• Journal of Microbiology
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• v.40 no.3
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• pp.211-218
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• 2002

A 4.8-kb DNA fragment encoding the P-450 type hydroxylase and ferredoxin genes was cloned from Pseudonocardia autotrophica IFO 12743 that can convert vitamin D$\_$3/ into its hydroxylated active forms. In order to isolate the P-450 gene cluster in this organism, we designed PCR primers on the basis of the regions of an oxygen binding site and a heme ligand pocket that are general characteristics of the P-450 hydroxylase. Sequencing analysis of the BamHI fragment revealed the presence of four complete and one incomplete ORFs, named PauA, PauB, PauC, and PauD, respectively. As a result of computer-based analyses, PauA and PauB have homology with enoyl-CoA hydratase from several organisms and the positive regulators belonging to the tetR family, respectively. PauC and PauD show similarity with SuaB/C proteins and ferredoxins, respectively, which are composed of P-450 monooxygenase systems for metabolizing two sulfonylurea herbicides in Streptomyces griseolus PauC shows the highest similarity with another CytP-450$\_$Sca2/ protein that is responsible for production of a specific HMG-CoA reductase inhibitor, pravastatin, in S. carbophilus. Cultures of Steptomyces lividans transformant, containing the P-450 gene cluster on the pWHM3 plasmid, was unable to convert vitamin D$\_$3/ to its hydroxylated forms.

• Reproductive and Developmental Biology
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• v.29 no.1
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• pp.57-62
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• 2005

One of the critical problems to be solved in transgenic animal production is uncontrollable constitutive expression of foreign genes, which usually results in serious physiological disturbances in the transgenic animal. To circumvent this problem, we constructed and tested two retrovirus vectors designed to express the GFP(green fluorescent protein) gene under the control of the tetracycline-inducible promoters. To maximize the GFP gene expression at turn-on state, WPRE(woodchuck hepatitis virus posttranscriptional regulatory element) sequence was introduced into the retrovirus vectors at downstream region of either the GFP gene or the sequence encoding rtTA(reverse tetracycline-controlled transactivator). Transformed cells were cultured in the medium supplemented with or without doxycycline(tetracycline derivative) for 48 hours, and induction efficiency was measured by comparing the GFP gene expression level using fluorometry and western blotting. Higher GFP expression was observed from the vector carrying the WPRE sequence at 3' side of the GFP gene, while tighter expression control(up to 20 fold) was obtained from the vector in which the WPRE sequence was placed at 3' side of rtTA sequence. The resulting tetracycline inducible vector system may be used in transgenic animal production and gene therapy.

• Proceedings of the KSAR Conference
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• 2003.06a
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• pp.54-54
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• 2003

The activation of protooncogenes or the inactivation of their gene products may be a specific and effective functional study for human neoplasia. To examine this possibility, we have used the tetracycline regulatory system to generate transgenic mice that conditionally express the HccR-2 protooncogene in vivo. The new human cervical cancer protooncogene (HccR-2) was detected from cervical cancer cell line. To elucidate its biological functions, we generated transgenic mice that expressed the HccR-2 gene. The sustained expression of the HccR-2 transgene culminated chronic neutrophilic leukemia (CNL). CNL is a rare chronic myeloproliferative disorder that presents as a sustained, mature neutrophilic leukocytosis with few or no circulating immature granulocytes, the absence of peripheral blood monocytosis, basophilia, or eosinophilia, and infiltration of neutrophils at the liver, spleen and kidney. Mice expressing the HccR-2 and tetracycline-transactivating protein (tTa) transgene were found to have altered myeloid development that was characterized by increased percentages of mature neutrophil and band form neutrophil in the peripheral blood, liver and spleen. Activation of the transgene causes CNL. In our model, expression of HccR-2 transgene mice was similar in many respects to the human CNL. This model will be valuable not only for investigating the biological properties of the HccR-2 and other protooncogenes in vivo but also for analyzing the mechanism involved in the progression of CNL.