Identification, Cloning and Nucleotide Sequencing of the Granulin Genes

Certain insect baculoviruses are occluded in a proteinaceous, crystalline structure which serves to protect the virus outside its insect host. Two groups of occluded viruses have been defined: the nuclear polyhedrosis viruses (NPVs) (Baculovirus Subgroup A), having many virions occluded per crystal, and the granulosis viruses (GVs) (Baculovirus Subgroup B), having only one virion per crystal. Using the cloned polyhedrin gene from the Orgyia pseudotsugata MNPV (OpMNPV) as a hybridization probe, a 2 kilobase Sal I fragment from the Trichoplusia ni granulosis virus (TnGV) was identified as the locus of the granulin gene. This fragment was cloned into pUC8 and mapped with restriction enzymes. Three fragments of about 400 base-pairs each were subcloned into reciprocal M13 vectors and their nucleotide sequences were determined by the dideoxy method. A 1.3 kilobase Eco RI clone containing most of the granulin gene from TnGV was used as a hybridization probe to identify the granulin gene from Pieris brassicae granulosis virus (PbGV). This gene was also cloned, mapped, subcloned and dideoxy sequenced. The amino acid sequences derived from both the PbGV and TnGV granulin gene sequences are about 70 % conserved with respect to each other and about 50 % conserved related to the Lepidopteran NPV poly-hedrins. This suggests that the GVs form a distinct branch of Baculoviruses which evolved before the extensive divergence of the Lepidopteran NPVs. Highly conserved sequences within these genes suggest regions of granulin and polyhedrin which might be determinants of higher-order structure. Comparison of the 5 ' flanking regions of both granulins, several NPVs and another hyperexpressed late gene, the "10k " protein of Autographa californica MNPV, reveals a highly conserved sequence which may be a regulatory element involved in governing the expression of these genes