• BMB Reports
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• v.33 no.6
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• pp.493-498
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• 2000

Aquifex pyrophilus, a hyperthermophilic bacterium, has a serine-type protease that is located at the cell wall fraction with a mature size of 43 kDa. Molecular cloning of the protease gene revealed that it has an ORF of 619 amino acids with homologous catalytic site of serine-type proteases [Choi, I.-G., Bang, W.-K., Kim, S.-H., Yu, G. Y., J. Biol. Chem. (1999), Vol. 274, pp. 881-888]. Constructs containing different regions of the protease gene, including a alanine-substituted mutant at the active site serine, were constructed, and the factors affecting the expression level of the cloned protease gene in E. coli were examined. The presence of the C-terminus hydrophobic region of the protease hindered over-expression in E. coli. Also, the proteolytic activity of the expressed protein appeared to toxic to E. coli. An inactive form that deleted both of the N-terminal signal sequence and the C-terminal polar residues was over-expressed in a soluble form, purified to homogeneity, and its thermostability examined. The purified protein showed three disulfide bonds and three free sulfhydryl group. The thermal denaturation temperature of the protein was measured around $90^{\circ}C$ using a differential scanning calorimeter and circular dichroism spectrometry. The disulfide bonds were hardly reduced in the presence of reducing agents, suggesting that these disulfide bonds were located inside of the protein surface.

• Korean Journal of Microbiology
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• v.45 no.4
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• pp.312-317
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• 2009

Genome analysis of a model filamentous fungus, Aspergillus nidulans, revealed that there are six putative forkhead genes. Among them, fkhF (AN8949.2) showed A. nidulans-specific. fkhF gene is located in chromosome VII and composed of 2,337 bp coding region for 778 amino acid. Since little is known about the involvement of the forkhead proteins in the developmental process of the filamentous fungi, including A. nidulans, we generated a deletion mutant of fkhF gene and analyzed. Deletion of fkhF resulted in less-dense conidiophore formation in a solid culture. However, the sexual developmental process or cleistothecia formation was normal. Furthermore, fkhF deletion mutant produced conidiophores and conidia under the submerged culture, suggesting that the fkhF gene is involved in repression of inappropriated induction and maturation of asexual developmental process but not in sexual development.

• Molecules and Cells
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• v.23 no.3
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• pp.304-315
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• 2007

Fusarium graminearum causes a serious scab disease of small grains in Korea. The nucleotide sequence of the genomic RNA of a double-stranded RNA (dsRNA) virus, Fusarium graminearum virus-DK21 (FgV-DK21), from F. graminearum strain DK21, which is associated with hypovirulence in F. graminearum, was determined and compared to the genome sequences of other mycoviruses, including Cryponectria hypoviruses. The FgV-DK21 dsRNA consists of 6,624 nucleotides, excluding the 3'-terminal poly(A) tail. The viral genome has 53- and 46-nucleotide 5' and 3' untranslated regions (UTRs), respectively, and five putative open reading frames. A phylogenetic analysis of the deduced amino acid sequence of ORF1, which encodes a putative RNA-dependent RNA polymerase, and those of other mycoviruses revealed that this organism forms a distinct virus clade with other hypoviruses, and is more distantly related to other mycoviruses (3.8 to 24.0% identity). However, pairwise sequence comparisons of the nucleotide and deduced amino acid sequences of ORFs 2 through 5 revealed no close relationships to other protein sequences currently available in GenBank. Analyses of RNA accumulation by Northern blot and primer extension indicated that these putative gene products are expressed from at least two different subgenomic RNAs (sgRNAs), in contrast to the cases in other hypoviruses. This study suggests the existence of a new, as yet unassigned, genus of mycoviruses that exhibits a potex-like genome organization and sgRNA accumulation.

• Journal of fish pathology
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• v.35 no.2
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• pp.141-155
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• 2022

Rock bream iridovirus (RBIV) causes high mortality and economic losses in the rock bream (Oplegnathus fasciatus) aquaculture industry in Korea. Viral open reading frames (ORFs) expression profiling at different RBIV infection stages was investigated using microarray approaches. Rock bream were exposed to the virus and held for 7 days at 23 ℃ before the water temperature was reduced to 17 ℃. Herein, 28% mortality was observed from 24 to 35 days post infection (dpi), after which no mortality was observed until 70 dpi (end of the experiment). A total of 27 ORFs were significantly up- or down-regulated after RBIV infection. In RBIV-infected rock bream, four viral genes were expressed after 2 dpi. Most RBIV ORFs (26 genes, 96.2%) were significantly elevated between 7 and 20 dpi. Among them, 12 ORF (44.4%) transcripts reached their peak expression intensity at 15 dpi, and 14 ORFs (51.8%) were at peak expression intensity at 20 dpi. Expression levels began to decrease after 25 dpi, and 92.6% of ORFs (25 genes) were expressed below 1-fold at 70 dpi. From the microarray data, in addition to the viral infection, viral gene expression profiles were categorized into three infection stages, namely, early (2 dpi), middle (7 to 20 dpi), and recovery (25 and 70 dpi). RBIV ORFs 009R, 023R, 032L, 049L, and 056L were remarkably expressed during RBIV infection. Furthermore, six ORFs (001L, 013R, 052L, 053L, 058L, and 061L) were significantly expressed only at 20 dpi. To verify the cDNA microarray data, we performed quantitative real-time PCR, and the results were similar to that of the microarray. Our results provide novel observations on broader RBIV gene expression at different stages of infection and the development of control strategies against RBIV infection.

• Korean Journal of Fisheries and Aquatic Sciences
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• v.57 no.1
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• pp.41-52
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• 2024

Serotransferrin cDNA from the Siberian sturgeon Acipenser baerii was isolated and its expression patterns during early life intervals were characterized. It contained an ORF encoding a 708-aa-long polypeptide, including a 19-aa signal peptide. Bioinfomatic analysis and 3D modeling indicated a typical bi-lobal structure with conserved iron-coordinating residues. During embryonic development, the potential transition of maternally provisioned transcripts to zygotically de novo transcribed ones occurred around blastula stage. The transferrin mRNA levels peaked at stages responsible for pronephros, heart and erythropoietic component differentiation. After hatching, the transferrin mRNA expression gradually increased at early ontogenic phases (0 to 3 DPH) corresponding to the periods in which prolarvae exhibited increased blood circulation and liver differentiation. The expression decreased at subsequent stages in which prolarvae exhibited benthic movement. The tissue distribution assay indicated liver-predominant expression at fingerling stage. From the microinjection-based challenge with Aeromonas hydrophila at day-0 and day-7, the transcriptional response was modulated toward upregulation, in which the amounts induced at 6, 12 and 24 HPI were greater in prolarvae injected at day-7 than at day-0. Therefore, transferrin plays important roles in both early development and host protective responses to pathogens in the Siberian sturgeon.

• Asian-Australasian Journal of Animal Sciences
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• v.20 no.9
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• pp.1319-1326
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• 2007

Fructose-1,6-bisphosphatase (FBP1) is a key regulatory enzyme of gluconeogenesis that catalyzes the hydrolysis of fructose-1,6-bisphosphate to generate fructose-6-phosphate and inorganic phosphate. Deficiency of fructose-1, 6-bisphosphatase is associated with fasting hypoglycemia and metabolic acidosis. The enzyme has been shown to occur in bacteria, fungi, plants and animals. The bovine FBP1 gene was cloned and characterized in this study. The full length (1,241 bp) FBP1 mRNA contained an open reading frame (ORF) encoding a protein of 338 amino acids, a 63 bp 5' untranslated region (UTR) and a 131 bp 3' UTR. The bovine FBP1 gene was 89%, 85%, 82%, 82% and 74% identical to the orthologs of pig, human, mouse, rat and zebra fish at mRNA level, and 97%, 96%, 94%, 93% and 91% identical at the protein level, respectively. This gene was broadly expressed in cattle with the highest level in testis, and the lowest level in heart. An intronic single nucleotide polymorphism (SNP) (A/G) was identified in the $5^{th}$ intron of the bovine FBP1 gene. Genotyping of 133 animals from four beef breeds revealed that the average frequency for allele A (A-base) was 0.7897 (0.7069-0.9107), while 0.2103 (0.0893-0.2931) for allele B (G-base). Our preliminary association study indicated that this SNP is significantly associated with traits of Average Daily Feed Intake (ADFI) and Carcass Length (CL) (p<0.01). In addition, the FBP1 gene was assigned on BTA8 by a hybrid radiation (RH) mapping method.

• Korean Journal of Microbiology
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• v.49 no.1
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• pp.8-16
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• 2013

A GAL4 type transcription factor gene (formally annotated as AN3912) locating downstream of sndA (AN3911) was characterized. The putative transcription factor carries both Zn(II)2Cys6 binuclear cluster DNA-binding domain and transcription activator domain. The gene named gtfA (gal4 type transcription factor) had an open reading frame which consisted of 762 amino acids and was disrupted by three introns. The deletion mutant produced reduced amount of conidia but increased amount of fruiting bodies, suggesting that the GtfA make function in decision of asexual preferential to sexual development. The forced over expression of gtfA caused the retardation of fruiting body formation on high glucose concentration. The transcript level of gtfA was kept constant through the life cycle except late vegetative stage and early sexual development stage during which slight increase was found. The expression of gtfA was not significantly affected by sexual or asexual development regulators, such as VeA, NsdD or FluG, FadA, and SfaD. The GtfA repressed the nsdC transcription, which suggested that GftA control sexual development negatively via negative regulation of nsdC expression.

• Journal of Microbiology and Biotechnology
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• v.17 no.1
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• pp.58-66
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• 2007

Isolation, expression, and characterization of a novel $endo-{\beta}-1,3(4)-D-glucanase$ with high specific activity and homology to Bacillus lichenases is described. One clone was screened from a genomic library of Paenibacillus sp. F-40, using lichenan-containing plates. The nucleotide sequence of the clone contains an ORF consisting of 717 nucleotides, encoding a ${\beta}-glucanase$ protein of 238 amino acids and 26 residues of a putative signal peptide at its N-terminus. The amino acid sequence showed the highest similarity of 87% to other ${\beta}-1,3-1,4-glucanases$ of Bacillus. The gene fragment Bg1 containing the mature glucanase protein was expressed in Pichia pastoris at high expression level in a 3-1 high-cell-density fermenter. The purified recombinant enzyme Bg1 showed activity against barley ${\beta}-glucan$, lichenan, and laminarin. The gene encodes an $endo-{\beta}-1,3(4)-D-glucanase$ (E. C. 3.2.1.6). When lichenan was used as substrate, the optimal pH was 6.5, and the optimal temperature was $60^{\circ}C$. The $K_m,\;V_{max},\;and\;k_{cat}$ values for lichenan are 2.96mg/ml, $6,951{\mu}mol/min{\cdot}mg,\;and\;3,131s^{-1}$, respectively. For barley ${\beta}-glucan$ the values are 3.73mg/ml, $8,939{\mu}mol/min{\cdot}mg,\;and\;4,026s^{-1}$, respectively. The recombinant Bg1 had resistance to pepsin and trypsin. Other features of recombinant Bg1 including temperature and pH stability, and sensitivity to some metal ions and chemical reagents were also characterized.

• Journal of Life Science
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• v.18 no.8
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• pp.1090-1095
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• 2008

Marbling (intramuscular fat) is an important factor in determining meat quality in Korean beef market. A grain based finishing system for improving marbling leads to inefficient meat production due to an excessive fat production. Identification of intramuscular fat-specific gene might be achieved more targeted meat production through alternative genetic improvement program such as marker assisted selection (MAS). We carried out ddRT-PCR in 12 and 27 month old Hanwoo steers and detected 300 bp PCR product of the inducible cAMP early repressor (ICER) gene, showing highly gene expression in 27 months old. A 1.5 kb sequence was re-sequenced using primer designed base on the Hanwoo EST sequence. We then predicted the open reading frame (ORF) of ICER gene in ORF finder web program. Tissue distribution of ICER gene expression was analysed in eight Hanwoo tissue using realtime PCR analysis. The highest ICER gene expression showed in Small intestine followed by Longissimus dorsi. Interestingly, the ICER gene expressed 2.5 time higher in longissimus dorsi than in same muscle type, Rump. For gene expression analysis in high- and low marbled individuals, we selected 4 and 3 animal based on the muscle crude fat contents (high is 17-32%, low is 6-7% of crude fat contents). The ICER gene expression was analysed using ANOVA model. Marbling (muscle crude fat contents) was affected by ICER gene (P=0.012). Particularly, the ICER gene expression was 4 times higher in high group (n=4) than low group (n=3). Therefore, ICER gene might be a functional candidate gene related to marbling in Hanwoo.

• IMMUNE NETWORK
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• v.4 no.1
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• pp.23-30
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• 2004

Background: A human orthologue of mouse S100A6-binding protein (CacyBP), Siah-1-interacting protein (SIP) had been shown to be a component of novel ubiquitinylation pathway regulating $\beta$-catenin degradation. The role of the protein seems to be important in cell proliferation and cancer evolution but the expression pattern of SIP in actively dividing cancer tissues has not been known. For the elucidation of the role of SIP protein in carcinogenesis, it is essential to produce monoclonal antibodies specific to the protein. Methods: cDNA sequence coding for ORF region of human SIP gene was amplified and cloned into an expression vector to produce His-tag fusion protein. Recombinant SIP protein and monoclonal antibody to the protein were produced. The N-terminal specificity of anti-SIP monoclonal antibody was conformed by immunoblot analysis and enzyme linked immunosorbent assay (ELISA). To study the relation between SIP and colon carcinogenesis, the presence of SIP protein in colon carcinoma tissues was visualized by immunostaining using the monoclonal antibody produced in this study. Results: His-tag-SIP (NSIP) recombinant protein was produced and purified. A monoclonal antibody (Korea patent pending; #2003-45296) to the protein was produced and employed to analyze the expression pattern of SIP in colon carcinoma tissues. Conclusion: The data suggested that anti-SIP monoclonal antibody produced here was valuable for the diagnosis of colon carcinoma and elucidation of the mechanism of colon carcinogenesis.