Inverted duplication-transposition event in mammalian cells at an illegitimate recombination join.

Illegitimate recombination events in mammalian cells often contain extraneous nucleotides or filler DNA at the recombinant joins. The polyomavirus-transformed cell line 7axB has previously been found to contain 37 base pairs (bp) of filler DNA at one virus-host join of the single insert of integrated viral DNA (A. Hayday, H. E. Ruley, and M. Fried, J. Virol. 44:67-77, 1982). By using a synthetic oligomer of these 37 bp as a probe, we demonstrated that this filler DNA is an inverted duplication of a single-copy rat sequence found 650 bp upstream from this virus-host join. The other virus-host join appears to be the result of a simple illegitimate recombination event between viral and host sequences. This is the first identification of filler DNA as a transposed copy of a chromosomal sequence. The relevance of the recombination events studied to cellular rearrangements and viral integration is discussed.     

Sequence and spacing requirements of a retrovirus integration site

Following infection, retroviruses insert a DNA copy of their RNA genome into the host cell genome. This integrative recombination reaction occurs at specific sites on the viral DNA: inverted repeat sequences near the termini of the linear DNA form of the viral genome. We have described elsewhere the generation and analysis of deletion mutations at one of the inverted repeat sequences in Moloney murine leukemia virus. We describe here the effects of insertion mutations made at this locus. Our results show that substantial sequence changes at the site of recombination can be tolerated, and that the spacing between the cleavage sites on the viral DNA can be expanded as well as contracted while still allowing efficient viral integration. After several rounds of virus replication, each of the insertion mutants gave rise to pseudorevertants with new alterations at the integration site.     

Frequent site-specific deletion of coliphage lambda murine sarcoma virus recombinants and its use in the identification of a retrovirus integration site.

Stocks of hybrid lambda phages carrying the complete integrated provirus of either m1 or HT1 Moloney murine sarcoma virus, as well as flanking host sequences, frequently contain significant numbers of phages carrying a specific deletion. This deletion arises from a recombination event between the terminally repeated sequences in the provirus that deletes the unique Moloney murine sarcoma virus sequences bracketed by the terminally repeated sequences. Physical mapping has shown that the deletion phage retains one complete copy of the terminally repeated sequence and the flanking mink host sequences. One such deletion, lambdaHT1r+, was used to characterize a mink genomic DNA sequence that contains an HT1 Moloney murine sarcoma virus integration site. This integration site sequence from normal mink cells was also cloned into phage lambda. An analysis of the heteroduplexes between the integration site and the lambdaHT1r+ deletion indicated that no major rearrangement of host sequences occurred upon integration of the Moloney murine sarcoma provirus.     

Human immunodeficiency virus type 1 DNA integration: fine structure target analysis using synthetic oligonucleotides.

The target specificity of DNA strand transfer mediated by human immunodeficiency virus type 1 integrase was examined in vitro with synthetic oligonucleotides. Although insertion occurred at most locations in the target, some sites were preferred over others by at least 15-fold. Changing the nucleotide sequence of the target changed the distribution of preferred sites in complex ways, some of which included changes in target preference distant from the sequence alteration. Alignment of target sequences revealed that adenosine is preferred adjacent to the insertion site. Strand transfer occurred to within 2 nucleotides of the 3' end and to within 3 nucleotides of the 5' end of the target. This suggests that only 2 or 3 nucleotides flanking the target site are required for integration; such restricted contact with target DNA would allow integrase to insert the two ends of viral DNA into two closely spaced sites in host DNA, consistent with the concerted in vivo integration reaction that generates a 5-bp target duplication.     

Arrangement of Integrated Avian Sarcoma Virus DNA Sequences Within the Cellular Genomes of Transformed and Revertant Mammalian Cells

We have examined the arrangement of integrated avian sarcoma virus (ASV) DNA sequences in several different avian sarcoma virus transformed mammalian cell lines, in independently isolated clones of avian sarcoma virus transformed rat liver cells, and in morphologically normal revertants of avian sarcoma virus transformed rat embryo cells. By using restriction endonuclease digestion, agarose gel electrophoresis, Southern blotting, and hybridization with labeled avian sarcoma virus complementary DNA probes, we have compared the restriction enzyme cleavage maps of integrated viral DNA and adjacent cellular DNA sequences in four different mouse and rat cell lines transformed with either Bratislava 77 or Schmidt-Ruppin strains of avian sarcoma virus. The results of these experiments indicated that the integrated viral DNA resided at a different site within the host cell genome in each transformed cell line. A similar analysis of several independently derived clones of Schmidt-Ruppin transformed rat liver cells also revealed that each clone contained a unique cellular site for the integration of proviral DNA. Examination of several morphologically normal revertants and spontaneous retransformants of Schmidt-Ruppin transformed rat embryo cells revealed that the internal arrangement and cellular integration site of viral DNA sequences was identical with that of the transformed parent cell line. The loss of the transformed phenotype in these revertant cell lines, therefore, does not appear to be the result of rearrangement or deletions either within the viral genome or in adjacent cellular DNA sequences. The data presented support a model for ASV proviral DNA integration in which recombination can occur at multiple sites within the mammalian cell genome. The integration and maintenance of at least one complete copy of the viral genome appear to be required for continuous expression of the transformed phenotype in mammalian cells.     

Rescue of silent integrated polyoma genomes suggests homologous recombination between resident and transfected DNA fragments

Two defective polyoma virus genomes, deleted in the nucleotide sequences coding the N-termini of the tumor antigens, were introduced into Fisher 3T3 rat cells by DNA-mediated gene transfer (transfection). The resulting integrated genomes were incapable of conferring a transformed phenotype to the cells. However, after transfection of these lines with small polyoma fragments overlapping the deleted sequences, transformed clones were isolated. These clones were analyzed by Southern genomic blot hybridization and by isolation in E. coli of plasmids containing viral sequences excised following fusion with mouse polyoma growth-permissive cells. In all cases at least one intact copy of the early region of the polyoma genome was found. Furthermore, restriction sites adjacent to the initial inactive insertion remained unchanged in many of the transformed lines. These results show that functional restoration of the defective polyoma early region involves homologous recombination between the deleted viral genomes integrated in the cellular DNA and the transfecting viral fragments.     

Mutant din-21, a variant of polyoma virus containing a mouse DNA sequence in the viral genome.

An unusual non-defective mutant of polyoma virus with an anomalously large genome, designated din-21, has been isolated. The viral chromosome lacks 49 base pairs of the putative control region between the origin of replication and the initiation codon for the early proteins, the T-antigens. In their stead , 95 base pairs, with limited homology to the deleted sequence and apparently of mouse origin, have been inserted. The primary sequence of the insert DNA has been determined and some of the biological properties of the mutant examined. It transforms rat-1 cells slightly better than wild-type virus and grows slightly less well in lytically infected mouse cells. It does not interfere with the growth of wild-type polyoma virus. The properties of this mutant suggest that it is a natural isolate of mouse cells. The mutant was presumably generated by reciprocal recombination between polyoma DNA and mouse host DNA. This could be associated with the integration of a viral DNA sequence into the host chromosome during the viral replicative cycle.     

Requirements for excision and amplification of integrated viral DNA molecules in polyoma virus-transformed cells

The integration of polyoma virus DNA into the genome of transformed rat cells generally takes place in a tandem head-to-tail arrangement. A functional viral large tumor antigen (T-Ag) renders this structure unstable, as manifested by free DNA production and excision or amplification of the integrated viral DNA. All of these phenomena involve the mobilization of precise genomic “units,” suggesting that they result from intramolecular homologous recombination events occurring in the repeated viral DNA sequences within the integrated structures. We studied polyoma ts-a-transformed rat cell lines, which produced large T-Ag but contained less than a single copy of integrated viral DNA. In all of these lines, reversion to a normal phenotype (indicative of excision) was extremely low and independent of the presence of a functional large T-Ag. The revertants were either phenotypic or had undergone variable rearrangements of the integrated sequences that seemed to involve flanking host DNA. In two of these cell lines (ts-a 4A and ts-a 3B), we could not detect any evidence of amplification even after 2 months of propagation under conditions permissive for large T-Ag. An amplification event was detected in a small subpopulation of the ts-a R5-1 line after 2 months of growth at 33°C. This involved a DNA fragment of 5.1 kilobases, consisting of the left portion of the viral insertion and about 2.5 kilobases of adjacent host DNA sequences. None of these lines spontaneously produced free viral DNA, but after fusion with 3T3 mouse fibroblasts, R5-1 and 4A produced a low level of heterogeneous free DNA molecules, which contained both viral and flanking host DNA. In contrast, the ts-a 9 cell line, whose viral insertion consists of a partial tandem of ~1.2 viral genomes, underwent a high rate of excision or amplification when propagated at temperatures permissive for large T-Ag function. These results indicate that the high rate of excision and amplification of integrated viral genomes observed in polyoma-transformed rat cells requires the presence of regions of homology (i.e., repeats) in the integrated viral sequences. Therefore, these events occur via homologous intramolecular recombination, which is promoted directly or indirectly by the large viral T-Ag.     

Analysis of stable and unstable viral forms in SV40-infected human keratinocytes

We analyzed the state of the genomic DNA of the papovavirus SV40 in human keratinocytes as viral-infected cells gradually acquired a transformed phenotype over time. Initially, the vast majority of the viral DNA is maintained either in a full-length supercoiled form or as truncated subgenomic fragments with little evidence of integration. However, analyses of clonal populations revealed great heterogeneity and instability of the viral DNA, and we were able to isolate one clonal subpopulation in which integrated forms of the virus appeared to predominate. Similarly, uncloned populations eventually ceased production of the "free" viral DNA after several years in culture and instead came to display tandemly repeated SV40 copies at a single host integration site. Interestingly, Bg1 II digestion of host DNA generated restriction fragments containing the integrated SV40 DNA, which were of differing sizes in cultures at the 144th vs the 163rd serial passage suggesting modification or rearrangement of sequences at or near the integration site. Host sequences flanking the integrated viral DNA at the 163rd serial passage have been isolated on restriction fragments generated by Eco RI, Bam HI, and Hpa II digestion. These analyses suggest that the integrated virus is linearized near the Bg1 I site and contains a large deletion in the SV40 early region at one of the viral-host junctions.     

Polyoma virus defective DNAs. II. Physical map of a molecule with rearranged and reiterated sequences (D74).

The structure of the polyoma virus defective species D74 (74% the size of full-length polyoma virus DNA) has been determined and compared with that of polyoma virus A2 DNA. D74 appears to be composed entirely of viral DNA sequences. (No host DNA sequences have been detected.) It is made up of three DNA segments, each about 24, 24 and 27% in size. The two 24% segments appear to be identical and the 27% segment contains one copy of all the sequences found in the 24% fragments as well as a duplication of some of the sequences. When related to the physical map of A2 DNA, each segment is found to be composed of viral sequences from 1 to about 19 map units, 67 to 69 map units and 70 to 72 map units.Three features found in other polyoma virus defective species (Lund et al., 1977) are also present in D74. (1) Sequences from the region around 67 map units are linked to other (non-contiguous) viral sequences. (2) Sequences at about 72 map units are linked to sequences at 1 map unit. (3) Multiple copies of sequences from around the origin of viral DNA replication are present. From studies on other polyoma defective molecules (Griffin &; Fried, 1975; Lund et al., 1977), the origin of DNA replication for polyoma virus has been defined to lie within the sequences from 67 to 72 map units. Since D74 replicates efficiently but lacks the sequences between 69 to 70 map units, the origin of DNA replication appears to be further defined as lying within 67 and 69 map units and/or 70 to 72 map units.     

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.     

Preferential integration of the Ad5/SV40 hybrid virus at the highly recombinogenic human chromosomal site 1p36

Human fibroblasts transformed with an adenovirus-5/simian virus 40 recombinant construct (Ad5/SV40) were analyzed to determine the chromosomal site(s) of virus integration. This was firstly done by in situ hybridization using metaphase and prometaphase chromosomes and 125I-labeled Ad5 DNA. Out of seven transformed cell lines (six of clonal origin and one uncloned), six were proven to have integrated the viral genome at the short- or the long-subtelomeric regions of autosome 1, two regions known to include chromosomal modification sites induced by acute infection with Ad12. Characterization of the integration sites was carried out by restriction analysis. Transformed cell lines with the same major chromosomal integration site were found to have the viral genome inserted in restriction fragments of different size, indicating that viral integration has occurred at different sites within a relatively small chromosomal region. Molecular studies carried out on one of the transformed cell lines (H13.1) gave an independent confirmation of the viral integration at the subterminal region of autosome 1 short arm. Nucleotide sequencing at this cellular-viral junction has shown that the virus has integrated within tandemly repeated Alu-like elements and that the cellular flanking sequences have several homologies with variable number of tandem repeats core sequences. Many possible open reading frames were identified in the DNA segment adjacent to the Alu-like elements.     

Characterization of unintegrated retroviral DNA with long terminal repeat-associated cell-derived inserts.

We have used a replication-competent shuttle vector based on the genome of Rous sarcoma virus to characterize genomic rearrangements that occur during retrovirus replication. The strategy involved cloning circular DNA that was generated during an acute infection. While analyzing a class of retroviral DNA clones that are greater than full length, we found several clones which had acquired nonviral inserts in positions adjacent to the long terminal repeats (LTRs). There appear to be two distinct mechanisms leading to the incorporation of cellular sequences into these clones. Three of the molecules contain a cell-derived insert at the circle junction site between two LTR units. Two of these molecules appear to be the results of abortive integration attempts, because of which, in each case, one of the LTRs is missing 2 bases at its junction with the cell-derived insert. In the third clone, pNO220, the cellular sequences are flanked by an inappropriately placed copy of the tRNA primer-binding site on one side and a partial copy of the U3 sequence as part of the LTR on the other side. A fourth molecule we characterized, pMD96, has a single LTR with a U5-bounded deletion of viral sequences spanning gag and pol, with cell-derived sequences inserted at the site of the deletion; its origin may be related mechanistically to pNO220. Sequence analysis indicates that all of the cellular inserts were derived from the cell line used for the acute infection rather than from sequences carried into the cell as part of the virus particle. Northern (RNA) analysis of cellular RNA demonstrated that the cell-derived sequences of two clones, pNO220 and pMD96, were expressed as polyadenylated RNA in uninfected cells. One mechanism for the joining of viral and cellular sequences suggested by the structures of pNO220 and pMD96 is recombination occurring during viral DNA synthesis, with cellular RNA serving as the template for the acquisition of cellular sequences.     

Isolation and analysis of retroviral integration targets by solo long terminal repeat inverse PCR

Upon retroviral infection, the genomic RNA is reverse transcribed to make proviral DNA, which is then integrated into the host chromosome. Although the viral elements required for successful integration have been extensively characterized, little is known about the host DNA structure constituting preferred targets for proviral integration. In order to elucidate the mechanism for the target selection, comparison of host DNA sequences at proviral integration sites may be useful. To achieve simultaneous analysis of the upstream and downstream host DNA sequences flanking each proviral integration site, a Moloney murine leukemia virus-based retroviral vector was designed so that its integrated provirus could be removed by Cre-loxP homologous recombination, leaving a solo long terminal repeat (LTR). Taking advantage of the solo LTR, inverse PCR was carried out to amplify both the upstream and downstream cellular flanking DNA. The method called solo LTR inverse PCR, or SLIP, proved useful for simultaneously cloning the upstream and downstream flanking sequences of individual proviral integration sites from the polyclonal population of cells harboring provirus at different chromosomal sites. By the SLIP method, nucleotide sequences corresponding to 38 independent proviral integration targets were determined and, interestingly, atypical virus-host DNA junction structures were found in more than 20% of the cases. Characterization of retroviral integration sites using the SLIP method may provide useful insights into the mechanism for proviral integration and its target selection.     

Characterization of endogenous and exogenous mouse mammary tumor virus proviral DNA with site-specific molecular clones.

Restriction fragments of the mouse mammary tumor virus (MMTV) proviral DNA were obtained by molecular cloning procedures. A 4-kilobase fragment delimited by two PstI sites was isolated from unintegrated, linear MMTV DNA and amplified in the pBr322 plasmid vector. EcoRI fragments of proviral DNA, integrated into the genome of a GR mammary tumor cell line, were isolated as lambda recombinant molecules. Five different recombinant phages which contained the 3' region of the MMTV proviral DNA and adjacent host DNA sequences were isolated. Heteroduplex analysis and S1 nuclease digestion suggested that there is no extensive sequence homology in the host DNA flanking the different proviral genes. The cloned DNA was fractionated into site-specific restriction fragments which served as molecular probes in the analysis of the endogenous MMTV proviral copies of C3H, GR, BALB/c, and feral mice. This allowed the correlation of MMTV-specific EcoRI fragments obtained from genomic DNA of these strains with the 5' and 3' ends of the proviral gene. Restriction fragments of two clones which contained the proviral sequences adjacent to the flanking host DNA as well as 1 to 2 kilobases of host DNA were used as hybridization probes, and the results allow the following conclusions: the proviral DNA of both clones contains nucleotide sequences complementary to the 5' and 3' ends of proviral DNA; and the host DNA flanking one clone belongs to the unique class of genomic DNA, whereas the DNA flanking the second clone is reiterated at least 15 times within the mouse genome.     

Two types of deletion within integrated viral sequences mediate reversion of simian virus 40-transformed mouse cells.

Simian virus 40 (SV40) DNA insertions from SV40-transformed mouse cell line W-2K-11 and its revertants M18, M31, and M42 were cloned. W-2K-11 cells contain 1.5 copies of the SV40 sequences in a partially tandem duplicated form. The endpoints of the viral sequences at the virus-host junctions are located very close to those reported by others, indicating that there are some preferred sites for integration and rearrangement in SV40 sequences. One flanking cellular sequence is a long stretch of adenine and thymine with repeated AAAT, and the other is a stretch of guanine and cytosine with repeated CCG. There are patchy homologies between the flanking cellular sequences and the corresponding parental SV40 sequences. The sequences around both junctions were retained in all the revertants, whereas most of the internal SV40 sequences coding for large T antigen were deleted. The coding sequences for small T antigen are intact, and small T antigen was expressed in all the revertants. The fragments cloned from M18 and M42 were identical and 3.9 kilobases of SV40 sequences were deleted. The parental SV40 sequences around the deletion site have sequences capable of forming a secondary structure which might reduce the effective distance between the two regions. The SV40 DNA retained in M31 is colinear with SV40 virion DNA, and a unit length of SV40 DNA was deleted within the SV40 sequences present in W-2K-11 cells. These results indicated that two types of deletion occurred during the reversion, one between homologous sequences and the other between nonhomologous sequences.     

Tagging the genome of the murine leukemia retrovirus SL3-3 by a bacterial lac operator sequence.

The bacterial lactose operator (lacO) was introduced into the PstI site of the long terminal repeat of the SL3-3 murine leukemia virus, generating a virus, SL3-3lacO, that can replicate in NIH3T3 cell cultures. DNA sequences harboring the lacO sequence might be recovered by molecular cloning in Escherichia coli lac+ lacZ+ using bacteriophage lambda or plasmid vectors. The high copy numbers of the lacO sequence titrate out the lac repressor, leading to the induction of the lac operon in the host. We show here that the lacO and the proviral sequences are carried stably together in the genomes of SL3-3lacO-infected cell cultures and in viral particles. This system is designed to facilitate studies on the provirus and the site of viral integration.     

Transformation of Chloroplast Ribosomal RNA Genes in Chlamydomonas: Molecular and Genetic Characterization of Integration Events

Transformation of chloroplast ribosomal RNA (rRNA) genes in Chlamydomonas has been achieved by the biolistic process using cloned chloroplast DNA fragments carrying mutations that confer antibiotic resistance. The sites of exchange employed during the integration of the donor DNA into the recipient genome have been localized using a combination of antibiotic resistance mutations in the 16S and 23S rRNA genes and restriction fragment length polymorphisms that flank these genes. Complete or nearly complete replacement of a region of the chloroplast genome in the recipient cell by the corresponding sequence from the donor plasmid was the most common integration event. Exchange events between the homologous donor and recipient sequences occurred preferentially near the vector:insert junctions. Insertion of the donor rRNA genes and flanking sequences into one inverted repeat of the recipient genome was followed by intramolecular copy correction so that both copies of the inverted repeat acquired identical sequences. Increased frequencies of rRNA gene transformants were achieved by reducing the copy number of the chloroplast genome in the recipient cells and by decreasing the heterology between donor and recipient DNA sequences flanking the selectable markers. In addition to producing bona fide chloroplast rRNA transformants, the biolistic process induced mutants resistant to low levels of streptomycin, typical of nuclear mutations in Chlamydomonas.