Epstein-Barr virus (B95-8) DNA VII: molecular cloning and detailed mapping.

Abstract

Two of the Sal I fragments and all of the internal BamHI fragments (with the exception of BamHI c, a 0.6 X 106 dalton fragment) of Epstein-Barr virus (EBV) DNA (2); DNA isolated from virus particles that have aged in the culture medium prior to harvest has frequent single-strand nicks and probably gaps arising from proteolytic degradation of virus and endonucleolytic degradation of the DNA (4-6). (iv) At both ends of the DNA there are from 1 or 2 to as many as 10-12 tandem repeats of a 500-base-pair sequence (5,6); the sequences at both ends of EBV DNA are direct repetitions of each other (6). (v) Beginning approximately 1.5 X 104 base pairs from one end of the DNA, there are from 1 or 2 to as many as 10 or 11 tandem reiterations of a 3000-base-pair sequence (7,8); these internal tandem reiterations connect the short unique region of 1.5 X 104 base pairs with the long unique region of 12.5 X 104 base pairs. (vi) The order of fragments generated by cleavage of the DNA with EcoRI, HindIII, Sal I, Xba I, Kpn I, and Hpa I is known in full or in part (5,8,9).More precise knowledge of the restriction enzyme sites in the DNA is needed for ongoing studies of EBV-specified mRNAs in growth-transformed human B lymphocytes (10) and in human Burkitt tumor tissue (11), for analysis of the more complex nuclear RNAs of growth-transformed cells (12, 13), for the mapping of virus-specified proteins in transformed and productively infected cells, for determination of whether EBV DNA is integrated into cellular DNA, and for comparison of EBV DNAs from different epidemiologic niches (5,14). For all of these purposes, it is necessary to obtain large quantities of specific viral DNA fragments. We therefore undertook to clone EBV DNA fragments in two vectors: the Col E1/pSC101The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "ad-

Spliced RNA from the IR1-U2 region of Epstein-Barr virus: presence of an open reading frame for a repetitive polypeptide.

“…About 30 000 colonies were screened by in situ hybridization. The viral BamHI-V, X and H restriction fragments (Figure iB), prepared respectively from the pDKI4, pDF307 and pDK286 recombinant plasmids (Dambaugh et al, 1980), were used as a probe. One positive clone, designated TI, was isolated.…”

Mapping genetic elements of Epstein-Barr virus that facilitate extrachromosomal persistence of Epstein-Barr virus-derived plasmids in human cells.

“…This plasmid, largely derived from pSV2neo (33), and which confers G-418r to animal cells, contains a single BamHI site at the 3' end of the G-418r gene and single EcoRI and XhoI sites 5' to the G-418r gene. A 5,097 bp EcoRI-to-PvuII segment of the strain B95-8 EBV genome, isolated from pDK10, which contains the BamHI-C fragment (2), was inserted between the EcoRI and XhoI sites of pSLneo, and the resultant plasmid was called p404 (Fig. 1A).…”

“…1A); these plasmids are named by the restriction site and end of their deletion. The BamHI-K fragment of the B95-8 EBV genome, isolated from pDF225 (2), was inserted at the single BamHI site of p404 to generate p410+ (Fig. 1B).…”

Epstein-Barr virus induces aggressive lymphoproliferative disorders of human B cell origin in SCID/hu chimeric mice.

“…20 ,Ag of tumor cell genomic DNA was digested with Eco RI or Msp I, electrophoresed in 0.7% or 1.7% agarose gels, respectively, and transferred by the alkaline blot method to nylon membranes, which were hybridized with the DNA probes. EBV DNA was detected with an Msp I fragment of the B95-8 Bam HI K region (coding for EBNA-1), from a pBR322-EBV recombinant library (14). Probes for c-myc and bc1-2 oncogenes were from Oncor, Gaithersburg, MD.…”