Synthesis of Avian Oncornavirus DNA in Infected Chicken Cells

The intracellular synthesis and integration of viral DNA (vDNA) into the host cell genome was studied in cultured chicken embryo fibroblasts infected with avian sarcoma or leukemia viruses. The newly synthesized vDNA was detected by hybridization with 70S viral RNA. Extraction of infected cell DNA by the selective procedure of Hirt resulted in the enrichment of newly synthesized vDNA in the low molecular weight supernatant fraction while leaving the bulk of cellular DNA containing integrated vDNA in the high molecular weight pellet fraction. This approach led to detection of intracellular vDNA synthesis within 1 h after infection and to vDNA integration into cellular DNA within 24 h. There was a several-fold increase in the vDNA content of infected cells during the initial phase of virus infection. But only a part of this newly synthesized vDNA appeared to become covalently linked with high molecular weight cellular DNA. Most of the remaining unintegrated vDNA gradually disappeared. The sedimentation profiles of minimally sheared cellular DNA in alkaline sucrose velocity gradients suggest that vDNA is synthesized as free linear molecules of approximately 3 x 10(6) daltons which subsequently are covalently linked to host cell DNA.     

Analysis of the Vicia faba genome by use of restriction endonucleases.

DNA of the broad bean, Vicia faba, was cleaved by the restriction endonucleases endoR . EcoRI, endoR . HindIII, endoR . HincII, endoR . BamI, and endoR . BspRI. Separation in agarose gels of the resulting fragments revealed, in addition to the bulk DNA, an enzyme-specific pattern of bands composed of restriction fragments of 300 to more than 30,000 base pairs in length. Bulk DNA was characterized by an unusual size distribution which significantly deviated from that expected according to the random fragmentation theory. It is argued that the observed distribution is due to the high proportion of repetitive DNA within this species (approximately equal to 75%). In all digests, a class of high-molecular-weight restriction fragments of more than 30,000 base pairs in length was observed which comprised 5-8% of the genome. It showed hybridization with highly repetitive DNA (c0t less than or equal to 2 x 10(-2) M . s) and included a fraction (2-3% of the genome) highly resistant to the activity of all the enzymes tested. The buoyant density in CsCl of this resistant DNA was not different from that of the total DNA (36% dG + dC). In endoR . EcoRI digests, the high-molecular-weight fragment class contained, in addition to the resistant DNA, a fraction of relatively high buoyant density (calculated dG + dC content: 61%) containing cleavage sites for the other enzymes used.     

Restriction endonuclease cleavage of linear and closed circular murine leukemia viral DNAs: discovery of a smaller circular form.

Both linear (form III) and closed circular (form I) viral DNAs obtained from mouse cells infected with Moloney murine leukemia virus were cleaved by Sal I, Sma I, Bam HI and Pst I restriction endonucleases. DNA fragments generated by these cleavages were ordered with respect to the 5' and 3' ends of the RNA genome by several techniques, including comparisons of the DNA fragments from cleavages of the linear and closed circular forms, double digestions using different combinations of enzymes and the use of an RNA probe specific for the 3' end. DNA from Hirt extractions of infected cells yielded a discrete species of linear viral DNA whose size was determined by agarose gel electrophoresis to be 5.7 x 10(6) daltons. In the course of characterizing the closed circular DNA, we observed two form I DNA molecules. The larger molecule was the same size as the linear DNA. The second molecule migrated faster on agarose gels and was the predominant species of the two closed circular DNAs. Using the restriction endonuclease maps which we derived, we demonstrate that this novel form I DNA is a smaller homogeneous species of viral DNA, missing about 600 nucleotides found in the linear and larger closed circular DNA molecules. We have localized the site of this missing DNA piece to be at either one or both ends of the linear viral DNA.     

Oncogen localization in the DNA composition of simian adenoviruses. II. The identification in SV20 virus DNA of a specific fragment possessing transforming activity

Simian adenovirus 20 DNA was specifically cleavered by restriction endonucleases EcoRI, BamHI, XbaI and HindIII. The transformation activity of the DNA digest was investigated. BamHI, XbaI, and HindII DNA digests were able to transform the primary rat kidney cell culture (Wistar) as well as the native SV20 DNA. The transforming activity was revealed in a specific fragment of the viral DNA, obtained after the treatment of the DNA with BamHI (fragment B), with molecular weight 5.4 x 10(6) dalton. This fragment is located in the left end of the viral genome. The lack of cell transformation by the EcoRI-hydrolysate of viral DNA may serve a proof of the extremely left position of the oncogene in the viral genome, since of EcoRI-fragment chips off a fragment with molecular weight 3 x 10(5) dalton fr om the left side of DNA molecule.     

Helper-independent transformation by unintegrated Harvey sarcoma virus DNA.

We have studied the unintegrated infectious DNA of Harvey sarcoma virus (Ha-SV) and Moloney leukemia virus (Mo-MuLV). The source of infectious viral DNA was the Hirt supernatant fraction from cells acutely infected with Ha-SV and Mo-MuLV. To obtain a direct quantitative assay for infectious viral DNA, recipient mouse cells were first exposed to calcium phosphate-precipitated viral DNA and then treated with dimethyl sulfoxide. Infectivity was monitored by focus formation for Ha-SV and XC plaque formation for Mo-MuLV. The viral DNA titration pattern followed single-hit kinetics for both foci and plaques, indicating that a single molecule carried information for each function. Focus-forming and plaque-forming activity were present in different molecules, since these two biological activities could be separated from each other by agarose gel electrophoresis. The focus-forming molecule was linear DNA with a molecular weight of about 4 x 10(6) daltons. The focus-forming activity of the viral DNA was sensitive to EcoRI and resistant to XhoI restriction endonucleases, whereas the plaque-forming activity was resistant to EcoRI and sensitive to XhoI. The generation of helper-independent foci indicates that Ha-SV DNA can transform mouse cells in the absence of helper virus or its proteins.     

The kinetoplast DNA of Trypanosoma equiperdum

We have analyzed the kinetoplast DNA for Trypanosoma equiperdum (American Type Culture Collection 30019) and two dyskinetoplastic strains derived from it. The DNA networks from the kinetoplastic strain are made up of catenated mini-circles and maxi-circles, like the networks from the closely-related Trypanosoma brucei. The mini-circles of T. equiperdum lack the pronounced sequence heterogeneity of T. brucei mini-circles, as shown by the fragment distribution of restriction digests and by the predominance of well-matched duplexes in electron micrographs of renatured DNA. The electrophoretic analysis of kinetoplast DNA digested with various restriction endonucleases shows the maxi-circle of T. equiperdum to consist of circular DNA molecules of 8.4 x 10(6) daltons, without size or sequence heterogeneity or repetitious segments. A comparison of the sequence by restriction endonuclease fragmentation and hybridization shows extensive sequence homology. The size difference between both maxi-circles is due to the deletion of one continuous segment of 5.10(6) daltons. In the two dyskinetoplastic strains, we cannot detect DNA sequences that hybridize with kinetoplast DNA from T. brucei or from the kinetoplastic strain of T. equiperdum. In one of these strains, a 'low-density' DNA fraction contained a simple sequence DNA, cleaved by restriction endonuclease HindIII into fragments of 180 base-pairs and multimers of this. The relation of this DNA to kinetoplast DNA, if any, is unknown.     

Hepatitis B virus contains protein attached to the 5′ terminus of its complete DNA strand

Hepatitis B virus DNA contains a tightly bound protein which was not removed by heating to 60°C with 2% SDS, 2% mercaptoethanol. The protein was indirectly demonstrated by the extraction of the DNA-protein complex with phenol before but not after its digestion with proteinase K. The DNA-protein complex had a lower buoyant density than protease-treated or free DNA; it was bound to glass fiber filters; it migrated at a slower rate in gel electrophoresis; and it could be radiolabeled by oxidative iodination. The binding site of the protein was mapped by extraction of restriction endonuclease digests with phenol and analysis of the digests for missing DNA fragments. The protein was localized to a site near the 5′ end of the complete viral DNA strand. It remained attached to this strand after heating with SDS to 90°C or treatment with 0.1 N NaOH, suggesting a covalent linkage. The 5′ end of neither viral DNA strand could be phosphorylated in a reaction with polynucleotide kinase, consistent with attachment of protein to the 5′ ends. The incomplete DNA strand, however, which is the strand elongated by the virion DNA polymerase reaction, did not contain a detectable amount of polypeptide as did the complete strand. The reasons for the apparent block of the 5′ end of the incomplete DNA strand is thus not known. The protein bound covalently to HBV DNA could be involved in the replication of the complete viral DNA strand and/or endonucleolytic generation of linear unit-length DNA pieces from replicative intermediates, although its function and origin are not yet known.     

Fragmentation of Bacillus bacteriophage phi105 DNA by complementary single-stranded DNA in the cohesive ends of the molecule.

The structure of DNA from the temperate Bacillus subtilis phage phi105 was examined by using the restriction endonuclease EcoRI and by sedimentation analysis. The DNA contains six EcoRI cleavage sites. Although eight DNA fragments were identified in the EcoRI digests, the largest of these was shown to consist of the two fragments that carry the cohesive ends of the phage DNA. In neutral gradients, the majority of whole phi105 DNA sedimented as nicked circles and the remainder as oligomers. No unit-length linear structures were detected. The associated cohesive ends could be sealed by DNA ligase from Escherichia coli and could be cleaved by S1 nuclease. On the basis of these results and previously reported studies, it appears that, as isolated from phage particles, phi105 DNA is a circular molecule that is formed from the linear structure by the association of complementary single-stranded DNA.     

The structure of the termini of the DNA of Epstein-Barr virus.

We have studied the DNA of Epstein-Barr virus (EBV) isolated from the B95-8 strain of that virus (Miller and Lipman, 1973). When EBV DNA is partially digested with lambda-exonuclease and allowed to reanneal, up to 50% of the full-length molecules circularize. The arrangements of nucleotide sequences containing the terminal repeats identified in this circularization experiment have been determined. Those fragments of viral DNA generated by digestion with restriction endonucleases which are terminal and contain the terminal repeats have been identified by their sensitivity to digestion of full-length DNA by lambda-exonuclease and by virtue of their being partially homologous to one another. The population of DNA molecules in the B95-8 strain of EBV was found to be nonuniform. The nonuniformity results from different molecules having different numbers of a 0.37 megadalton terminal repeat at each end. About 70% of molecules have four terminal repeats at one end, while four equal classes, each comprising approximately 25% of the population, have one, two, three or four repeats at the other end. The arrangements of nucleotide sequences identified as being terminal in virion DNA were studied in the intracellular circular viral DNA of cells transformed by a single particle on EBV. All fragments produced by digestion with endonucleases and scored as being terminal in virion DNA were absent from intracellular circular DNA. An additional fragment was identified in the digests of intracellular DNA of each transformed clone. The molecular weights of the new fragments equal the sum of the molecular weights of two terminal fragments which are joined upon intracellular circularization of viral DNA.     

Does simian virus 40 DNA integrate into cellular DNA during productive infection?

Late after infection of permissive monkey cells by simian virus 40 (SV40), large amounts of SV40 DNA (30,000 to 220,000 viral genome equivalents per cell) can be isolated with the high-molecular-weight fraction of cellular DNA. Hirai and Defendi (J. Virol.9:705-707, 1972) and H?lzel and Sokol (J. Mol. Biol. 84:423-444, 1974) suggested that this SV40 DNA is covalently integrated into the cellular DNA. However, our data indicate that the high-molecular-weight viral DNA is composed of tandem, "head-to-tail" repeats of SV40 DNA and that very little, if any, of this viral DNA is covalently joined to the cellular DNA. This was deduced from the following experimental findings. The size of the SV40 DNA associated with the high-molecular-weight cellular DNA fraction is greater than 45 kilobases, based on its electrophoretic mobility in agarose gels. In this form the SV40 DNA did not produce heteroduplex structures with a marker viral DNA (an SV40 genome with a characteristic deletion and duplication). After the high-molecular-weight DNA was digested with EcoRI or HpaII endonucleases, enzymes which cleave SV40 DNA once, more than 95% of the SV40 DNA migrated as unit-length linear molecules and, after hybridization with the marker viral DNA, the expected heteroduplex structures were easily detected. Digestion of the high-molecular-weight DNA fraction with restriction endonucleases that cleave cellular, but not SV40. DNA did not alter the electrophoretic mobility of the polymeric SV40 DNA, nor did it give rise to molecules that form heteroduplex structures with the marker viral DNA. Polymeric SV40 DNA molecules produced after coinfection by two physically distinguishable SV40 genomes contain only a single type of genome, suggesting that they arise by replication rather than by recombination. The polymeric form of SV40 DNA is highly infectious for CV-1P monolayers (6.5 X 10(4) PFU per microgram of SV40 DNA), yielding virtually exclusively normal, covalently closed circular, monomer-length DNA. Quite clearly these cells have an efficient mechanism for generating monomeric viral DNA from the SV40 DNA polymers.     

Herpes simplex virus amplicon: cleavage of concatemeric DNA is linked to packaging and involves amplification of the terminally reiterated a sequence.

Herpes simplex virus-infected cells contain large concatemeric DNA molecules arising from replication of the viral genome. The large concatemers are cleaved to generate unit-length molecules terminating at both ends with the a sequence. We have used constructed defective virus vectors (amplicons) derived from herpes simplex virus to study the mechanism of cleavage of viral DNA concatemers and the packaging of viral DNA into nucleocapsids. These studies revealed that (i) a 248-base-pair a sequence contained the signal(s) required for cleavage-packaging, (ii) the cleavage of viral DNA concatemers was coupled to packaging, (iii) the a sequence contained the information required for its own amplification, and (iv) cleavage-packaging occurred by a novel process involving the amplification of the a sequence.     

The rat liver mitochondrial DNA-protein complex: displaced single strands of replicative intermediates are protein coated

Mitochondrial DNA (mtDNA)-protein complexes were released from the organelles by sodium dodecyl sulfate-lysis and purified by Phenyl-Sepharose CL-4B chromatography. The mitochondrial DNA-binding protein P16 was the only detectable protein in the complex. Treatment of the complex with proteinase K, or subtilisin, revealed the presence of a protease-insensitive, submolecular domain (Mr approximately equal to 6,000) that retained the capacity to bind tenaciously to the DNA. Analysis of chemically fixed complexes by CsCl isopycnic gradient centrifugation showed that P16 was bound to a large subpopulation of mtDNA enriched in displacement loops (D-loops). Based upon the effective buoyant density of the complex in CsCl gradients and the molecular weights of P16 and mtDNA, it was estimated that a mean of 49 P16 molecules were bound per mtDNA. For this measurement, the variation in hydration of protein and DNA at different CsCl concentrations was ignored. Analysis of restriction endonuclease-digested complexes by glass fiber filters that bind only protein-associated DNA resulted in the retention of a single fragment regardless of the enzyme, or enzymes, used. In each case, the retained fragment was the D-loop-containing fragment. With direct electron microscopy, the protein was readily visualized on the displaced single strand portions of D-loops and expanding D-loops. The nucleoprotein fibers were approximately 12 nm in diameter without correcting for the thickness of tungsten coating and roughly 1/3 the length of the double strand segment of the corresponding D-loop structure. In addition, occasional molecules with the characteristics of gapped circles were seen exhibiting a nucleoprotein fibril, presumably containing the single strand gap segment, linking the ends of double strand DNA. P16 was not seen on the double strand portions in any of the complexes.     

Isolation and Partial Characterization of Different Defective DNA Molecules Derived from Polyoma Virus

Supercoiled DNA molecules purified from mouse cells infected with high-multiplicity-passaged polyoma virus has a broader size distribution and sediments more slowly than DNA derived from low-multiplicity-passaged virus. The shorter DNA molecules are predominately noninfectious. Virus populations containing distinct size classes of defective virus DNA were isolated by growing virus from single cells infected by a defective and nondefective helper virus (infectious center). This technique probably results in the cloning of defective virus particles. By applying the infectious center method to DNA from various fractions of sucrose gradients it has been possible to obtain shorter circular DNA molecules ranging in size from 50 to 95% of the unit-length polyoma DNA molecule. The shorter molecules in any one preparation are homogeneous in size. This class size is retained upon repeated passage of crude viral lysates at high multiplicity. Thus far, all the purified shorter DNA molecules tested appear to be noninfectious and largely resistant to cleavage by the R(1) restriction enzyme. Some of the purified defective molecules have been found to interfere with the production of infectious virus upon co-infection with unit-length infectious polyoma DNA.     

Fusion of the termini of the murine cytomegalovirus genome after infection.

The genome of murine cytomegalovirus, extracted from extracellular virions, is a linear double-stranded DNA molecule ca. 240 kilobase pairs long. In our initial cloning of subgenomic fragments of the murine cytomegalovirus genome, we obtained a HindIII clone which contained fused HindIII-terminal fragments. By hybridizing this cloned DNA fragment to infected-cell DNA, we identified an intracellular restriction fragment which was the length of the sum of the two authentic termini. This fusion fragment was not present in virion DNA but could be detected as early as 2 h postinfection and reached its highest level shortly after the onset of DNA replication at 16 h postinfection. The prereplicative increase of fused ends was not inhibited by a level of phosphonoacetic acid which effectively shut off viral DNA synthesis, nor was the early conversion from free to fused ends prevented by inhibitors of protein or RNA synthesis. The results are consistent with the fused state of viral DNA being a replicative intermediate and precursor to DNA synthesis.     

Automatic construction of restriction site maps.

A computer program is described which constructs maps of restriction endonuclease cleavage sites in DNA molecules, given only the fragment lengths. The program utilizes fragment length data from single and double restriction enzyme digests to generate maps for linear or circular molecules. The search for a map can be limited to the unknown (insert) region of a recombinant phage or plasmid. Typical restriction maps with four or five enzymes which cut at three to five unknown sites can be calculated in a few minutes.     

Construction of a cloned library of the EcoRI fragments from the human cytomegalovirus genome (strain AD169).

The DNA genome of human cytomegalovirus (HCMV) strain AD169 is 158 x 10(6) Mr. Cleavage of the HCMV DNA with the restriction endonuclease EcoRI yields 35 major fragments ranging in size from 0.54 x 10(6) Mr. We have constructed a cloned library of the EcoRI fragments of this strain of HCMV, using the plasmid pACYC184 and the recipient bacterium Escherichia coli strain HB101 RecA-. The viral origin of the cloned inserts was determined by hybridization to viral DNA. The fragments were characterized further by digestion with other restriction enzymes. Several clones were obtained which contained sequences spanning the junction between the long (L) and short (S) components of the viral DNA sequences. These clones differed in molecular weight by multiples of 0.3 x 10(6) to 0.4 x 10(6) Mr. The variability found in the clones was also reflected in the genome. Each clone containing a junction sequence hybridized to a series of bands on Southern filters of EcoRI-digested HCMV DNA. This "ladder effect" provided evidence for a region of heterogeneity within the L-S junction.     

Genetic analysis of type 5 capsular polysaccharide expression by Staphylococcus aureus.

Capsules are produced by over 90% of Staphylococcus aureus strains, and approximately 25% of clinical isolates express type 5 capsular polysaccharide (CP5). We mutagenized the type 5 strain Reynolds with Tn918 to target genes involved in CP5 expression. From a capsule-deficient mutant, we cloned into a cosmid vector an approximately 26-kb EcoRI fragment containing the transposon insertion. In the absence of tetracycline selection, Tn918 was spontaneously excised, thereby resulting in a plasmid containing 9.4 kb of S. aureus DNA flanking the Tn918 insertion site. The 9.4-kb DNA fragment was used to screen a cosmid library prepared from the wild-type strain. Positive colonies were identified by colony hybridization, and a restriction map of one clone (pJCL19 with an approximately 34-kb insert) carrying the putative capsule gene region was constructed. Fragments of pJCL19 were used to probe genomic DNA digests from S. aureus strains of different capsular serotypes. Fragments on the ends of the cloned DNA hybridized to fragments of similar sizes in most of the strains examined. Blots hybridized to two fragments flanking the central region of the cloned DNA showed restriction fragment length polymorphism. A centrally located DNA fragment hybridized only to DNA from capsular types 2, 4, and 5. DNA from pJCL19 was subcloned to a shuttle vector for complementation studies. A 6.2-kb EcoRI-ClaI fragment complemented CP5 expression in a capsule-negative mutant derived by mutagenesis with ethyl methanesulfonate. These experiments provide the necessary groundwork for identifying genes involved in CP5 expression by S. aureus.     

Structure of the baboon endogenous virus genome: cloning of circular virus DNA in bacteriophage lambda.

Linear, small and large circular forms of unintegrated viral DNAs were detected in Hirt supernatant fraction of human cultured cells infected with baboon endogenous virus M7. The circular M7 DNAs were cloned in bacteriophage lambda, Charon 28. Seventeen independent clones were isolated and analyzed by restriction endonuclease mapping. Nine clones were carrying a viral sequence of 8.6 kilobase pairs (kb) with two tandem repeats of 0.6 kb, which correspond to the large circular form of the unintegrated M7 DNA. Eight other clones had the viral insert of 8.0 kb, i. e., the small circular form, and were deleted one of the repeated sequences. The repeated sequences correspond to the long terminal repeats of 0.6 kb, located at both ends of the linear M7 DNA of 8.6 kb. One of the recombinants of the large circular M7 DNA had an inversion of 2.5 kb. One end of the inverted sequence was near the terminus of the long terminal repeats and the other in the gag gene region. The inversion seems to be occurred by integration of a viral DNA within itself during early periods of infection. The mechanism of the processes leading to integration is discussed from the structure of these unintegrated M7 DNAs as the precursors.     

Resolution of linear minichromosomes with hairpin ends from circular plasmids containing vaccinia virus concatemer junctions

The junctions, separating unit-length genomes in intracellular concatemeric forms of vaccinia virus DNA, are duplex copies of the hairpin loops that form the ends of mature DNA molecules present in infectious virus particles. Circular E. coli plasmids with palindromic junction fragments were replicated in vaccinia virus-infected cells and resolved into linear minichromosomes with vector DNA in the center and vaccinia virus DNA hairpins at the two ends. Resolution did not occur when the concatemer joint was less than 250 bp or when plasmids were transfected into uninfected cells, indicating requirements for a specific DNA structure and viral trans-acting factors. These studies indicate that concatemers can serve as replicative intermediates and account for the generation of flip-flop sequence variation of the hairpins at the ends of the mature vaccinia virus genome.