• Title/Summary/Keyword: molecular biological experiment

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Construction of a Pure Cryparin-null Mutant for the Promoter Analysis of Cryparin Gene (Cryparin 유전자의 promoter 분석을 위한 cryparin 유전자 치환체의 순수 제조)

  • Kim, Myoung-Ju;Yang, Moon-Sik;Kim, Dae-Hyuk
    • The Korean Journal of Mycology
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    • v.26 no.4 s.87
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    • pp.450-457
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    • 1998
  • The cryparin of Cryphonectria parasitica belongs to a cell wall associated fungal hydrophobin. The cryparin, though it is encoded by a single copy gene, is known for the high expression during the liquid culture of C. parasitica, and it turns out that 22% of total mRNA was transcribed for cryparin at 48hr after the liquid culture. In addition, it is also known as one of down-regulated fungal proteins by the presence of double stranded RNA virus, Cryphonectria hypovirus 1. In previous studies (Kim et al., 1999), we have constructed a cryparin-null mutant by replacing the cryparin gene with hygromycin B resistance gene due to site directed homologous recombination. In order for the promoter analysis of cryparin which seems to be very strong as well as mycoviral specific, it is preferable to have a strain with only a target promoter replaced and a discernable target site for incoming vectors. However, the cryparin-null mutant revealed the presence of an additional copy of transforming vector except the one which replaced the cryparin gene. In addition, the cryparin-null mutant did not contain any markers for targeted integration of incoming vectors. This prompts us to design an experiment to obtain a strain for promoter analysis of cryparin gene. A different mating type strain EP6(Mata, $met^-$) was mated with the cryparin-null mutant ${\triangle}$Crp194-7(MatA, Crp${\triangle}$::hph) to make the progenies with only a single replacement vector and $met^-$ characteristic remained. Nutritional assay as well as Southern blot analysis revealed that the progeny, ${\triangle}$Crp194-a6, was the methionine auxotroph with a single replacing vector in genome. Northern blot analysis and PAGE showed that there was no cryparin produced in this bred strain either.

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Immunophenotype Characterization for Swine Selected Line, Which is Resistant for the Mycoplasma Pneumonia

  • Katayama, Masafumi;Fukuda, Tomokazu;Okamuara, Toshihiro;Suda, Yoshihito;Suzuki, Eisaku;Uenishi, Hirohide;Suzuki, Keiichi
    • Asian-Australasian Journal of Animal Sciences
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    • v.24 no.7
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    • pp.889-897
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    • 2011
  • Mycoplasma Pneumonia of swine (MPS) decreases the daily growth of pigs, and, co-infection with a virus sometimes causes severe pneumonia. Genetic selection of pigs resistant to the pulmonary MPS lesion might solve the economic loss due to MPS in animal production. Here, we examined the immunophenotype of Landrace line (Miyagino L2), genetically selected to reduce the incidence of pulmonary MPS lesion for 5 generations in Miyagi Prefecture Animal Industry Experiment Station. Although this line is expected to be resistant to the pulmonary MPS lesion, the biological characteristics of its immune function are not clear. We investigated details of the immunorelated phenotype of Miyagino L2 at the hematological and molecular biological level, including cytokine expression, and compared the results with that of non-genetically selected Landrace. Miyagino L2 showed decreased antigen-specific IgG and IgM production and increased CD8-positive T-cell population, and high levels of cortisol concentration, suggesting that the MPS-resistant phenotype is associated these immunological differences. Additionally, T-cell CD4 expression was highly correlated with the MPS expected breeding value. Although the detailed mechanisms underlying this high correlation remain unknown, our result suggested that the genetic selection of the expression level of CD4 might be useful to improve MPS resistance in pig production.

The Analysis of Ethanol Distribution Level in Biological Specimens (생체 시료중에서 Ethanol 농도 분포에 관한 연구)

  • Hong, Hoon-Gi;Kim, Dong-Wook
    • Analytical Science and Technology
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    • v.11 no.3
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    • pp.222-228
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    • 1998
  • Five kinds of packed column and two kinds of capillary column were used to get optimum condition for ethanol analysis by using fifteen different volatile, low molecular weight organic substances. Only two columns, Gaskuropack 54 and DB-1, showed good separation efficiency. In the adding salt-effect experiment 0.6N - perchloric acid, 1M - meta-phosphoric acid and saturated NaCl solution were used for alcohol concentration measurement of biological fluids and tissue specimens. Among adding salt experiment, adding saturated NaCl solution showed the most stable value of alcohol concentration. This fact might be due to the increased vaporization of alcohol in the saturated NaCl solution. In the time-course of blood alcohol concentration, the alcohol level was lineary decreased to the diameter of vessel containing specimens. This result was interpreted in view of ethanol level, weight, water content, and hematocrit value. The ethanol distribution levels were measured from samples of blood and tissue obtained from 25 postmortems cases investigated by NISI. This study showed that the distribution level was decreased in order of brain, blood, kidney, spleen, liver, and lung.

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Characterization of Doxorubicin-nonproducing Mutant, Nu3 of Streptomyces peucetius ATCC27952

  • Kyu, Hwang-Cheol;Lee, Hong-Sub;Hong, Young-Soo;Paek, Nam-Soo;Kim, Tae-Han;Lee, Jung-Joon
    • Journal of Microbiology and Biotechnology
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    • v.7 no.5
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    • pp.363-366
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    • 1997
  • A doxorubicin-nonproducing mutant, Nu23 was selected from the mutagenesis of Streptomyces peucetius ATCC27952. Chemical and molecular biological analysis suggested that the mutant was blocked at the step between polyketide synthase and aklaviketon reductase in the biosynthesis of doxorubicin. Furthermore, the bioconversion experiment with the mutant revealed that 13-dihydrodaunorubicin is likely to be a biosynthetic intermediate.

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Characteristics of Gas Sensor in Langmuir-Blodgett Ultrathin Films (Langmuir-Blodgett 초박막의 gas 센서 특성)

  • Choi, Yong-Sung;Kim, Chul-Hong;Chang, Sang-Mok;Kwon, Young-Soo
    • Proceedings of the Korean Institute of Electrical and Electronic Material Engineers Conference
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    • 1992.11a
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    • pp.55-58
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    • 1992
  • It was attempted to investigate the reaction of LB biological membrane in the gas surrounding by the use of LB method. The performance of the experiment was based on the idea that the adhesion of gas molecular on the surface of LB membrane, which induces the change of electrical properties, may make it possible to develop various sensor system modified from the sensor organs. The experimental results showed that for the acetone gas, the frequency changes in the proportional to the concentration of the gas. The reappearance of the gas reaction vs. frequency change was also able to be obtained properly.

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Purification and Characterization of a New κ-Carrageenase from the Marine Bacterium Vibrio sp. NJ-2

  • Zhu, Benwei;Ning, Limin
    • Journal of Microbiology and Biotechnology
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    • v.26 no.2
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    • pp.255-262
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    • 2016
  • The carrageenan-degrading marine bacterium Vibrio sp. strain NJ-2 was isolated from rotten red algae, and κ-carrageenase with high activity was purified from the culture supernatant. The purified enzyme with molecular mass of 33 kDa showed the maximal activity of 937 U/mg at 40℃ and pH 8.0. It maintained 80% of total activity below 40℃ and between pH 6.0 and 10.0. The kinetics experiment showed the Km and Vmax values were 2.54 g/ml and 138.89 mmol/min/mg, respectively. The thin layer chromatography and ESI-MS analysis of hydrolysates indicated that the enzyme can endolytically depolymerize the κ-carrageenan into oligosaccharides with degrees of depolymerization of 2-8. Owing to its high activity, it could be a valuable tool to produce κ-carrageenan oligosaccharides with various biological activities.

Purification of Extracellular Enzyme Produced by Vibrio sp. AL-145 (Vibrio sp. AL-145가 생산하는 균체외 효소의 정제 (I))

  • 주동식;이응호
    • Journal of the Korean Society of Food Science and Nutrition
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    • v.22 no.2
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    • pp.234-239
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    • 1993
  • The alginate degrading bacteria have been screened from the marine environment. Sodium alginate and NaCl were required for cell growth and enzyme production of 145-C strain and the adequate concentrations were 0.7 and 2.5%, respectively. The effective nitrogen source was peptone and adequate temperature was 28$\pm$2$^{\circ}C$. The 145-C strain was identified as Vibrio sp. from biochemical and biological experiment. The extracellular enzyme produced by Vibrio sp. was purified and the molecular weight was estimated to be 27, 000.

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The treatment effect of novel hGHRH homodimer to male infertility hamster

  • Zhang, Xu-Dong;Guo, Xiao-Yuan;Tang, Jing-Xuan;Yue, Lin-Na;Zhang, Juan-Hui;Liu, Tao;Dong, Yu-Xia;Tang, Song-Shan
    • The Korean Journal of Physiology and Pharmacology
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    • v.22 no.6
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    • pp.637-647
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    • 2018
  • Extra-hypothalamic growth hormone-releasing hormone (GHRH) plays an important role in reproduction. To study the treatment effect of Grin (a novel hGHRH homodimer), the infertility models of 85 male Chinese hamsters were established by intraperitoneally injecting 20 mg/kg of cyclophosphamide once in a week for 5 weeks and the treatment with Grin or human menopausal gonadotropin (hMG) as positive control was evaluated by performing a 3-week mating experiment. 2-8 mg/kg of Grin and 200 U/kg of hMG showed similar effect and different pathological characteristics. Compared to the single cyclophosphamide group (0%), the pregnancy rates (H-, M-, L-Grin 26.7, 30.8, 31.3%, and hMG 31.3%) showed significant difference, but there was no difference between the hMG and Grin groups. The single cyclophosphamide group presented loose tubules with pathologic vacuoles and significant TUNEL positive cells. Grin induced less weight of body or testis, compactly aligned tubules with little intra-lumens, whereas hMG caused more weight of body or testis, enlarging tubules with annular clearance. Grin presented a dose-dependent manner or cell differentiation-dependentincrease in testicular GHRH receptor, and did not impact the levels of blood and testicular GH, testosterone. Grin promotes fertility by proliferating and differentiating primitive cells through up-regulating testicular GHRH receptor without triggering GH secretion, which might solve the etiology of oligoasthenozoospermia.

Differential Subcellular Localization of Ribosomal Protein L7 Paralogs in Saccharomyces cerevisiae

  • Kim, Tae-Youl;Ha, Cheol Woong;Huh, Won-Ki
    • Molecules and Cells
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    • v.27 no.5
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    • pp.539-546
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    • 2009
  • In Saccharomyces cerevisiae, ribosomal protein L7, one of the ~46 ribosomal proteins of the 60S subunit, is encoded by paralogous RPL7A and RPL7B genes. The amino acid sequence identity between RPl7a and RPl7b is 97 percent; they differ by only 5 amino acid residues. Interestingly, despite the high sequence homology, Rpl7b is detected in both the cytoplasm and the nucleolus, whereas Rpl7a is detected exclusively in the cytoplasm. A site-directed mutagenesis experiment revealed that the change in the amino acid sequence of Rpl7b does not influence its subcellular localization. In addition, introns of RPL7A and RPL7B did not affect the subcellular localization of Rpl7a and Rpl7b. Remarkably, Rpl7b was detected exclusively in the cytoplasm in rpl7a knockout mutant, and overexpression of Rpl7a resulted in its accumulation in the nucleolus, indicating that the subcellular localization of Rpl7a and Rpl7b is influenced by the intracellular level of Rpl7a. Rpl7b showed a wide range of localization patterns, from exclusively cytoplasmic to exclusively nucleolar, in knockout mutants for some rRNA-processing factors, nuclear pore proteins, and large ribosomal subunit assembly factors. Rpl7a, however, was detected exclusively in the cytoplasm in these mutants. Taken together, these results suggest that although Rpl7a and Rpl7b are paralogous and functionally replaceable with each other, their precise physiological roles may not be identical.

Expression of γ-Tocopherol Methyltransferase Transgene Improves Tocopherol Composition in Lettuce (Latuca sativa L.)

  • Cho, Eun Ae;Lee, Chong Ae;Kim, Young Soo;Baek, So Hyeon;de los Reyes, Benildo G.;Yun, Song Joong
    • Molecules and Cells
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    • v.19 no.1
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    • pp.16-22
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    • 2005
  • A cDNA encoding ${\gamma}-tocopherol$ methyltransferase (${\gamma}-TMT$) from Arabidopsis thaliana was overexpressed in lettuce (Latuca sativa L.) to improve the tocopherol composition. Seven lines of lettuce ($T_0$) containing the ${\gamma}-TMT$ transgene were produced by Agrobacterium-mediated transformation. The inheritance and expression of the transgene were confirmed by DNA and RNA gel blot analyses as well as quantification of tocopherols and ${\gamma}-TMT$ activities. The ratio of ${\alpha}-/{\gamma}-tocopherol$ content (TR) varied from 0.6 to 1.2 in non-transformed plants, while the $T_0$ plants had ratios of 0.8 to 320. The ratio ranged from 0.4 to 544 in 41 $T_1$ progenies of the $T_0$ transgenic line gTM3, and the phenotypic segregation indicated monogenic inheritance of the transgene (i.e., 3:1 = dominant:wild-type classes). There was a tight relationship between the TR phenotype and ${\gamma}-TMT$ activity, and enzyme activities were affected by the copy number and transcript levels of the transgene. The TR phenotype was stably expressed in $T_2$ progenies of $T_1$ plants. The results from this study indicated that a stable inheritance and expression of Arabidopsis ${\gamma}-TMT$ transgene in lettuce results in a higher enzyme activity and the conversion of the ${\gamma}-tocopherol$ pool to ${\alpha}-tocopherol$ in transgenic lettuce.