Submitted to: Entomological Society of America Annual Meeting
Publication Type: Abstract Only
Publication Acceptance Date: 10/29/2003
Publication Date: 10/27/2003
Citation: Girsch, K.M., Behle, R.W. 2003. Exploring restriction endocuclease inhibition by UV-induced pyrimidine dimers as a means of showing AfMNPV inactivation after exposure to UV energy [abstract]. Entomological Society of America 51st Annual Meeting. Abstract No. D0120. p. 67.
Technical Abstract: The inhibition of restriction endonuclease activity through the formation of UV-induced pyrimidine dimers in Anagrapha falcifera nucleopolyhedrovirus (AfMNPV) DNA was explored as a molecular method for showing UV damage. Cytosine and thymine have been shown to form dimers with adjacent pyrimidine bases upon exposure to UV energy. These dimers can potentially disrupt restriction endonuclease activity, resulting in a loss of expected bands and an appearance of novel bands during gel electrophoresis. In vivo produced viral occlusion bodies (OBs) were purified on a sucrose gradient, suspended in water, and exposed to a Xenon lamp designed to mimic sunlight. Wet virus samples were exposed to 0, 9.2 x 10(5), 1.14 x 10(6), 1.37 x 10(6), 1.60 x 10(6), 1.83 x 10(6) Ws/m of energy in the 300-400 nm range, determined using an LI-1800 spectroradiometer (LiCor). DNA was isolated using an Easy-DNA(TM) Kit (Invitrogen) and digested with Dra I (AAA/TTT) and Rsa I (GT/AC), as a control with no adjacent pyrimidines. Our insecticidal assay against Trichoplusia ni (Hübner) showed a loss of viral activity at all exposure levels, yet our electrophoresis showed no evidence of endonuclease activity disruption. This molecular technique was not as sensitive as our insect assay, and therefore not effective for correlating dimer formation from UV exposure with a loss of insecticidal activity. However, it was noted that less DNA was isolated from samples exposed to more light energy. This might suggest that some mechanism of UV degradation may be affecting our isolation of damaged DNA.