MIYAGI Mobuaki Faculty of Bioresource Sciences, Akita Prefectural University, Post-doctoral fellow, 附属生物工学研究所, 流動研究員
UEDA Kenji Faculty of Bioresource Sciences, Akita Prefectural University, Assistant Professor, 附属生物工学研究所, 助手 (80279504)
ONO Michiyuki Faculty of Bioresource Sciences, Akita Prefectural University, Associate Professor, 附属生物工学研究所, 助教授 (50201405)
|Budget Amount *help
¥1,800,000 (Direct Cost : ¥1,800,000)
Fiscal Year 2000 : ¥500,000 (Direct Cost : ¥500,000)
Fiscal Year 1999 : ¥600,000 (Direct Cost : ¥600,000)
Fiscal Year 1998 : ¥700,000 (Direct Cost : ¥700,000)
To identify nuclear matrix proteins in higher plants, we have constructed a library of monoclonal antibodies raised against carrot nuclear matrix proteins. In this study, we describe the characteristics of three cDNA clones isolated using these antibodies.
The cDNA clone, 8G4-66b, is 1,467 bp in length and encodes a deduced protein, deisignated p48, of 418 amino acids with a molecular weight of 48.2 kDa and four putative nuclear localization signals(NLSs). No protein was found in the databases with a high level of sequence identity. Antiserum raised against the recombinant p48 recognized not only carrot nuclei but also nuclei from tobacco and rice. These results suggest that the p48 is a novel and highly-conserved internal matrix protein in higher plants.
The cDNA clone, 8G4-50, is 2,060 bp long and encodes a protein, p64, of 581 amino acids with a molecular weight of 64.4 kDa. Primary structure analysis of the deduced protein revealed two thioredoxin-like active sites and an endoplasmic
reticulum(ER)-retention signal at its C-terminus, as also found in protein disulfide isomerases(PDIs) in plants and animals. Although the carrot protein and plant PDIs show only a 30% identity over their complete length, their active site regions are almost identical. The recombinant p64 had a similar level of enzymatic activity as commercially-available calf PDI.Therefore, our results suggest that p64 is a novel member of the PDI family in plants. An examination of the subcellular localization of this novel PDI using specific antibodies, is currently under way.
The cDNA clone, 2A7-85, is 2,203 bp in length and encodes a protein, p69, of 602 amino acids with a molecular weight of 69.4 kDa and two typical putative NLSs. Immunocytochemical analysis using a specific antiserum raised against the recombinant p69 revealed that the protein was localized only to the nucleolus. However, the deduced protein did not show a high level of sequence identity to known nuclear or nucleolar proteins in the databases. We therefore conclude that p69 is a novel nucleolar protein. Less