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Restriction Mapping of Lymantria dispar Nuclear Polyhedrosis Virus DNA: Localization of the Polyhedrin Gene and Identification of Four Homologous Regions

J. Thomas McClintock

Insect Pathology Laboratory, Agricultural Research Service, U.S. Department of Agriculture, Beltsville, Maryland 20705, U.S.A.

The genome of the multiple-embedded nuclear polyhedrosis virus (MNPV) of Lymantria dispar (LdMNPV) was partially characterized by restriction endonuclease analysis and a physical map was constructed using cosmid cloning and Southern cross blot hybridization. Using BamHI, BglII, EcoRI and HindIII, the size of the genome was estimated to be 88.5 x 10⁶ M_r or 134.04 kbp. LdMNPV DNA was also analysed using methylation-sensitive restriction enzymes. The resulting restriction profiles suggested that extensive methylation did not occur at the nucleotide sequence recognized by HpaII and MspI. A BamHI restriction map was constructed by comparing overlapping BamHI fragments between cosmid clones containing partial digests of viral DNA. The positions of the BglII, EcoRI and HindIII sites were determined by Southern cross blot hybridizations and aligned to the BamHI restriction map. At least four homologous regions were identified by cross blot hybridizations of BglII-digested LdMNPV DNA and such regions were found to be interspersed along the genome in a fashion similar to that reported for other baculoviruses. Using recombinant plasmids containing the HindIII-V fragment of Autographa californica MNPV to probe Southern blots of LdMNPV DNA, the restriction fragment(s) that contain the polyhedrin gene were identified. Based on these findings the map was oriented with the polyhedrin gene of LdMNPV as the zero point.

Keywords: baculovirus, polyhedrin, L. dispar

Present address: Digene Diagnostics Inc., Building 334, University of Maryland, College Park, Maryland 20742, U.S.A.

Received 21 December 1988; accepted 20 May 1988.