoplasmic RNA. Data from hybridization of fragments HindIII-C, HpaI-C and HpaI-F, when taken together, suggested the increased complexity introduced in the map shown in Figure 4(c). The h-strand hybridization of Hpd-C with late cytoplasmic RNA corresponds to 0.07 G. Of these sequences, 0.04 G are localized within the h-strand of HindIIT-C.

HYHRTDIZATIOK

XIAl’S

OF

mR5C.A

OF

ADEPI;OVIRUS

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The difference, 0.03 G, is clearly insufficient to bridge the gap (0.08 G) between the right, end of HindIII-C and the left end of HpaI-F. The latter fragment also has h-strand sequences which hybridize wit,h late cytoplasmic RNA (Table 2). Because HpaI-X h-strand probe DNA gave near background hybridization with late cytoplasmic RNA. and since some HlpaI-C h-strand sequences which hybridize with late RNA cannot be accounted for by sequences common to HindIII-C, it is proposed that the h-strand block of hybridizable sequences in HpaI-F is cont’inuous with a small h-strand block of HpaI-C sequences. The results of hybridization with a fragment obtained by cleavage of HindZll-B \rith endoR.H~~aI (HindIII-B/HpaI-a, see Results, Fig. 2(c) and Table 4) are compat’ible with the map as presented in Figure 4(c). The previous version (Fig. 4(b)) would lead to the prediction that only h-strand sequences of (HindIII-B/HpaI-x) would hybridize with late cytoplasmic RNA. This contradicts the finding that SSq;, of the l-strand and 30% of the h-strand sequences of this fragment hybridized witjh late a,vt(oplasmic RNA (Table 4) and is consistent w&h the map as shown in Figurr 4(c). It should. however, be emphasized that Figure 4(c) represents the least complicat,ed interpretation of our experimental data. Due to the lack of enzymes which cleave fragment HindIII-C into smaller segments it’ is impossible to establish the exact) position of the h-strand block within fragment HindITl-C.

(ii) illqping

within

EcoRI-B

Significant amounts of hybridization of cytoplasmic RNA with both h-strand (early and late RNA) and l-strand (late RNA only) sequences of EcoRI-B were observed (Table 1). The l-strand hybridization at’ the right-hand end of EcoRI-A (fragment EcoRI-A/HpaI-a, see Table 4) and the h-strand hybridization (10 to 15q/:,) of EcoRI-F probe DNA suggested the arrangement of sequences corresponding to early and late mRNA as shown in Figure 5. As discussed earlier, HindIII-A h-strand probe showed t’he same extent of hybridization with early and late cytoplasmic RNA (0.062 G) as seen with the h-st’rand probes of EcoRI-B and -F (0.057 G).

H

I 0.5

(Eco RI-A/r‘fpoI-a)

I O-6

I 07

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FIG. 5. Location of mRNA sequences which hybridize to the h-strand of fragment The map is based on the finding that late mRNA hybridizes exclusively to the l-strand of EcoRI-A/H@-a and that early as well as late mRNA hybridizes to 10 to 15% of the of fragment EcoRI-F. The bars indicate the location of fragment, EcoRT-A/H@-a and HindllI-A.

EcoRI-B. fragment h-strand fragment

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(iii) Mapping

I’E’l”I’b~tKSSOS,

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‘l’t tarly snd late RNA preparations (Table 5). Taken together these results indicate that the block of early and late mRNA sequences which is complementary to about 50°,b of the h-strand from fragment EcoRT-C is located near but not at the very right-hand terminus of the Ad2 genome, as indicated in Figure 6. Additional RNA sequences seem to be present late after infection, which hybridize with the l-strand of a DNA segment which is located between positions 0.98 and 1.00. This small block of l-strand sequences at the righthand end of the Ad2 genome represents less than IT,, of t’he Sd2 genome (0.007 G or about 250 nucleotides) and its functional significance as mRn’A, if any, is unclear. A possible explanation is that, hybridization in this region is due to nuclear RNA contaminat,ion. This explanation is compatible with the results of Pettersson & Philipson (1974), in which tra,nscription late aft,cr infection is primarily l-strandspecific and includes regions complementa,ry t’o early h-strand-specific cytoplasmic RNA. As shown in Figure 6 it is not necessary to conclude that the small region of l-strand hybridization extends to the right into t,he sequences corresponding t’o the inverted t,erminal repetition of Ad2 DNA. From the maps presented in .Figures 6 and

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FJC:. 7. A composite map of early and late mRNA sequences. The map was deduced from data presented in Tables 1 to 5. Early regions are indicated with open bars and regions which are expressed exclusively late are indicated with solid bars. The assignment of the 5’ and 3’ ends stems from data presented by Sharp et al. (1974a). The order and relative size of cleavage fragments generated by 3 different restriction enzymes are also indicated. The left-hand border of the block of early sequences which covers fragments EcoRl-D and parts of fragments EcoRI-F and -E is not firmly established. Another uncertainty pertains to the 2 late-specific h-strand blocks in the left-hand part of the genome. The exact location within fragment HindIII-C and the relative size of the 2 blocks cannot be established from available data. The positions of DNA fragments which were generated by cleavage with 2 or more rest,riotion enzymes are indicated on the top of the Figure: a, HindIII-B/H@-cr; b, EcoRI-A/HpnI-a; c, HSndIII-K/HjnnI-a.

1%

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PETTERXSOS,

(‘.

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7 it is not possible to explain why a small fraction EcoRI-E and HindIII-E hybridized with late mRNA not’cworthy that this hybridization is not detectable

(c) Regions in adenovirus

2 DNA

1,. PHILIPSON

of the h-strand from fragment (Tables 1 and 3). It is? however. with early mRKA (Table 1).

corresponding to early- and late-speci$c RNA: a composite map

cytoplasmic

Maps representing the combined data presented and discussed in the previous sections are shown in Figure 7. Since it is known that sequences of early cytoplasmic RNA persist in the cytoplasm late after infection (Tibbetts et al.. 1974; Green et al.. 1970), comparison of maps constructed with data for early and late cytoplasmic RNA led to identification of those sequences specifically represented late after infection. At this stage additional arbitrary assumptions would be required to construct alternative maps consistent wit’h all of the data now available. The maps presented in Figure 7 differ in some respects from maps suggested earlier on the basis of more limited data (Tibbetts Q Pattersson, 1974; Philipson et al.. 1974; Sharp et al.. 1974a). The differences reflect the limitation of data and assumptions required earlier for arrangement of sequences within large cleavage fragments. In this study we have employed overlapping sets of cleavage fragments of Ad2 DNA in order to specify the locations of sequences which hybridize with early or late cytoplasmic RNA. A complementary approach to hybridization mapping of the Ad2 genome has been pursued by Raskas and co-workers. Labeled viral RNA, sized by gel electrophoresis, has been localized by hybridization with denatured cleavage fragments of Ad2 DNA (Craig et al., 1974,1975a; Craig & Raskas, 1974). There appear to be two distinct classes of early cytoplasmic mRNA which are distinguished by their relative abundance late after infection. Class I is synthesized at greatly reduced rates, if at all, late after infection, and is much less abundant t’han late-specific and class II early RNA species. Class II early sequences are synthesized throughout infection. The localizat’ion of class I and II regions relative to the endoR.EcoRI cleavage fragments of Ad2 DNA (Craig et al., 1975a) is in general accord \vith the four blocks of early RNA as shown in Figures 3 and 7. The l-strand block in the region of EcoRI-F. -D: a,nd -E corresponds to class II RNA synthesized throughout infecbion. The h-strand block of early genes on EcoRI-C was found to be class I RNA, consistent with the lack of h-strand t’ranscription late after infection (Pettersson & Philipson, 1974). The block of early genes on EcoRI-B now also appears to be expressed via class 1 mRNA (Craig et al.. 1975a; H. Raskas, personal communication) in accord with our identification of the RNA as h-strand transcripts and the results of Pettersson & Philipson (1974). Labeled class I and II early mRNA which hybridize with EcoRI-A DNA have more recently been observed to hybridize selectively with DNA from Hsu-G(==NindlII-G) and -C (=HindIII-C): and providing more specific localizat’ion, fragments Smu-J and -E (H. Raskas, personal communication). Fragments J and E from endoR.Sma (from Rerratia marcescens) digestion of Ad2 correspond to the left-hand 120/, of the viral genome. a region only slightly larger t’han the block of early l-strand-specific RNA shown in Figure 3 or Figure 7. The two small blocks of h-strand late-specific sequences within EcoRI-A (Fig. 4(c) or Fig. 7) were rather surprising. The results of Pettersson & Philipson (1974) showed that less than 19: of mRNA which is synthesized late (16 to 18 h post-infection) is complementary to the h-strand. The observed late-specific hybridization of

HYBRIDIZATION

MAPS

OF

mRIS.1

OF

ADESOVIRUS

197

these h-strand sequences must represent species of RNA synthesized prior to the interval selected for labeling of late nuclear or cytoplasmic RNA. Perhaps these RNA sequences are derived during a transient period of h-strand transcription following the onset of DNA-replication but ending shortly thereafter. A more interesting possibility is that these species are synthesized early after infection and are subject to a controlled dela,y of their processing or transport t’o t*he cyt,oplasm. (d) tlrmaitt.tkg

ambiguities

in the hybri&atio?t

map of fhP adenovirur

2 genomu

‘l’hc observation that the nucleus both early and late after adenovirus infection contains sequences which are restricted to the nucleus (Sharp et al.. 1974a; Pettersson & Philipson. 1974) may limit the accuracy of the hybridization map. Because of t’he assay even a’ minute cont’amination of nuclear RNA sensitivity of the hybridization in our mRNA preparations could be of importance. This may be part,icularly relevant early after infection since early mRNA gives hybridization levels slightly above background with a number of fragments which are not included in the early regions of the genome (Tables 1 and 2). It seems likely t’hat adenovirus mRNA is derived by a mechanism which includes polyadenylation of the 3’ end of the nascent t’ranscript and subsequent, cleavage a,nd degradation of the 5’ end. Therefore, contaminating nuclear RNA sequences would most likely affect the boundaries at the 6’ ends of early mRNS blocks. Thus it is conceivable that the observed hybridization bet’ween early mRNA and the l-strand of fragment EcoRI-F is caused by an incompletely processed mRNA since we fail t,o completely saturate tfhe l-strand of the adjacent fragment EcoRT-D (Table 1). Thus the right-hand block of early l-strand-specific mRNS sequences may start within fragment EcoRT-D at position 0.79 rat,her than within fragment EcoRT-17. Nuclear RNA contamination may also explain why early mRNA saturat(es a slightly larger fraction of the h-strand from fragment EcoRI-C than late mRNA. 4not~her uncertainty pert,ains to t’he late-specific h-strand blocks that, we have identified in the left-hand part of the genome. The present data are clearly incompatible with previous tentative maps reported hy us and others (Tibbetts & P&ersson, 1974; Sharp et al.. 1974a; Philipson et al.. 1974). And two separate blocks havtl to be present to account for the hybridization between lat)e mRNA and h-strand prohcs from fragments Hindl Il-C and Hpal-F. Holvever, the exact location w&hin fragment HindIIl -C and the relative sizes of the two blocks cannot be accurately det,ermined unt’il additional cleavage fragments hecome availahlc. It was recently shown that the adenovirus-specifc 55 S RNA (VA-RNA) maps in a DN=\ segment close to position 0.30 on the unit map of the Ad2 genome (Pettersson & Philipson 1975; Mathews, 1975). The 5.5 S RNA is probably t’ranscribed by a different RN.\ polymerase tha,n that which transcribes precursors t’o adenovirus mRNA (Price & Penman, 1972 ; Weinmann et al., 1974) and this region may therefore be of particular importance for transcriptional regulat,ion. It is thus possible that hyhridizat#ion analysis with smaller rest,riction fragment’s will reveal further details in this part of the genome. ((3) Conapa~ixotL Detween the hybridizatiott. map attd genetic ntrd functional gethome

maps of

‘I’hc results of physical mapping studies with viral mRNA have provided considerable information regarding the complex organizat,ion of t,he Ad2 genome. Efforts to

49x

C.

PETTERSSON,

C. TIBBETTS

AND

I,.

PHILIPSON

reveal the genetic functions associated with specific locations in the Ad2 genome are in progress on two major fronts: complementation and recombination analysis using conditional lethal mutants (Grodzicker et al., 1974; Begin & Weber, 1975; Williams et al., 1975 ; Mautner et al., 1975) and identification of in vitro translation products of isolated Ad2 mRNA (Anderson et al., 1974; Eron et al.. 1974a,b; ijberg et al., 1975; Lewis et al., 1975). Species of mRNA are now being isolated by selective hybridization wit’h denatured cleavage fragment,s of Ad2 DNA, permitting localizat,ion of the Ad2 genome of regions coding for specified polypeptides (Lewis et al., 1975). ‘l!he region coding for the fiber polypeptide has been identified as the lat’e-specific block encompassing the EcoRI-E/C junction (Lewis et aZ.. 1975: Mautner et al., 1975). Zt is likely that, this block corresponds to the most apparent region of interserotypic heterology in adenovirus DNA heteroduplex structures (Garon et al., 1973). The polypeptide coding for type-specific determinant in the major capsid protein. the hexon, has been assigned to a region from about 0.44 to 0.59 on the Ad2 genome by Mautner et al. (1975), and Lewis et al. (1975) have shown that hexon mRNA can be selected by hybridization with both fragments EcoRI-A and -B, indicating that t,he structural gene for the hexon overlaps the cleavage site between these two fragment’s, This is compatible with our hybridization map (Fig. 5). Lewis et al. (1975) have also shown that fragment EcoRI-A includes the genes for a number of structural proteins: polypeptides III, IIIa, V? P-VII and IX. It seems likely that these are derived from the major l-strand late gene block in our hybridization map (Fig. 7). The gene for the late protein 100 K has been assigned to fragments EcoRI-F and D (Lewis et al.. 1975). In our hybridization map there is not enough space for a gene which codes for a polypeptide of this size. A possible explanation is that some of the sequences which code for this protein are transcribed early and thus included in the righthand block of l-strand-specific early sequences. The elegant mapping of Ad2 DNA sequences consistently associat’ed with Ad2transformed rat fihroblasts (Sharp et al.. 1974h; Gallimore et al.. 1974) defined a biologically selected group of viral genes. localized within the block of early l-strand transcription of Hpa-E and -C. This region corresponds to at least two early genes since it is expressed in productive infection by two distinct 13 S mRNA species and perhaps an additional 11 S mRNA (Craig et al., 19756). These genes may, in fact, code for viral T and TSTA antigens associated with Ad2-t’ransformed cells, but this has not yet been established. Three groups of defective and non-defective AdB-simian virus (SV) 40 hybrid viruses have been studied and found to contain significant deletions of Ad2 DNA which were mapped using electron microscope heteroduplex techniques (Morrow $ Berg, 1972; Kelly & Lewis, 1973; Kelly et aE.: 1974). The deletions corresponding to are shown in Figure 8 with bhe series Ad2+ND,-,, Bd2++HEY, and Bd2 + ‘LEY the relevant, portion of our map for regions of Ad2 DNA represent,ed in early- and late-specific cytoplasmic RNA. The first st,riking observation is t)hat the right’-hand end position of each set of delet~ions appears to coincidt: (wit’hin 0.01 G) with an earl>. gene-late gene transition. Could the DNA sequence at such points of transition be predisposed to recombination events such as those which lead to formation of the Bd 2-SV40 hybrid genomes? The deletions of the non-defective group of hybrid viruses effect removal of significant portions of the class II early region on EcoRI-E and -D. Considering the variable and consistently smaller amount of SV40 DNA inserted in the place of the deleted Ad2 DNA it appears that much of this early block

0.25

I

I

0 50

0 75

Posmon

100

on Ad 2 genome mop

and “late” genes on the Ad2 genome and Fro. 8. Relationship between “early” DNAs from simian virus 40-adenovirus 2 hybrids. The bars indicate the size and deletions in the non-defective hybrid viruses (ND,_,) and in strains Ad2+ +LEY Data from Kelly & Lewis (1973) and Kelly et nl. (1974). Solid bars represent late ripen bars correspond to early regions.

deletions in location of and HEY. regions and

of genes is not essential for productive Ad2 infection. Another Ad2 deletion 11% recently been mapped (O-83to 0.85 G) which lacks SV40 DNA sequences (N. Newell & T. Kelly, personal communication). This deletion at the EcoRI-D/E junction confirms that some of t’he early gene block in this region is not essential for productive Ad2 infection in human cells. Hybrid viruses Ad2 + +HEY and Ad2 + +LEY contain deletions of Ad2 DNA which include regions of late genes (l-strand blocks of EcoRlA-B and EcoRI-E-C, respectively). Deletion of such regions coding for major virus capsid proteins can explain the fact that these two sets of viruses are defective. The complex organization of the Ad2 genome and the extensive transcription of the viral DNA, including sequences complementary to mRNA species (Pettersson & Philipson, 1974), emphasize the role of post-transcriptional controls in the selection of specific mRNA species from a broader population of nuclear transcripts. The differentiation of class I and II early RNA and late-specific mRNA species suggests that there are several ways in which Ad2 can modi[y and exploit the regulatory mechanisms of gene expression in the infected cell. With information now availa,ble on the physical location of DNA sequences destined to be represented in mRNA, efforts will be directed towards a structural analysis of the regions which play a role in the control of specificity and regulation of viral genome expression. This investigation has received support from the Swedish Medical Research Council, the Swedish Cancer Society and the Wallenberg Foundation, Stockholm, Sweden. One of us (C.T.) was supported by a post-doctoral fellowship from the Jane Coffin Childs Memorial Fund for Medical Research. We express our gratitude to Drs R. &men and IX. Winnacker for generous gifts of the restriction enzymes and Miss Gun-Inger Lindh and Mr Hans-Jiirg Monstein for excellent technical assistance. We are also indebted to Dr Richard Roberts and other colleagues at the Cold Spring Harbor Laboratory for making unpublished information about cleavage maps available to us prior to publication.

500

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1’I~:‘I”I’EKSSON.

(‘. TIKHE’I’TS

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REFERENCES Anderson, (1. \V., Lrswis, .I. B.. Atkins, .I. I;. & G&eland, K. F. (1!174). J’roc. Xat. Acatl. i7.’CL., U.S.A. 71, 2756-2760. Ando, T. (1966). Biochim. Biophys. Beta, 114, 158168. Begin, M. & Weber, J. (1975). .J. Viral. 15, lL7. Bhaduri. S., Raskas H. & Green, M. (1972). J. Viral. 10, 1126-1129. c’raig. E. A. & Raskas, H. J. (1974). J. Viral. 14, 751-757. (!rai~, E. A., Twl, ,J., Nishimoto, T., McGrogan, M., Zimmer, S. & Raskas, H. .J. (1974). Cold Spring Harbor Symp. Quant. Biol. 39, 483-493. Craig, E. A., Zimmer, S. & Raskas, H. J . (1975a).