Biomedical subjects

Takefumi Doi

Publications and source records attributed to Takefumi Doi.

21 records · Page 2Linked to original sources

PubMed2002

Hepatitis C virus core protein binds to a C-terminal region of NS5B RNA polymerase.

Hepatitis C virus (HCV) NS5B has been shown to exhibit RNA-dependent RNA polymerase activity for its viral RNA replication. In this study, we demonstrated the formation of a complex between NS5B and the core protein (NS5B-core protein complex) in mammalian cells, as determined by indirect immunofluorescence and immunoprecipitation analyses. The localization of the core protein was observed to change to the same locus in ER as NS5B locates by its coexpression with NS5B, indicating that the localization of the core protein is determined by NS5B. The truncated NS5B molecule lacking the C-terminal region did not form a complex with the core protein, suggesting that the C-terminal region of NS5B is essential for its interaction with the core protein. Moreover, the change in NS5B localization because of C-terminal deletion indicates that this region includes a certain signal for NS5B retention in ER.

Journal Article↗

PubMed2002

Identification of a chromosome-targeting domain in the human condensin subunit CNAP1/hCAP-D2/Eg7.

CNAP1 (hCAP-D2/Eg7) is an essential component of the human condensin complex required for mitotic chromosome condensation. This conserved complex contains a structural maintenance of chromosomes (SMC) family protein heterodimer and three non-SMC subunits. The mechanism underlying condensin targeting to mitotic chromosomes and the role played by the individual condensin components, particularly the non-SMC subunits, are not well understood. We report here characterization of the non-SMC condensin component CNAP1. CNAP1 contains two separate domains required for its stable incorporation into the complex. We found that the carboxyl terminus of CNAP1 possesses a mitotic chromosome-targeting domain that does not require the other condensin components. The same region also contains a functional bipartite nuclear localization signal. A mutant CNAP1 missing this domain, although still incorporated into condensin, was unable to associate with mitotic chromosomes. Successful chromosome targeting of deletion mutants correlated with their ability to directly bind to histones H1 and H3 in vitro. The H3 interaction appears to be mediated through the H3 histone tail, and a subfragment containing the targeting domain was found to interact with histone H3 in vivo. Thus, the CNAP1 C-terminal region defines a novel histone-binding domain that is responsible for targeting CNAP1, and possibly condensin, to mitotic chromosomes.

Adenosine Triphosphatases↗

PubMed2002

The generation of monoclonal antibodies against human peroxisome proliferator-activated receptors (PPARs).

Monoclonal antibodies (Mabs) are valuable reagents for the purification, characterization and immunolocalization of proteins. In this study, we raised Mabs against human peroxisome proliferator-activated receptors (PPARs) using baculovirus particles displaying surface glycoprotein gp64-fusion proteins as the immunizing agent. In this system, to display fusion proteins on the viral surface, the amino terminal sequences of human PPARd and PPARg2 are inserted in-frame between the signal sequence and the mature domain of the gp64 nucleotide sequence.Mabs were raised by immunization with whole virus without a purification of the target antigens. The Mabs generated by this novel method were shown to recognize not only the gp64-PPARs fusion protein, but also mature, expressed proteins by a wide variety of techniques, including immunohistochemistry, immunoblotting, and electrophoretic mobility shift assays (EMSAs). Transfection of the transfer vector containing a nucleotide sequence encoding less than 30 amino acids along with linearized baculovirus DNA allows for the production of a high affinity antibody against the corresponding mature form. This method is of potential utility in that it allows the production of valuable antibodies without the requirement of a protein purification step.

Animals↗