Restriction endonuclease mapping of linear unintegrated proviral DNA of bovine leukemia virus.

A detailed restriction map was deduced for the genome of the exogenous bovine leukemia virus. The cleavage sites for nine restriction enzymes were mapped. The unintegrated linear viral DNA intermediate that is produced by infection of permissive cells with bovine leukemia virus was isolated. The linear viral DNA had a unique restriction map, indicating that it is not a set of random circular permutations of the RNA genome. From hybridization with a 3'-enriched probe, the DNA restriction map was aligned relative to the 5'-to-3' orientation of the viral RNA. Restriction enzyme analysis of integrated bovine leukemia virus information present in animals with enzootic bovine leukosis provided evidence for the existence of genetic variants of the virus.     

Molecular cloning of the unintegrated squirrel monkey retrovirus genome: organization and distribution of related sequences in primate DNAs

The closed circular form of the endogenous squirrel monkey type D retrovirus (SMRV) was molecularly cloned in a bacteriophage vector. The restriction map of the biologically active clone was determined and found to be identical to that of the parental SMRV linear DNA except for the deletion of one long terminal repeat. Restriction enzyme analysis and Southern blotting indicated that the SMRV long terminal repeat was approximately 300 base pairs long. The SMRV restriction map was oriented to the viral RNA by using a gene-specific probe from baboon endogenous virus. Restriction enzyme digests of a variety of vertebrate DNAs were analyzed for DNA sequence homology with SMRV by using the cloned SMRV genome as a probe. Consistent with earlier studies, multiple copies of SMRV were detected in squirrel monkey DNA. Related fragments were also detected in the DNAs from other primate species, including humans.     

Organization of type C viral DNA sequences endogenous to baboons: analysis with cloned viral DNA.

Unintegrated linear and circular forms of baboon endogenous type C virus M7 DNA were prepared from M7-infected cells by chromatography on hydroxyapatite columns, and the circular DNAs were purified in cesium chloride-ethidium bromide equilibrium density gradients. The circular DNAs were linearized by digestion with EcoRI, which had a unique site on the viral DNA. The linearized DNA was then inserted into lambda gtWES. lambda B at the EcoRI site and cloned in an approved EK2 host. Molecularly cloned full-length M7 DNA was restricted with BamHI, and the resulting five subgenomic fragments were then subcloned individually in plasmid pBR322. The organization and sites of integration of the approximately 100 copies of M7 DNA sequences endogenous to baboons were investigated by digesting the DNA with restriction enzymes and identifying the virus-specific fragments by hybridization to labeled probes made by using the molecularly cloned full-length and subgenomic fragments of the viral DNA. We found that most of the endogenous sequences had sizes and organizations similar to those of the unintegrated viral DNA and therefore approximately similar to the RNA of the infectious virus. A few of the multiple sequences had deletions in the 3' end (envelope region), and some of the sequences either lacked or contained modified BamHI restriction sites on the 5' end of the viral DNA. The endogenous viral DNA sequences were nontandem, uninterrupted, and colinear with the DNA of the infectious virus, and they were integrated at different sites in the baboon DNA, like the M7 proviral DNA sequences acquired upon infection.     

Restriction endonuclease mapping of unintegrated viral DNA of B- and N-tropic BALB/c murine leukemia virus.

Unintegrated linear and closed circular DNAs of B- and N-tropic endogenous BALB/c murine leukemia virus (MuLV) were extracted from newly infected mouse cells and cleaved with EcoRI, XhoI, PvuI, HindIII, SalI, XbaI, KpnI, SmaI, and PstI restriction endonucleases. The DNA fragments were separated by electrophoresis and analyzed by the Southern blot hybridization procedure. EcoRI did not cleave the two genomes. A physical map of 15 cleavage sites on B- and N-tropic genomes was constructed with the other restriction endonucleases. Identical cleavage sites of B- and N-tropic MuLV DNAs were found with all these enzymes. However, the N-tropic linear genome was found to lack about 75 base pairs at each end of the molecule. PstI, KpnI, and SmaI recognize a cleavage site at both ends of the linear molecules. And sequences derived from the 5' end of the RNA genome were found in the third left end of the linear DNA and at its extreme right-end terminus, suggesting the presence of redundant sequences. Two species of closed circular viral DNA were observed. The larger species has the same size as the linear molecule and appears to be a circularized form of linear DNA. The smaller species contains sequences common to both the linear and the larger circular viral DNA but seems to be deleted from sequences present at either one or both ends of the linear DNA. Therefore, the general structure of the linear and circular DNA species of these B- and N-tropic endogenous BALB/c MuLV appears analogous to the structure found with other retroviruses.     

Structural organization and biological activity of molecular clones of the integrated genome of a BALB/c mouse sarcoma virus.

BALB/c mouse sarcoma virus (BALB-MSV) is a spontaneously occurring transforming retrovirus of mouse origin. The integrated form of the viral genome was cloned from the DNA of a BALB-MSV-transformed nonproducer NRK cell line in the Charon 9 strain of bacteriophage lambda. In transfection assays, the 19-kilobase-pair (kbp) recombinant DNA clone transformed NIH/3T3 mouse cells with an efficiency of 3 X 10(4) focus-forming units per pmol. Such transformants possessed typical BALB-MSV morphology and released BALB-MSV after helper virus superinfection. A 6.8-kbp DNA segment within the 19-kbp DNA possessed restriction enzyme sites identical to those of the linear BALB-MSV genome. Long terminal repeats of approximately 0.6 kbp were localized at either end of the viral genome by the presence of a repeated constellation of restriction sites and by hybridization of segments containing these sites with nick-translated Moloney murine leukemia virus long terminal repeat DNA. A continuous segment of at least 0.6 and no more than 0.9 kbp of helper virus-unrelated sequences was localized toward the 3' end of the viral genome in relation to viral RNA. A probe composed of these sequences detected six EcoRI-generated DNA bands in normal mouse cell DNA as well as a smaller number of bands in rat and human DNAs. These studies demonstrate that BALB-MSV, like previously characterized avian and mammalian transforming retroviruses, arose by recombination of a type C helper virus with a well-conserved cellular gene.     

Identification of the avian myeloblastosis virus genome. II. Restriction endonuclease analysis of DNA from lambda proviral recombinants and leukemic myeoblast clones.

Two lambda proviral DNA recombinants were characterized with a number of restriction endonucleases. One recombinant contained a complete presumptive avian myeloblastosis virus (AMV) provirus flanked by cellular sequences on either side, and the second recombinant contained 85% of a myeloblastosis-associated virus type 1 (MAV-1)-like provirus with cellular sequences adjacent to the 5' end of the provirus. Comparing the restriction maps for the proviral DNAs contained in each lambda hybrid showed that the putative AMV and MAV-1-like genomes shared identical enzyme sites for 3.6 megadaltons beginning at the 5' termini of the proviruses with respect to viral RNA. Two enzyme sites near the 3'-end of the MAV-1-like provirus were not present in the putative AMV genome. We also examined a number of leukemic myeloblast clones for proviral content and cell-provirus integration sites. The presumptive AMV provirus was present in all the leukemic myeloblast clones regardless of the endogenous proviral content of the target cells or the AMV pseudotype used for conversion. Multiple cellular sites were suitable for integration of the putative AMV genome and the helper genomes. The proviral genomes were all integrated colinearly with respect to linear viral DNA.     

Restriction map of the single-stranded DNA genome of Kilham rat virus strains 171, a nondefective parvovirus.

We constructed a physical map of Kilham rat virus strains 171 DNA by analyzing the sizes and locations of restriction endonuclease-generated fragments of the replicative-form viral DNA synthesized in vitro. BglI, KpnI, BamHI, SmaI, XhoI, and XorII did not appear to have any cleavage sites, whereas 11 other enzymes cleaved the genome at one to eight sites, and AluI generated more than 12 distinct fragments. The 30 restriction sites that were mapped were distributed randomly in the viral genome. A comparison of the restriction fragments of in vivo- and in vitro-replicated replicative-form DNAs showed that these DNAs were identical except in the size or configuration of the terminal fragments.     

Physical map of infectious baboon type C viral DNA and sites of integration in infected cells.

Three species of unintegrated viral DNAs were found in permissive cells infected with baboon type C virus. The major species was a 9.0-kilobase (kb) linear DNA that was infectious. A restriction endonuclease map of this DNA was constructed and oriented with respect to the viral RNA. The linear DNA had a 0.6-kb sequence repeated at each terminus. These terminal repeat sequences were required for infectivity of the viral DNA. The minor species of the unintegrated viral DNAs were covalently closed circles of 9.0 and 8.4 kb. The smaller circle was in two- to threefold excess over the larger circle. The difference appeared to be that the smaller circle lacked one of the two 0.6-kb repeat sequences found in the larger circle. Restriction endonuclease maps of the integrated viral DNAs were constructed, and the sequences on both viral DNA and cellular DNA that are involved in integration were determined. The integrated viral DNA map was identical to that of the unintegrated infectious 9.0-kb linear DNA. Therefore, a specific site in the terminal repeat sequence of the viral DNA was used to integrate with the host cell DNA. The sizes of the cellular DNA fragments were different from clone to clone but stable with cell passage. Therefore, many sites in the cell DNA can recombine with the viral DNA.     

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.     

Two strains of baboon endogenous virus demonstrate a high degree of genetic conservation

Molecular clones of the baboon endogenous viruses 455K, from Papio anubis, and M7, from Papio cynocephalus, have been constructed from unintegrated viral DNA. Although two specific restriction sites do distinguish the two, detailed restriction maps are nearly identical and heteroduplex molecules show complete homology. The latter observations indicate that this endogenous retrovirus genome has been strongly conserved among different species of baboon.     

Detailed physical map and set of overlapping clones covering the genome of the archaebacterium Haloferax volcanii DS2.

An integrated approach of "bottom up" and "top down" mapping has produced a minimal set of overlapping cosmid clones covering 96% of the 4140 kilobase-pairs (kbp) Haloferax volcanii DS2 genome and a completely closed physical map. This genome is partitioned into five replicons: a 2920 kbp chromosome and four plasmids, of 690 kbp (pHV4), 442 kbp (pHV3), 86 kbp(pHV1) and 6.4 kbp (pHV2). A restriction map for six infrequently-cutting restriction enzymes was constructed, representing a total of 903 sites in the cloned DNA. We have placed the two ribosomal RNA operons, the genes for 7 S RNA and for RNaseP RNA and 22 protein-coding genes on the map. Restriction site frequencies show significant variation in different portions of the genome. The regions of high site density correspond to halobacterial satellite or FII DNA which includes two small regions of the chromosome, the plasmids pHV1 and pHV2, and half of pHV4, but not pHV3.     

Tumorigenic poxviruses: construction of the composite physical map of the Shope fibroma virus genome.

The sites for the restriction enzymes BamHI, Bg/I, HindIII, PstI, PvuII, and SstI on the linear DNA genome of Shope fibroma virus, a tumorigenic poxvirus of rabbits, have been determined by digestions of the cloned BamHI and HindIII restriction fragments and by hybridization of 32P-labeled cloned fragments to Southern blots of Shope fibroma virus DNA cleaved partially or completely with the various enzymes. The linear genome is shown to be 160 kilobases in length and to possess terminal inverted repeat sequences of between 12.2 and 12.5 kilobases extending inwards from the cross-linked DNA telomeres. The fine map of the Shope fibroma virus terminal inverted repeats has been constructed and shown to be distinctly different from that of members of the orthopoxvirus group, such as vaccinia, by the absence of detectable tandemly repeated sequences near the termini and by the lack of detectable sequence homology with vaccinia termini.     

Colobus type C virus: molecular cloning of unintegrated viral DNA and characterization of the endogenous viral genomes of Colobus.

The unintegrated viral DNA intermediates of colobus type C virus (CPC-1) were isolated from infected human cells that were permissive for viral growth. There were two major species of DNA, linear molecules with two copies of the long terminal repeat and relaxed circles containing only a single long terminal repeat. In addition, there was a minor species (approximately 10%) composed of relaxed circles with two copies of the long terminal repeat. A restriction endonuclease map of the unintegrated DNA was constructed. The three EcoRI fragments of circular CPC-1 DNA were cloned in the EcoRI site of lambda gtWES . lambda B and then subcloned in the EcoRI site of pBR322. Using these subgenomic fragments as probes, we have characterized the endogenous viral sequences found in colobus cellular DNA. They are not organized in tandem arrays, as is the case in some other gene families. The majority of sequences detected in cellular DNA have the same map as the CPC-1 unintegrated DNA at 17 of 18 restriction endonuclease sites. There are, however, other sequences that are present in multiple copies and do not correspond to the CPC-1 map. They do not contain CPC-1 sequences either in an altered form or fused to common nonviral sequences. Instead, they appear to be derived from a distinct family of sequences that is substantially diverged from the CPC-1 family. This second family of sequences, CPC-2, is also different from the sequences related to baboon endogenous type C virus that forms a third family of virus-related sequences in the colobus genome.     

Characterization of an infective molecular clone of the B-tropic, ecotropic BL/Ka(B) murine retrovirus genome.

Using molecular cloning techniques, we amplified the unintegrated, linear proviral DNA of the BL/Ka(B) virus, a non-leukemogenic retrovirus of mouse strain C57BL/Ka. Two independent clones in lambda phage vector 607 and one subclone in pBR322 were infective when transfected into mouse fibroblasts. Analysis of the progeny virus revealed biological properties and a restriction map identical to those of the parental viral shock. Comparison of the restriction map with the maps of other ecotropic murine viruses reveals many similarities. Particularly interesting is the comparison of the N-tropic Akv virus and the B-tropic BL/Ka(B) virus. The long terminal repeats of the two viruses are virtually identical, as are 22 of 23 restriction sites located outside of the region which spans from 1.8 to 3.8 kilobases from the left end of the genome. Within this region, however, only three of nine sites examined are shared. This suggests that the BL/Ka(B) virus was derived from an endogenous N-tropic virus closely related to Akv by recombinational events which altered the sequence in the last half of the gag gene and the first third of the pol gene. This change is probably responsible for the observed difference in the Fv-1 tropism of the two viruses.     

Evidence for a chromosomal location of polydnavirus DNA in the ichneumonid parasitoid Hyposoter fugitivus.

Evidence is presented in support of a chromosomal location for sequences homologous to polydnavirus DNA in the ichneumonid parasitoid Hyposoter fugitivus. In this study, four different viral genome segments were cloned and used as probes against genomic DNA extracted from male parasitoids and digested with a variety of restriction enzymes. Each probe typically identified a single off-size fragment (OSF) in the case of enzymes not cutting viral genome segments, while two OSFs were generated by enzymes cutting at one and two sites. While extra OSFs were occasionally observed, these were invariably found to be due to the presence of polymorphic restriction sites in flanking chromosomal DNA. Analysis of these data suggests that a single, stable chromosomal locus exists for sequences homologous to each viral genome segment; the data also indicate that viral and cognate parasitoid genomic DNAs are largely if not entirely colinear.     

Integration site of polyoma virus DNA in the inducible LPT line of polyoma-transformed rat cells

The structure of the polyoma virus (Py) integration site in the inducible LPT line of Py-transformed rat cells was determined by biochemical methods of gene mapping. LPT cell DNA was digested with various restriction enzymes. The digestion products were electrophoresed in agarose gels and transferred onto nitrocellulose sheets by Southern blotting. Fragments containing viral or cell DNA sequences, or both, were identified by hybridization with Py DNA or with a cloned flanking cell DNA probe. Cleavage of LPT DNA with enzymes that restrict the Py genome once generated linear Py DNA molecules and two fragments containing both cell and viral DNA sequences. Cleavage of LPT DNA with enzymes which do not restrict Py DNA generated series of fragments whose lengths were found to differ by increments of a whole Py genome; the smallest fragment in each series was found to be longer than the viral genome. These data indicate that LPT cultures contain Py insertions of various lengths integrated into the same chromosomal site in all the cells. The length heterogeneity of the viral insertions is due to the presence of 0, 1, 2, 3. . . Py genomes arranged in a direct tandem repeat within invariable sequences of viral DNA. Double-digestion experiments were also carried out with the above enzymes and with enzymes that cleave the Py genome at multiple sites. The data obtained in these experiments were used to construct a physical map of the integration site. This map showed that the early region of the virus remained intact even in the smallest insertion (which contains no whole duplicated genomes), whereas the late region was partially duplicated and split during integration. The smallest insertion is colinear with the Py physical map over a region including the entire Py genome and at least a part of the duplicated segment. This structure could give rise to nondefective circular viral DNA molecules by single homologous recombination events. Similar recombination events may occur at a higher frequency in the longer insertions, which include longer regions of homology, and may yield many more free viral genomes. The presence of these insertions in LPT cells could thus be one of the factors which account for the high inducibility of the LPT line.     

The translational map of the Autographa californica nuclear polyhedrosis virus (AcNPV) genome.

We have mapped early and late viral gene products expressed in Autographa californica nuclear polyhedrosis virus ( AcNPV )-infected Spodoptera frugiperda cells by cell-free translation of virus-specific RNA which was selected by hybridization to cloned restriction endonuclease fragments of AcNPV DNA. Proteins synthesized in vitro were labeled with [35S]methionine and analyzed by SDS-polyacrylamide gel electrophoresis followed by fluorography. At least four early AcNPV -specific polypeptides were found which mapped in two regions of the genome (9-25 and 43-59 map units). These early mRNAs are also synthesized at late times in the infection cycle. Cell-free translation of restriction fragment-selected late AcNPV -specific RNA (24 h post-infection) resulted in the identification and mapping of 24 viral proteins. Curiously, the region between approximately 70 and 80 map units on the viral genome has been found silent with respect to mRNA which is translatable in a cell-free system. However, there may be RNA transcribed from this viral DNA segment.     

Sites in simian virus 40 chromatin which are preferentially cleaved by endonucleases.

Simian virus 40 (SV40) chromatin isolated from infected BSC-1 cell nuclei was incubated with deoxyribonuclease I, staphylococcal nuclease or an endonuclease endogenous to BSC-1 cells under conditions selected to introduce one doublestrand break into the viral DNA. Full-length linear DNA was isolated, and the distribution of sites of initial cleavage by each endonuclease was determined by restriction enzyme mapping. Initial cleavage of SV40 chromatin by deoxyribonuclease I or by endogenous nuclease reduced the recovery of Hind III fragment C by comparison with the other Hind III fragments. Similarly, Hpa I fragment B recovery was reduced by comparison with the other Hpa I fragments. When isolated SV40 DNA rather than SV40 chromatin was the substrate for an initial cut by deoxyribonuclease I or endogenous nuclease, the recovery of all Hind III or Hpa I fragments was approximately that expected for random cleavage. Initial cleavage by staphylococcal nuclease of either SV40 DNA or SV40 chromatin occurred randomly as judged by recovery of Hind III or Hpa I fragments. These results suggest that, in at least a portion of the SV40 chromatin population, a region located in Hind III fragment C and Hpa I fragment B is preferentially cleaved by deoxyribonuclease I or by endogenous nuclease but not by staphylococcal nuclease.Complementary information about this nuclease-sensitive region was provided by the appearance of clusters of new DNA fragments after restriction enzyme digestion of DNA from viral chromatin initially cleaved by endogenous nuclease. From the sizes of new fragments produced by different restriction enzymes, preferential endonucleolytic cleavage of SV40 chromatin has been located between map positions 0.67 and 0.73 on the viral genome.