Molecular Cloning and Characterization of Avian ... - Journal of Virology

JOURNAL

OF VIROLOGY, Oct. 1980, 0022-538X/80/10-0050/12$02.00/0

p.

50-61

Vol. 36, No. 1

Molecular Cloning and Characterization of Avian Sarcoma Virus Circular DNA Molecules WILLIAM J. DELORBE,l* PAUL A. LUCIW,' HOWARD M. GOODMAN,2 HAROLD E. VARMUS,' AND J. MICHAEL BISHOP'

Department of Microbiology and Immunology' and The Howard Hughes Medical Institute, Department of Biochemistry and Biophysics,2 University of California, San Francisco, California, 94143

Supercoiled DNA molecules were used for the molecular cloning of full-length avian sarcoma virus (ASV) DNA. Viral DNA produced by the Schmidt-Ruppin A (SR-A) strain of ASV was isolated from acutely infected transformed quail cells. Supercoiled DNA was separated from linear and open circular DNA by acid phenol extraction, opened into a full-length linear form by cleavage with the restriction endonuclease Sacd, and cloned into XgtWES * AB. Four different cloned viral DNA molecules were isolated: SRA-1 contains two copies of the 330-base pair terminal redundancy normally found at each end of the linear DNA molecules, but harbors a 63-base pair deletion that spans the site at which the two copies of the terminal redundancy are joined in circular DNA molecules; SRA-2 contains two complete copies of the terminal redundancy; SRA-3 probably contains only one copy of the terminal redundancy but in all other respects appears to be similar to SRA-2; SRA-4 contains a 2,500-base pair deletion that removes all of the src gene (the gene responsible for transformation by ASVs) plus additional nucleotides adjacent to the src gene whose precise locations have not been determined. Transfection of chicken embryo fibroblasts by either SRA1 or SRA-2 resulted both in the appearance of transformed cells and in the production of infectious virus. These results demonstrate that the cloned DNA molecules are functionally identical to viral DNA produced in vivo; therefore, molecular cloning did not cause any major alterations of the DNA. The infectivity of SRA-1 DNA indicates that the 63 base pairs missing from that molecule are not required for the initiation of viral RNA synthesis, even though the deletion is located in a copy of the terminal redundancy thought to carry a promoter for RNA synthesis. This suggests that the deletion does not remove any sequences required for the initiation of transcription.

Avian sarcoma virus (ASV) is an RNA tumor virus whose genome consists of two copies of a 9- to 10-kilobase RNA molecule (1, 2, 7). After infection of permissive cells, the viral RNA is transcribed into double-strand DNA by the enzyme reverse transcriptase (see Fig. 1) (for reviews, see references 26 and 28). Both linear and circular (supercoiled) forms of viral DNA have been detected in permissive cells (10, 13, 19), and it has been shown that the linear molecules are the precursors of the supercoiled forms (20). The linear DNA molecule contains two copies of a 300- to 350-base pair (bp) segment, composed of sequences from both the 3' and 5' ends of viral RNA, in the same orientation at each end of the DNA molecule (see Fig. 1) (13, 19). Due to its locations at the ends of the DNA, the repeated segment generally is referred to as the terminal redundancy. Approximately half of the circular DNA molecules contain both copies of the terminal redundancy, but the rest of the circular DNAs contain only a single copy of the terminal

redundancy; the other copy apparently is lost during circularization (13, 19). Viral DNA can be isolated as free (nonintegrated) DNA from infected cells, but only small (less than microgram) amounts can be obtained routinely in this way and it is always contaminated with host DNA. Large quantities of highly purified viral DNA would be useful for a variety of studies, among them sequencing of the viral genome, studies of viral gene expression, and site-specific mutagenesis within the viral genome. Molecular cloning of ASV DNA molecules presents a simple and efficient method of amplifying the synthesis of viral DNA so that milligram quantities can be obtained free from contaminating host sequences. The small size of full-length ASV DNA (slightly less than 10 kilobase pairs [kbp]) made it possible to clone the entire DNA molecule in the bacteriophage XgtWES * XB. By cleaving supercoiled ASV DNA with the restriction endonuclease SacI (which recognizes only one site on the DNA of the 50

VOL. 36, 1980

Schmidt-Ruppin A [SR-A] strain of ASV), we were able to produce permuted, full-length linear DNA molecules that were cloned as a single piece. We have isolated and characterized four different cloned DNA molecules from cells infected with the SR-A strain of ASV. Two of these molecules probably are representative of the majority of supercoiled DNA molecules found in vivo: one molecule (SRA-2) contains two copies of the terminal redundancy, and the other molecule (SRA-3) contains only one copy. The other two cloned DNA molecules are aberrant: one (SRA-1) contains a small deletion (63 bp) in the terminal redundancy, and the other (SRA-4) contains a large deletion that encompasses all of the src (transforming) gene of ASV plus some additional DNA sequences whose precise location has not been determined. We have tested the biological activity of SRA-1 and SRA-2 DNA by transfection and have found that both are able to initiate productive infection and to transform chicken embryo fibroblasts in culture. MATERIALS AND METHODS Cells and virus. QT-6 cells, a continuous line derived from methylcholanthrene-induced fibrosacromas of Japanese quail (17), were propagated as monolayers at 37°C in medium 199 (GIBCO Laboratories) supplemented with 10% tryptose phosphate broth, 0.1% sodium bicarbonate, 4% fetal calf serum (GIBCO), 1% heat-inactivated chick serum (GIBCO), and 1% dimethyl sulfoxide. SR-A virus was originally a gift from P. Vogt and was propagated in chicken embryo fibroblasts at 41°C. For virus stocks, medium from infected chicken embryo fibroblasts was harvested every 24 h. Escherichia coli strains DP50supF, NS428, and dg805 and the bacteriophage XgtWESAXB were gifts from F. Blattner. Sonicated extract and freeze-thaw lysate were prepared from NS428 and lambda A protein was prepared from dg805 exactly as described by Blattner et al. (3). XgtWES.XB was purified as described (3) except that the phage were routinely propagated in DP50supF. 32P-labeled DNAs complementary to the entire ASV genome (cDNA,,p) or to only the 3' portion of the genome (cDNAO) were prepared as previously described (18, 24). We have adopted the following terminology to distinguish in vivo and cloned DNA molecules: SR-A defines the virus particle (DNA produced in vivo during productive infection is referred to as SR-A DNA); A* SRA or AgtWES * SRA refers to recombinant cloned molecules (e.g., AXSRA-2); and SRA refers to cloned ASV DNA, separated from A DNA by SacI cleavage and sucrose gradient purification (e.g., SRA2). Analysis of restriction digests of ASV DNA. DNA samples were digested with various restriction endonucleases, using conditions prescribed by the supplier (New England BioLabs). Digested DNA was

CLONING OF ASV DNA MOLECULES

51

subjected to electrophoresis through 0.8 to 3% agarose gels (Seakem), depending on the size of the DNA fragments to be studied, and was transferred to nitrocellulose filters (Schleicher & Schuell Co.) as described by Southern (22). ASV DNA fragments on the filters were detected by hybridization to 3P-labeled ASVspecific cDNA's (19). Transfer of cloned DNA to nitrocellulose filters was allowed to proceed for 12 to 14 h. Hybridization of 106 cpm of cDNA probe to the nitrocellulose filter was for 12 to 24 h. Transfers and hybridizations for longer periods of time generally resulted in high background; recently, we have shortened the transfer and hybridization steps to 3 h each and find that background is reduced significantly. Subsequent washing and exposure of the filters were as described previously (19). Purification of closed circular ASV DNA. Monolayers of QT-6 cells grown in roller bottles (Corning 25140) at 37°C were infected with SR-A ASV (in the presence of 4 ,ug of polybrene per ml) at a multiplicity of infection of 1 to 2 focus-forming units per cell when the monolayers were approximately three-quarters confluent. At 18 to 24 h after infection, the cells were removed from the roller bottles by treatment with trypsin, collected by centrifugation for 10 min at 800 x g, washed with Tris-glucose (0.14 M NaCl-5 mM KCI-5.5 mM glucose-25 mM Tris-HCl, pH 7.4), and again pelleted. High-molecular-weight cellular DNA was separated from viral DNA by the method of Hirt (12). Infected QT-6 cells were resuspended in TE buffer (10 mM Tris-hydrochloride, pH 7.4-10 mM EDTA) to a final concentration of 5 x 106 to 1 x 107 cells/nil. Cells were lysed by the addition of sodium dodecyl sulfate to 1% and incubated at room temperature for 20 min; 5 M NaCl was then added dropwise with gentle mixing to a final concentration of 1 M. The mixture was poured into centrifuge bottles for a Beckman L19 rotor and kept at 4°C overnight. The precipitate (high-molecular-weight DNA and protein) was then collected by centrifugation at 17,000 rpm for 45 min at 4°C, and the supernatant (containing viral DNA) was removed and treated with pronase (predigested for 1.5 h at 37°C), at a final concentration of 500 ug/ml, for 1 h at 37°C. The mixture was extracted several times with phenol-chloroform (1:1), and nucleic acid was precipitated by the addition of ammonium acetate (to 0.2 M) and 2 volumes of ethanol. Precipitated nucleic acid was collected by centrifugation (10,000 rpm, 30 min; Sorvall GSA rotor), resuspended in TE buffer, treated with pancreatic RNase (100 Lg/ml) for 30 min at 37°C, and again digested with pronase, extracted with phenol-chloroform, and precipitated with ethanol as described above. For analytical purposes, portions were resuspended in 10 mM Tris-hydrochloride, pH

7.4-1 mM EDTA and either run directly on 0.8% agarose gels or treated with restriction endonucleases before agarose gel electrophoresis. High-molecular-weight linear DNA was removed from the purified Hirt supernatant by acid phenol extraction essentially as described by Zasloff et al. (28). Ethanol-precipitated Hirt supernatant DNA from 10'° cells was resuspended in 9 ml of water; to this was added 0.5 ml of 1.5 M sodium acetate (pH 4.0) and 0.5 ml of 1.5 M NaCl. The DNA was extracted

52

DeLORBE ET AL.

one time with phenol that had been redistilled and equilibrated with 50 mM sodium acetate (pH 4.0). The aqueous phase was removed and extracted once with an equal volume of chloroform. Tris-hydrochloride (pH 7.4) was added to a final concentration of 50 mM, sodium chloride was added to a final concentration of 200 mM, and nucleic acid was precipitated by the addition of 2 volumes of 95% ethanol. Precipitated nucleic acid was collected by centrifugation and resuspended in 2 ml of TE buffer. Sodium chloride was added to a final concentration of 0.5 M, and the solution was passed through a Bio-Gel A50m agarose column (2.5 by 40 cm; Bio-Rad Laboratories) to remove small nucleic acids. Material eluting in the leading peak (monitored by absorbance at 260 nm) was pooled and ethanol precipitated. Molecular cloning of ASV DNA. Partially purified ASV supercoiled DNA was resuspended in 10 mM Tris-hydrochloride (pH 7.4)-i mM EDTA, and the nucleic acid concentration was determined by absorbance at 260 mm. Then 5 ,ug of DNA was incubated with 1 U ofthe restriction endonuclease SacI (New England BioLabs) for 3 h at 370C. Digestion was termiinated by the addition of 0.1 volume of 1% diethylpyrocarbonate (in 95% ethanol) followed by incubation at 370C for 10 min. The DNA was then placed under a vacuum for 5 min in a desiccator to remove CO2 and ethanol. SacIdigested ASV DNA (0.4 fig) was mixed with AgtWESAB DNA (2 ,ug) in ligation buffer (66 mM Tris-hydrochloride, pH 7.4-6.6 mM MgCl2-10 mM dithiothreitol), 0.2 U of T4 DNA ligase (New England BioLabs) was added, and the reaction (final volume, 50 1d) was incubated overnight at 40C. Twenty-five microliters of the ligation mixture (i.e., 1 jig of AgtWES.*XB and 0.2 ug of ASV DNA) was packaged into phage as described by Blattner et al. (3) in a final volume of 250 pi. The resulting phage titer was 2 x 105 per ml. Phage were mixed with E. coli DP50supF and plated on 150-mm plates at a phage concentration of 10,000 plaques per plate. After overnight growth of the phage at 370C, 139.5-mm-diameter nitrocellulose filters (Schleicher & Schuell), moistened in 6x SSC (SSC = 0.15 M NaCl plus 0.015 sodium citrate), were placed on the plate for 5 min. The filters were then processed by successive 5-min incubations in denaturing buffer (0.2 M NaOH-1.5 M NaCl) and neutralizing buffer (0.5 M Trs-chloride, pH 7.0-3 M NaCi) and a short rinse in 2x SSC. The filters were allowed to air dry and then were baked in vacuo at 800C for 2 h. The filters were then hybridized with 106 cpm of cDNAr.p for 48 h and subsequently processed for autoradiography as described (19). Putative recombinant plaques were picked, replated, and rescreened to verify the presence of ASV DNA and to plaque purify the phage. Large-scale growth and purification of recombinant DNA. Large-scale lytic growth and purification of AgtWES ASV recombinant phage in DP50supF was carried out in 500-ml volumes essentially as described by Blattner et al. (3). In brief, lysates were clarified by centrifugation; then phage were precipitated by the addition of polyethylene glycol, collected by centrifugation, suspended in 20 ml of 10 mM Trishydrochloride (pH 7.4)-10 mM MgCl2, extracted one time with an equal volume of chloroform, and banded twice in cesium chloride. Purified phage were dialyzed

J. VIROL. to remove cesium chloride, extracted several times with phenol, and dialyzed exhaustively against 10 mM Tris-hydrochloride (pH 7.4)-i mM EDTA. DNA concentration was determined by absorbance at 260 mm, and DNA was digested for 3 h at 370C with Sacl (1 U of enzyme per 10 ,ug of DNA). ASV DNA was separated from phage DNA by centrifugation through 15 to 30% linear sucrose gradients (in 10 mM Tris-hydrochloride, pH 7.4-1 mM EDTA-0.5 M NaCl) in a Beckman SW27 rotor at 25,000 rpm for 24 to 36 h at 40C. We loaded between 100 and 400,ug of DNA per gradient. Equal portions of gradient fractions were analyzed by agarose gel electrophoresis, and those fractions containing only ASV DNA were pooled and ethanol precipitated. Transfection assay of cloned SRA DNA. A transfection assay was used to test for the ability of cloned DNA to transform chicken embryo fibroblasts in culture and to produce infectious viral particles. Transfections were performed according to the method of Graham and Van der Eb (9) as modified by Stowe and Wilkie (23). Twenty-four hours before transfection, secondary or tertiary chicken embryo fibroblasts were seeded at a density of 7 x 105 cells per 60-mm tissue culture plate in 5 ml of medium 199 containing 5% fetal calf serum plus 1% chicken serum. Different amounts of cloned SRA DNA were mixed with high-molecular-weight carrier DNA (prepared from NIH 3T3 cells) at a final total DNA concentration of 20 itg/ml in N-2-hydroxyethylpiperazine-N'-2ethanesulfonic acid (HEPES)-buffered saline (HBS; 137 mM NaCl-5 mM KC1-7 mM Na2HPO4-6 mM dextrose-20 mM HEPES, pH 7.05). To precipitate the DNA, 0.1 volume of 1.25 M CaCl2 was added. After 30 min, 0.5 ml of the mixture was applied directly to the medium (5 ml) in each 60-mm culture plate. After 4 to 6 h of incubation at 370C, the medium was decanted and the cell monolayers were treated with 1 ml of 30% dimethyl sulfoxide in HBS for 4 min, washed once with medium (199 containing 5% calf serum plus 1% chicken serum), and subsequently maintained in fresh medium at 370C. Foci of transformed cells were observed and counted within 6 to 10 days after transfection. In some experiments, medium from plates containing transformed cells was harvested and assayed for the presence of infectious virus. Biological and physical containment. All packaging, growth, and DNA isolation involving recombinant phage were carried out in a P2 containment facility, using the EK2 certified vector XgtWESB-X and its certified host E. coli DP50supF in accordance with the National Institutes of Health Guidelines for Recombinant DNA Research.

RESULTS Some strains of ASV (e.g., Prague C and Prague A) contain a single recognition site for the restriction endonuclease Sacl, encoded approximately 3,400 bp from the left end of the DNA (19). Restriction mapping of SR-A DNA (data not shown) demonstrated that it also contains a single SacI site; this site, however, is located only about 400 bp from the left end of SR-A

VOL. 36, 1980


53

1 2 3 4 6 5 DNA (the left end of the DNA molecule as drawn in Fig. 1 corresponds to the 5' end of the RNA molecule). Thus, digestion of supercoiled SR-A DNA by SacI results in permuted fulllength linear molecules that contain either one -a or two copies of the terminal redundancy located - b near the right end of the DNA molecule along -c with an additional 100-bp segment from the left end of the genome (see Fig. 1). The size of these -d full-length molecules (slightly less than 10 kbp) - e makes them suitable for cloning in the EK2 certified vector XgtWES . XB, which can accommodate inserts of 0 to 10 kbp after digestion with Sacl (15). Molecular cloning of SR-A DNA. Electrophoretic analysis of Hirt supernatant DNA prepared from SR-A-infected QT-6 cells demonstrated that the majority of DNA observed by ethidium bromide staining was contaminating cellular DNA migrating at the exclusion limit of the gel (Fig. 2, lane 1) and that ASV DNA could be detected only by transfer to nitrocellulose filters and hybridization to ASV-specific probes (Fig. 2, lane 3). FIG. 2. Acidphenol extraction of Hirt supernatant To ensure that there would be sufficient viral SR-A DNA. Hirt superntants from QT-6 DNA for cloning, Hirt supernatant DNA was containing cells acutely infected with SR-A were prepared as prepared from 35 roller bottles of acutely in- described in Materials and Methods. Portions of Hirt fected QT-6 cells (-7 x l09 cells), resulting in a supernatant DNA were electrophoresed through 0.8% yield of 2.1 mg of DNA. The Hirt supernatant agarose gels before (lanes 1 and 3) and after (lanes 2 was enriched for supercoiled SR-A DNA by and 4) acid phenol extraction. Lanes I and 2 illusextraction with acid phenol (29). This procedure trate the ethidium bromide stain patterns before (lane (see Materials and Methods) removes linear 1) and after (lane 2) acid phenol extraction. Lanes 3 DNA molecules larger than 106 daltons from the and 4 are an autoradiograph of the DNA from lanes I and 2 transferred to a nitrocelulose filter (22) and pOd to 3P-labeled ASV cDNA,,,. Lane 5 congoo hybridized RNA 0tains Hirt supernatant DNA purified by gel filtration .rc

through agarose A50m after acid phenol extraction. Lane 6 is a SacI digestion of the ASVDNA shown in lane 5. The ASV-specific bands visible in this figure are: (a) open circle; (b) nondefective (ful-length) linear; (c) td linear; (d) nondefective supercoil; (e) td supercoil.

DNA

aqueous phase but does not remove supercoiled molecules as large as 25 x 106 daltons (29). For enrichment of SR-A supercoiled DNA, the Hirt supernatant (which contained 2.1 mg of DNA) FIG. 1. Schematic representation of the uninte- was resuspended in a total volume of 10 ml. At grated forms of SR-A DNA found in the Hirt super- this DNA concentration, a single extraction with natant fraction of acutely infected QT-6 cells. The acid phenol removed almost all high-moleculartriangle indicates the position of the SacI restriction weight linear DNA (Fig. 2, lane 2), including endonuclease recognition site. Each copy of the ter- linear SR-A DNA (Fig. 2, lane 4). Supercoiled minal redundancy is represented by a single open box SR-A DNA remained in the aqueous phase (Fig. in the supercoiled (depicted here as a circle) and 2, lane 4). Note that the two types of supercoils SacI-digested DNA molecules. The relative positions (containing either one or two copies of the terof the four known genes of ASV are shown: gag, minal redundancy) were not separated from encoding a polyprotein that contains the structural proteins found in the interior of the virus; pol, encod- each other on this gel; a small amount of transformation-defective (td) supercoiled DNA moling reverse transcriptase; env, encoding the glycoprotein(s) of the viral envelope; and src, the transforming ecules were present in this preparation (Fig. 2, lane 4) (td ASV arises by a deletion of approxigene. 4.saci

4 *s.a

gag

pol

e

rc

54

DeLORBE ET AL.

mately 2,000 bp encompassing the src gene). Of the original 2.1 mg of DNA, 240 ,Lg remained after acid phenol extraction. We observed that even the relatively mild acid phenol extraction procedure described here resulted in the breakdown of high-molecularweight DNA into smaller fragments (compare lanes 3 and 4, Fig. 2). These small fragments, as well as residual small RNA molecules, could be removed by filtration through an A50m agarose column (Fig. 2, lane 5). The final DNA yield after filtration through the column was 120 Mg. The major contaminant remaining was supercoiled mitochondrial DNA, which could be seen in the gels by ethidium bromide staining (not shown). The enriched supercoil fraction was subsequently digested with SacI (Fig. 2, lane 6) to produce full-length permuted linear SR-A DNA as depicted in Fig. 1. A portion of this DNA was ligated to SacI-digested XgtWES * XB DNA. The ligated DNA (1 Mg of XWES and 0.2 Mug of partially purified SR-A DNA) was then packaged in vitro and plated at a density of 10,000 plaques per plate (150-mm diameter). Of 50,000 plaques screened, 6 appeared to hybridize to cDNArep. These six were picked, replated, and rescreened; four of them proved to be recombinants containing SR-A DNA. In preparing the XgtWES.AB DNA for ligation, no attempt was made to separate the lambda arms from the small piece of DNA that is excised by SacI digestion. Since the lambda arms produced are large enough to be packaged even without an insert, the purity of the SR-A DNA fraction could not be estimated simply by comparing the number of recombinant plaques obtained with the total number of plaques screened, because many of the plaques contained either self-ligated lambda arms packaged without any insert or DNA in which the original lambda Sacl fragment had been reinserted between the two arms. Moreover, the contaminating mitochondrial DNA was cleaved by SacI into an undetermined number of fragments, the largest of which was about 10 kbp (data not shown). Since mitochondrial DNA was present in great excess over SR-A DNA (determined by ethidium bromide staining of agarose gels), a large number of plaques probably contained mitochondrial DNA. The 0.2 Mug of DNA used for packaging was less than 0.2% of the total supercoiled DNA fraction (120 Mg) prepared from the original 35 roller bottles. Thus the four recombinant SR-A clones that were found were derived from the equivalent of about 2 x 107 cells. Characterization of cloned SR-A DNA. The SR-A DNA present in each of the four recombinant phage was characterized by diges-

.:

J. VIROL.

tion of the recombinant DNA by Sacl (not shown). Three of the recombinants (A * SRA-1, XASRA-2, and X.SRA-3) contained inserts that comigrated with full-length linear SR-A DNA in agarose gels and hybridized to ASV cDNArep; the fourth recombinant (XA SRA-4) contained an insert that migrated faster than full-length DNA and appeared to be slightly smaller than the DNA of td deletion mutants of ASV DNA. Viral DNA was excised from XA SRA-2 by SacI digestion and was separated from the lambda arms on sucrose gradients. This purified viral DNA was subsequently digested with various restriction endonucleases known to differentiate among viral strains (19) and electrophoresed in agarose gels in parallel with similarly treated supercoiled SR-A DNA from infected QT-6 cells. There were no major differences between the cloned DNA and supercoiled viral DNA molecules produced in QT-6 cells (Fig. 3), indicating

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L 9000F, FIG. 3. Comparison of restriction patterns of SRA DNA produced in vivo and ofSRA-2 DNA. Various restriction endonucleases were used to digest: (a) supercoiled SR-A DNA, from acutely infected QT-6 cells, obtained by Hirt fractionation and acid phenol extraction; (b) SRA-2 DNA separated from the arms as described in Materials and Methods. All digestions also contained SacI to linearize the supercoiled DNA molecules so they would be structurally equivalent to the permuted cloned molecule. The lines to the right of the figure indicate the positions of HindIII fragments run in parallel as size standards. The sizes of the fragments are given in kilobase pairs. Smmm

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VOL. 36, 1980

that a full-length SR-A molecule had been cloned and that SRA-2 was apparently representative of the majority of supercoiled molecules produced during acute infection of QT-6 cells. The stock of in vivo supercoiled DNA used for these digests was a mixture of molecules containing either one or two copies of the terminal redundancy. The additional band seen after digestion of the in vivo molecules came from those supercoils that harbored a single copy of the terminal redundancy and therefore yielded a fragment about 330 bp smaller than the corresponding fragment from a supercoil that contained two copies of the terminal redundancy. The best example of an extra fragment is seen in the XbaI digest. Each of the other three digests of in vivo DNA also contained an extra fragment that is difficult to detect in Fig. 3 because it ran only slightly faster than the fragment above it and the two fragments appear as one in a long exposure of the autoradiogram. A restriction map of SRA-2 DNA is presented in Fig. 4. The map is presented with the permuted orientation of the DNA as it is obtained from the recombinant phage by cleavage with SacI. Because the precise limits of the ASV

55

genes are as yet unknown, no attempt has been made to indicate the locations of their boundaries. We have made no effort to look for fragments smaller than 200 bp; therefore, it is possible that there may be additional restriction fragments not reported here. Since the sum of the sizes of the fragments varies slightly for different enzymes, the map has been drawn so that the total number of base pairs for any given restriction endonuclease is considered to be 100% genome length. The positions of the restriction sites have been placed primarily to show the relative positions of sites for various restriction enzymes, even if this meant making a particular fragment appear slightly longer or shorter than its measured size indicated in Fig. 4. For instance, the SmaI B fragment (1,850 bp) is drawn smaller than the combined PvuI D (1,000 bp) plus E (800 bp) fragments because the PvuII E fragment extends into the SmaI F fragment. Precise sizes of the fragments will be determined from the sequence of the DNA, which is now being elucidated. Clones SRA-1 and SRA-3 have not been mapped as extensively as SRA-2, but so far no major differences have been observed. The differences that we have discovered are discussed

SRA-2 DNA - gag Enzymes

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Sac I

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C 1350

1

9480

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9575

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9250

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II

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B 2400

A 3300

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20

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FIG. 4. Restriction map of clone SRA-2 DNA. The DNA molecule is presented in the permuted form that is obtained by SacI digestion of the recombinant phage A DNA molecule. The approximate positions of the four ASV genes are shown in the line drawing above the restriction map; each box represents a complete copy of the terminal redundancy. Fragment sizes are given in base pairs.

56

DeLORBE ET AL.

below. SRA-4 DNA contains a large deletion in the 2,950-bp EcoRI B fragment. A typical td deletion reduces this fragment to about 1,000 bp; the deletion in SRA-4 reduces the fragment to about 500 bp. We have not yet mapped the boundaries of this deletion. As shown in Fig. 1, some of the in vivo supercoiled DNA contained but a single copy of the terminal redundancy that was found at each end of the in vivo linear DNA. We have found that SRA-1, SRA-2, and SRA-4 each contain two copies of the terminal redundancy; SRA-3 apparently contains only one copy of the redundancy. This was determined by cleavage of the XgtWES - SRA recombinant DNA molecules with either EcoRI or PvuI. There is an EcoRI site and a PvuI site in the terminal redundancy; double copies of the terminal redundancy in the cloned ASV DNA molecule are contiguous (as shown for SRA-2 in Fig. 4), and digestion by either EcoRI or PvuI will cut out a fragment about 330 bp long that contains a portion of each terminal redundancy (the PvuI C and EcoRI D fragments shown in Fig. 4). Since the recombinant molecules were not digested with SacI, the right terminal PvuI B or EcoRI E SRA fragment remains covalently bound to a portion of the lambda EcoRI B fragment; these chimeric fragments are at least 600 bp long and therefore do not appear in the critical area of the gel where the 320-bp terminal redundancy should be. Figure 5 shows an analysis of EcoRI, PvuI, and EcoRI plus Sacl digests of SRA-1, SRA-2, and SRA-3 on a 1.5% agarose gel, using a cDNA probe specific for sequences from the 3' end of viral RNA (cDNA3-). Both EcoRI and PvuI cleave in a region of the terminal redundancy that is complementary to the 3' end of viral RNA (see Fig. 5B); if a single copy of the terminal redundancy is present, the terminal redundancy will be split by EcoRI or PvuI digestion into two DNA fragments that hybridize to the cDNA3 probe (Fig. 5A, lane 3). If two copies of the terminal redundancy are present, a third fragment (PvuI-C or EcoRI-D) will hybridize to cDNA3 (Fig. 5A, lanes 1 and 2). The terminal redundancy fragment excised from SRA-1 ran considerably faster than that from SRA-2. The SRA-2 fragment (running at position "a" in Fig. 5A) was approximately 330 bp, in good agreement with the size previously determined for the terminal redundancy from the Prague A strain of ASV (19). DNA sequencing of the terminal redundancy fragment from SRA-1 (running at position "c" in Fig. 5A) demonstrates that it is 63 bp shorter than the corresponding fragment from SRA-2 (R. Swanstrom, manuscript in preparation). The exact boundaries of the deletion have been determined by comparison with the sequence of SRA-2 and

J. VIROL.

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FIG. 5. Determination of the number of copies of the terminal redundancy in SRA-1, SRA-2, and SRA3. AXSRA-1, -2, and -3 were cleaved with EcoRI, PvuI, or EcoRI plus SacI and electrophoresed on a 1.5% agarose gel. DNA was transferred to nitrocellulose paper (22), and fragments containing terminal redundancy sequences were detected by hybridization to

32P-labeled cDNA3. (A) 1, AXSRA-1; 2, A*SRA-2; 3, A*SRA-3; a, terminal redundancy of SRA-2; b, 280bp EcoRI E fragment (released only after digestion with SacI) (see Fig. 4); c, terminal redundancy of SRA-1. Note that SacI failed to cleave AXSRA-3 DNA appreciably in the EcoRI + SacI digestion. This is evidenced by the small amount of the EcoRI E fragment and the presence of an additional high-molecular-weight band. The lines to the left of the figure indicate the positions of A HindIII fragments run in parallel as size standards. The sizes of the A fragments are given in kilobase pairs. The sizes of the small ASV fragments could not be determined accurately from this gel but were determined more carefully on polyacrylamide gels (data not shown). (B) An expanded view of the terminal redundancy regions of SRA-1 and SRA-2 showing the position of the 63 bp deleted from in SRA-1 (shown as an open box). are shown in 5B; two bases are missing from one terminal redundancy, and 61 bases are miss-

Fig. ing from the adjacent terminal redundancy. Cleavage of the cloned DNAs by both EcoRI and Sacl reveals the small (280 bp) EcoRI E fragment (running at position "b" in Fig. 5A). This fragment is obscured in Fig. 5A by the SRA-2 and SRA-1 terminal redundancy frag-

VOL. 36, 1980


ments (at positions "a" and "c," respectively). The EcoRI E fragment can be seen in the digest of SRA-3 by EcoRI and Sacl, but it is very faint because SacI digestion was incomplete (as indicated by the failure of Sacl to cleave the large X-ASV hybrid fragment of approximately 4 kbp). SRA-4 contains two copies of the terminal redundancy (data not shown). Orientation of SRA DNA in lambda. The differences in the mobilities of the higher-molecular-weight bands of the various cloned DNAs observed in Fig. 5 results from the insertion of the cloned DNA into the lambda DNA in either of two possible orientations. In one orientation, the left end of the SRA DNA is joined to the left arm of lambda; in the other, it is joined to the right arm of lambda. Since the SacI sites in lambda are eccentrically located in the EcoRI B fragment, it is easy to determine the orientation of the SRA DNA by digesting the entire chimeric DNA molecule with EcoRI. The triangles in Fig. 6 indicate the positions of the two EcoRI sites in XgtWES *XB and demonstrate the eccentricity of the SRA DNA molecule within the lambda EcoRI B fragment. If the right end of SRA DNA is joined to the right arm of lambda, then the SRA EcoRI E fragment will be joined to a large piece of the lambda EcoRI B fragment and the resulting chimeric fragment will contain about 4,000 bp. If, on the other hand, the SRA DNA is inserted in the reverse orientation (right xgtWES-ASV W- E-

SRA 1

unbda

la

gag

SacI I

pad

gag

SRAl

n9

pol

I

env

src

SRA 2 SRA 3

Sa

.raAmVda env

env

*env

s-

sic

pol

n gag

6. Comparison of four recombinant DNA molecules. The upper drawing depicts the general form of recombinant DNA molecule obtained by ligation of SacI-digested XgtWES.AB DNA to SacIdigested ASV supercoiled DNA. The triangles indicate the positions of the EcoRI sites in phage lambda. Each open box represents a copy of the terminal redundancy; note that the terminal redundancies in SRA-1 have been drawn slightly smaller to account for the 63-bp deletion. The boundaries of the 2,500-bp deletion (hatched box) in SRA-4 have not been located precisely. FIG.

end joined to the left arm of lambda), then the SRA EcoRI C fragment will be attached to the small segment of the lambda EcoRI B fragment and the resulting chimeric fragment will contain only about 600 bp. The right end of the SRA DNA can be detected by the hybridization of cDNA3' to the EcoRI E fragment (cDNA3' also hybridizes to the EcoRI B and D fragments). The EcoRI digestions shown in Fig. 5A demonstrate that the right ends of SRA-1 and of SRA3 are joined to the right arn of A; SRA-2 is in the reverse orientation. Figure 6 summarizes the orientations of the four SRA clones in lambda. Infectivity of cloned DNA. Although restriction mapping indicated that SRA-2 DNA molecules had not incurred any major structural anomalies, the mapping could not exclude the presence of small deletions or other genetic aberrations. To test for these, we used a transfection assay to determine the biological activity of the SRA DNA, that is, the ability of the cloned DNA to transform cells and subsequently produce infectious viral particles. We also tested these same parameters with SRA-1 DNA to determine the effect of the 63-bp deletion on the infectivity of the DNA. Various amounts of SRA DNA were used to infect chicken embryo fibroblasts as described in Materials and Methods (Table 1). At the lower concentrations of SRA DNA, there was an approximately linear relationship between the amount of SRA DNA used for transfection and the number of foci produced in the permissive chicken cells. Hence, each focus was probably the result of a single infectious molecule; we have no evidence that intermolecular reactions between cloned DNA molecules can facilitate transfection. The number of foci produced per plate did not increase appreciably when more than 10 ng of cloned DNA was used: the maximum number of foci obtained was generally between 10 and 30 per 106 cells, when either

gog

src

pol

57

TABLE 1. Transfection of chicken embryo fibroblasts by cloned ASV DNAa Cloned ASV DNA

ASVFoin Total Total Foci/ Foci/ng

DNA/ plate

foci

plates plate

(ng) SRA-1

100 10 1 0.1 100 10 1 0.1

27 30 4 0 35 117 12 2

4 12 12 12 4 12 12 12

6.8 2.5 0.4 0 8.9 9.8 1.0 0.2

DNASV

0.068 0.25 0.36 0 0.09 SRA-2 0.98 1.0 2 a Transfection was carried out as described in Materials and Methods, using the indicated amounts of cloned SR-A DNA.

58

DeLORBE ET AL.

J. VIROL.

cloned DNA (Table 1) or DNA prepared from ASV-infected cells (P. Luciw, unpublished data) was used for transfection. However, the specific infectivity of cloned DNA (one focus per 10-3 jig) was considerably less than the infectivity of DNA prepared from ASV-infected cells (one focus per 3 x 10-6 to 30 x 106 jig of ASV DNA; see Discussion). Both clones SRA-1 and SRA-2 were able to transform cells, indicating that molecular cloning had no deleterious effects on the transforming ability of viral DNA. SRA-1 may be slightly less efficient than SRA-2, but most likely this apparent decrease in efficiency is merely a reflection of the variability inherent in the transfection procedure. We also assayed for the production of infectious viral particles in cells transformed by cloned SRA DNA. Medium from a plate containing several foci produced by transfection either with SRA-1 or SRA-2 was harvested and placed either on chicken embryo fibroblasts or on QT-6 cells. The presence of viral particles in the medium was confirmed by the appearance of newly transformed chicken embryo fibroblasts. Moreover, DNA isolated from Hirt supernatant fractions of the QT-6 cells contained both supercoiled and linear forms of ASV DNA that were shown by restriction mapping to be identical to the cloned SRA DNA (data not shown). These findings demonstrate that both SRA-1 and SRA-2 contain all the information required to produce infectious viral particles.

cerned that these small DNA contaminants (and small RNA contaminants that were also present) might interfere with efficient ligation of SR-A DNA to the lambda DNA; we therefore removed the majority of the low-molecular-weight nucleic acids by filtration through an agarose column. We use this same combination of steps (acid phenol extraction, gel filtration) routinely to purify plasmid DNA. Acid phenol extraction resulted in almost a 10-fold purification of supercoiled DNA (240 jig) from the original Hirt supernatant (2.1 mg); gel filtration increased that purification by another factor of two (final yield, 120 jig), resulting in a total purification nearly 20-fold greater than that obtained by Hirt fractionation alone. The major species of DNA remaining after acid phenol extraction and gel filtration was supercoiled mitochondrial DNA, which could be detected easily by ethidium bromide staining when fractions from the filtration column were analyzed by agarose gel electrophoresis (data not shown). The phage DNA arms produced by SacI digestion of XgtWES * XB are large enough to be packaged without the insertion of additional DNA. As a result, an undetermined number of the phage particles produced by the in vitro packaging of our ligation mixture do not contain recombinant DNA molecules but simply contain self-ligated lambda DNA. Thus we are unable to estimate the fraction of total recombinants that contain SR-A DNA. Characterization of cloned SR-A DNA. SRA-2 DNA is the largest of the cloned DNA molecules. The presence of two complete copies DISCUSSION of the terminal redundancy and the biological Molecular cloning of SR-A DNA. We have activity of the molecule as measured by transused the EK2 certified bacteriophage XgtWES. fection (Table 1) have led us to consider this as XB, digested with the restriction endonuclease the prototype for SR-A DNA. The restriction Sac, as a vector for cloning ASV circular DNA enzyme digests of SR-A DNA and SRA-2 DNA molecules. We were able to clone the entire ASV shown in Fig. 3 demonstrate the similarity of the genome by converting circular DNA molecules cloned molecule to the majority of DNA moleinto full-length linear molecules by digestion cules produced in vivo. The enzymes used for with Sac. A similar approach has been used to this comparison were chosen because they give clone Harvey sarcoma virus DNA (11) murine different restriction patterns with other strains sarcoma virus DNA (25), and td ASV DNA (14). of avian sarcoma viruses (19); thus, we are conWe used the acid phenol extraction procedure fident that we have cloned the DNA of Schmidtof Zasloff et al. (29) to purify supercoiled forms Ruppin A virus, and not that of some other ASV of DNA for molecular cloning. At a DNA con- strain. centration of 200 jig/nil, a single extraction with Recently it has been reported (14) that clones acid phenol was sufficient to remove virtually all of Schmidt-Ruppin B DNA containing two cophigh-molecular-weight linear DNA from the ies of the terminal redundancy can delete one aqueous phase (Fig. 2). We consistently found copy of the terminal redundancy at a rather high that even a single extraction with acid phenol frequency during growth of the recombinant. resulted in the appearance of additional low- These clones were in the bacteriophage charon molecular-weight DNA which may be the result 21A. We have not observed this deletion pheof acid-catalyzed depurination of some of the nomenon with any of our cloned molecules, perhigh-molecular-weight DNA. We were con- haps because our vector is XgtWES AB. -

VOL. 36, 1980

Each of the other three cloned DNAs are different from SRA-2. Although we have not done extensive restriction endonuclease mapping of SRA-3, the available information indicates that SRA-3 DNA is identical to SRA-2 except that SRA-3 contains a single copy of the terminal redundancy. Since this type of molecule is found in vivo (19), we believe that SRA2 and SRA-3 represent the two predominant types of supercoiled DNA molecules produced during acute infection of quail cells by ASVs. Like SRA-2, SRA-1 contains two copies of the terminal redundancy. However, of the 660 bases contained in the region of DNA defined by the two adjacent terminal redundancies, 63 bases are deleted. DNA sequencing has shown that the deletion is of 63 contiguous bases, 2 from one terminal redundancy and 61 from the adjacent redundancy (see Fig. 5B). We are unable to tell whether SRA-1 represents a type of molecule found in vivo or whether the deletion occurred as an artifact of molecular cloning. Initial screening of the recombinant DNA molecules suggested that SRA-4 might represent one of the td DNA molecules from which 2,000 bp encompassing the src gene have been deleted. Analysis of SRA-4 DNA by cleavage with EcoRI (not shown) demonstrated a deletion of 2,500 bp from the EcoRI B fragment, a deletion approximately 500 bp larger than that observed in the majority of td DNA molecules produced in vivo (13, 19). The boundaries of the SRA-4 deletion have not been determined, but there is one PvuI site present, indicating that the right end of the deletion must be to the left of the PvuI site located in the terminal redundancy. The relative proportion of td DNA molecules to nondefective (full-length) molecules increases with each successive passage of the virus through host cells in culture (8, 27). For acute infection of QT-6 cells to produce supercoiled viral DNA for molecular cloning, we had used a stock of freshly cloned SR-A virus that had been passaged only once or twice in chicken embryo fibroblasts. As a result, our viral stock contained a very small amount of td virus, as evidenced by the amount of td supercoiled DNA present in the DNA (Fig. 2, last columns), and we did not expect to find cloned td molecules. As with SRA-1, we do not know whether SRA-4 represents a type of td molecule produced during infection in vivo or whether it is an artifact of the molecular cloning procedure. Subelones of SRA DNA. Both SRA-1 and SRA-2 have been subcloned into the plasmid pBR322 by self-ligating the cloned DNA to form closed circles and subsequently digesting the circular DNA with Sail to produce linear DNA


59

that could be ligated to SalI-digested pBR322. Several restriction fragments of SRA-2 have been subcloned into XgtWES * AB or pBR322: the BamHI fragments (B. Baker, personal communication), the PvuII fragments (R. Parker, personal communication), and the EcoRI B, D, and E fragments (EcoRI-B contains src, the gene responsible for transformation by ASV). Several of these subclones are useful as gene-specific probes and are available upon request. Infectivity of cloned SRA DNA. Both SRA-1 and SRA-2 are able to transform permissive cells and produce infectious viral particles. As noted previously, there is a linear relationship between the amount of transfecting viral DNA and the number of foci produced, at least at the lower concentrations of SRA DNA used. This linear dose-response relationship is also observed when ASV DNA partially purified from infected cells (i.e., in vivo DNA) is used for transfection (5, 6; P. Luciw, unpublished data). A similar dose-response relationship has been reported for focus-forming ability of unintegrated species of Harvey sarcoma virus and Moloney murine leukemia virus DNAs (16, 21) and also for cloned Harvey sarcoma virus DNA (11). Thus, it appears that in all of these cases each focus arises from a single infectious DNA molecule. The maximum number of foci observed after transfection of 106 cells is usually between 10 and 30, with either cloned or in vivo DNA. This upper limit probably indicates that only about 1 in 105 cells is competent for transformation; however, it is also possible that at high concentrations ofviral DNA transformation is hindered by interaction between viral DNA molecules. The efficiency of transfornation by cloned DNA is considerably less than that of in vivo DNA. We have obtained approximately one focus per ytg of DNA from chronically infected chick cells. Viral DNA has a molecular weight of approximately 6 x 106; each avian cell contains about 2 x 1012 daltons of DNA. If we assume that there are 1 to 10 copies of viral DNA per cell (28), then one focus obtained by transfection with 1 jig of DNA from chronically infected cells arises from 3 x 10' to 30 x 10-6 Lg of ASV DNA. This is about 300- to 3,000-fold greater than the efficiency of transfection obtained with cloned DNA (one focus per l0' pg). We suspect that this significant difference in infectivity is due to the permutation of the ASV genome that occurs as a result of molecular cloning of the DNA. By contrast, transfection of monkey cells with permuted linear simian virus 40 is only two- to threefold less efficient than

60

J. VIROL.

DeLORBE ET AL.

transfection with supercoiled simian virus 40 DNA (4). At the present time we are unable to explain the vast differences in the infectivity of cloned and in vivo ASV DNA. It is interesting to note that SRA-1 is able to produce infectious virus after transfection of permissive cells, despite the loss of 61 bp from the rightmost terninal redundancy. This copy of the terminal redundancy corresponds to the 5' end of the viral genome and, at least under normal conditions, is thought to contain the promoter for ASV RNA synthesis. The infectivity of SRA1 indicates that the deletion does not prohibit the synthesis of viral RNA and suggests that the deletion does not extend into the promoter/initiator region of the terminal redundancy. We hasten to point out that only one round of transcription ofthe deleted DNA would be necessary to initiate productive infection; if the initiation site lies to the right of the deletion, then the RNA produced during this first round would not include the region affected by the deletion and could be used subsequently as a substrate for reverse transcriptase to produce nondeleted DNA molecules as outlined in Fig. 1. These predictions are supported by experiments in which we used virus produced by cells transfected with SRA-1 to infect QT-6 cells. Cleavage of the DNA produced during this infection with EcoRI indicated the presence of two complete copies of the terminal redundancy in the DNA (data not shown). Thus, the deletion in SRA-1 was not perpetuated after transfection, presumably because transcription of SRA-1 DNA initiated somewhere to the right of the deletion in the terminal redundancy. ACKNOWLEDGMENTS We thank R Parker for assistance with restriction endonuclease mapping and helpful discussion and also R. Swanstrom, J. Majors, and B. Vennstrom for helpful discussions. We also thank B. Cook for excellent stenographic assistance. This work was supported by Public Health Service grants CA 12705, CA19287, and CA 14026 and training grant 1T32 CA 09043, all from the National Cancer Institute, and by American Cancer Society grant VC-70. W.J.D. holds a fellowship from the Leukemia Society of America; P.A.L. is supported by a fellowship from National Institutes of Health. LITERATURE CITED 1. Beemon, K., P. Duesberg, and P. Vogt. 1974. Evidence for crossing over between avian tumor viruses based on

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