• Journal of Microbiology
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• v.44 no.5
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• pp.515-522
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• 2006

A cDNA library of Ganoderma lucidum has been constructed using a Zap Express cloning vector. A glyceraldehyde-3-phosphate dehydrogenase gene (gpd) was isolated from this library by hybridization of the recombinant phage clones with a gpd-specific gene probe generated by PCR. By comparison of the cDNA and the genomic DNA sequences, it was found that the complete nucleotide sequence encodes a putative polypeptide chain of 338 amino acids interrupted by 6 introns. The predicted amino acid sequence of this gene shows a high degree of sequence similarity to the GPD proteins from yeast and filamentous fungi. The promoter region contains a CT-rich stretch, two CAAT boxes, and a consensus TATA box. The possibility of using the gpd promoter in the construction of new transformation vectors is discussed.

• KSBB Journal
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• v.10 no.1
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• pp.38-45
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• 1995

The expression of Aspergillus nidulans glyceraldehyde-3-phosphate dehydrogenase (gpdA) and trpC promoters in A. oryzae were compared using E. coli lacZ gents fusions. The specific activities of the expressed E. coli $\beta$-galactosidase in A. oryzae transformants containing the A. nidulans gpdA promoter were around 2,000 units per ug of protein. The specific activities of transformants containing the A. nidulans trpC promoter were very low, ranging from 10.5 to 52.3 units per ug of protein. These results showed that the expression of the A. nidulans gpdA promoter in A. oryzae was approximately 70 times greater than the A. nidulans trpC promoter. In western blot analysis, immunoreactive bands of a imlilar molecular weight as the E. coli $\beta$-galactosidase were observed in A. oryzae carrying the gpdA-lacZ fusion and to a lesser intensity in those carrying the tvpC-lacZ fusion. Southern analysis showed that the higher expression of the gpdA-lacZ fusion as compared to the trpC-lacZ fusion was not due a greater number of integrated plasmids.

• Biotechnology and Bioprocess Engineering:BBE
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• v.6 no.3
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• pp.173-178
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• 2001

Food yeast, Saccharomyces cerevisiae, is a safe organism with a long history of use for the production of biomass rich in high quality proteins and vitamins. AmA1, a seed storage albumin from Amaranthus hypochondriacus, has a well-balanced amino acid composition and high levels of essential amino acids and offers the possibility of further improving food animal feed additives. In order to find an effective means of expressing AmA1 in yeast, the gene was cloned into an episomal shuttle vector. Four different promoters were tested: the glyceraldehyde-3-phosphate dehydrogenase promoter, galactose dehydrogenase 10 promoter, alcohol dehydrogenase II promoter, and a hybrid ADH2-GPD promoter. The recombinant AmA1 genes were then introduced into the yeast Saccharomyces cerevisiae 2805. Northern and Western blot analyses of the yeast under appropriate conditions revealed that AmA1 was expressed by all four promoters at varying levels. An enzyme-linked immunosorbent assay demonstrated that the amount of AmA1 protein in the recombinant yeast was 1.3-4.3% of the total soluble proteins. The highest expression level was obtained from the hybrid ADH2-GPD promoter.

• Biotechnology and Bioprocess Engineering:BBE
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• v.9 no.4
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• pp.292-296
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• 2004

The recombinant soluble human tumor necrosis factor-alpha (hTNF-$\alpha$) was expressed in a yeast Saccharomyces cerevisiae and its cytotoxicity was evaluated. A cDNA encoding hTNF-$\alpha$ was placed under the control of two different promoters: a glyceraldehyde-3-phosphate dehydrogenase (GPD) promoter and a yeast hybrid ADH2-GPD promoter, consisting of alcohol dehydrogenase II (ADH2) and the GPD promoter. A Northern blot analysis revealed that, although variation in the expression level of hTNF-$\alpha$ existed among transformants, the higher expression was obtained with the GPD promoter. Expressed hTNF-$\alpha$ protein (rhTNF-$\alpha$) was successfully secreted into the culture medium, producing 2.5 mg per liter of culture filtrate, with no changes in cell growth. The bioassay for observing the cytotoxicity to the murine L929 fibroblast cell line, with serial dilution of rhTNF-$\alpha$, indicated that the secreted rhTNF-$\alpha$ was bioactive and its dose-response was improved eight to ten times over that of the E. coli-derived rhTNF-$\alpha$.

• Journal of Microbiology and Biotechnology
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• v.13 no.4
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• pp.537-543
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• 2003

The mouse interferon gene (MuIFN-${\gamma}$) was cloned and then used to transform Saccharomyces cerevisiae. Expressed MuIFN-$\{gamma}$ protein (MuIFN-${\gamma}$) was successfully secreted into culture medium due to the presence oi the signal peptide of rice amylase 1A. Two different promoters fused to MuIFN-${\gamma}$ were tested: glyceraldehyde-3-phosphate dehydrogenase (GPD) promoter and a yeast hybrid ADH2-GPD (AG) promoter consisting of alcohol dehydrogenase II (ADH2) and GPD promoter. Using the hybrid promoter, the accumulation of MuIFN-${\gamma}$transcript was the highest after the 24 h cultivation, and then gradually decreased as the cultivation proceeded. However, both cell growth and recombinant MuIFN-${\gamma}$production reached their peaks after the 4-day cultivation. It was possible to produce 6.5 mg/l of MuIFN-${\gamma}$ without any changes in cell growth. Using GPD promoter, the MuIFN-${\gamma}$ transcript accumulation and the recombinant MuIFN-${\gamma}$ production followed the same pattern as the cell growth. However. compared to that of the hybrid promoter, the production of recombinant MuIFN-${\gamma}$ was 0.2 mg/l. The secreted MuIFN-${\gamma}$ had estimated molecular masses of 21 kDa and 23 kDa, which were larger than that of the encoded size due to glycosylation. The protection assay against the viral infection indicated that the recombinant MuIFN-${\gamma}$ was bioactive.

• The Korean Journal of Mycology
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• v.51 no.3
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• pp.245-249
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• 2023

Prickly lettuce (Lactuca serriola), an invasive annual plant, poses a significant threat to the agricultural systems of many countries, including Korea. In 2020, leaf blight symptoms were observed in the prickly lettuce populations of various farms across Korea. Detailed morphological and molecular sequence analyses revealed that the disease was caused by the fungus, Curvularia intermedia. A pathogenicity test confirmed that the fungus can cause the same symptoms in healthy prickly lettuce, thereby fulfilling Koch's postulates. To the best of our knowledge, this is the first report of C. intermedia causing leaf blight on L. serriola in Korea, suggesting its potential as a biocontrol agent for this weed. However, further investigations are necessary to determine its ecological impact to prevent any non-target effects.

• Journal of Microbiology and Biotechnology
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• v.11 no.6
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• pp.915-921
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• 2001

Phytase improves the bioavailability of phytate phosphorus in plant foods to humans and animals, and reduces the phosphorus pollution of animal waste. In order to express a high level of fungal phytase in Saccharomyces cerevisiae, various expression vectors were constructed with different combinations of promoters, translation enhancers, signal peptides, and terminator. Three different promoters fused to the phytase gene (phyA) from Aspergillus niger were tested: a galactokinase (GAL1) promoter, glyceraldehyde-3-phosphate dehydrogenase (GPD) promoter, and yeast hybrid ADH2-GPD promoter consisting of alcohol dehydrogenase II (ADH2) and a GPD promoter. The signal peptides of phytase, glucose oxidase (GO), and rice amylase 1A(RAmy1A) were included. Plus, the translation enhancers of the ${\Omega}$ sequence and UTR70 from the tobacco mosaic virus (TMV) and spinach, respectively, were also tested. Among the recombinant vectors, pGphyA06 containing the GPD promoter, the ${\Omega}$ sequence, RAmy1A, and GAL7 terminator expressed the highest phytase activity in a culture filtrate, which was estimated at 20 IU/ml. An intracellular localization of the expressed phytase activity in a culture filtrate, which was estimated at 20 IU/ml. An intracellular localization of the expressed phytase was also performed by inserting an endoplasmic reticulum (ER) retention signal, KDEL sequence, into the C-terminus of the phytase within the vector pHphyA-6. It appeared that the KDEL sequence directed most of the early expression of phytase into the intracellular compartment yet more than $60\%$ of the total phytase activity was still retained within the cell even after the prolonged (>3 days) incubation of the transformant. However, the intracellular enzyme activity of the transformant without a KDEL sequence was as high as that of the extracellular one, thereby strongly suggesting that the secretion of phytase in S. cerevisiae appeared to be the rate-limiting step for the expression of a large amount of extracellular recombinant phytase, when compared with other yeasts.

• Journal of Microbiology and Biotechnology
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• v.10 no.3
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• pp.287-292
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• 2000

Recombinant Aspergillus niger was constructed to express and secrete a biologically active murine granulaocyte macrophage-colony stimulating factor (mGM-CSF). A 500 bp fragment encoding the signal peptide and terminator of glyceraldehyde-3-phosphate dehydrogenase (gpd). The hygromycin phosphotrasferase gene (hph) was used as a selection marker for the fungal transformants. An expression vector was introduced into A. niger ATCC 9642, and a Northern blot analysis indicated the presence of a considerable amount of transcripts from the introduced mGM-CSF. The biological activity of recombinant mGM-CSF (rmGM-CSF) isolated from the culture filtrate was confirmend by measuring the proliferationof the GM-CSF dependent FDC-P1 cell line. It appeared that rmGM-CSF was amenable to the proteolytic activity produced by A. niger, since biological actibity was only observed when the transformants were grown in a protease-repressing medium, and the activity of rmGM-CSF dramatically decreased with an increase of age of the culture. The yield of rmGM-CSF, as determined by ELISA. was 640 ng/l of culture filtrate. Accordingly, its specific activity is estimated to be approximately two-and-a-half times higher than that of a commercial preparation from E. coli.

• KSBB Journal
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• v.17 no.2
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• pp.162-168
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• 2002

Human ferritin H- and L-chain genes(hfH and hfL) were cloned into the yeast shuttle vector YEp352 with various promoters, and the vectors constructed were used to transform Saccharomyces cerevisiae 2805. Three different promoters fused to hfH and hfL were used: galactokinase 1 (GAL1) promoter, glyceraldehyde-3-phosphate dehydrogenase(GPD) promoter and alcohol dehydrogenase 1(ADH1 ) promoter. SDS-polyacrylamide gel electrophoresis and Western blotting analyses displayed expression of the introduced hfH and hfL. In the production of both ferritin H and L subunits GAL1 promoter was more effective than GPD promoter or ADH1 promoter. Ferritin H and L subunits produced in S. cerevisiae were spontaneously assembled into its holoproteins as proven on native polyacrylamide gels. Both recombinant H and L-chain ferritins were catalytically active in forming iron core. When the cells were cultured in the medium containing 10 mM ferric citrate, the cell-associated concentration of iron was 174.9 $\mu\textrm{g}$ Per gram(dry cell weight) for the recombinant yeast YG-L and 148.8 $\mu\textrm{g}$ Per gram(dry cell weight) for the recombinant yeast YG-L but was 49.4 $\mu\textrm{g}$ Per gram(dry cell weight) in the wild type, indicating that the iron contents of yeast is improved by heterologous expression of human ferritin H-chain or L-chain genes.

• Mycobiology
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• v.46 no.2
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• pp.172-176
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• 2018

A new species belonging to the genus Alternaria was isolated from the necrotic leaf spots of Brassica rapa subsp. pekinensis in Yuseong district, Daejeon, Korea. It is an occasional isolate, not an etiological agent, which is morphologically similar to A. broccoli-italicae, but differs in conidial size and conidiophore shape. Phylogenetic analysis using the sequence datasets of the internal transcribed spacer (ITS) region of the rDNA, glyceraldehyde-3-phosphate dehydrogenase (gpd), and plasma membrane ATPase genes showed that it is distantly related to A. broccoli-italicae and closely related to Alternaria species in the section Pseudoalternaria, which belonged to a clade basal to the section Infectoriae. Morphologically, the species is unique because it produces solitary conidia or conidial chains (two units), unlike the four members in the section Pseudoalternaria that produce conidia as short branched chains. It exhibits weak pathogenicity in the host plant. This report includes the description and illustration of A. brassicifolii as a new species.