A physical map of the Mamestra configurata nucleopolyhedrovirus genome and sequence analysis of the polyhedrin gene.

Abstract

The genome structure of a nucleopolyhedrovirus (NPV) isolated from the bertha armyworm, Mamestra configurata Walker (Lepidoptera: Noctuidae) (MacoNPV) was analysed with six restriction endonucleases (REN): BamHI, EcoRI, HindIII, PstI, SmaI and Xhol. More than 70 MacoNPV REN fragments were cloned into plasmids pUC18 and pBluescript SK(+). The physical map with 112 restriction sites for the above REN was constructed using double digests and Southern blot hybridization analysis of the MacoNPV DNA clones. The size of the DNA genome of the MacoNPV-90/2 isolate used for this study was estimated at 156 kbp based on REN fragment sizes. The position of the polyhedrin gene, which has by convention been used as the zero point of the REN maps of NPV, was determined by hybridizing the Autographa californica multicapsid nucleopolyhedrovirus HindIII-V fragment clone, which contains most of the polyhedrin gene, with genomic blots of MacoNPV. The cloned MacoNPV fragments identified as containing the polyhedrin gene were sequenced and an ORF coding for a 246 amino acid polypeptide with 98.7% sequence identity with Panolis flammea nucleopolyhedrovirus (PaflNPV) polyhedrin protein was identified. The putative polyhedrin gene sequence had 97.2% and 91.2% identity with the PaflNPV and Mamestra brassicae multicapsid nucleopolyhedrovirus polyhedrin gene sequences, respectively, and also contained an upstream region identical to the highly conserved 12 bp consensus sequence TGTAAGT-AATTT typical of NPV very late genes.