Characterization of Hyphantria cunea nucleopolyhedrovirus gp64 gene and analysis of elements regulating its early promoter activity

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  • Alves Cristiano A. Felipe
    Laboratory of Biodynamics, Graduate School of Bioagricultural Sciences, Nagoya University
  • Ikeda Motoko
    Laboratory of Sericulture and Entomoresources, Graduate School of Bioagricultural Sciences, Nagoya University
  • Kobayashi Michihiro
    Laboratory of Biodynamics, Graduate School of Bioagricultural Sciences, Nagoya University

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  • Characterization of <i>Hyphantria cunea</i> Nucleopolyhedrovirus <i>gp64</i> Gene and Analysis of Elements Regulating Its Early Promoter Activity

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Hyphantria cunea nucleopolyhedrovirus (HycuNPV) homologue (hycu-gp64) of baculovirus gp64 gene was identified and characterized. The hycu-gp64 open reading frame encoded a polypeptide of 509 amino acids with a predicted molecular weight (MW) of 58, 378, that shared 72 to 82% amino acid sequence identities with GP64's from other group I NPVs. The Hycu-GP64 possessed ten putative N-glycosylation sites that represented the largest number among GP64's so far sequenced. Analyses of the hycu-gp64 promoter region revealed a number of elements responsible for transcriptional regulation, that included an early transcription start motif (CAGT), three late transcription start motifs (A/T/GTAAG), an IE1 binding motif (IBM: 5′-ACBYGTAA-3′)-like sequence, a TATA box, two GATA elements, and a heat shock factor motif. Northern blot analysis revealed the presence of two major transcripts (1.7 and 1.6kb) that were detectable from 2h postinfection (pi), whereas Western blot analysis showed a polypeptide with an approximate MW of 68, 000 that was detected from 4h pi onward. Transient expression assays suggested that basal activity of the hycu-gp64 early promoter depended on the presence of the TATA box, whereas full activity of the hycu-gp64 early promoter on a HycuNPV homologous repeated sequence, hycu-hr6, that was located immediately upstream of the hycu-gp64 promoter. Cotransfection experiments with a plasmid containing the HycuNPV ie1(hycu-ie1) showed that Hycu-IE1 stimulated the transcription from the hycu-gp64 early promoter, although gene expression from the full hycu-gp64 promoter was two times lower than that from the construct carrying the hycu-gp64 promoter with a deletion of an IBM-like and GATA sequences. The transient expression assays also demonstrated that hycu-gp64 contained a secretory signal sequence that was active in uninfected Splm cells.

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