Flanking Cellular Sequences Research Articles

Linear Lepidopteran ambidensovirus 1 sequences drive random integration of a reporter gene in transfected Spodoptera frugiperda cells.

BackgroundThe Lepidopteran ambidensovirus 1 isolated from Junonia coenia (hereafter JcDV) is an invertebrate parvovirus considered as a viral transduction vector as well as a potential tool for the biological control of insect pests. Previous works showed that JcDV-based circular plasmids experimentally integrate into insect cells genomic DNA.MethodsIn order to approach the natural conditions of infection and possible integration, we generated linear JcDV-gfp based molecules which were transfected into non permissive Spodoptera frugiperda (Sf9) cultured cells. Cells were monitored for the expression of green fluorescent protein (GFP) and DNA was analyzed for integration of transduced viral sequences. Non-structural protein modulation of the VP-gene cassette promoter activity was additionally assayed.ResultsWe show that linear JcDV-derived molecules are capable of long term genomic integration and sustained transgene expression in Sf9 cells. As expected, only the deletion of both inverted terminal repeats (ITR) or the polyadenylation signals of NS and VP genes dramatically impairs the global transduction/expression efficiency. However, all the integrated viral sequences we characterized appear “scrambled” whatever the viral content of the transfected vector. Despite a strong GFP expression, we were unable to recover any full sequence of the original constructs and found rearranged viral and non-viral sequences as well. Cellular flanking sequences were identified as non-coding ones. On the other hand, the kinetics of GFP expression over time led us to investigate the apparent down-regulation by non-structural proteins of the VP-gene cassette promoter.ConclusionAltogether, our results show that JcDV-derived sequences included in linear DNA molecules are able to drive efficiently the integration and expression of a foreign gene into the genome of insect cells, whatever their composition, provided that at least one ITR is present. However, the transfected sequences were extensively rearranged with cellular DNA during or after random integration in the host cell genome. Lastly, the non-structural proteins seem to participate in the regulation of p9 promoter activity rather than to the integration of viral sequences.

Human papillomavirus (HPV) insertional mutation as a dynamic & specific tumour biomarker in HPV-associated carcinoma.

The work reported by Das et al1 in this issue is focused on the analysis of the physical status of human papillomavirus (HPV) genomes in cervical carcinoma, in comparison with the course of the disease. It is necessary to recall that the HPV genome, a 8-Kb circular molecule, may exist in two different physical forms according to the type of lesions. In benign papillomas as in most pre-invasive neoplasia, HPV DNA molecules are present as free episomes in the nucleus of keratinocytes whereas in most invasive carcinoma, viral DNA is integrated into the tumour cells genome2. Free molecules, heterogeneously distributed in tumour cells, may also be present in variable amount according to different cases. Integration disrupts the circular viral chromosome which is generally partially deleted. However, the viral E6 and E7 oncogenes are constantly conserved and expressed in tumour cells3. Das et al1 have shown that in cervical cancers with integrated forms of HPV DNA, the disease outcome is worse than in cases harbouring only free molecules. How to explain this result and why is it an important one? The analysis of different cellular models has shown that the integration of HPV DNA frequently interrupts the viral E2 gene which has negative regulation properties on expression level of the E6 and E7 viral oncogenes. Integration, per se, leads to the constitutive expression of the viral oncogenes and can be an important step in tumour progression. In addition, insertion of HPV DNA may also lead to a deregulation of cellular genes located at the vicinity of the integration locus. This deregulation may be secondary to structural changes such as focal amplification4 or losses5 of genes implied in tumour progression, or to the influence of the viral regulatory region on cellular genes located in the vicinity of the viral genome. Finally, it cannot be excluded that in certain cases, integration is a mere evidence of instability of the tumour genome and represents a biological parameter of the severity of the disease. In the perspective of the current progress in the molecular characterization of solid tumours, three important points can be derived from the analysis of HPV genome status in tumours: (i) HPV DNA is a tumour marker that should facilitate the detection of circulating tumour DNA (ctDNA) and improve the biological follow up of patients. Circulating viral DNA is an indirect tumour marker but the use of mutational insertion for the detection of ctDNA provides a more specific tumour marker6. Using HPV and cellular flanking sequences as a molecular marker, the detection of ctDNA will thus allow a highly sensitive and specific diagnosis of infra-clinical relapses, and provides a tool for an optimal biological “personalized monitoring of patients”. (ii) HPV is not only a tumour marker but also a therapeutic target. This has been shown in immunotherapy protocols7. Moreover, recent data on in vitro genetic engineering show that the specific destruction of HPV oncogenes leads to a reversion of the tumour state8. These innovative treatments will be more efficient in cases with minimal tumour mass. And thus, the early detection of tumour relapse will be of paramount importance in the perspective of specific therapy, and the specificity of the alteration detected will be of crucial importance. (iii) The advances on cervical cancer patients monitoring and treatment will be easily extended to other HPV-associated disease. In particular, HPV has been found to be associated with 88 per cent of carcinoma of the anal canal9 and 40 per cent of head and of neck10. This latter tumour type will be particularly important since it is the world's seventh most common type of cancer11, and HPV status is of strong prognostic significance12. On the whole, the findings of Das et al1 are important as these imply that teams of researchers and clinicians are together involved in the development of tumour markers. These teams will be able to include the leads obtained in their practice and in new therapeutic approaches of HPV-associated cancer in the very next future.

Genomic Organization of Amplified MYC Genes Suggests Distinct Mechanisms of Amplification in Tumorigenesis

Integration of the human papillomavirus (HPV) genome into the host genome is associated with the disruption of the HPV E2 gene and with amplification and rearrangement of the viral and flanking cellular sequences. Molecular characterization of the genomic structures of coamplified HPV sequences and oncogenes provides essential information concerning the mechanisms of amplification and their roles in carcinogenesis. Using fluorescent hybridization on stretched DNA molecules in two cervical cancer-derived cell lines, we have elucidated the genomic structures of amplified regions containing HPV/myc genes over several hundreds of kilobases. Direct visualization of hybridization signals on individual DNA molecules suggests that overreplication and breakage-fusion-bridge-type mechanisms are involved in the genomic instability associated with HPV cervical cancers. Further analysis from two other genital cancer-derived cell lines reveals a recurrent motif of amplification, probably generated by a common mechanism involving overreplication upon viral integration. Interestingly, different amplification patterns seem to be correlated with the disease outcome, thus providing new insights into HPV-related cancer development and tumor progression.

The human L1 promoter: Variable transcription initiation sites and a major impact of upstream flanking sequence on promoter activity

Human L1 elements are non-LTR retrotransposons that comprise approximately 17% of the human genome. Their 5'-untranslated region (5'-UTR) serves as a promoter for L1 transcription. Now we find that transcription initiation sites are not restricted to nucleotide +1 but vary considerably in both downstream and upstream directions. Transcription initiating upstream explains additional nucleotides often seen between the 5'-target site duplication and the L1 start site. A higher frequency of G nucleotides observed upstream from the L1 can be explained by reverse transcription of the L1 RNA 5'-CAP, which is further supported by extra Gs seen for full-length HERV-W pseudogenes. We assayed 5'-UTR promoter activities for several full-length human L1 elements, and found that upstream flanking cellular sequences strongly influence the L1 5'-UTR promoter. These sequences either repress or enhance the L1 promoter activity. Therefore, the evolutionary success of a human L1 in producing progeny depends not only on the L1 itself, but also on its genomic integration site. The promoter mechanism of L1 is reminiscent of initiator (Inr) elements that are TATA-less promoters expressing several cellular genes. We suggest that the L1 5'-UTR is able to form an Inr element that reaches into upstream flanking sequence.

Selection for c-myc integration sites in polyclonal T-cell lymphomas.

Type B leukemogenic virus (TBLV) is highly related to mouse mammary tumor virus but induces rapidly appearing T-cell lymphomas in mice. Unlike other T-cell tumors induced by retroviruses, only 5 to 10% of TBLV-induced lymphomas have detectable viral integrations near c-myc by Southern blotting, whereas Northern blotting has shown that most tumors have two- to sixfold overexpression of c-myc RNA. In this report, PCR was used to demonstrate that at least 30% of these lymphomas have TBLV insertions near c-myc. Some tumors contained multiple TBLV proviruses in different locations and orientations, suggesting that the tumors are polyclonal. The integrated proviruses near c-myc had different numbers (two to four) of long terminal repeat (LTR) enhancer repeats, although LTRs with three-repeat enhancers dominated the proviral population. Passage of polyclonal tumors in immunocompetent mice and semiquantitative PCR revealed that only cells with particular integrations were selected for growth. In three of six tumors tested, proviruses containing four-repeat enhancers near c-myc were selected during tumor passage. Since tumor cell selection may be accomplished by overexpression of c-myc RNA due to proximity to the unique TBLV LTR enhancer, we inserted LTRs at various locations within a plasmid containing the entire c-myc locus and cellular flanking sequences. To quantitatively measure effects on transcription, the Renilla luciferase gene was substituted for most of c-myc exon 2, and transient transfections were performed with c-myc reporter constructs in two different T-cell lines. As expected, insertion of a TBLV LTR with three-repeat enhancers in either orientation, 5" and 3", of the myc gene elevated reporter activity from 2- to 160-fold, consistent with enhancer function, but four-repeat LTRs had lower levels of expression compared to three-repeat LTRs. Surprisingly, LTR insertions that gave maximal c-myc expression in transient-transfection assays declined in tumor cells selected for growth in vivo. Selection for clonal growth may occur in tumor cells that have modest c-myc overexpression after proviral insertion to prevent apoptosis.

Somatic mutation in human T-cell leukemia virus type 1 provirus and flanking cellular sequences during clonal expansion in vivo.

Human T-cell leukemia virus type 1 (HTLV-1), the causative agent of adult T-cell leukemia/lymphoma, shows intrapatient genetic variability. Although HTLV-1 can replicate via the reverse transcription of virion RNA to a double-stranded DNA provirus (the conventional manner for retroviruses), its predominant mode of replication is via the clonal expansion (mitosis) of the infected cell. This expansion is achieved by the viral oncoprotein Tax, which keeps the infected CD4 T lymphocyte cycling. Because Tax also interferes with cellular DNA repair pathways, we investigated whether somatic mutations of the provirus that occur during the division of infected cells could account for HTLV-1 genetic variability. An inverse polymerase chain reaction strategy was designed to distinguish somatic mutations from reverse transcription-associated substitutions. This strategy allows the proviral sequences to be isolated together with flanking cellular sequences. Using this method, we sequenced 208 HTLV-1 provirus 3' segments, together with their integration sites, belonging to 29 distinct circulating cellular clones from infected individuals. For 60% of the clones, 8%-80% of infected cells harbored a mutated HTLV-1 provirus, without evidence of reverse transcription-associated mutations. Mutations within flanking cellular sequences were also identified at a frequency of 2.8 x 10(-4) substitution per base pair. Some of these clones carried multiple discrete substitutions or deletions, indicating progressive accumulation of mutations during clonal expansion. The overall frequency of somatic mutations increased with the degree of proliferation of infected T cells. These data indicate that, in vivo, HTLV-1 variation results mainly from postintegration events that consist of somatic mutations of the proviral sequence occurring during clonal expansion. The finding of substitutions in flanking sequences suggests that somatic mutations occurring after integration, presumably coupled with selection, help move the cellular clones toward a transformed phenotype, of which adult T-cell leukemia/lymphoma is the end point.

Characterization of hepatitis B virus integrant that results in chromosomal rearrangement.

A hepatitis B virus (HBV) integrant was cloned from the genomic DNA library of human hepatocellular carcinoma cell line, Hep3B. Sequence analysis of the restriction fragment bearing the virus-host junction revealed that its integration pattern was the common type, with the right junction located at the cohesive region. The open reading frame of the major viral surface antigen was intact with rearranged preS1 and core sequences. The X protein, although truncated, maintained the trans-activating activity to simian virus 40 enhancer/promoter. S1 nuclease mapping showed that 4.0-, 2.9-, and 2.2-kb HBV RNAs detected in Hep3B cells were transcribed from this integrant using preS2/S promoter. By somatic-cell hybrid mapping, the left and right cellular flanking sequences were assigned to chromosomes 13 and 4, respectively. The results of this study support the notion that integrated hepatitis B virus, resulting in chromosomal rearrangement as well as the production of the carboxy-terminal truncated X protein with trans-activating activity, is important for viral hepatocarcinogenesis.

Detection of Hepatitis B Virus x Transcripts in Human Hepatocellular Carcinoma Tissues

Background: The hepatitis B virus (HBV) or hepatitis C virus (HCV) sequences were frequently detected in hepatocellular carcinoma (HCC) tissues by the method of polymerase chain reaction (PCR). However, the expression levels of HBV and HCV in HCC tissues remain to be documented. This study evaluates the mRNA expression levels of those virus sequences in the human HCC tissues through Northern blot hybridization. Methods: We performed Northern blot hybridization to identify the HBV x mRNA and HCV RNA in HCC tissues of 14 cases excised at Chiba University Hospital. Results: The HBV x transcripts could be identified in the tumor tissues from all three serum HBsAg-positive cases and two cases among 11 HBsAg-negative patients. One HBsAg-negative case showed the most intensive signal in those five cases. However, HCV RNA could not be identified by this method. The HBV x gene transcript is predicted to have a size of about 0.7 kb; however, the molecular weight of HBV x specific mRNA is not consistent and is distributed between approximately 2.0 and 9.0 kb. The hybridization signal intensity is also variable according to the cases. Conclusion: These observations suggest that HBV x gene may be transcribed with flanking cellular sequences and the carcinogenic activity may be different according to the expressed levels. The identification of HBV x transcripts in HCC tissues on the Northern blot level also suggests that HBV x mRNA expression might be significant and concerned with the development of human HCC even in HBsAg-negative cases.

Deletion of integrated hepatitis B virus genome and cellular flanking sequences in hepatocellular carcinoma cells in BALB/c mice

We have previously reported the establishment of well-differentiated BALB/c mouse liver (ML) cell lines. Transfection of these cell lines with hepatitis B virus (HBV) DNA led to the expression of HBV-specific antigens and integration of HBV sequences in the cellular genome. Two cloned HBV-transfected ML cell lines, ML2(HBV) and ML-3(HBV), expressed viral antigens and were highly tumorigenic in nude mice. However, the tumorigenicity of the two cell lines was significantly reduced in BALB/c mice. Southern blot analyses showed that the integrated HBV sequences were retained in tumors growing in nude mice but deleted in tumors growing in BALB/c mice. Furthermore, the deletion of HBV DNA was accompanied by deletion of chromosomal sequences flanking the HBV integration sites. In ML-2(HBV) cells, a significant reduction in chromosomal number was also observed. These results suggest that the immune response of BALB/c mice selected against hepatocellular carcinoma (HCC) cells expressing viral antigens and led to the proliferation of cells with deleted HBV sequences and concomitant chromosome aberrations. By using this mechanism, HCC cells escape the immune surveillance and gain the advantage of cell growth.

Deletion of integrated hepatitis B virus genome and cellular flanking sequences in hepatocellular carcinoma cells in BALB/c Mice

We have previously reported the establishment of well-differentiated BALB/c mouse liver (ML) cell lines. Transfection of these cell lines with hepatitis B virus (HBV) DNA led to the expression of HBV-specific antigens and integration of HBV sequences in the cellular genome. Two cloned HBV-transfected ML cell lines, ML-2(HBV) and ML-3(HBV), expressed viral antigens and were highly tumorigenic in nude mice. However, the tumorigenicity of the two cell lines was significantly reduced in BALB/c mice. Southern blot analyses showed that the integrated HBV sequences were retained in tumors growing in nude mice but deleted in tumors growing in BALB/c mice. Furthermore, the deletion of HBV DNA was accompanied by deletion of chromosomal sequences flanking the HBV integration sites. In ML-2(HBV) cells, a significant reduction in chromosomal number was also observed. These results suggest that the immune response of BALB/c mice selected against hepatocellular carcinoma (HCC) cells expressing viral antigens and led to the proliferation of cells with deleted HBV sequences and concomitant chromosome aberrations. By using this mechanism, HCC cells escape the immune surveillance and gain the advantage of cell growth.

Inverse polymerase chain reaction for cloning cellular sequences adjacent to integrated hepatitis B virus DNA in hepatocellular carcinomas

Integration of hepatitis B virus (HBV) DNA is found in most HBV-related human hepatocellular carcinomas (HCCs). In the past, construction of genomic libraries was mainly employed to study the role of viral integration. However, large amounts of tissue DNA and a laborious screening procedures were required. Inverse polymerase chain reaction (IPCR) is based on the simple procedures of digestion of DNA with restriction enzymes and circularization of cleavage products before amplification using primers synthesized in the opposite orientations to those normally employed for PCR. This technique allows the in vitro amplification of DNA flanking a region of known sequence. By employing this method, starting from nanograms of hepatoma DNA, two adjacent cellular sequences were cloned from 11 HBV integrants in three HCCs. The original configurations in the chromosomes were further confirmed. One of the flanking cellular sequences was identified as the human 28S rRNA gene, the other was not found homologous to any known human sequences. This method appears to be practical and can be improved further to clone more flanking cellular sequences, especially in early and small HCCs.

Analysis of integrated hepatitis B virus DNA and flanking cellular sequences in the hepatocellular carcinoma cell line HCC36.

A hepatocellular carcinoma cell line, HCC36, was established from an adult HBV carrier in Taiwan. From Southern blot analysis, there were at least four sites of integration of HBV DNA, and no viral replicative intermediates were detected. A genomic library was constructed from HCC36 DNA, and two phage clones, designated lambda 36A and lambda 36B, were shown to contain HBV DNA and flanking cellular sequences. In lambda 36A, HBV DNA sequences were quite conserved, and 7.4% base variation was detected. The viral sequences in lambda 36A and lambda 36B differed in only four bases, in addition to the microdeletion and -insertion observed in lambda 36B. The flanking cellular sequences identified in lambda 36A were human Alu sequences and in lambda 36B satellite sequences.

Integration site of human papillomavirus type-18 DNA in chromosome band 8q22.1 of C4-I cervical carcinoma: DNase I hypersensitivity and methylation of cellular flanking sequences.

The C4-I cell line derived from a non-keratinizing squamous cell carcinoma of the uterine cervix contains integrated human papillomavirus-18 DNA. Fluorescence in situ hybridization of C4-I cells demonstrated a single viral integration site at 8q22.1 on a derivative chromosome originating from an 8q;12q translocation. 8q22 is a site of chromosome fragility and is also recombinogenic in several human malignancies. DNase I hypersensitivity of the integration site was studied with a cellular flanking probe. A hypersensitive site was detected within 3 kb from viral DNA. The integration sites are undermethylated in C4-I and HeLa cells and fully methylated in tumor cell lines of other origin, such as lymphoid cells.

Transactivation of Cellular Promoters by an Integrated Hepatitis B virus DNA

A new Hepatitis B virus (HBV) DNA integrant clone DA2-6, isolated from a human hepatocellular carcinoma (HCC) genomic library, was tested for its ability to transactivate expression of other genes. DA2-6 consists of 3.7 kb flanking cellular sequences and an integrated 2.8 kb HBV DNA which covers the region of preS, S, and the 3′ truncated X. Using a chloramphenicol acetyltransferase (CAT) assay, a number of cellular and viral promoters were transactivated by DA2-6,and the spectrum of transactivational effect was the same as that by the wild type X gene of the virus. Deletion mutant analyses indicated that the transactivation function of DA2-6 is expressed by the region that encodes a truncated X-cell fusion product.

Scheme for the generation of a truncated endogenous murine leukaemia virus, the Fv-4 resistance gene.

The Fv-4 resistance (Fv-4r) gene is a truncated endogenous murine leukaemia virus (MuLV) containing a 3' portion of pol, the entire env gene and the 3' long terminal repeat. Env expression renders mice resistant to infection by ecotropic MuLVs, probably via receptor interference. Previous studies have suggested that the flanking cellular sequences are also important for Fv-4 env gene expression. To establish how the truncated retrovirus was generated and the nature of the cellular sequences involved, the Fv-4 susceptible (Fv-4s) allele DNA was cloned, and its restriction map and nucleotide sequence were compared with those of the Fv-4r allele. A likely mechanism for generation of the truncated endogenous MuLV is suggested by the results; integration of a prototype MuLV provirus at a site within the Fv-4s allele about 6 to 8 kb downstream of a non-retroviral promoter region, followed by deletion of the 5' half of the provirus, with an accompanying loss of only 7 or 10 bp of cellular flanking sequences. The deletion may have led to the expression of the Fv-4r env gene under control of the non-retroviral promoter.

A Chromosome 17:7 Translocation Is Associated With A Hepatitis B Virus Dna Integration in Human Hepatocellular Carcinoma Dna

Chronic hepatitis B virus infection is often associated with major structural rearrangements of both the integrated viral DNA and the associated cellular sequences. We present here the structure of a single-copy hepatitis B virus insert cloned from human hepatocellular carcinoma DNA recently reported to encode a novel transcriptional trans-activator function. The hepatitis B virus portion of the clone consists of two colinear fragments covering the X gene with its promoter and enhancer (nucleotides 717 to 1796) and a 3' truncated pre-S/S gene (nucleotides 2703 to 423). The lack of the entire pre-C/C gene caused a fusion of the 3' end of the X gene with sequences upstream from the pre-S gene. The structure of the integrated viral DNA fragments suggests insertion of hepatitis B virus replication intermediates into cellular DNA and subsequent recombination between these primary integrations to generate the final structure of the clone. The 5' and 3' cellular flanking sequences mapped to the centromeric alpha-satellite DNA of chromosome 17 and to the short arm of chromosome 7 (p14-pter), respectively, indicating that chromosomal translocation was associated with the hepatitis B virus DNA integration. Because this is the fourth case reported in which hepatitis B virus-associated rearrangements have affected chromosome 17, it is conceivable that a loss of important cellular genes (such as the p53 antioncogene on chromosome 17) may be a crucial step in hepatitis B virus-related hepatocarcinogenesis.

Expression of an activated Notch-related int-3 transgene interferes with cell differentiation and induces neoplastic transformation in mammary and salivary glands.

Expression of the int-3 locus is activated in mouse mammary tumors as a consequence of insertional mutagenesis by the mouse mammary tumor virus (MMTV). Integration of the MMTV provirus into the int-3 locus promotes the transcription and translation of flanking cellular int-3 sequences sharing significant homology with the intracellular domain of the neurogenic Notch gene of Drosophila, and with the yeast cell cycle regulatory genes cdc10 and SWI6. To determine the in vivo consequences of activated int-3 expression, transgenic mice were generated harboring a genomic tumor DNA fragment consisting of the MMTV LTR and the flanking cellular int-3 sequences. All six int-3 founder transgenic mice and the progeny of one established line exhibited similar dramatic phenotypic abnormalities in tissues in which the transgene was expressed. Focal and often multiple poorly differentiated mammary and salivary adenocarcinomas appeared in the majority of transgenic mice between 2 and 7 months of age. Significantly, mammary glands were arrested in development and were lactation deficient in all female int-3 mice. The salivary glands, glands of the nasal mucosa and maxillary sinus, the extraorbital lacrimal glands, and the Harderian glands of juvenile and adult transgenic mice all contained proliferating immature ductule cells and were incompletely differentiated. In addition, all male int-3 transgenic mice were sterile, apparently the result of severe hyperplasia of the epididymis. These findings demonstrate in vivo that expression of the activated Notch-related int-3 gene causes deregulation of normal developmental controls and hyperproliferation of glandular epithelia.

Integration of bovine papillomavirus type 1 DNA and analysis of the amplified virus-cell junctions in transformed primary mouse fibroblasts.

We have analysed the site of bovine papillomavirus type 1 (BPV-1) DNA integration in clones originating from a transformed primary mouse fibroblast cell line established by transfection of linear BPV-1 DNA. Viral DNA was integrated at a single site in the host genome with an intact early region and an almost complete long control region. Sequence analysis showed that the BPV-1 DNA was integrated at the HindIII site (the enzyme used to linearize the BPV-1 DNA for transfection) with short deletions at both ends. These deletions correspond to a 534 bp segment spanning the 3' end of the L1 open reading frame and the replication enhancer element in the BPV-1 genome. The cellular sequences 5' to the viral integration site exhibited 85 to 97% identity to several sequences belonging to the mouse L1 family of long interspersed repetitive sequences. Cellular sequences 3' to the viral DNA exhibited no significant similarity to any known sequence. The BPV-1 sequences and the cellular flanking sequences were found to be amplified 45- to 50-fold. All the cell clones shared an identical integration site but one of the clones had an additional population of amplified and integrated BPV-1 DNA molecules with an internal deletion of 1136 bp in the late region. The significance of viral DNA integration at a murine long interspersed repetitive sequence containing an amplification-promoting sequence is discussed.

A putative approach for cloning ‘silent’ genes using retroviral vectors

An alternative approach is described for cloning ‘silent’ or poorly expressed genes that might be activated as a result of proviral DNA integration. Generally, proviral DNA integrates randomly, in a single copy per cell, although some preferred integration sites exist. Thus, each cell infected by a retroviral vector should represent a different clone. These clones would have different phenotypes depending on the site of proviral DNA integration. Those expressing a desired phenotype would be screened for by using an assay system that depends on the gene of interest. The 5′ and 3′ flanking cellular DNA sequences, responsible for the observed phenotype in such mutants, could be cloned. Screening of these clones should be relatively simple due to the presence of a selectable marker in the proviral DNA. These sequences would then be used to isolate the wild type (wt) copy of the insertionally activated gene. Packaging retroviral vectors could lead to insertional activation of cellular genes and packaging of insertionally activated RNAs into vector particles. RNA extracted from the particles released into the culture medium would greatly facilitate cDNA cloning of ‘silent’ genes activated as a result of proviral DNA integration.

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.