Physical mapping and field inversion gel electrophoresis of Amsacta moorei entomopoxvirus DNA

Agarose in situ digestion was used to prepare intact Amsacta moorei entomopoxvirus (AmEPV) DNA from embedded occlusion bodies (OBs). Direct dissolution of OBs in agarose eliminated the necessity for separate purification of virions. A physical map of AmEPV DNA was constructed for five restriction enzymes (BamHI, EcoRI, HindIII, PstI, and XhoI) using single and multiple digests, and isolated fragment digestions. End fragments were identified by Bal31 digestion and snap-back analysis. A least squares procedure was used to reconcile fragment lengths. AmEPV genome size estimates were based on restriction enzyme (REN) fragment length totals (222 kb), reconciled physical map distance (225 kb), and field inversion gel electrophoresis (FIGE) (about 242 kb). Presumably due to the high A + T content (18.5% G + C) of AmEPV DNA, FIGE values for the intact genome and large REN fragments were about 6 to 10% higher than expected. Preparative FIGE was used to concentrate AmEPV DNA from agarose microbead encapsulated insect cells (Estigmene acrea, BTI-EAA). REN digests of this DNA were identical to those from OBs from caterpillars.