By Bernard N. Fields and Rudolf Jaenisch (Eds.)
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D. thesis, Mass. Institute of Technology. 10. , and Schaefer, F. ), Cold Spring Harbor Laboratory, pp. 469-475. 11. , and Hendrix, R. (1974) J_. Mol. Biol. 90:20-25. 12. A. Jr. (1975) J. Mol. Biol. 9L· 439-462. 13. M. (1975) J. Mol. Biol. 9L· 421-438. 14. M. S. and Fox, C F . A. Benjamin, pp. 363-384. 15. M. , and Botstein, D. (1975) J_. Mol. Biol. 98_: 413-424. 16. , Langenaur, C , and Agabian, N. (1976) J_. Virol. 17: 568-575. MOLECULAR CLONING OF THE HUMAN CYTOMEGALOVIRUS GENOME (STRAIN AD169) 1 Joyce C.
MOLECULAR CLONING OF THE HUMAN CYTOMEGALOVIRUS GENOME (STRAIN AD169) 1 Joyce C. Tamashiro and Deborah H. Spector Department of Biology, C-016; University of California, San Diego; La Jolla, CA 92093 ABSTRACT Human Cytomegalovirus (HCMV), one of the herpesviruses, is medically significant both as a cause of birth defects and as a source of problems in immunosuppressed individuals. Because the virus is highly cellassociated, it is difficult to obtain large quantities of pure viral DNA. For this reason we have sought to construct a cloned library of the HCMV genome to aid in further studies of the molecular biology of HCMV infections.
Viral DNA for the cloning was obtained from a low multiplicity infection of human embryonic lung (HEL) cells with a plaquepurified stock of the HCMV strain AD169. We routinely use low multiplicity infections in the propagation of virus to minimize the number of defective particles produced. The extracellular virus was harvested from the medium and isolated by centrifugation through a pre-formed cesium chloride gradient. After treatment with SDS and pronase, the DNA was purified twice by isopycnic cesium chloride centrifugation.