• Mycobiology
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• 제43권1호
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• pp.31-36
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• 2015

We have previously isolated ${\varepsilon}$-COP, the ${\alpha}$-COP interactor in COPI of Aspergillus nidulans, by yeast two-hybrid screening. To understand the function of ${\varepsilon}$-COP, the $aneA^+$ gene for ${\varepsilon}$-COP/AneA was deleted by homologous recombination using a gene-specific disruption cassette. Deletion of the ${\varepsilon}$-COP gene showed no detectable changes in vegetative growth or asexual development, but resulted in decrease in the production of the fruiting body, cleistothecium, under conditions favorable for sexual development. Unlike in the budding yeast Saccharomyces cerevisiae, in A. nidulans, over-expression of ${\varepsilon}$-COP did not rescue the thermo-sensitive growth defect of the ${\alpha}$-COP mutant at $42^{\circ}C$. Together, these data show that ${\varepsilon}$-COP is not essential for viability, but it plays a role in fruiting body formation in A. nidulans.

• 미생물학회지
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• 제48권2호
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• pp.171-174
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• 2012

THOC1/Hpr1는 mRNA가 전사되는 동안 mRNP의 포장과 mRNA 방출에 관여하는 진화적으로 잘 보존된 THO 복합체의 한 소단위이다. 분열효모 Schizosaccharomyces pombe에서도 THOC1/Hpr1과 유사한 단백질을 암호화하는 유전자(sphpr1로 명명)를 찾아 그 특성을 조사하였다. 이배체 S. pombe 균주에 하나의 sphpr1 유전자만을 결실시킨 후 4분체 분석을 수행한 결과, 이 유전자는 생장에 필수적이었다. 티아민에 의해 발현이 조절되는 강력한 nmt1 프러모터를 이용하여 sphpr1를 과발현시키더라도 세포의 생장과 mRNA 방출에는 전혀 영향이 없었다. 하지만, sphpr1의 발현을 억제하면 생장이 억제되었으며 poly$(A)^+$ RNA가 핵 안에 축적되었다. 이와 같은 결과들은 sphpr1 유전자가 생장과 mRNA의 핵에서 세포질로의 방출에 관여하고 있음을 시사한다.

• The Plant Pathology Journal
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• 제35권6호
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• pp.564-574
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• 2019

Like many fungal pathogens, the conidium and appressorium play key roles during polycyclic dissemination and infection of Magnaporthe oryzae. Ran-binding protein microtubule-organizing center (RanBPM) is a highly conserved nucleocytoplasmic protein. In animalia, RanBPM has been implicated in apoptosis, cell morphology, and transcription. However, the functional roles of RanBPM, encoded by MGG_00753 (named MoRBP9) in M. oryzae, have not been elucidated. Here, the deletion mutant ΔMorbp9 for MoRBP9 was generated via homologous recombination to investigate the functions of this gene. The ΔMorbp9 exhibited normal conidial germination and vegetative growth but dramatically reduced conidiation compared with the wild type, suggesting that MoRBP9 is involved in conidial production. ΔMorbp9 conidia failed to produce appressoria on hydrophobic surfaces, whereas ΔMorbp9 still developed aberrantly shaped appressorium-like structures at hyphal tips on the same surface, suggesting that MoRBP9 is involved in the morphology of appressorium-like structures from hyphal tips and is critical for development of appressorium from germ tubes. Taken together, our results indicated that MoRBP9 played a pleiotropic role in polycyclic dissemination and infection-related morphogenesis of M. oryzae.

• 한국응용약물학회:학술대회논문집
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• 한국응용약물학회 1994년도 제2회 추계심포지움
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• pp.69-80
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• 1994

Although ureases play important roles in microbial nitrogen metabolism and in the pathogenesis of several human diseases, little is known of the mechanism of metallocenter biosynthesis in this Ni-Containing enzyme. Klebsiella aerogenes urease apo-protein was purified from cells grown in the absence of Ni. The purified apo-enzyme showed the same native molecular weight, charge, and subunit stoichiometry as the holo-enzyme. Chemical modification studies were consistent with histidinyl ligation of Ni. Apo-enzyme could not be activated by simple addition of Ni ions suggesting a requirement for a cellular factor. Deletion analysis showed that four accessory genes (ureD, ureE, ureF, and ureG) are necessary for the functional incorporation of the urease metallocenter. Whereas the $\Delta$ureD, $\Delta$ureF, and $\Delta$ureG mutants are inactive and their ureases lack Ni, the $\Delta$ureE mutants retain partial activity and their ureases possess corresponding lower levels of Ni. UreE and UreG peptides were identified by SDS-polyacrylamide gel comparisons of mutant and wild type cells and by N-terminal sequencing. UreD and UreF peptides, which are synthesized at ve교 low levels, were identified by using in vitro transcription/translation methods. Cotransformation of E. coli cells with the complementing plasmids confirmed that ureD and ureF gene products act in trans. UreE was purified and characterized. immunogold electron microscopic studies were used to localize UreE to the cytoplasm. Equilibrium dialysis studies of purified UreE with $^{63}$ NiC1$_2$ showed that it binds ～6 Ni in a specific manner with a $K_{d}$ of 9.6 $\pm$1.3 $\mu$M. Results from spectroscopic studies demonstrated that Ni ions are ligated by 5 histidinyl residues and a sixth N or O atom, consistent with participation of the polyhistidine tail at the carboxyl termini of the dimeric UreE in Ni binding. With these results and other known features of the urease-related gene products, a model for urease metallocenter biosynthesis is proposed in which UreE binds Ni and acts as a Ni donor to the urease apo-protein while UreG binds ATP and couples its Hydrolysis to the Ni incorporation process.ouples its Hydrolysis to the Ni incorporation process.s.

• Asian-Australasian Journal of Animal Sciences
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• 제30권5호
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• pp.743-748
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• 2017

Objective: Based on rapid advancement of genetic modification techniques, genomic editing is expected to become the most efficient tool for improvement of economic traits in livestock as well as poultry. In this study, we examined and verified the nickase of mutated CRISPR-associated protein 9 (Cas9) to modulate the specific target gene in chicken DF1 cells. Methods: Chicken myostatin which inhibits muscle cell growth and differentiation during myogenesis was targeted to be deleted and mutated by the Cas9-D10A nickase. After co-transfection of the nickase expression vector with green fluorescent gene (GFP) gene and targeted multiplex guide RNAs (gRNAs), the GFP-positive cells were sorted out by fluorescence-activated cell sorting procedure. Results: Through the genotyping analysis of the knockout cells, the mutant induction efficiency was 100% in the targeted site. Number of the deleted nucleotides ranged from 2 to 39 nucleotide deletion. There was no phenotypic difference between regular cells and knockout cells. However, myostatin protein was not apparently detected in the knockout cells by Western blotting. Additionally, six off-target sites were predicted and analyzed but any non-specific mutation in the off-target sites was not observed. Conclusion: The knockout technical platform with the nickase and multiplex gRNAs can be efficiently and stablely applied to functional genomics study in poultry and finally adapted to generate the knockout poultry for agribio industry.

• Asian Pacific Journal of Cancer Prevention
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• 제16권10호
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• pp.4291-4295
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• 2015

Background: Chemokines and their receptors influence carcinogenesis and cysteine-cysteine chemokine receptor 5 (CCR5) directs spread of cancer to other tissues. A 32 base pair deletion in the coding region of CCR5 that might alter the expression or function of the protein has been implicated in a variety of immune-mediated diseases. The action of antiviral drugs being proposed as adjuvant therapy in cancer is dependent on CCR5 wild type status. In the present study, distribution of CCR5${\Delta}32$ polymorphism was assessed in North Indian esophageal cancer patients to explore the potential of using chemokine receptors antagonists as adjuvant therapy. Materials and Methods: DNA samples of 175 sporadic esophageal cancer patients (69 males and 106 females) and 175 unrelated healthy control individuals (69 males and 106 females) were screened for the CCR5${\Delta}32$ polymorphism by direct polymerase chain reaction (PCR). Results: The frequencies of wild type homozygous (CCR5/CCR5), heterozygous (CCR5/${\Delta}32$) and homozygous mutant (${\Delta}32/{\Delta}32$) genotypes were 96.0 vs 97.72%, 4.0 vs 1.71% and 0 vs 0.57% in patients and controls respectively. There was no difference in the genotype and allele frequencies of CCR5${\Delta}32$ polymorphism in esophageal cancer patients and control group. Conclusions: The CCR5${\Delta}32$ polymorphism is not associated with esophageal cancer in North Indians. As the majority of patients express the wild type allele, there is potential of using antiviral drug therapy as adjuvant therapy.

• Journal of Microbiology and Biotechnology
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• 제19권11호
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• pp.1456-1463
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• 2009

Vibrio parahaemolyticus possessing urease-positive property is relatively rare, but such strains consistently exhibit the TDH-related hemolysin (TRH) gene. In this study, we examined the effects of incubation temperature on urease activity expression, using the TH3996 and AQ4673 strains where the enzyme activity is known to be temperature-dependent and -independent, respectively. In the TH3996 strain, $\beta$-galactosidase activity was 4.4-fold lower after $30^{\circ}C$ cultivation than after $37^{\circ}C$ in a ureR-lacZ fusion strain, but temperature dependency was not found in ureD- or nikA-lacZ fusion strains. However, ureR-, ureD-, and nikA-lacZ fusions of the AQ4673 strain was not influenced by incubation temperature. We compared the promoter sequences of ureR between the above two strains. Intriguingly, we detected mismatches of two nucleotides between the two strains located at positions -66 and -108 upstream of the methionine initiation codon for UreR. Additionally, urease activity was not affected by culture temperature at either $30^{\circ}C$ or $37^{\circ}C$ by allelic introduction of the AQ4673 ureR gene into the TH3996 ureR deletion mutant. Taken together, our study demonstrates that the transcriptional factor UreR is involved in the temperature dependency of urease activity, and two nucleotides within the ureR promoter region are of particular importance for the urease activity dependency of V. parahaemolyticus.

• 한국생명과학회:학술대회논문집
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• 한국생명과학회 2005년도 국제학술심포지움 The 44th Annual Meeting of Korean Society for Life Science
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• pp.23-36
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• 2005

Bax, a mammalian pro-apoptotic member of the Bcl-2 family, induces cell death when expressed In yeast. To investigate whether .Bax expression can induce cell death in plant, we produced transgenic Arabidopsis plants that contained murine Bax cDNA under control of a glucocorticoid-inducible promoter. Transgenic plants treated with dexamethasone, a strong synthetic glucocorticoid, induced Bax accumulation and cell death, suggesting that some elements of cell death mechanism by Bax may be conserved among various orgarusms. Therefore, we developed novel yeast genetic system, and cloned several Plant Bax Inhibitors (PBIs). Here, we report the function of two PBIs In detail. PBIl is ascorbate peroxidase (sAPX). Fluorescence method of dihydrorhodamine123 oxidation revealed that expression of Bax in yeast cells generated reactive oxygen species (ROS), and which was greatly reduced by co-expression with sAPX. These results suggest that sAPX inhibits the generation of ROS by Bax, which in turn suppresses Bax-induced cell death in yeast. PBI2 encodes nucleoside diphosphate kinase (NDPK). ROS stress strongly induces the expression of the NDPK2 gene in Arabidopsis thaliana (AtNDPK2). Transgenic plants overexpressing AtNDPK2 have lower lovels of ROS than wildtype plants. Mutants lacking AtNDPK2 had higher levels of ROS than wildtype. H$_{2O2}$ treatment induced the phosphorylation of two endogenous proteins whose molecular weights suggested they are AtMPK3 and AtMPK6. In the absence of H2O2 treatment, phosphorylation of these proteins was slightly elevated in plants overexpressing AtNDPK2 but markedly decreased In the AtNDPK2 deletion mutant. Yeast two-hybrid and in vitro protein pull-down assays revealed that AtNDPK2 specifically interacts with AtMPK3 and AtMPK6. Furthermore, AtNDPK2 also enhances the MBP phosphorylation activity of AtMPK3 i'n vitro. Finally, constitutive overexpression of AtNDPK2 in Arabidopsis plants conferred an enhanced tolerance to multiple environmental stresses that elicit ROS accumulation In situ. Thus, AtNDPK2 appears to play a novel regulatory role in H2O2-mediated MAPK signaling in plants.

• 환경생물
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• 제20권4호
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• pp.316-324
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• 2002

To investigate the mutational spectrum of laccase (Lac) genes (lac -A and lac -B) involved in degrading lignin which is the recalcitrant cell wall polymer, the genes of the Pleurotus ostreatus mutants induced by gamma ray radiation were amplified by PCR and were cloned. All partial lac-A genes of 4 mutants (PO-6, -7, -14 and -15) consisted of 1763 base pairs due to the deletion of two bases (491-nt and 492-nt) and addition of one base (875-nt) in 1764 base pairs of lac -A gene of PO-1. Totally 36 mutational hot spots were detected and 32 positions were mutated in all of those 4 mutants simultaneously. These mutations were predominantly A : T -> G : C transitions (40%). Putative amino acid sequences of lac -A genes of mutants have one simultaneous mutated residue (from Thr-44 to Ala-44). The 1764 bp of partial lac -B gene was cloned only in PO -5 mutant and contained 19 mutated bases. These mutations were predominantly G : C->A : T transitions (45%). Lac-B protein of PO-5 has two mutated residues of Glu-290 and His-363 from Ala-290 and Phe-363, respectively. The hyper-mutational positions were concentrated in specific regions of between 50-nt and 900-nt in lac genes. These results suggested that the mutational hotspots responded to gamma radiation could be in some genes, at least lac -A and lac -B of p. ostreatus.

• 한국생물물리학회:학술대회논문집
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• 한국생물물리학회 2003년도 정기총회 및 학술발표회
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• pp.50-50
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• 2003

We have identified and analysed a putative response regulator two-component gene (CaSKN7) from Candida albicans and its encoding protein (CaSkn7). CaSKN7 has an open reading frame of 1677bp. CaSKN7 encodes a 559 amino acid protein (CaSkn7) with an estimated molecular mass of 61.1 kDa. CaSKN7 is a homologue of a Saccharomyces cerevisiae SKN7 that is the regulator involved in the oxidative stress response. To study the role of CaSKN7, we constructed a CAI4-derived mutant strain carrying a homozygous deletion of the CaSKN7 gene. In the caskn7 disruptant cells, the formation of germ tube require shorter time than that in the congenic wild-type strain but the growth of mycelium delayed in liquid media. In contrast, the caskn7 disruptant cells attenuate the differentiation in solid media and the virulence in mouse model system. Expression level of hypha-specific and virulence genes - HYR1, ECE1, HWP1, and ALS1 - in the caskn7 disruptant cells increased as compared with that in the congenic wild-type strain in 10％ serum YPD. Skn7 in 5. cerevisiae was found to bind the HSE element from the SSA promoter, Also, CaSkn7 contains heat shock factor DNA-binding domain and the promoters of these genes have HSE-like sties. Therefore these results show that CaSKN7 regulate the differentiation and virulence of C. albicans.