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Title: Identification, characterization and functional studies of serine/threonine kinase of spodoptera litura nucleopolyhedrovirus - I (SpltNPV-I)
Researcher: Mishra, Gourav
Guide(s): Hari Das, Rakha
Keywords: Biotechnology
Upload Date: 26-Aug-2011
University: University of Pune
Completed Date: September, 2007
Abstract: An open reading frame (ORF) of 819 nt to code for a 272 amino acids protein is identified in the genome of Spodoptera litura nucleopolyhedrovirus (SpltNPV-I). Nucleotide and nucleotide sequence derived amino acid sequence analysis of this ORF suggested it to be a eukaryotic type protein kinase having conserved I-XI subdomains of Hanks kinase. In addition to kinase catalytic domains, this putative protein has two bromo-domains which could play regulatory role in transcription. The ORF expressed as ~31 kDa apoprotein in E. coli and ~33 kDa glycoprotein in Sf9 cells, the expressed protein is designated as SpltNPV-I pk1 or pk1. The protein is localized in the nucleus of the SpltNPV-I infected permissive cell line NIV-HA-197. The recombinant protein has auto-phosphorylation and substrate phosphorylation (MBP and Histone H1) activities in presence of Mn+2 or Mg+2, and these activities are inhibited by staurosporine. Mutation of Lys-50 to Met but not Lys-44 to Gln of the protein abolished its kinase activity. Kinetics of pk1 showed that rate of phosphorylation of SpltNPV-I pk1gt MBPgt Histone H1, and both MBP and Histone H1 have the Km of 3 µM. Analysis of phosphorylated protein showed the phosphorylation of serine and threonine residues but not tyrosine. All these results suggested that identified SpltNPV-I ORF codes for a serine/threonine kinase. Polyhedrin (polh) and p10 are the two hyper-expressed very late genes of nucleopolyhedroviruses. Alpha amanitin resistant transcription from SpltNPV-I polh promoter occurred with virus infected nuclear extract of NIV-HA-197 cells but not with that from uninfected nuclear extract. Anti-pk1 antibody inhibited the transcription and the inhibition reversed on addition of pk1, however, pk1 mutant protein, K50M having no phosphorylation activity did not overcome the transcription inhibition.
Pagination: 110p.
Appears in Departments:Institute of Genomics and Integrative Biology

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01_title.pdfAttached File154.64 kBAdobe PDFView/Open
02_certificate.pdf145 kBAdobe PDFView/Open
03_declaration.pdf107.81 kBAdobe PDFView/Open
04_dedication.pdf235.16 kBAdobe PDFView/Open
05_acknowledgements.pdf113.48 kBAdobe PDFView/Open
06_contents.pdf111.4 kBAdobe PDFView/Open
07_list of figures and tables.pdf165.54 kBAdobe PDFView/Open
08_abstract.pdf176.8 kBAdobe PDFView/Open
09_preface.pdf136.55 kBAdobe PDFView/Open
10_abbreviations.pdf160.95 kBAdobe PDFView/Open
11_chapter 1.pdf887.26 kBAdobe PDFView/Open
12_chapter 2.pdf314.06 kBAdobe PDFView/Open
13_chapter 3.pdf8.75 MBAdobe PDFView/Open
14_chapter 4.pdf157.77 kBAdobe PDFView/Open
15_chapter 5.pdf193.94 kBAdobe PDFView/Open
16_bibliography.pdf162.05 kBAdobe PDFView/Open
17_appendix.pdf187.7 kBAdobe PDFView/Open

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