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Characterization and Isolation of Mutants Involved in Cell Cycle Progression and Regulation in Saccharomyces cerevisiae (Saccharomyces cerevisiae에서 세포주기의 진행과 조절에 관련된 변이주들의 분리 및 특성화)

  • 박정은;임선희;선우양일
    • Korean Journal of Microbiology
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    • v.37 no.1
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    • pp.28-36
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    • 2001
  • These studies were carried out to understand the mechanisms of genes which are related in cell cycle progression at G1/S phase. Mutants involved in cell cycle progression and regulation in Saccharomyces cerevisiae were isolated and characterized. To isolate new mutants, we screened the sensitivity to ciclopirox olamine (CPO) which inhibits the cell cycle traverse at or very near the G1/S phase boundary in HeLa cell and budding yeast. As results, we isolated 30 mutants and named cos(ciclopirox olamine sensitivity: cos27∼cos57) mutants. To determine the phenotype of mutants, we examined the sensitivity to methyl-methane sulfonate (MMS) and hydroxyurea (HU). Several mutants were sensitive to MMS and HU. According to these Phenotypes, cos mutants were grouped into four. Group I mutants are cos27, cos28, cos32, cos33, cos36, cos37, cos40, cos42, cos46, cos50, cos52 and cos53 which show MMS, HU sensitivities and might act at a checkpoint pathway during S phase. Group II mutants are cos43 and cos48 which show MMS sensitivities and might act at a checkpoint pathway during Gl or G2 phase. Group III mutants are cos35, cos47, cos54, cos55 and cos56 which show HU sensitivities and might act at a progress pathway during S phase. Finally, Group IV mutants are cos29, cos30, cos31, cos34, cos38, cos39, cos41, cos44, cos45, cos49, cos51 and cos57 which show only CPO sensitivities. Moreover, we examined the terminal phenotype of mutants under fluorescent microscope and then found one of S phase checkpoint related mutant(cos37). Furthermore, we constructed the heterozygote strain between mutant and wild type haploid strains to study their genetic analysis of cos mutants.

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Expression of a $\beta$-1,3-Glucanase Gene from Bacillus circulans in B. subtilis and B. megaterium (Bacillus subtilis와 Bacillus megaterium에서의 $\beta$-1,3-glucanase 유전자의 발현)

  • 김기훈;김지연;김한복;이동석
    • Korean Journal of Microbiology
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    • v.37 no.4
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    • pp.253-258
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    • 2001
  • A Bacillus circulans KCTC3004 $\beta$-1,3-glucanase gene contained in a recombinant plasmid pLM460 derived from subcloning the original recombinant plasmid pLM530 was trasferred into a new shuttle vector plasmid pLMS1180 by ligating linearized DNAs of pLM460 and pUB110. B. subtilis RM125 and B. megaterium ATCC14945 transformed with pLMS1180 produced the $\beta$-1,3-glucanase substantially. Most of the enzyme was produced during the exponential growth period. The maxium activities of the $\beta$-1,3-glucanase produced by the Bacillus transformants were compared with that of the B. circulans gene donor strain. The B. subtilis RM125 (pLM1180) enzyme showed the activity 14 times higher than that of the gene donor cells, followed by the B. megaterium ATCC14945 (pLMS 1180) enzyme with activity 5 times higher than that of the gene donor cells. While E. coli secreted about 7% of the produced enzyme, B. subtilis excreted the enzyme into the medium wholly and B. megaterium about 97% of the total product. The SDS-PAGE of this enzyme produced in E. coli (pLMS1180), B subtilis (pLMS1180) or B. megaterium (pLMS1180) indicated a molecular weight of 38,000. The enzymes overproduced in three different host cells hydrolyzed laminarin to produce mainly laminaribiose, laminaritriose, and laminarioligosaccharides. The plasmid pLMS1180 was stable in B. megaterium, E. coli, but was unstable in B. subtilis.

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Ecological Characteristics of Actinomycetes from Mercury and Chrome Polluted Soil (수은, 6가크롬 오염토양으로부터 분리된 방선균군의 생태학적 특성)

  • Cho Min-Hye;Han Sang-Mi;Baek Ha-Ju;Whang Kyung-Sook
    • Korean Journal of Environmental Biology
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    • v.24 no.1 s.61
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    • pp.38-45
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    • 2006
  • Ecological characteristics of microbial populations inhabiting heavy metal polluted soil were investigated. The samples were collected from 293 sites around an factory and industry at Gyeoungsangbuk-do. We measured the contents of seven heavy metal elements (Cd, Cu, As, Hg, Pb, $Cr^{6+}$, CN), seven sites have been seriously contaminated by mercury and chrome. A quantitative evaluation of microbial populations in mercury and chrome contaminated soil was examined by using plate count method. Bacterial numbers in polluted soil samples ranged from $7.4X10^5\;to\;9.3X10^7\;cfu\;g^{-1}$, about $10\sim100$ fold less than the count for the unpolluted soil. Moulds were not detected in chrome polluted soil. The log values of actinomycetes of each contaminated soil samples were log ranged from 6.18 to 7.52. The ratio of actinomycetes was similar to unpolluted soil. The investigation showed actinomycetes to be the major microbial population inhabiting the mercury and chrome polluted soil. Thirty-one isolates among the total isolates were examined for antibacterial activity. These isolates were identified based on a phylogenetic analysis using 16S rRNA gene nucleotide sequences, they were categorized in three major phylogenetic groups, belong to the Streptomyces (6 strains), Saccharopolyspora (3 strains), Nocardiodes (1 strain). On the phylogenetic tree, the clade consisting of five isolates were distantly related to all of the established Streptomycetes genera, indicating the possibility as members of new species.

Physiological Characteristics and ACE Inhibitory Activity of Lactobacillus zeae RMK354 Isolated from Raw Milk (원유에서 분리한 Lactobacillus zeae RMK354의 생리적 특성 및 ACE 억제능)

  • Lim, Sang-Dong;Kim, Kee-Sung;Do, Jeong-Ryong
    • Food Science of Animal Resources
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    • v.28 no.5
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    • pp.587-595
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    • 2008
  • In order to develop a new starter for fermented milk, 1037 bacterial strains were isolated from raw milk. The strain that showed excellent acid producing and angiotensin converting enzyme (ACE) inhibitory activity (88.6%) was selected and identified as a Lactobacillus zeae based on the result of API carbohydrate fermentation pattern and 16S rDNA sequence. Lactobacillus zeae RMK354 was investigated further to study its physiological characteristics. It showed strong ACE inhibitory activity compared with commercial LAB starters tested. The optimum growth temperature of L. zeae RMK354 was $40^{\circ}C$ and it took 10 hr to reach pH 4.3 under this condition. L. zeae RMK354 showed more sensitive to penicillin-G, bacitracin, novobiocin, in a comparison of 14 different antibiotics, and showed most resistance to polymyxin B and vancomycin. It showed higher esterase and leucine arylamidase activities compared with 16 other enzymes. It was comparatively tolerant to bile juice and able to survive at pH 2 for 3 hr. It showed inhibitory activity against Salmonella Typhimurium with the rate of 60%. Based on these and previous results, L. zeae RMK354 could be an excellent starter culture for fermented milk with high level of ACE inhibitory activity.

Isolation of Pseudoalteromonas sp. HJ 47 from Deep Sea Water of East Sea and Characterization of its Extracellular Protease (동해 심층수로부터 Pseudoalteromonas sp. HJ 47의 분리 및 체외단백질분해효소 특성)

  • Cha, In-Tae;Lim, Hayung-Joon;Roh, Dong-Hyun
    • Journal of Life Science
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    • v.17 no.2 s.82
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    • pp.272-278
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    • 2007
  • Proteases are enzymes that break peptide bonds between amino acids of other proteins and occupy a crucial position with respect to their applications in both physiological and commercial fields. In order to screen new source of protease, bacteria producing extracellular proteases at low temperature were isolated from deep sea water of East Sea, Korea. A bacterium showing the best growth rate and production of an extracellular protease at low temperature was designated HJ 47. The DNA sequence analysis of the 16S rRNA gene, phenotypic tests and morphology led to the placement of this organism in the genus Pseudoalteromonas. Although maximal growth was observed at $37^{\circ}C$, enzyme production per culture time was maximum at $20^{\circ}C$. At this temperature, extracellluar protease production was detected from the end of the exponential phage to stationary phase, and maximal at 15 hours after initial production. The optimum temperature and pH of the protease were found to be $35^{\circ}C$ and 8.

Examination of Antimicrobial Activity by Phaeobacter inhibens KJ-2 Isolated from a Marine Organism (해양 생물에서 분리된 Phaeobacter inhibens KJ-2의 항균 활성)

  • Kim, Yun-Beom;Kim, Dong-Hwi;Heo, Moon-Soo
    • Journal of Life Science
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    • v.27 no.10
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    • pp.1161-1167
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    • 2017
  • In order to find a new antimicrobial bacterium, we performed screening for antimicrobial activity of bacteria isolated from the eggs of a sea hare. The newly identified strain was designated as Phaeobacter inhibens KJ-2, based on the biochemical characterization and 16S rRNA gene sequence analysis. A colony of P. inhibens KJ-2 showed a circular and ruler-like smooth form at the edge, and a brown color. However, when maintained with a longer incubation time, its coloring was transformed into dark brown. From the result of SEM, P. inhibens KJ-2 is a bacillus which has a length of $0.8{\sim}1.0{\mu}m$ and a width of $0.4{\sim}0.6{\mu}m$. The optimal growth and antimicrobial activity were observed by shaking the culture for 24 hr at $20^{\circ}C$, which showed potent activity against pathogenic bacteria including Vibrio logei, Vibrio campbellii, Vibrio mimicus, Vibrio vulnificus, and Vibrio salmonicida. The antimicrobial activity was proportional to the amount of produced acylated homoserine lactones (AHLs). Therefore, we suggest that production of antimicrobial materials from P. inhibens KJ-2 is regulated by Quorum sensing (QS).

Characterization of the Antagonistic Activity against Lactobacillus plantarum and Induction of Bacteriocin Production (김치로부터 Lactobacillus plantarum 생육저해 박테리오신 생산균주의 분리 및 박테리오신 생산의 유도효과)

  • Yang, Eun-Ju;Chang, Ji-Yoon;Lee, Hyong-Joo;Kim, Jeong-Hwan;Chung, Dae-Kyun;Lee, Jong-Hoon;Chang, Hae-Choon
    • Korean Journal of Food Science and Technology
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    • v.34 no.2
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    • pp.311-318
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    • 2002
  • A new bacteriocin producing lactic acid bacteria having antagonistic activity against Lactobacillus plantarum, was isolated from Kimchi. It was identified as Leuconostoc mesenteroides, and designated as Leuconostoc mesenteroides B7. The bacteriocin from Leuconostoc mesenteroides B7 named as bacteriocin B7 was stable in the pH range $2.5{\sim}9.5$. Bacteriocin B7 was active over a wide temperature range from $4^{\circ}C$ to $120^{\circ}C$. It was inactivated by proteinase K, trypsin, ${\alpha}-chymotrypsin$, and protease treatments indicating its proteinous nature. Tricine-SDS-PAGE of the purified bacteriocin B7 showed the presence of a single band, having a molecular mass of about 3,500 dalton. Mixed culture of the producer and the indicator, Lb. plantarum KFRI 464 or Lb. delbruekii KFRI 347, increased production of bacteriocin B7. This result suggested the presence of bacteriocin inducing factor in the indicator strain. The inducing factor was localized in cell debris and intracellular faction of the indicator cell, Lb. plantarum KFRI 464. Treatment of the inducing factor with proteinase K destroyed inducing activity. This result strongly suggested that the inducing factor is a protein.

Shear Behavior of Sands Depending on Shear Box Type in Direct Shear Test (직접전단실험시 전단상자의 종류에 따른 모래시료의 전단거동)

  • Hong, Young-Ho;Byun, Yong-Hoon;Chae, Jong-Gil;Lee, Jong-Sub
    • Journal of the Korean Geotechnical Society
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    • v.31 no.3
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    • pp.51-62
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    • 2015
  • Shear behavior obtained by direct shear tests is dependent on shear box and boundary condition. The objective of this study is to analyze problems of conventional direct shear test (type-A) and provide the reliable results by developing type-C direct shear apparatus. Experimental tests are carried out for Ulleung sand by using type-A and -C direct shear devices. The soil specimens, which are prepared at the relative density of 60%, and are applied to vertical confining stresses of 50, 100, 200, 300, and 400 kPa, are sheared at a constant shear strain rate of 0.5 mm/min. By comparing the results obtained by type-A and -C direct shear apparatus under constant normal load (CNL) condition, the performance of new one is verified. In addition, two constrained conditions including constant normal load (CNL) and constant pressure (CP) are applied to type-C one. Experimental results show that type-A direct shear apparatus has some problems such as rotating of loading plate and upper shear box, and the frictional forces between soil and inner wall of upper shear box. Thus, the shear strengths obtained by type-A device are overestimated or underestimated depending on shear box and boundary condition. On the other hand, type-C device produces clear and consistent test results regardless of constrained conditions. This study represents that type-C direct shear apparatus not only can solve the problems of type-A direct shear apparatus but provide the reliable results.

Geotechnical Characteristics of the Ulleung Basin Sediment, East Sea (1) - Cosolidation and Shear Waves Velocity (동해 울릉분지 심해토의 지반공학적 특성(1) - 압밀 특성, 전단파 특성에 관한 연구)

  • Kim, Youngmoon;Lee, Jongsub;Lee, Jooyong;Lee, Changho
    • Journal of the Korean GEO-environmental Society
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    • v.14 no.5
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    • pp.33-39
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    • 2013
  • A drilling exploration in deep sea is being processed to develop new energy resource in the world. In 2007, the presence of the gas hydrate had been confirmed during the UBGH1 (Ulleung Basin Gas Hydrate Expedition 1) in the Ulleung Basin. Geotechnical properties of the deep marine sediment are important factors for assessing the safety of gas production facility and productivity from the hydrate bearing sediment. In this study, comprehensive laboratory tests are conducted to investigate the geotechnical engineering characteristics of the deep marine sediments recovered from the hydrate occurrence regions during the UBGH2 (Ulleung Basin Gas Hydrate Expedition 2) in the Ulleung Basin, East Sea, Korea. The index properties of the specimens including the specific gravity, atterberg limits, specific surface, and particle size distribution are measured, and these are compared to the results reported by previous studies. A zero-lateral strain cell, which houses bender elements, is used to determine stress-dependant characteristics and shear wave velocities with the vertical effective stresses. Furthermore, the hydraulic conductivity is calculated based on the consolidation test results.

Studies on Breeding and Cultivation Characteristics of Lentinula edodes Strains for Sawdust Cultivation (표고 톱밥재배용 균주의 육종과 재배특성에 관한 연구)

  • Ryu, Sung-Ryul;Bak, Won-Chull;Koo, Chang-Duck;Lee, Bong-Hun
    • The Korean Journal of Mycology
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    • v.37 no.1
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    • pp.65-72
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    • 2009
  • For the breeding purpose, in this study we attempted to select a new Lentinula edodes strain by comparing mycelial growth rate, fruit-body quality and mushroom productivity per substrate weight of hybrid strains with parent strains. Nineteen hybrid strains were made by cross of di-karyotic and mono-karyotic to develop strains suitable for sawdust cultivation of L. edodes. Among the nineteen strains, ten were selected by comparing mycelial growth rate on PDA media and wood rot on sawdust-based substrates. The most mushroom harvesting was achieved by 100 days incubation from KFRI 960, KFRI 961, KFRI 962, KFRI 963, KFRI 964, KFRI 971, KFRI 973 and 125 days from KFRI 968, 970. Experiment of incubation periods with the ten strains showed that there were productivity differences between 100 and 150 days incubations. Through comparison of hybrid strains' mushroom productivity it was found that there was no difference in mushroom productivity through crossing high temperature types with high temperature types, crossing low temperature types with high temperature types and crossing unknown temperature types with high temperature types, but there was difference through crossing middle temperature types with high temperature types. Hybrid strains showed better productivity compared with parent strains. Fruiting temperature type analysis of hybrid strains confirmed that strains of high temperature types have a dominant character.