Characterization of the ecdysteroid UDP-glucosyltransferase (egt) gene of Anticarsia gemmatalis nucleopolyhedrovirus.
The Anticarsia gemmatalis nucelopolyhedrovirus (AgMNPV) egt gene was cloned, sequenced and its expression characterized by RT-PCR and western blot analysis. Sequence analysis of the gene indicated the presence of an open reading frame (ORF) of 1482 nucleotides, which codes for a polypeptide of 494 amino acids. ATATA box and a conserved regulatory sequence (CATT) found in other baculovirus early genes were present in the promoter region of the egt gene. A poly-A consensus sequence was present in the 3' untranslated region (3'-UTR) of the gene. Homology comparisons showed that the EGT protein of AgMNPV is most closely related (95.9% amino acid sequence identity) to the EGT from the Choristoneura fumiferana DEF nucleopolyhedrovirus (CfDEF). Transcriptional analysis of the AgMNPV egt gene showed that egt-specific transcripts can be detected both early and late in infection. The EGT protein was detected, by western blot analysis, in the intra- (from 12 to 48 h post-infection) and extra-cellular (from 12 to 96 h post-infection) fractions of infected insect cells. The AgMNPV Bgl II-F fragment, which has homology to the AcMNPV ie-1 gene, was cloned and used to cotransfect SF21 cells with the cloned AgMNPV egt gene. EGT activity was observed, suggesting that AgMNPV ie-1 can transactivate egt expression.
Rodrigues
J C
JC
Embrapa Genetic Resources and Biotechnology, Brasília, Brasil. carlyle@cenargen.embrapa.br
De Souza
M L
ML
O'Reilly
D
D
Velloso
L M
LM
Pinedo
F J
FJ
Razuck
F B
FB
Ribeiro
B
B
Ribeiro
B M
BM
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D015153:Blotting, Western; D002478:Cells, Cultured; D003001:Cloning, Molecular; D005964:Glucosyltransferases / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D007915:Lepidoptera / Q000821:virology; D008969:Molecular Sequence Data; D017924:Nucleopolyhedroviruses / Q000201:enzymology* / Q000235:genetics; D020133:Reverse Transcriptase Polymerase Chain Reaction; D017422:Sequence Analysis, DNA; D018411:Spodoptera / Q000821:virology; D014158:Transcription, Genetic
D005964:Glucosyltransferases; C086659:ecdysteroid UDP-glucosyltransferase