"Foreign Citizens" and Public Sphere in Japan

departmental bulletin pape

Observation of the Color-Suppressed Decay B̅ 0→D0π0

journal articl

データベースガク ブンヤ ガイヨウ

video/mp4講演場所: バイオサイエンス研究科大講義室講演者所属: 奈良先端科学技術大学院大学教授, 助教授および助手vide

Quantitative analysis of vRNA, cRNA, and mRNA levels for wild-type and mutant PB1 proteins.

<p>293T cells were transfected with plasmids expressing PB2, PA, NP, and wild-type or mutant PB1 protein, along with a plasmid transcribing a viral RNA. The levels of vRNA, cRNA, and mRNA were assessed by quantitative RT-PCR and compared to those obtained with wild-type PB1 protein. The results shown represent the average levels of RNAs ± standard deviations from a representative experiment carried out in triplicate. As a negative control (Mock), the PB1 protein expression plasmid was omitted.</p

Western blot analysis of wild-type and mutant PB1 proteins.

<p>293T cells were transfected with plasmids expressing wild-type or mutant PB1 protein. Western blot analysis was carried out with antibodies to PB1 and beta-actin (control).</p

Effects of PB1 mutation K480R on influenza polymerase activity in human and avian cells.

<p>The effects of PB1 mutation K480R were evaluated in the background of the A/California/04/09 (H1N1) virus replication complex in human 293T and chicken DF-1 cells. The experiments were carried out as described in the legend to <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0036113#pone-0036113-g001" target="_blank">Figure 1</a>. Luciferase activity levels were normalized by the <i>Renilla</i> internal control and expressed as a percentage relative to wild-type PB1 activity in the particular cell line. *P<0.07 by Student’s t-test.</p

Mutations in the conserved motifs I – IV of the PB1 protein and their effects on replicative ability in human and chicken cells.

1<p>The indicated mutations were introduced into the PB1 protein of A/bar-headed goose/Qinghai/1/2005 (H5N1; BHG) virus. Polymerase activity in human 293T and avian DF-1 cells is indicated as percentage relative to wild-type PB1 activity.</p>2<p>For some of these isolates, different sequences are reported in the Los Alamos National Laboratory and NIH NCBI databases.</p

Avian virus PB1 proteins with mutations in the conserved motifs I – IV.

*<p>Percentage of total changes.</p

Influenza A virus PB1 proteins with mutations in the conserve motifs I – IV.

*<p>Percentage of total changes.</p

Effects of mutations in the four conserved motifs of PB1 on influenza polymerase activity.

<p>(<b>A</b>) Motif I; (<b>B</b>) Motif II; (<b>C</b>) Motif III; (<b>D</b>) Motif IV. Consensus sequences are listed by motif with numbers at each end indicating the position of the first and last amino acid residue. Invariant amino acids are underlined. Selected mutations found in the four conserved motifs of natural isolates (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0036113#pone-0036113-t003" target="_blank"><b>Table 3</b></a>) were introduced into the PB1 protein of A/bar-headed goose/Qinghai/1/2005 (H5N1) virus. The polymerase activity of each PB1 mutant was assessed in human 293T cells by quantifying luciferase activity in the cell lysates of cells transfected with PB1- (wild type or mutant), PB2-, PA- and NP-protein expression plasmids along with a reporter plasmid expressing an influenza virus-like RNA construct for luciferase. Luciferase activity levels were normalized by the <i>Renilla</i> internal control and expressed as a percentage relative to wild-type PB1 activity set as 100%. Data values are averaged over three independent transfection experiments where each experiment was run in triplicate. Error bars represent one standard deviation.</p