Epstein-Barr virus (EBV) infects human B lymphocytes, transforming the infected cells into dividing blasts that can proliferate indefinitely. The viral genome of 172 kilobase pairs (kbp) is a plasmid in most transformed cells. We have identified a region of EBV DNA, termed oriP (nucleotides 7,333-9,109 of strain B95-8), which acts in cis to permit linked DNAs to replicate as plasmids in cells containing EBV DNA. We have postulated the existence of a trans-acting gene allowing oriP function. Here we report that this gene lies in a 2.6-kbp region of the viral genome (nucleotides 107, 567-110, 176) which encodes the EBNA-1 antigen. We show that circular DNAs containing oriP, the EBNA-1 gene and a selectable marker replicate autonomously in cells derived from at least four developmental lineages and from at least three species. We also find that the one-third of the EBNA-1 gene repetitive in sequence is not essential for the trans-acting function that EBNA-1 gives oriP.
The Epstein-Barr viral (EBV) genome of -170 kilobase pairs (kbp) is maintained as a plasmid in human B lymphoblasts transformed by the virus. We have identified a cis-acting element within 1.8 kbp of the viral genome that allows recombinant plasmids carrying it to be selected at high frequency and maintained as plasmids in cells latently infected by EBV. This functional element(s) requires a segment of DNA at least 800 bp and at most 1800 bp long, which contains a family of 30-bp tandem repeats at one end. Since this region confers efficient stable replication only to plasmids transfected into cells containing EBV genomes, its function probably requires trans-acting products encoded elsewhere in the viral genome.Epstein-Barr virus (EBV) is a human lymphotropic herpesvirus that causes infectious mononucleosis and lymphoproliferative disorders in immunosuppressed individuals. It is also probably a causative agent of Burkitt lymphoma and nasopharyngeal carcinoma in some parts of the world (for a review, see ref. 1). The causal association of EBV to diseases is likely a result of its capacity to induce unlimited proliferation in infected cells (2). Progress in understanding cell transformation by EBV has been limited by its large genome [172 kilobase pairs (kbp)], the likelihood that more than one gene is required for transformation (3), and the difficulty of introducing DNA stably into lymphocytes.Our approach in studying transformation by EBV has been to seek to define the genomic location of functions required for viral DNA replication in transformed cells. Since the viral genome is maintained as an independently replicating plasmid (4) in most cells transformed in vivo or in vitro (4-7), plasmid replication functions are probably required for efficient transformation. In addition, mapping the location of viral replication functions would allow the construction of a small EBV plasmid replicon suitable for the manipulations of molecular cloning, which in turn could aid in locating the remaining genes involved in transformation. Using a method described below, we scanned the EBV genome for cis-acting functions that would permit stable plasmid replication, and we found one such element in the short unique region of the genome. MATERIALS AND METHODSCell Lines. D98/Raji Cl 5 and D98/HR1 Cl 1 are hybrids between the human epithelial cell line D98 and the EBV genome-positive lymphoma lines Raji and P3JHR1, respectively (8,9). Both contain multiple copies of the EBV genome (ref. 10; unpublished results). These lines were grown in RPMI 1640 medium except during transfections, when they were in Dulbecco's modified Eagle's medium.Human fibrosarcoma 143 cells (11) and mouse BALB 3T3 fibroblasts (12) were grown in Dulbecco's modified Eagle's medium. Both media were supplemented with 10% fetal bovine serum.Transfections. About 1 x 106 cells in 6-cm dishes were transfected without carrier DNA, essentially as described by Graham and Van der Eb (13), with plasmid DNAs at 10 ,g/ml in Hepes-buffered saline (14). After 4 to ...
A viral promoter that functions on recombinant plasmids in cells immortalized by Epstein-Barr virus was identified and characterized. It is identical to that mapped on the viral genome by Bodescot et al. (M. Bodescot, M. Perricaudet, and P. J. Farrell, J. Virol. 61:3424-3430, 1987) which functions during the latent phase of the viral life cycle in some but not all cells to encode several latent viral gene products. Experiments with these plasmids indicated that this promoter requires the enhancer within oriP of Epstein-Barr virus in cis to function efficiently. They also indicated that it requires the EBNA-1 gene in trans to function efficiently. The EBNA-1 gene therefore positively affects both viral DNA replication (J.
Hybrid plasmids containint the bacterial resistance-transfer factor pBR322 and the yeast leu2+ gene have been used to isolate DNA fragments of Physarum that are capable of initiating DNA replication in a yeast host. Five of forty hybrid plasmids containint Physarum sequences transform leu2- yeast to Leu+ at high frequency. The resulting Leu+ transformants are characterized by phenotypic instability. Supercoiled plasmid molecules containing pBR322 sequences can be detected in the transformed yeast, indicating that the transforming DNA replicates autonomously. Plasmid DNA isolated from Leu+ yeast can transform leuB bacteria. The hybrid plasmid recovered from the Leu+ bacterial transformants is identical to the original plasmid, indicating structural integrity is maintained during passage through the yeast host. These hybrid plasmids containing Physarum sequences have the same characteristics as those containing autonomously replicating yeast chromosomal sequences. As the temporal sequence of DNA replication is particularly accessible to study in Physarum plasmodia, the functional significance of these segments should be amenable to study.
Using a selfing strain of Physarumpolycephalum that forms haploid plasmodia, we have isolated temperature-sensitive growth mutants in two ways. The negative selectant, netropsin, was used to enrich for temperature-sensitive mutants among a population of mutagenized amoebae, and, separately, a nonselective screening method was used to isolate plasmodial temperature-sensitive mutants among clonal plasmodia derived from mutagenized amoebae. Complementation in heterokaryons was used to sort the mutants into nine functional groups. When transferred to the restrictive temperature, two mutants immediately lysed, whereas the remainder slowed or stopped growing. Of the two lytic mutants, one affected both amoebae and plasmodia, and the other affected plasmodia alone. The growth-defective mutants were examined for protein and deoxyribonucleic acid synthesis and for aberrations in mitotic behavior. One mutant may be defective in both protein and deoxyribonucleic acid synthesis, and another only in deoxyribonucleic acid synthesis. The latter shows a striking reduction in the frequency of postmitotic reconstruction nuclei at the restrictive temperature. We believe that this mutant, MA67, is affected in a step in the nuclear replication cycle occurring late in G2. Execution of this step is necessary for both mitosis and chromosome replication.The regulation of cell replication has been under investigation in a number of laboratories. The genetic approach to this problem has centered on the isolation and characterization of temperature-sensitive mutants defective in the cell division cycle. Such mutational analysis has been extensively exploited in bakers' yeast by Hartwell and co-workers (11) and in fission yeast (21), Aspergillus (18), and Chiamydomonas (13). Most mutants have been genetically studied in regard to dominance and epistasis (11,12), and a few defects are understood at the molecular level (6,20). Ideally, a system should facilitate molecular as well as genetic analysis. Some desirable properties are the availability of synchronous cultures, radioactive labeling of macromolecular synthesis, and morphological landmarks for cell cycle analysis. Physarum polycephalum has the potential for all of the above: several grams of synchronous plasmodia are readily obtained; radioactive precursors are quickly incorporated into DNA, RNA, and protein; and mitosis and nucleolar reconstruction are easily observed. t It is a pleasure to dedicate this paper to Dr. Harold P. Rusch on the occasion of his 70th birthday. We particularly note his signal contributions in establishing the biochemical analysis of growth in Physarum polycephauwn and in maintaining the esprit of many Physarum workers.Also, complementation and the coordination of the nuclear replication cycle can be studied in easily prepared heterokaryons.In this publication we report the results of a study designed to identify mutants defective in nuclear replication. Such mutants are expected to arrest an asynchronous culture at a unique point in the nuclear replication cy...
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