• 한국식물병리학회:학술대회논문집
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• 한국식물병리학회 2003년도 정기총회 및 추계학술발표회
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• pp.66.2-66
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• 2003

A gain-of-function mutant, SHM-11 obtained through gamma-ray mutagenesis, is resistant to rice blast caused by Magnaporthe grisea while wild type Sanghaehyanghyella is highly susceptible to the same disease. The resistance in the mutant was not race-specific when we tested with four races (KJ-201, KI-1113a, KI-313, KI-409) of M. grisea. To identify genes involved disease resistance in the gain-of-function mutant, genes specifically expressed in the mutant were selected by suppression subtractive hybridization using cDNAS of blast-inoculated mutant and wild type as a tester and a driver, respectively, Random 200 clones from the subtracted library were selected and analyzed by DNA sequencing. The sequenced genes represented three major groups related with disease resistance； genes encoding PR proteins, genes probably for phytoalexin biosynthesis, and genes involved in disease resistance signal transduction. A gene encoding a putative receptor-like protein kinase was identified as highly expressed only in the gain-of-function mutant after blast infection. The role of the putative receptor-like protein kinase gene during blast resistance will be further studied.

• Journal of Crop Science and Biotechnology
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• 제11권4호
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• pp.237-242
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• 2008

A liguleless mutant, which showed complete loss of lamina joint region at the junction between leaf blade and leaf sheath, was isolated from a Ds insertional mutants derived from the source cultivar, Dongjin. This mutant could not affect other developmental patterns like phyllotaxis. Southern blot analysis, using GUS as a probe, revealed that the liguleless mutant contained three Ds copies transposed in the rice genome. Among the four genomic sequences flanking the Ds, one was mapped in the intergenic region (31661640 - 31661759), and the other two predicted a protein kinase domain (12098980 - 12098667) as an original insertion site within a starter line used for massive production of Ds insertional mutant lines. Another predicted and inserted in first exon of liguleless 1 protein (OsLG1) that was mapped in coding region (LOC_Os04g56170) of chromosome 4. RT-PCR revealed that the OsLG1 gene was not expressed liguleless mutants. Structure analysis of OsLG1 protein revealed that it predicted transcription factor with a highly conserved SBP domain consisting of 79 amino acids that overlapped a nuclear localization signal (NLS). RT-PCR revealed that OsLG1 is mainly expressed in vegetative organs.

• Journal of Microbiology and Biotechnology
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• 제15권5호
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• pp.1022-1027
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• 2005

Based on plasmid display technology by the complexes of fusion protein and the encoding plasmid DNA, an in vitro selection method for high affinity DNA-binding protein was developed and experimentally demonstrated. The GAL4 DNA-binding domain (GAL4 DBD) was selected as a model DNA-binding protein, and enhanced green fluorescent protein (EGFP) was used as an expression reporter for the selection of target proteins. Error prone PCR was conducted to construct a mutant library of the model. Based on the affinity decrease with increased salt concentration, mutants of GAL4 DBD having high affinity were selected from the mutant protein library of protein-encoding plasmid complex by this method. Two mutants of (Lys33Glu, Arg123Lys, Ile127Lys) and (Ser47Pro, Ser85Pro) having high affinity were obtained from the first generation mutants. This method can be used for rapid in vitro selection of high affinity DNA-binding proteins, and has high potential for the screening of high affinity DNA-binding proteins in a sequence-specific manner.

• BMB Reports
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• 제44권2호
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• pp.102-106
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• 2011

Thermodynamic stability and refolding kinetics of firefly luciferase and three representative mutants with depletion of negative charge on a flexible loop via substitution of Glu by Arg (ER mutant) or Lys (EK mutant) as well as insertion of another Arg in ER mutants (ERR mutant) was investigated. According to thermodynamic studies, structural stability of ERR and ER mutants are enhanced compared to WT protein, whereas, these mutants become prone to aggregation at higher temperatures. Accordingly, it was concluded that enhanced structural stability of mutants depends on more compactness of folded state, whereas aggregation at higher temperatures in mutants is due to weakening of intermolecular repulsive electrostatic interactions and increase of intermolecular hydrophobic interactions. Kinetic results indicate that early events of protein folding are accelerated in mutants.

• Mycobiology
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• 제38권2호
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• pp.108-112
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• 2010

To investigate the possible roles of LAMMER kinase homologue, Lkh1, in Schizosaccharomyces pombe, whole proteins were extracted from wild type and lkh1-deletion mutant cells and subjected to polyacrylamide gel electrophoresis. Differentially expressed proteins were identified by tandem mass spectrometry (MS/MS) and were compared with a protein database. In whole-cell extracts, 10 proteins were up-regulated and 9 proteins were down-regulated in the mutant. In extracellular preparations, 6 proteins were up-regulated in the lkh1+ null mutant and 4 proteins successfully identified: glycolipid anchored surface precursor, $\beta$-glucosidase (Psu1), cell surface protein, glucan 1,3-$\beta$-glucosidase (Bgl2), and exo-1,3 $\beta$-glucanase (Exg1). These results suggest that Lkh1 is involved in regulating cell wall assembly.

• Journal of Microbiology
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• 제44권6호
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• pp.641-648
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• 2006

We previously reported that Skp1, a component of the Skp1-Cullin-F-box protein (SCF) complex essential for the timely degradation of cell cycle proteins by ubiquitination, physically interacts with Bfa1, which is a key negative regulator of the mitotic exit network (MEN) in response to diverse checkpoint-activating stresses in budding yeast. In this study, we initially investigated whether the interaction of Skp1 and Bfa1 is involved in the regulation of the Bfa1 protein level during the cell cycle, especially by mediating its degradation. However, the profile of the Bfa1 protein did not change during the cell cycle in skp1-11, which is a SKP1 mutant allele in which the function of Skp1 as a part of SCF is completely impaired, thus indicating that Skp1 does not affect the degradation of Bfa1. On the other hand, we found that the skp1-12 mutant allele, previously reported to block G2-M transition, showed defects in mitotic exit and cytokinesis. The skp1-12 mutant allele also revealed a specific genetic interaction with ${\Delta}bfa1$. Bfa1 interacted with Skp1 via its 184 C-terminal residues (Bfa1-D8) that are responsible for its function in mitotic exit. In addition, the interaction between Bfa1 and the Skp1-12 mutant protein was stronger than that of Bfa1 and the wild type Skp1. We suggest a novel function of Skp1 in mitotic exit and cytokinesis, independent of its function as a part of the SCF complex. The interaction of Skp1 and Bfa1 may contribute to the function of Skp1 in the mitotic exit.

• KSBB Journal
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• 제15권2호
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• pp.181-187
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• 2000

본 연구에서 갈락토즈를 거의 사용하지 않고 glucose repression 정도가 줄어든 변이주를 이용하여 fed-batch를 통한 발현최적화를 수행하였다. 두 균주에서의 GAL promoter에 의한 외래단백질 생산시 glucose repression 정도에 대해 조사하였는데 대조구 Y2805는 글루코즈가 다 소비된 후 2∼3시간 지난 후 발현이 시작되나 변이주 Y334는 약 0.5 g/L 글루코즈 농도에서 25%정도의 발현이 이루어짐에 따라 변이주 Y334는 GAL promoter에 미치는 glucose repression 정도가 매우 약한 장점을 확인하였다. 배양 중 재조합 미생물인 두균주 변이주 Y334와 대조구 효모 Y2805의 plasmid stability에 대해 안정한 균주임을 알 수 있었으며 대조구 Y2805의 경우도 plasmid stability는 90%로 유지됨을 알 수 있었다. GAL promoter에 의한 외래 단백질 생산시 글루코즈와 갈락토즈, 에탄올의 소비속도를 조사하였는데, 글루코즈와 에탄올의 소비속도는 거의 비슷하였으나 갈락토즈 소비속도는 Y2805는 0.1232 g/L/hr/O.D.이고 변이주 Y334는 0.0131 g/L/hr/O.D. 이다. 또한 mutant Y334와 대조구 효모 Y2805의 Fed-batch 시의 올바른 feeding 방법을 구하기위한 실험을 수행하여 각 균주의 Fed-batch 실험에서의 최적 발현 방법을 획득하였다.

• The Plant Pathology Journal
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• 제37권6호
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• pp.555-565
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• 2021

Bacteriophages infecting Acidovorax citrulli, the causal agent of bacterial fruit blotch, have been proven to be effective for the prevention and control of this disease. However, the occurrence of bacteriophage-resistant bacteria is one of hurdles in phage biocontrol and the understanding of phage resistance in this bacterium is an essential step. In this study, we aim to investigate possible phage resistance of A. citrulli and relationship between phage resistance and pathogenicity, and to isolate and characterize the genes involved in these phenomena. A phage-resistant and less-virulent mutant named as AC-17-G1 was isolated among 3,264 A. citrulli Tn5 mutants through serial spot assays and plaque assays followed by pathogenicity test using seed coating method. The mutant has the integrated Tn5 in the middle of a cupin protein gene. This mutant recovered its pathogenicity and phage sensitivity by complementation with corresponding wild-type gene. Site-directed mutation of this gene from wild-type by CRISPR/Cas9 system resulted in the loss of pathogenicity and acquisition of phage resistance. The growth of AC-17-G1 in King's B medium was much less than the wild-type, but the growth turned into normal in the medium supplemented with D-mannose 6-phosphate or D-fructose 6-phosphate indicating the cupin protein functions as a phosphomannos isomerase. Sodium dodecyl sulfa analysis of lipopolysaccharide (LPS) extracted from the mutant was smaller than that from wild-type. All these data suggest that the cupin protein is a phosphomannos isomerase involved in LPS synthesis, and LPS is an important determinant of pathogenicity and phage susceptibility of A. citrulli.

• IMMUNE NETWORK
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• 제9권2호
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• pp.58-63
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• 2009

Background: T cell immunoglobulin and mucin domain containing 3 protein (Tim-3) expressed on terminally differentiated Th1 cells plays a suppressive role in Th1-mediated immune responses. Recently, it has been shown that N-glycosylation affects the binding activity of the Tim-3-Ig fusion protein to its ligand, galectin-9, but the binding properties of non-glycosylated Tim-3 on $CD4^+CD25^+$T cells has not been fully examined. In this study, we produced recombinant Tim-3-Ig fusion proteins in different cellular sources and its N-glycosylation mutant forms to evaluate their binding activities to $CD4^+CD25^+$T cells. Methods: We isolated and cloned Tim-3 cDNA from BALB/C mouse splenocytes. Then, we constructed a mammalian expression vector and a prokaryotic expression vector for the Tim-3-Ig fusion protein. Using a site directed mutagenesis method, plasmid vectors for Tim-3-Ig N-glycosylation mutant expression were produced. The recombinant protein was purified by protein A sepharose column chromatography. The binding activity of Tim-3-Ig fusion protein to $CD4^+CD25^+$T cells was analyzed using flow cytometry. Results: We found that the nonglycosylated Tim-3-Ig fusion proteins expressed in bacteria bound to $CD4^+CD25^+$T cells similarly to the glycosylated Tim-3-Ig protein produced in CHO cells. Further, three N-glycosylation mutant forms (N53Q, N100Q, N53/100Q) of Tim-3-Ig showed similar binding activities to those of wild type glycosylated Tim-3-Ig. Conclusion: Our results suggest that N-glycosylation of Tim-3 may not affect its binding activity to ligands expressed on $CD4^+CD25^+$T cells.

• 통합자연과학논문집
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• 제4권3호
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• pp.222-226
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• 2011

Cyclic AMP receptor protein(CRP) is involved in the activation of many genes corresponding to catabolite enzymes in Escherichia coli. In this study, mutant CRP(S128A) was used to elucidate the effect of Ser 128 on the cAMP-induced structural change. Based on the protease digestion and thermal analysis, serine 128 in CRP affects the cAMP binding capability and then structural change of CRP protein. In addition, CD spectra in near UV region revealed that S128A CRP retained the sensitive conformation to thermal effect relative to that of wild-type CRP, in spite of identical Tm values in the absence of cAMP.