• Journal of Microbiology and Biotechnology
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• v.15 no.3
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• pp.477-483
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• 2005

Corynebacterium ammoniagenes ATCC 6872, which does not accumulate pyrimidine nucleoside or nucleotide, was metabolically engineered to secrete a large amount of thymidine. Characteristics of 5-fluorouracil resistance ($FU^r$), hydroxyurea resistance ($HU^r$), trimethoprim resistance ($TM^r$), thymidylate phosphorylase deficiency ($deoA^-$), inosine auxotrophy ($ino^-$), 5-fluorocytosine resistance ($FC^r$), thymidine kinase deficiency, and thymidine resistance ($thym^r$) were successively introduced into mutant strains KR3 and DY5T9-5, and shake-flask cultures were able to accumulate 408.1 mg/l and 428.2 mg/l of thymidine, respectively, as a major product. The mutant strains did not accumulate thymine at all and accumulated less than 10 mg/l of other pyrimidine nucleosides, such as cytosine, cytidine, and deoxycytidine, as byproducts.

• 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.

• Korean Journal of Microbiology
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• v.38 no.2
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• pp.96-102
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• 2002

The S-phase checkpoint mechanisms response to DNA damage or inhibition of DNA replication for maintenance of genetic stability in eukaryotic cells. These roles include cell cycle control arrest at S-phase and Iranscriptional induction of repair genes. To characterize the defects of dpbll mutant for both these responses, we examined the over-expression effect of DPBll gene, the sensitivity to HU, MMS, and the transcriptional pattern by DNA damage agent for RNRS mRNA. RNRS transcript is induced in response to a wide variety of agents that either damage D7A directly through chemical modification or induce stress by blocking DNA synthesis. As results, dpbll-1 cells are sensitive to DNA damage agents and the level of RNR3 mRNA is reduced approximately 40% than wild type cells. Moreover, we found the same results in dpb2-1 cells. Therefore, we propose that DPB2 and DPBll act as a sensor of replication that coordinates the transcriptional and cell cycle responses to replication blocks.

• Toxicological Research
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• v.33 no.2
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• pp.107-118
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• 2017

Although alternative test methods based on the 3Rs (Replacement, Reduction, Refinement) are being developed to replace animal testing in reproductive and developmental toxicology, they are still in an early stage. Consequently, we aimed to develop alternative test methods in male animals using mouse spermatogonial stem cells (mSSCs). Here, we modified the OECD TG 489 and optimized the in vitro comet assay in our previous study. This study aimed to verify the validity of in vitro tests involving mSSCs by comparing their results with those of in vivo tests using C57BL/6 mice by gavage. We selected hydroxyurea (HU), which is known to chemically induce male reproductive toxicity. The 50% inhibitory concentration ($IC_{50}$) value of HU was 0.9 mM, as determined by the MTT assay. In the in vitro comet assay, % tail DNA and Olive tail moment (OTM) after HU administration increased significantly, compared to the control. Annexin V, PI staining and TUNEL assays showed that HU caused apoptosis in mSSCs. In order to compare in vitro tests with in vivo tests, the same substances were administered to male C57BL/6 mice. Reproductive toxicity was observed at 25, 50, 100, and 200 mg/kg/day as measured by clinical measures of reduction in sperm motility and testicular weight. The comet assay, DCFH-DA assay, H&E staining, and TUNEL assay were also performed. The results of the test with C57BL/6 mice were similar to those with mSSCs for HU treatment. Finally, linear regression analysis showed a strong positive correlation between results of in vitro tests and those of in vivo. In conclusion, the present study is the first to demonstrate the effect of HU-induced DNA damage, ROS formation, and apoptosis in mSSCs. Further, the results of the current study suggest that mSSCs could be a useful model to predict male reproductive toxicity.