• Molecules and Cells
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• v.27 no.3
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• pp.359-363
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• 2009

Chfr, a checkpoint with FHA and RING finger domains, plays an important role in cell cycle progression and tumor suppression. Chfr possesses the E3 ubiquitin ligase activity and stimulates the formation of polyubiquitin chains by Ub-conjugating enzymes, and induces the proteasome-dependent degradation of a number of cellular proteins, including Plk1 and Aurora A. While Chfr is a nuclear protein that functions within the cell nucleus, how Chfr is localized in the nucleus has not been clearly demonstrated. Here, we show that nuclear localization of Chfr is mediated by nuclear localization signal (NLS) sequences. To reveal the signal sequences responsible for nuclear localization, a short lysine-rich stretch (KKK) at amino acid residues 257-259 was replaced with alanine, which completely abolished nuclear localization. Moreover, we show that nuclear localization of Chfr is essential for its checkpoint function but not for its stability. Thus, our results suggest that NLS-mediated nuclear localization of Chfr leads to its accumulation within the nucleus, which may be important in the regulation of Chfr activation and Chfr-mediated cellular processes, including cell cycle progression and tumor suppression.

• Molecules and Cells
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• v.24 no.2
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• pp.194-199
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• 2007

GCN4 is a typical eukaryotic transcriptional activator that is implicated in the expression of many genes involved in amino acids and purine biosyntheses under stress conditions. It is degraded by 26S proteasomes following ubiquitination. However, the immediate receptor for ubiquitinated Gcn4p has not yet been identified. We investigated whether ubiquitinated Gcn4p binds directly to Rpn10p as the ubiquitinated substrate receptor of the 26S proteasome. We found that the level of Gcn4p increased in cells deleted for Rpn10p but not in cells deleted for RAD23 and DSK2, the other ubiquitinated substrate receptors and, unlike Rpn10p, neither of these proteins recognized ubiquitinated Gcn4p. These results suggest that Rpn10p is the receptor that binds the polyubiquitin chain during ubiquitin-dependent proteolysis of Gcn4p.

• Journal of Microbiology and Biotechnology
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• v.31 no.5
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• pp.740-746
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• 2021

Efficient cellulolytic enzyme production is important for the development of lignocellulose-degrading enzyme mixtures. However, purification of cellulases from their native hosts is time- and labor-consuming. In this study, a constitutive expression system was developed in Penicillium oxalicum for the secreted production of proteins. Using a constitutive polyubiquitin gene promoter and cultivating with glucose as the sole carbon source, nine cellulolytic enzymes of different origins with relatively high purity were produced within 48 h. When supplemented to a commercial cellulase preparation, cellobiohydrolase I from P. funiculosum and cellobiohydrolase II from Talaromyces verruculosus showed remarkable enhancing effects on the hydrolysis of steam-exploded corn stover. Additionally, a synergistic effect was observed for these two cellobiohydrolases during the hydrolysis. Taken together, the constitutive expression system provides a convenient tool for the production of cellulolytic enzymes, which is expected to be useful in the development of highly efficient lignocellulose-degrading enzyme mixtures.

• Journal of Plant Biotechnology
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• v.34 no.2
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• pp.145-152
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• 2007

Lactoferrin is an 80-kDa iron-binding glycoprotein known to exert many biological activities, such as facilitating iron absorption and having antimicrobial and anti-inflammatory effects. Rice can be a useful target for edible food plants to introduce human lactoferrin, because it has lower allergenicity and is likely to be safer than microorganisms or transgenic animals. A cDNA fragment encoding human lactoferrin (HLF) driven by the maize polyubiquitin promoter, along with herbicide resistance gene (bar) driven by CaMV 35S promoter, was introduced into rice (Oryza sativa L. cv. Dong Jin) using the Agrobacterium -mediated transformation system. Putative transformants were initially selected on the medium containing bialaphos. The stable integration of the bar and HLF genes into transgenic rice plants was further confirmed through polymerase chain reaction (PCR) and Southern blot analyses. The expression of the full length HLF protein from various tissues such as grains and young leaves of transgenic rice was verified by Western blot analysis. Analysis of progeny also demonstrated that introduced genes were stably inherited to the next generation at the Mendelian fashion.

• ALGAE
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• v.31 no.2
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• pp.167-174
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• 2016

Biological response of cells to variable conditions should affect the expression level of certain genes. Quantification of these changes in target genes needs stable internal controls. Real-time quantitative polymerase chain reaction (PCR) has traditionally used reference or ‘housekeeping’ genes, that are considered to maintain equal expression in different conditions, to evaluate changes in target genes between samples and experimental conditions. Recent studies showed that some housekeeping genes may vary considerably in certain biological samples. This has not been evaluated in red algae. In order to identify the optimal internal controls for real-time PCR, we studied the expression of eleven commonly used housekeeping genes; elongation factor 1-alpha, glyceraldehyde-3-phosphate dehydrogenase, β-actin, polyubiquitin, 30S ribosomal gene, 60S ribosomal gene, beta-tubulin, alpha-tubulin, translation initiation factor, ubiquitin-conjugating enzyme, and isocitrate dehydrogenase in different life-history stages of Bostrychia moritziana. Our results suggest that glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and 30S ribosomal gene, have the most stable gene expression levels between the different life history stages (male, female, carposporophyte, and tetrasporophyte), while the other genes are not satisfactory as internal controls. These results suggest that the combinations of GAPDH and 30S would be useful as internal controls to assess expression level changes in genes that may control different physiological processes in this organism or that may change in different life history stages. These results may also be useful in other red algal systems.