Construction of a PAC vector system for the propagation of genomic DNA in bacterial and mammalian cells and subsequent generation of nested deletions in individual library members

The BAC and PAC cloning systems allow investigators to propagate large genomic DNA fragments up to 300 kb in size in E. colicells.We describe a new PAC shuttle vector that can be propagated in both bacterial and human cells. Specifically, the P1 cloning vector pAd10sacBII was modified by the insertion of a puromycin-resistance gene (pac), the Epstein-Barr Virus (EBV) latent replication origin oriP,and the EBV EBNA1 gene. Transfection studies in HEK 293 cells demonstrated that the modified vector was stably maintained as an episome for at least 30 generations. And since pJCPAC-Mam1 contains a loxP site, genomic DNA cloned into this vector can be subjected to loxP-Cre -mediated deletion events. The transposon vector pTnPGKpuro/loxP was modified to make this system amenable to propagation in human cells by inserting pac, oriP, and EBNA1 elements into the vector (Chatterjee, P.K., Coren, J.C., 1997. Isolating large nested deletions in PACs and BACs by in vivo selection of P1 headful-packaged products of Cre-catalyzed recombination between the loxP site in PAC and BAC and one introduced in transposition. NAR 25, 2205-2212.). pTnPGKpuro/loxP-EBV was then used to generate deletions in an individual library member to demonstrate that all of the deletions still contain the required eukaryotic elements and that they were nested. All library members constructed in pJCPAC-Mam1 can be directly transformed into human cells to assess function. And the deletion technology can be used to aid in delineating the boundaries of genes and other cis-acting elements.     

Construction and properties of an Epstein-Barr-virus-derived cDNA expression vector for human cells

A cDNA expression vector containing the element oriP and the sequence encoding the Epstein-Barr virus (EBV) nuclear antigen 1 (EBNA-1) as well as the hygromycin B-resistance dominant marker gene has been constructed. Its characteristics have been compared to a similar vector lacking the EBV sequences. (a) The EBV+ vector is maintained as an episome with a copy number of approx. 50 per cell, whereas the number of the integrated EBV- copies is in general smaller than 10, when simian virus 40-transformed xeroderma pigmentosum fibroblasts (XP20S-SV) constitute the recipient cell line. (b) The presence of the EBV sequences in the vector resulted in a five- to ten-fold higher transfection efficiency with the Ca.phosphate precipitation technique. (c) cDNA inserts in the EBV+ vector are shown to be efficiently and properly expressed in the recipient cell. (d) If transfection is performed with a mixture of EBV+ vectors with different inserts, transfectants are shown to harbour different plasmids within one cell. (e) The ratio between these plasmids in one cell can be shifted in favour of a vector with a particular insert, when selection for this insert is performed. (f) Reconstruction experiments indicated that isolation of a low-abundance sequence from a mixture of vectors is at least 100-fold more efficient with the EBV+ system, than with the EBV- system. (g) Rescue of the episomal vector from transfected cells can be readily achieved.     

Development of a novel helper-dependent adenovirus-Epstein-Barr virus hybrid system for the stable transformation of mammalian cells

Epstein-Barr virus (EBV) episomes are stably maintained in permissive proliferating cell lines due to EBV nuclear antigen 1 (EBNA-1) protein-mediated replication and segregation. Previous studies showed the ability of EBV episomes to confer long-term transgene expression and correct genetic defects in deficient cells. To achieve quantitative delivery of EBV episomes in vitro and in vivo, we developed a binary helper-dependent adenovirus (HDA)-EBV hybrid system that consists of one HDA vector for the expression of Cre recombinase and a second HDA vector that contains all of the sequences for the EBV episome flanked by loxP sites. Upon coinfection of cells, Cre expressed from the first vector recombined loxP sites on the second vector. The resulting circular EBV episomes expressed a transgene and contained the EBV-derived family of repeats, an EBNA-1 expression cassette, and 19 kb of human DNA that functions as a replication origin in mammalian cells. This HDA-EBV hybrid system transformed 40% of cultured cells. Transgene expression in proliferating cells was observed for over 20 weeks under conditions that selected for the expression of the transgene. In the absence of selection, EBV episomes were lost at a rate of 8 to 10% per cell division. Successful delivery of EBV episomes in vivo was demonstrated in the liver of transgenic mice expressing Cre from the albumin promoter. This novel gene transfer system has the potential to confer long-term episomal transgene expression and therefore to correct genetic defects with reduced vector-related toxicity and without insertional mutagenesis.     

Differences in human cell lines to support stable replication of Epstein-Barr virus-based vectors

Vectors carrying the origin of replication, ori-P, of the Epstein-Barr virus (EBV) are maintained extrachromosomally in human cells expressing the EBV nuclear antigen 1 (EBNA-1). We have studied the EBV vectors p201 and p292 in which both ori-P and EBNA-1 functions are present using the human cell lines A431 and HeLa. The two lines showed differences in their transfectability by the EBV vectors. Thousands of HeLa transfectants were obtained with either vector and these remained intact as episomes. A431 could only be efficiently transfected with p292 and a high ratio of chromosomal integrations and rearrangements were observed. The vector p292 expressed the EBNA-1 gene more efficiently than p201 and this was found to be associated with a harmful effect on the grown of both HeLa and A431 lines. These results indicate that EBV vectors behave differently, depending on the cell line and that over-expression of EBNA 1 from these vectors may be detrimental to the cells.     

Maintenance of Epstein-Barr virus-derived episomal vectors in the murine Sp2/0 myeloma cell line is dependent upon exogenous expression of human EBP2.

Vectors carrying the origin of replication (oriP) and driving expression of the EBNA-1 protein from Epstein-Barr virus (EBV) replicate as extrachromosomal episomes in human cells. Whether these vectors can be maintained as episomes in murine cells is still controversial. Here we demonstrate that EBNA-1 expression alone was unable to maintain episomal expression of an EBV-based vector in the murine Sp2/0 cell line. However, we were able to obtain long-term episome maintenance in Sp2/0 cells after exogenously expressing human EBP2 by genetic engineering. Our results provide further evidence for the fundamental role of human EBP2 in episomal maintenance of EBV-based vectors. Moreover, we demonstrate that EBV-based vectors can be successfully used in cells presumably incompetent for episomal maintenance.     

Epstein-Barr virus nuclear antigen 2 transactivates latent membrane protein LMP1.

Several lines of evidence are compatible with the hypothesis that Epstein-Barr virus (EBV) nuclear antigen 2 (EBNA-2) or leader protein (EBNA-LP) affects expression of the EBV latent infection membrane protein LMP1. We now demonstrate the following. (i) Acute transfection and expression of EBNA-2 under control of simian virus 40 or Moloney murine leukemia virus promoters resulted in increased LMP1 expression in P3HR-1-infected Burkitt's lymphoma cells and the P3HR-1 or Daudi cell line. (ii) Transfection and expression of EBNA-LP alone had no effect on LMP1 expression and did not act synergistically with EBNA-2 to affect LMP1 expression. (iii) LMP1 expression in Daudi and P3HR-1-infected cells was controlled at the mRNA level, and EBNA-2 expression in Daudi cells increased LMP1 mRNA. (iv) No other EBV genes were required for EBNA-2 transactivation of LMP1 since cotransfection of recombinant EBNA-2 expression vectors and genomic LMP1 DNA fragments enhanced LMP1 expression in the EBV-negative B-lymphoma cell lines BJAB, Louckes, and BL30. (v) An EBNA-2-responsive element was found within the -512 to +40 LMP1 DNA since this DNA linked to a chloramphenicol acetyltransferase reporter gene was transactivated by cotransfection with an EBNA-2 expression vector. (vi) The EBV type 2 EBNA-2 transactivated LMP1 as well as the EBV type 1 EBNA-2. (vii) Two deletions within the EBNA-2 gene which rendered EBV transformation incompetent did not transactivate LMP1, whereas a transformation-competent EBNA-2 deletion mutant did transactivate LMP1. LMP1 is a potent effector of B-lymphocyte activation and can act synergistically with EBNA-2 to induce cellular CD23 gene expression. Thus, EBNA-2 transactivation of LMP1 amplifies the biological impact of EBNA-2 and underscores its central role in EBV-induced growth transformation.     

Amplification of Epstein-Barr virus-based shuttle vectors by ultraviolet light in human cells.

In order to approach the mechanism of gene amplification, we have developed a model system in human cells based on the use of episomally-replicating shuttle vectors. Shuttle vectors carrying the replication origin of the Epstein-Barr virus can be stably maintained in human cells. These vectors replicate as an episome with a low copy number. We also constructed hybrid plasmids containing both the EBV and the SV40 replication origins. These molecules are able to replicate episomally either like an EBV vector or like SV40 if the SV40 large T antigen is provided at the same time. UV irradiation of both human adenovirus transformed 293 or SV40-transformed MRC5 host cells leads to vector amplification whatever the type of replication origin used for the episomal maintenance. Our result clearly shows that the EBV latent replication origin (OriP), in the presence of the Epstein-Barr nuclear antigen-1 (EBNA-1) and the SV40 large T antigen, is sensitive to over-replication in UV-irradiated human cells. Since the UV doses were small enough to induce very little damage, if any, on the plasmid sequences, this amplification should be mediated through a cellular factor acting in trans. The interest in using shuttle vectors for this kind of study lays in the easy analysis of the amplified vectors in rescued bacterial colonies. The accuracy of the amplification process can be monitored by studying restriction maps of individual plasmid molecules or more precisely the integrity of a target gene, such as the lacZ' sequence, carried by our vectors.     

Live-cell imaging reveals multiple interactions between Epstein-Barr virus nuclear antigen 1 and cellular chromatin during interphase and mitosis

Epstein-Barr virus (EBV) establishes a life-long latent infection in humans. In proliferating latently infected cells, EBV genomes persist as multiple episomes that undergo one DNA replication event per cell cycle and remain attached to the mitotic chromosomes. EBV nuclear antigen 1 (EBNA-1) binding to the episome and cellular genome is essential to ensure proper episome replication and segregation. However, the nature and regulation of EBNA-1 interaction with chromatin has not been clearly elucidated. This activity has been suggested to involve EBNA-1 binding to DNA, duplex RNA, and/or proteins. EBNA-1 binding protein 2 (EBP2), a nucleolar protein, has been proposed to act as a docking protein for EBNA-1 on mitotic chromosomes. However, there is no direct evidence thus far for EBP2 being associated with EBNA-1 during mitosis. By combining video microscopy and Förster resonance energy transfer (FRET) microscopy, we demonstrate here for the first time that EBNA-1 and EBP2 interact in the nucleoplasm, as well as in the nucleoli during interphase. However, in strong contrast to the current proposed model, we were unable to observe any interaction between EBNA-1 and EBP2 on mitotic chromosomes. We also performed a yeast double-hybrid screening, followed by a FRET analysis, that led us to identify HMGB2 (high-mobility group box 2), a well-known chromatin component, as a new partner for EBNA-1 on chromatin during interphase and mitosis. Although the depletion of HMGB2 partly altered EBNA-1 association with chromatin in HeLa cells during interphase and mitosis, it did not significantly impact the maintenance of EBV episomes in Raji cells.     

Construction of an EBV-derived shuttle vector for studying the influence of transcription on mutagenesis

We have constructed an EBV-derived shuttle vector, pF1-EBV, which replicates in human cells as an extrachromosomal element. The structural sequences of the gene encoding the bacterial xanthine-guanine-phosphoribosyltransferase (gpt) were fused to the promoter and presumptive control region of the mouse metallothionein I (MT-I) gene. Human 293 cells transformed with the recombinant plasmid synthesized gpt mRNA and the expression of the gene was inducible by zinc. The gpt gene offers a convenient system of selection for mutant plasmids by transformation into the appropriate gpt- E. coli strain. A clonal cell line created by establishment of the pF1-EBV shuttle vector showed a spontaneous gpt- frequency of 2.10(-5). An increase in mutation frequency above background was induced by mutagenizing this cell line with the alkylating agent N-methyl-N-nitrosourea (MNU). The recombinant molecule that we have constructed should provide a tool for studying the role of gene expression in DNA repair and mutagenesis.     

Epstein-Barr virus (EBV) recombinants: use of positive selection markers to rescue mutants in EBV-negative B-lymphoma cells.

The objective of these experiments was to develop strategies for creation and identification of recombinant mutant Epstein-Barr viruses (EBV). EBV recombinant molecular genetics has been limited to mutations within a short DNA segment deleted from a nontransforming EBV and an underlying strategy which relies on growth transformation of primary B lymphocytes for identification of recombinants. Thus, mutations outside the deletion or mutations which affect transformation cannot be easily recovered. In these experiments we investigated whether a toxic drug resistance gene, guanine phosphoribosyltransferase or hygromycin phosphotransferase, driven by the simian virus 40 promoter can be recombined into the EBV genome and can function to identify B-lymphoma cells infected with recombinant virus. Two different strategies were used to recombine the drug resistance marker into the EBV genome. Both utilized transfection of partially permissive, EBV-infected B95-8 cells and positive selection for cells which had incorporated a functional drug resistance gene. In the first series of experiments, B95-8 clones were screened for transfected DNA that had recombined into the EBV genome. In the second series of experiments, the transfected drug resistance marker was linked to the plasmid and lytic EBV origins so that it was maintained as an episome and could recombine with the B95-8 EBV genome during virus replication. The recombinant EBV from either experiment could be recovered by infection and toxic drug selection of EBV-negative B-lymphoma cells. The EBV genome in these B-lymphoma cells is frequently an episome. Virus genes associated with latent infection of primary B lymphocytes are expressed. Expression of Epstein-Barr virus nuclear antigen 2 (EBNA-2) and the EBNA-3 genes is variable relative to that of EBNA-1, as is characteristic of some naturally infected Burkitt tumor cells. Moreover, the EBV-infected B-lymphoma cells are often partially permissive for early replicative cycle gene expression and virus replication can be induced, in contrast to previously reported in vitro infected B-lymphoma cells. These studies demonstrate that dominant selectable markers can be inserted into the EBV genome, are active in the context of the EBV genome, and can be used to recover recombinant EBV in B-lymphoma cells. This system should be particularly useful for recovering EBV genomes with mutations in essential transforming genes.     

Analysis of mutation in human cells by using an Epstein-Barr virus shuttle system.

We developed highly sensitive shuttle vector systems for detection of mutations formed in human cells using autonomously replicating derivatives of Epstein-Barr virus (EBV). EBV vectors carrying the bacterial lacI gene as the target for mutation were established in human cells and later returned to Escherichia coli for rapid detection and analysis of lacI mutations. The majority of the clonal cell lines created by establishment of the lacI-EBV vector show spontaneous LacI- frequencies of less than 10(-5) and are suitable for studies of induced mutation. The ability to isolate clonal lines represents a major advantage of the EBV vectors over transiently replicating shuttle vectors (such as those derived from simian virus 40) for the study of mutation. The DNA sequence changes were determined for 61 lacI mutations induced by exposure of one of the cell lines to N-nitroso-N-methylurea. A total of 33 of 34 lacI nonsense mutations and 26 of 27 missense mutations involve G X C to A X T transitions. These data provide support for the mutational theory of cancer.     

Factor VIII mRNA expression from a BAC carrying the intact locus made by homologous recombination

Hemophilia A is caused by mutations in the gene encoding factor VIII (F8) and is an important target for gene therapy. The F8 gene contains 26 exons spread over approximately 186 kb and no work using the intact genomic locus has been carried out. We have constructed a 250-kb BAC carrying all 26 exons, the introns, and more than 40 kb of upstream and 20 kb of downstream DNA. This F8 BAC was further retrofitted with either the oriP/EBNA-1 elements from Epstein-Barr virus, which allow episomal maintenance in mammalian cells, or alphoid DNA, which allows human artificial chromosome formation in some human cell lines. Lipofection of the oriP/EBNA-1-containing version into mouse Hepa1-6 cells resulted in expression of F8 mRNA spanning the F8 gene. The >300-kb BAC carrying alphoid DNA was successfully delivered to 293A and HT1080 cells using bacterial delivery, resulting in greater than endogenous levels of F8 mRNA expression.     

Mammalian assay for site-specific DNA damage processing using the human H-ras proto-oncogene.

The human genomic H-ras proto-oncogene was inserted into an Epstein-Barr virus (EBV) vector (p220.2) that replicates synchronously with the cell cycle. Unique restriction enzyme sites, 30 bp apart, were created on either side of codon 12 to enable the construction of gapped heteroduplex (GHD) DNA. Depending upon experimental protocol, the gap could be located either on the coding (non-transcribed) strand or the non-coding (transcribed) strand. GHD DNA was created using a 1.8 kb segment of H-ras DNA containing exon 1, into which a synthetic 30 nucleotide oligomer containing a strand- and site-specific mismatched nucleotide was annealed. The 1.8 kb segment of H-ras DNA containing a codon 12; middle G:T, A:C or T:C mismatch has been religated with high efficiency into the EBV vector and transfected into NIH 3T3 cells using a mild liposome-mediated protocol. Subsequent hygromycin resistant NIH 3T3 colonies have been PCR amplified and sequenced. In this study, codon 12; middle nucleotide mismatch correction rates to wild-type G:C during replication in NIH 3T3 cells were 96.4% of G:T mismatches, 87.5% of A:C mismatches and 67% of T:C mismatches.     

Induction of anti-EBNA-1 protein by 12-O-tetradecanoylphorbol-13-acetate treatment of human lymphoblastoid cells.

Binding of the Epstein-Barr virus (EBV) nuclear antigen (EBNA-1) to BamHI-C DNA was studied by affinity column chromatography followed by immunoblotting with human serum specific for EBNA-1. Two species of EBNA-1 (68 and 70 kilodaltons) were identified in nuclear extracts of the EBV-positive Burkitt's lymphoma cell line Raji and not in nuclear extracts of the EBV-negative Burkitt's lymphoma cell line BJAB. Both EBNA-1s bound specifically to the region required for EBV plasmid DNA maintenance (oriP) located in the BamHI-C fragment. Upon treatment with 12-O-tetradecanoylphorbol-13-acetate, which activates latent EBV genome in Raji cells, the 68-kilodalton EBNA-1 was uncoupled from binding to EBV oriP. Nuclear extracts from 12-O-tetradecanoylphorbol-13-acetate-treated BJAB cells also uncoupled the binding of both EBNA-1s to oriP. DNA-cellulose column chromatography identified two protein species which competed for and uncoupled the binding of EBNA-1 to oriP. The two cellular competitors we called anti-EBNA-1 proteins had molecular masses of 60 and 40 kilodaltons, respectively. They were not found in nuclear extracts of BJAB cells not activated by 12-O-tetradecanoylphorbol-13-acetate.     

Recovery and potential utility of YACs as circular YACs/BACs

A method has been established to convert pYAC4-based linear yeast artificial chromosomes (YACs) into circular chromosomes that can also be propagated in Escherichia coli cells as bacterial artificial chromosomes (BACs). The circularization is based on use of a vector that contains a yeast dominant selectable marker (G418R), a BAC cassette and short targeting sequences adjacent to the edges of the insert in the pYAC4 vector. When it is introduced into yeast, the vector recombines with the YAC target sequences to form a circular molecule, retaining the insert but discarding most of the sequences of the YAC telomeric arms. YACs up to 670 kb can be efficiently circularized using this vector. Re-isolation of megabase-size YAC inserts as a set of overlapping circular YAC/BACs, based on the use of an Alu-containing targeting vector, is also described. We have shown that circular DNA molecules up to 250 kb can be efficiently and accurately transferred into E.coli cells by electroporation. Larger circular DNAs cannot be moved into bacterial cells, but can be purified away from linear yeast chromosomes. We propose that the described system for generation of circular YAC derivatives can facilitate sequencing as well as functional analysis of genomic regions.     

Production of high-titer Epstein-Barr virus recombinants derived from Akata cells by using a bacterial artificial chromosome system

An Epstein-Barr virus (EBV) genome in Burkitt's lymphoma-derived cell line Akata was cloned into a bacterial artificial chromosome (BAC) vector. The BAC clone, designated AK-BAC, was rapidly and precisely modified by means of efficient homologous recombination in Escherichia coli. This system was used to produce recombinant EBVs with transgenes. An expression cassette of green fluorescent protein (GFP) was inserted into AK-BAC, and the resultant BAC clone, AK-BAC-GFP, was transfected into Akata cells. We found that transfected BAC plasmids efficiently formed episomes in EBV-positive Akata cells. Mixtures of wild-type and AK-BAC-GFP viruses were then produced and used to infect EBV-negative Akata cells. We obtained cell clones that harbored only AK-BAC-GFP but no wild-type episome. These cell clones produced infectious viruses after stimulating virus production, and the recombinant viruses of AK-BAC-GFP efficiently immortalized primary B lymphocytes. We further revised the method so that any kind of cDNA could be rapidly inserted into the unique I-PpoI site that had been artificially introduced into AK-BAC. The AK-BAC system will have a broad range of applications, such as genetic analyses of various viral gene products and development of viral vectors for human gene therapy.