Identification of a Tolerant Locus on Arabidopsis thaliana to Hypervirulent Beet Curly Top Virus CFH Strain

Seong-Hee,Park,Jinkyung,Hur,Jongbum,Park,Sangseob,Lee,Taek-Kyun,Lee,Man,Chang,Keith,R.,Davis,Jeongha,Kim,이석찬 한국분자세포생물학회 2002 Molecules and cells Vol.13 No.2

The infection of hosts by the geminivirus depends on the interactions between host and viral factors for viral DNA replication, viral gene expression, and the movement of virus throughout the hosts. This work reports that a hypervirulent strain of Beet curly top virus (BCTV) is different in its ability to infect several ecotypes of Arabidopsis thaliana. Symptoms appeared on Arabidopsis ecotypes around 7 to 10 d after inoculation with BCTV-CFH. Symptoms were more severe in ecotype SKKU including severe leaf curling and development of severely deformed and stunted bolting compared to Col-O as a lab standard ecotype. One ecotype Cen-O was asymptomatic to BCTV-CFH infection. Studies of viral DNA replication and virus movement in three excised organs of asymptomatic Cen-O demonstrated that BCTV-CFH could replicate viral DNA and move systemically in this ecotype, suggesting that tolerance was due to the blocks of interactions between host and viral factors on symptom development. This asymptomatic phenotype is similar to the mutation of leftward ORFs, especially ORF R2. Genetic analysis of this ecotype Cen-O indicated that tolerance is due to a single recessive locus.

Crystal Structure of the Regulatory Domain of MexT, a Transcriptional Activator of the MexEF-OprN Efflux Pump in Pseudomonas aeruginosa

Nam-Chul,Ha,김수현,Songhee,H.,Kim,Jinsook,Ahn,조인성,이지원,Sang,Ho,CHOI 한국분자세포생물학회 2019 Molecules and cells Vol.42 No.12

The Gram-negative opportunistic pathogen, Pseudomonas aeruginosa, has multiple multidrug efflux pumps. MexT, a LysR-type transcriptional regulator, functions as a transcriptional activator of the MexEF-OprN efflux system. MexT consists of an N-terminal DNA‑binding domain and a C‑terminal regulatory domain (RD). Little is known regarding MexT ligands and its mechanism of activation. We elucidated the crystal structure of the MexT RD at 2.0 Å resolution. The structure comprised two protomer chains in a dimeric arrangement. MexT possessed an arginine-rich region and a hydrophobic patch lined by a variable loop, both of which are putative ligand‑binding sites. The three-dimensional structure of MexT provided clues to the interacting ligand structure. A DNase I footprinting assay of full-length MexT identified two MexT-binding sequence in the mexEF-oprN promoter. Our findings enhance the understanding of the regulation of MexT-dependent activation of efflux pumps.

Development of Research into Autophagic Lysosome Reformation

Yang,Chen,Li,Yu 한국분자세포생물학회 2018 Molecules and cells Vol.41 No.1

Autophagy is a lysosome-dependent degradation process that is essential for maintaining cellular homeostasis. In recent years, more studies have focused on the late stages of autophagy. Our group discovered and studied the terminal step of autophagy, namely autophagic lysosome reformation (ALR). ALR is the process that regenerates functional lysosomes from autolysosomes, thus maintaining lysosome ho-meostasis. ALR involves clathrin-mediated membrane budding from autolysosomes, elongation of membrane tubules along microtubules with the pulling force provided by the motor protein KIF5B, proto-lysosome scission by dynamin 2, and finally maturation of proto-lysosomes to functional lysosomes. In this review, we will summarize progress in unveiling the molecular mechanisms underlying ALR and its potential pathophysiological roles.

Novel Discovery of LINE-1 in a Korean Individual by a Target Enrichment Method

신원석,문세영,김준세,이우석,박동국,최승규,이태윤,차승희,한규동 한국분자세포생물학회 2019 Molecules and cells Vol.42 No.1

Long interspersed element-1 (LINE-1 or L1) is an autonomous retrotransposon, which is capable of inserting into a newregion of genome. Previous studies have reported that these elements lead to genomic variations and altered functions byaffecting gene expression and genetic networks. Mounting evidence strongly indicates that genetic diseases or variouscancers can occur as a result of retrotransposition events that involve L1s. Therefore, the development of methodologies tostudy the structural variations and interpersonal insertion polymorphisms by L1 element-associated changes in an individual genome is invaluable. In this study, we applied a systematic approach to identify human-specific L1s (i.e., L1Hs)through the bioinformatics analysis of high-throughput nextgeneration sequencing data. We identified 525 candidatesthat could be inferred to carry non-reference L1Hs in a Korean individual genome (KPGP9). Among them, we randomlyselected 40 candidates and validated that approximately 92.5% of non-reference L1Hs were inserted into a KPGP9 genome. In addition, unlike conventional methods, our relatively simple and expedited approach was highly reproduciblein confirming the L1 insertions. Taken together, our findings strongly support that the identification of non-reference L1Hsby our novel target enrichment method demonstrates its future application to genomic variation studies on the risk of cancer and genetic disorders.

Histone Deacetylase 701 (HDT701) Induces Flowering in Rice by Modulating Expression of OsIDS1

조래현,윤진미,Antt,Htet,Wai,안진흥 한국분자세포생물학회 2018 Molecules and cells Vol.41 No.7

Rice is a facultative short-day (SD) plant in which flowering is induced under SD conditions or by other environmental factors and internal genetic programs. Overexpression of Histone Deacetylase 701 (HDT701) accelerates flowering in hybrid rice. In this study, mutants defective in HDT701 flowered late under both SD and long-day conditions. Expression levels of florigens Heading date 3a (Hd3a) and Rice Flowering Locus T1 (RFT1), and their immediate upstream floral activator Early heading date 1 (Ehd1), were significantly decreased in the hdt701 mutants, indicating that HDT701 functions upstream of Ehd1 in controlling flowering time. Transcript levels of OsINDETERMINATE SPIKELET 1 (OsIDS1), an upstream repressor of Ehd1, were significantly increased in the mutants while those of OsGI and Hd1 were reduced. Chromatinimmunoprecipitation assays revealed that HDT701 directly binds to the promoter region of OsIDS1. These results suggest that HDT701 induces flowering by suppressing OsIDS1.

OASL1 Traps Viral RNAs in Stress Granules to Promote Antiviral Responses

강지선,황윤상,김락균,이수정,이욱빈,Jeongsil,Kim-Ha,김영준 한국분자세포생물학회 2018 Molecules and cells Vol.41 No.3

Oligoadenylate synthetase (OAS) protein family is the major interferon (IFN)-stimulated genes responsible for the activation of RNase L pathway upon viral infection. OAS-like (OASL) is also required for inhibition of viral growth in human cells, but the loss of one of its mouse homolog, OASL1, causes a severe defect in termination of type I interferon production. To further investigate the antiviral activity of OASL1, we examined its subcellular localization and regulatory roles in IFN production in the early and late stages of viral infection. We found OASL1, but not OASL2, formed stress granules trapping viral RNAs and promoted efficient RLR signaling in early stages of infection. Stress granule formation was dependent on RNA binding activity of OASL1. But in the late stages of infection, OASL1 interacted with IRF7 transcripts to inhibit translation resulting in down regulation of IFN production. These results implicate that OASL1 plays context dependent functions in the antiviral response for the clearance and resolution of viral infections.

Characterization of Lipopolysaccharide Responsive Proteins that Binds to the 5'Flanking Regions of Mouse Rantes

Youn-Uck,Kim,Youn-Hwoan,Kim,Duek-Jun,An,Hyuk-Chu,Kwon 한국분자세포생물학회 2002 Molecules and cells Vol.13 No.1

When macrophage (like the RAW264.7 cell line) was stimulated with lipopolysaccharide (LPS), factors that bind specifically to the LPS responsive element (LRE) of murine Rantes gene appeared in the nucleus. An electrophoretic mobility shift assay (EMSA) detected 2 specific bands, designated as S (slow) and M (middle). The S band appeared within 15 min of LPS stimulation, and reached its highest intensity within 2 h. The M band was present in unstimulated cells, but after stimulation its intensity increased and reached its highest intensity also in about 2 h. Significantly, in LPS hyporesponsive 10-9 macrophage like cells, the S band was absent. The M band was present in equal amounts in stimulated and unstimulated cells. The results suggest that the S band was induced by LPS stimulation. In the nuclear extract, the native molecular weight of the S band-forming factor was approximately 270 kDa, and that of the M bands-forming factor was approximately 140 kDa. U.V. cross linking studies consistently showed at least 2 different polypeptides of approximate molecular mass of 70 kDa, both in the S band-forming factor (complex) and the M band-forming factor (complex). In the nuclear extracts of both the LPS stimulated and unstimulated cells, we detected a factor with approximate molecular mass of 120 kDa that could convert the S band-forming complex to the M bandforming complex. This factor, designated as a converting factor, is a protein phosphatase since its activity was blocked by okadaic acid, an inhibitor of Ser/Thr protein phosphatase. Also, purified protein phosphatase type 1 (PP-1) could convert the S bandforming complex to the M band-forming complex.

Characterization of Mycoplasma Arginine Deiminase Expressed in E. coli and Its Inhibitory Regulation of Nitric Oxide Synthesis

Eun,Joo,Noh,Sang,Wook,Kang,신용재,Dong,Chung,Kim,In,Sun,Park,Min,Young,Kim,Boe,Gwun,Chun,Bon,Hong,Min 한국분자세포생물학회 2002 Molecules and cells Vol.13 No.1

We previously reported that a cytostatic protein that is found in ASC-17D Sertoli cell-conditioned media was Mycoplasma arginine deiminase (ADI), which hydrolyzes L-arginine into L-citrulline and ammonia. Here, we report the over-expression of recombinant ADI (rADI) in E. coli and the down-regulation of lipopolysaccharide (LPS) induced-nitric oxide (NO) production by rADI treatment. We cloned the ADI gene from Mycoplasma arginini genomic DNA by a polymerase chain reaction, and changed five TGA tryptophan codons (stop codon in E. coli) to TGG codons in the coding region by site-directed mutagenesis in order to express in E. coli. The rADI was purified to apparent homogeneity by DEAE-Sepharose and arginine-affinity chromatography. The rADI expressed in E. coli was identified as 45 kDa on SDS-PAGE and 90 kDa on native PAGE, implying that it exists as a dimer like ADI of M. arginini. The Km for arginine of rADI was approximately 370 ± 50 mM. Its optimal temperature and pH were 41oC and pH 6.4, respectively, and enzyme activity remained ³ 50% for 5 d at physiological temperature and pH. Treatment of purified rADI suppressed NO production in macrophage-like RAW 264.7 and primary glial cells that were exposed to LPS. Furthermore, an intraperitoneal injection of rADI significantly suppressed the rise of blood nitrite/nitrate levels that were induced by the systemic administration of bacterial endotoxin LPS to mice, resulting in an improvement in their survival rate. These results suggest that the depletion of blood arginine with an arginine- metabolizing enzyme, such as ADI, could suppress excessive production of NO that is caused by inducible NOS (iNOS) during the endotoxemia. Also, rADI may be used as a new approach to control NO-related diseases, such as sepsis.

Osteopontin in Kainic Acid-induced Microglial Reactions in the Rat Brain

김성윤,Yun-Sik,Choi,최정선,차정호,OkNyu,Kim,SangBok,Lee,정진웅 한국분자세포생물학회 2002 Molecules and cells Vol.13 No.3

The present study was performed to investigate the spatial and temporal expression of osteopontin (OPN) mRNA in the rat brain after kainic acid-induced seizures, and to determine whether this phenomenon is associated spatiotemporally with the microglial reaction. The expression of OPN mRNA was detected using an in situ hybridization technique and Northern blot analysis. Following intraperitoneal injection of kainic acid (10 mg/kg), OPN mRNA was expressed in selective vulnerable areas, including the hippocampus, thalamus, hypothalamus, amygdala, and entorhinal cortex. Comparison of the morphology and localization with the established microglial marker OX-42 in the adjacent sections positively identified the OPNexpressed cells as microglia. Furthermore, double labeling experiments revealed that OPN mRNA expression was confined to ameboid-like cells among microglia stained with GSI-B4, an another microglial marker. These findings from a rat model of seizure support the notion that OPN can be synthesized in a subpopulation of reactive microglial cells. It can therefore be assumed that in the response of the brain to excitotoxic injury, synthesis of OPN occurs generally in a subset of activated microglia.

Involvement of Subcomplexes of 32 and 14 kDa Subunits in RPA's DNA Binding Activity through Redox Change

김안드레,박장수 한국분자세포생물학회 2002 Molecules and cells Vol.13 No.3

The eukaryotic replication protein A (RPA) is a heterotrimeric protein complex. It consists of 70, 32, and 14 kDa subunits that are involved in DNA replication, repair, and genetic recombination. RPA is a 4-cysteine type zinc-finger protein. RPA's zinc-finger domain is not essential for DNA binding activity, but it is involved in the regulation of RPA's DNA binding activity through reduction-oxidation (redox). In this study, we show that yeast RPA's ssDNA binding activity is regulated by redox potential through its subcomplexes of 32 and 14 kDa subunits. In contrast, the subunits' complex, RPA70, formed a stable complex with ssDNA, even under non-reducing conditions. The addition of DTT and H2O2 had no effect on its DNA binding activity. In RPA70, since the addition of the subcomplexes of the 32 and 14 kDa subunits, it restored the modulating ssDNA binding activity to native RPA's DNA binding activity. These results suggest that the subcomplexes of the 32 and 14 kDa subunits may be involved in the modulating RPA's DNA binding activity through redox change. These studies, therefore, show the novel structure and function relationship of a multiprotein complex in that the role of a specific domain (or one subunit) is regulated by the other subunits.