• Journal of Life Science
• /
• v.21 no.12
• /
• pp.1666-1677
• /
• 2011

The regulation of gene expression plays an important role in cell cycle controls. In this study, a novel gene, the $mas1^+$($\underline{mi}$tosis $\underline{as}$sociated protein) gene, a homolog of human CIP29/Hcc1, was isolated and characterized from fission yeast Schizosaccharomyces pombe (S. pombe) using a gene-specific polymerase chain reaction. The isolated gene contained a complete open reading frame capable of encoding 245 amino acid residues with a typical promoter, as judged by nucleotide sequence analysis. It was also found that a PCB ($\underline{p}$ombe cell $\underline{c}$ycle $\underline{b}$ox) is located in the promoter region, which controls M-$G_1$ specific transcription in S. pombe. The quantitative analysis of the $mas1^+$ transcript against $adh1^+$ showed that the pattern of expression is similar to that of the septation index. Cytokinesis of mas1 mutant was greatly delayed at $25^{\circ}C$ and $36^{\circ}C$, and a large number of multi-septate cells were produced. The mas1 mutant had 2C, 4C and 6C DNA contents, as determined by FACS analysis. In addition, the number of multi-septate cells significantly increased. When cells were cultured in nitrogen starvation medium to increase proliferation, the abnormal phenotypes of mas1 mutant dramatically increased. These phenotypes could be rescued by an overexpression of the $mas1^+$ gene. The mas1 protein localized in the nuclei of S. pombe and human HeLa cells, as evidenced by Mas1-EGFP signals. The abnormal growth pattern and the morphology of mas1 mutant were complemented by a plasmid carrying human CIP29/Hcc-1cDNA. In addition, CIP29 /Hcc-1 transcript level increased in active cell proliferation stages in the developing mouse embryos. These results indicate that the $mas1^+$ ishomologous to the human CIP29/Hcc1 gene and is involved in cytokinesis and cell shape control.

• Applied Biological Chemistry
• /
• v.38 no.1
• /
• pp.55-62
• /
• 1995

To understand the molecular structure of Korean garlic viruses, cDNA cloning of virus genomic RNA was attempted. Virus particles were isolated from virus-infected garlic leaves and a cDNA library was constructed from garlic virus RNA. One of these clones, S81, selected by random sequencing has been identified as a member of potexvirus group other than potyvirus and carlavirus. The clone is 873 bp long contains most of the coat protein (CP) coding region and 3'-noncoding region including poly(A) tail. A putative polyadenylation signal sequence (AAUAAA) and the hexanucleotide motif (ACUUAA), a replicational cis-acting element conserved in the 3'-noncoding region of potexvirus RNAs are noticed. The clone S81 shows about 30-40% identity in both nucleotide and amino acid sequences with CPs of potexviruses. The genome size of the virus was analysed to be 7.46 knt by Northern blot analysis, which was longer than those of other potexviruses. The open reading frame encoding CP was expressed as a fusion protein (S81CP) in Escherichia coli and the recombinant protein was purified by immobilized metal binding affinity chromatography. Polyclonal antibody was raised against S81CP in rabbit to examine the occurrence of garlic potexvirus in Korean garlic plants by immunoblot analysis. Two virus protein bands of Mr 27,000 and 29,000 from garlic leaf extract of various cultivars reacted with the antibody. It was shown that Mr 27,000 band might not be a degradation product of Mr 29,000 band, suggesting that two types of potexvirus different in size of coat protein could exist in Korean garlic plants.

• Microbiology and Biotechnology Letters
• /
• v.14 no.6
• /
• pp.495-500
• /
• 1986

Enzymatic micro-assay methods were studied those were capable of determining ammonia down to 10$^{-5}$M(0.01 $\mu$mole/ml) in the presence of other nitrogenous compounds such as protein and amino acid. Microquantities of ammonia (0.01-0.1 $\mu$mole) could be determined indirectly by measuring phosphorous, one of the products of the enzymatic reaction catalyzed by glutamine synthetase. In this reaction, L-glutamate, ATP and ammonium chloride were used as substrates, and phosphorous was formed in propotion to the concentration of ammonium chloride In the reaction mixture. Another procedure was examined in which glutamine synthetase reaction coupled with pyruvate kinase and lactate dehydrogenase reactions was used. One mililiter of the assay mixture contained; phosphoenol pyruvate, 3 mM, L-glutamate, 10 mM; ATP, 1mM: MgSO$_4$, 20 mM: KCl, 75mM: NADH, 0.2mM: Tris-HCl buffer(pH 7.0), 100mM; pyruvate kinase, 10 U: lactate dehydrogenase, 12 U and glutamine synthetase, 4 U. After preincubation for 20 min at 3$0^{\circ}C$, NH$_4$Cl was added and the rates of NADH oxidation were followed at 340nm. The effective range of this method was proved to be from 0.01 to 0.05 $\mu$mole/$m{\ell}$. Glutamine synthetase reaction coupled with glutamate synthase reaction could also be effectively used for determining microquantities of ammonia. The one mililiter assay mixture contained; ATP, 5mM: L-glutamate, 5mM; L-ketoglutarate, 5mM; MgCl$_2$, 15mM; NADPH, 0.15mM; Tris-HCl buffer(pH 7.0); 100mM; glutamine synthetase, 1U and glutamate synthase, 0.5U. After preincubation for 20min at 3$0^{\circ}C$ NH$_4$Cl was added and the rates of NADPH oxidation were followed at 340nm. The effective range of this procedure was appeared to be from 0.01 to 0.05$\mu$mole/$m{\ell}$.

• Journal of Life Science
• /
• v.19 no.9
• /
• pp.1218-1225
• /
• 2009

Difractose anhydrides (DFAs) is studied as a sweetener for diabetics because of its structural property. DFAs have four types: DFA I, III, IV (degradation of levan) and V (degradation of inulin). Especially, DFA IV has been shown to enhance the absorption of calcium in experiments using rats. Levan fructotransferase is an enzyme for producing di-d-fructose-2,6':6,2-dianhydride (DFA IV). To identify structural characterization, we purified wild-type and mutants (D63A, D195N and N85S) of levan fructotransferase (LFTase) from Microbacterium sp. AL-210. These proteins were purified to apparent homogeneity by Ni-NTA affinity column, Q-sepharose ion exchange and gel filtration chromatography and detected by SDS-PAGE. They were also analyzed by circular dichroism (CD) measurements, JNET secondary structure prediction, activity measurements at various temperatures, and pH analysis. The optimum pH for the enzyme-catalyzed reaction was pH 7.5 and optimum temperature was observed at $55^{\circ}C$. Along with wild-type LFTase, mutants were analyzed by CD measurement, fluorescence analysis and differential scanning calorimetry (DSC). N85S showed less $\alpha$-helix and more $\beta$ strand than others. Also, N85S showed almost the same curve as wild-type in their steady-state fluorescence spectra, whereas mutant D63A and D195N showed higher intensity than wild-type. The amino acid sequence of wild-type LFTase was compared to the sequences of exo-inulinase from Aspergillus awamori, a plant fructan 1-exohydrolase from Cichorium intybus, and Thermotogo maritime (Tm) invertase and showed a high identity with Exo-inulinase from Aspergillus awamori.

• Journal of Animal Science and Technology
• /
• v.49 no.6
• /
• pp.737-744
• /
• 2007

An experiment was conducted to compare the ME values of imported corns measured by In vivo(TME) and In vitro(MEn, ME and MEpc) methods and to investigate the true amino acid availability(TAAA) and the true nutrient availability(TNA) of imported corns. For TME assay, twenty four 57-weeks-old Hy-Line roosters were assigned to fasting group(4 roosters) and four corn groups(5 roosters each): USA; corn produced in the United States, ARG; corn produced in Argentina, CHN; regular corn produced in China, CHNP; premium corn produced in China. The MEn, ME and MEpc values were determined by equations based on chemical analysis. The TME value of USA(3,745kcal/kg) in as fed basis was significantly (P<0.01) higher than ARG(3,555kcal/kg) and CHNP(3,518kcal/kg) but was not significantly different from CHN(3,671kcal/kg). The TME value of USA(4,144kcal/kg) in DM basis was not significantly different from CHN(4,060kcal/kg) and CHNP(4,008kcal/kg) but was significantly(P<0.05) higher than ARG(4,001kcal/kg). There were significant differences in TAAA of phenylalanine, histidine and arginine among imported corns. Those of USA were highest but overall TAAA was not significantly different among imported corns. True availability of NFE of USA, ARG and CHN was significantly(P<0.05) higher than that of CHNP. However, true availability of crude protein, crude fat, crude fiber and crude ash were not significantly different among corns. The correlation coefficient between TME and MEn value was 0.91 which was significant at P<0.1 but correlation coefficient between TME and ME value and between TME and MEpc value was 0.90 and 0.83, respectively which was not significant at P<0.1. In conclusion, US corn was highest in TME values and Chinese premium corn was not significantly different from regular Chinese corn. The MEn value obtained by equation based on chemical analysis may be used as a tool to evaluate TME value of corn.

• Journal of Life Science
• /
• v.19 no.1
• /
• pp.101-110
• /
• 2009

The regulation of gene expression plays an important role in cell cycle controls. In this study, a novel $mas2^+$ (mitosis associated protein) gene, a homolog of human SMARCAD1 was isolated and characterized from a fission yeast Schizosaccharomyces pombe (S. pombe) using gene-specific polymerase chain reaction. The isolated gene contained a complete open reading frame capable of encoding 922 amino acid residues with a typical promoter, as judged by nucleotide sequence analysis. It was also found that an SNF2 domain is located, which is involved in the chromosome remodeling. The quantitative analysis of the $mas2^+$ transcript against $adh1^+$ showed that the expression level of $mas2^+$ is high before septum formation in S. pombe. When $mas2^+$ null mutant cells were grown at 27 and $35^{\circ}C$, the cytokinesis of $mas2^+$ null mutant was greatly delayed and a large number of multi-septate and mis-segregated cells were produced. In addition, the number of multi-septate cells significantly increased. When cells were cultured in YES rich medium to increase proliferation, the abnormal phenotypes $mas2^+$ null mutant dramatically increased. These phenotypes could be rescued by an over-expression of the mast gene. The Mas2 protein localized in the nuclei of S. pombe, as evidenced by Mas2-EGFP signals. These results suggest that the $mas2^+$ is homologous to human SMARCAD1 gene and involved in septum formation and chromosome remodeling control.

• Microbiology and Biotechnology Letters
• /
• v.35 no.2
• /
• pp.163-172
• /
• 2007

This study was designed to investigate that inorganic germanium $(GeO_2)$ did not exist in germanium-fortified yeast or obtained to non-detectable value by current analytical methods and equipments. For this purpose, we achieved $GeO_2$ qualitative analysis protocol which could be the scientific basis of the study. Since reddish brown precipitate was formed from the reaction of $GeO_2$ with 1 equiv $NaBH_4$, and dark brown precipitate was also formed from the reaction of $GeO_2$ with 2 equiv $NaBH_4$, $GeO_2$ was qualitatively analyzed by observing these particular colored-precipitates. Because no color change was showed from the reaction between $NaBH_4$ and $SiO_2$, the color change could be caused by charge transfer transition on Ge-O and B binding properties. The reaction between $NaBH_4$ and germanium-fortified yeast did not show any color change and precipitate formation which meant no $GeO_2$ existed in germanium-fortified yeast. The reaction between $NaBH_4$ and supernatant specimen collected from the outside of dialysis membrane (MWCO 1,200 dalton) did not show any color change and precipitate formation. Therefore, we considered that the both germaniums in and outside of the dialysis membrane were organic germaniums. Germanium-fortified yeast which was biosynthesized organic germanium can be applied not only as a new functional material for improving health, prevention and treatment of chronic degenerative diseases including cancers, and the regulation of immune system, but also as a new materials.

• The Korean Journal of Malacology
• /
• v.32 no.4
• /
• pp.263-268
• /
• 2016

Metallothionein (MT) family of metal-binding proteins are involved in maintaining homeostasis and heavy metal poisoning. Recently, MT has been considered as a biomarker that can identify a particular species, very similar to the use of cytochrome oxidase I (COI) gene. Satsuma myomphala species of land snails have been reported from North-East Asia, including South Korea and Japan. In particular, the land snail species have been known from only a limited area of Geoje Island, Gyeongsangnam-do province of South Korea. Genetic studies of S. myomphala has been limited with only 6 nucleotide, 2 protein registered on the NCBI server. For elucidating the genetic information of S. myomphala, we conducted RNA sequencing analysis using Illumina HiSeq 2500 next-generation platform. We screened the MT gene from the RNA-Seq database to confirm the molecular phylogenetic relationship. After sequencing, the de novo analysis and clustering generated 103,774 unigenes. After annotation against PANM database using BLAST program, we obtained MT sequence of 74 amino acid residues containing the coding region of 222 bp. Based on this sequence, we found about 53 sequences using the BLAST program in NCBI nr database. Using ClustalX alignment, Maximum-Likehood Tree of MEGA program, we confirmed the molecular phylogenetic relationships that showed similarity with mollusks such as Helix pomatia and H. aspersa, Megathura crenulata.

• Food Science and Preservation
• /
• v.15 no.3
• /
• pp.477-482
• /
• 2008

The quality characteristics of Goami by-product under the mixed enzyme treatment condition of $\alpha$-amylase and cellulase have been compared, and found the highest amount of soluble solids and reducing sugars at the $\alpha$-amylase treated group (A), and the contents revealed to show gradual decrease with the increase of cellulase content. The amounts of total dietary fiber and total sugars did not show large difference by both of enzyme concentration. The result of sugar analysis revealed the presence of all $G{\sim}G5$ in all treatment groups, and the content of malto-oligosaccharide recorded the highest content of 2,200 mg% at the $\alpha$-amylase treatment group (A). When the quality characteristic of the hydrolyzed powders manufactured by the optimum hydrolysis condition was compared, no significant color difference was found between samples. Among the contents of dietary fibers, insoluble dietary fiber was found to present in the lowest content of 6.95% at the Goami flour (GF) and the Goami by-product powder (GBPP) and Goami by-product hydrolysate powder (GBPHP) resulted the similar content around 14% and the highest soluble dietary fibers content was found in Goami by-product hydrolysate powder (GBPHP), which was followed by in the order of Goami by-product powder (GBPP) and Goami flour (GF), but the content variation was not large. The free amino acid was found to be highest in Goami by-product hydrolysate powder (GBPHP) followed by in the order of Goami by-product powder (GBPP) and Goami flour (GF). In the sugar analysis, the Goami by-product hydrolysate powder (GBPHP) was found with all $G{\sim}G5$ sugars by showing the highest amount of 1,800 mg% At the Goami by-product powder (GBPP), $G{\sim}G2$ sugars were detected with about 66 mg% and malto-oligosaccharides were not detected at the Goami flour (GF). Based upon the results, the functionality of Goami by-product hydrolysate powder (GBPHP) was found to be enforced compared to Goami flour (GF) and Goami by-product powder (GBPP), which allow us to expect it to be used as the various rice processing food source.

• Korean Journal of Microbiology
• /
• v.52 no.2
• /
• pp.192-201
• /
• 2016

Recently, Psychrobacter sp. ArcL13 strain showing the extracellular lipase activity was isolated from the Chuckchi Sea of the Arctic Ocean. However, due to the low expression levels of the enzyme in the natural strain, the production of recombinant lipase is crucial for various applications. Identification of the gene for the enzyme is prerequisite for the production of the recombinant protein. Therefore, in the present study, a novel lipase gene (ArcL13-Lip) was isolated from Psychrobacter sp. ArcL13 strain by gene prospecting using PCR, and its complete nucleotide sequence was determined. Sequence analysis showed that ArcL13-Lip has high amino acid sequence similarity to lipases from bacteria of some Psychrobacter genus (84-90%) despite low nucleotide sequence similarity. The lipase gene was cloned into the bacterial expression plasmid and expressed in E. coli. SDS-PAGE analysis of the cells showed that ArcL13-Lip was expressed as inclusion bodies with a molecular mass of about 35 kDa. Refolding was achieved by diluting the unfolded protein into refolding buffers containing various additives, and the highest refolding efficiency was seen in the glucose-containing buffer. Refolded ArcL13-Lip showed high hydrolytic activity toward p-nitrophenyl caprylate and p-nitrophenyl decanoate among different p-nitrophenyl esters. Recombinant ArcL13-Lip displayed maximal activity at $40^{\circ}C$ and pH 8.0 with p-nitrophenyl caprylate as a substrate. Activity assays performed at various temperatures showed that ArcL13-Lip is a cold-active lipase with about 40% and 73% of enzymatic activity at $10^{\circ}C$ and $20^{\circ}C$, respectively, compared to its maximal activity at $40^{\circ}C$.