Gene, 27 (1984) 279-288 Elsevier 279 GENE 957 A complete set of overlapping cosmid clones of M-ABA virus derived from nasopharyngeal carcinoma and its similarity to other Epstein-Barr virus isolates (Recombinant DNA; ,? Charon cloning vector; tandem repeats; Escherichiu zyxwvutsrqponmlkjihgfedcbaZYXW coli; herpesvirus; plasmid; blot hybridization) Axe1 Polack, Gabriele Hartl, Ursula Zimber, Ulrich-Karl Freese, Gerhard Laux, Kazutaka Takaki, Barbara Hohn *, Lutz Gissmann and Georg W. Bornkamm ** Institutftir Virologie,Zentrum ftir Hygiene, Universitiit Freiburg, D 7800 Freiburg (F.R. G.) Tel. (0761) 203-3253, and * Friedrich Miescher Institut, CH 4002 Base1 (Switzerland) Tel. (0041) 61376682 (Received August 5th, 1983) (Revision received November 20th, 1983) (Accepted November 23rd, 1983) SUMMARY DNA of the transforming, nondefective Epstein-Barr virus (EBV) strain M-ABA, which is derived from nasopharyngeal carcinoma cells, was cloned as large overlapping pieces into the cosmid pHC79. The termini were cloned from closed circular virus DNA molecules out of M-ABA cell DNA in phage 1L47. The large overlapping clones were used to prepare a library of subclones with inserts of 1-15 kb. A detailed restriction enzyme map of M-ABA virus DNA reveals the close similarity to isolates from other sources. The high number of tandem repeats in EBV DNA stresses the importance of using cloning vectors that can be propagated in recA - Escherichia coli hosts. INTRODUCTION The EBV, a lymphotropic human herpesvirus, is the causative agent of infectious mononucleosis. Ad- ditionally, the virus is associated with two human malignancies, Burkitts lymphoma and nasopharyn- ** To whom all correspondence and reprint requests should be directed. Abbreviations: bp, base pairs; c, cosmid; DS,, DS,, duplicated sequences left and right, respectively (see Fig. 1); DTT, dithio- threitol; EBNA, Epstein-Barr nuclear antigen; EBV, Epstein- Barr virus; kb, kilobase pairs; p, plasmid; U,, Us, long and short unique regions (see Fig. 1). 0378-I 119/84/$03.00 0 1984 Elsevier Science Publishers geal carcinoma (for review see Epstein and Achong, 1979). Infection of lymphocytes of human origin or of new world monkey species induces unlimited pro- liferation of B lymphocytes in vitro. EBV-transform- ed lymphocytes, as well as tumor cells from Burkitts lymphoma and nasopharyngeal carcinoma, contain multiple copies of predominantly circular EBV ge- nomes (zur Hausen et al., 1970; Lindahl et al., 1976; Kaschka-Dierich et al., 1976) and express the viral nuclear antigen EBNA (Reedman and Klein, 1973). In cell lines carrying EBV usually only a small and variable number of cells produces virus particles. The DNA isolated from virus particles is a double- stranded linear DNA molecule of about 175 kb with identical tandem repeats of about 450 bp at both