Characterization of the Chaetoceros salsugineum nuclear inclusion virus coat protein gene.
Analysis of the genome of Chaetoceros salsugineum nuclear inclusion virus (CsNIV) revealed the presence of six putative open reading frames (ORFs) in the genome. We further characterized ORF3, which encodes a putative coat protein. Polymerase chain reaction (PCR) using ORF3 gene-specific primers amplified a single DNA band nearly 1.2kb. This amplified product was gel-purified, cloned, sequenced, and expressed in Escherichia coli. Specific antiserum was raised against the recombinant protein and used for Western blotting to test whether the ORF3 protein is the CsNIV coat protein. One major CsNIV protein of approximately 46kDa reacted positively with the antiserum, suggesting that this antiserum is specific for the CsNIV coat protein. Matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) analysis of the 46kDa structural band revealed 14 peptide sequences that matched the ORF3 regions of CsNIV. The expression of ORF3 in host cells was examined by constructing a cDNA library of CsNIV-infected cells. Nucleotide sequences of the cDNA clones were complementary to various regions of both CsNIV ORF3 and ORF4; however, no clones containing only the ORF3 region were identified. Also, Northern blotting revealed a single 2.5-kb transcript, indicating that ORF3 could be transcribed together with ORF4.
Park
Yunjung
Y
Department of Microbiology, Pukyong National University, 599-1, Daeyeon 3-Dong, Nam-Gu, Busan 608-737, South Korea.
Jung
Sang-Eun
SE
Tomaru
Yuji
Y
Choi
Woobong
W
Kim
Yoonhee
Y
Mizumoto
Hiroyuki
H
Nagasaki
Keizo
K
Choi
Tae-Jin
TJ
D000595:Amino Acid Sequence; D001483:Base Sequence; D036022:Capsid Proteins / Q000737:chemistry / Q000235:genetics*; D017377:Diatoms / Q000821:virology*; D008969:Molecular Sequence Data; D016366:Open Reading Frames; D014764:Viral Proteins / Q000737:chemistry / Q000235:genetics; D014780:Viruses / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification*
D036022:Capsid Proteins; D014764:Viral Proteins