• Title/Summary/Keyword: cleavage site

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Genetic Characterization of H7-subtype Avian Influenza Viruses (H7 아형 조류인플루엔자 바이러스의 유전자 특성)

  • Yeo, Jiin;Kwon, Hyuk-Moo;Sung, Haan-Woo
    • Korean Journal of Poultry Science
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    • v.46 no.3
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    • pp.173-183
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    • 2019
  • Based on their virulence, the avian influenza viruses (AIVs) are classified into two pathotypes: low pathogenic avian influenza (LPAI) virus and highly pathogenic avian influenza (HPAI) virus. Among the 16 HA subtypes of AIV, only the H5 and H7 subtypes are classified as HPAI. Some AIVs, including H5 and H7 viruses, can infect humans directly. Six H7 subtype isolates from wild birds of the H7N7 (n=4) and H7N1 (n=2) subtypes were characterized in this study. Phylogenetic analysis showed that eight viral genes (HA, NA, PB2, PB1, PA, NP, M, and NS) of the H7 isolates clustered in the Eurasian lineage, the genetic diversity of which is indicated by its division into several sublineages. The Korean H7 isolates had two motifs, PEIPKGR and PELPKGR, at the HA cleavage site, which have been associated with LPAI viruses. Six H7 isolates encoded glutamine (Q) and glycine (G) at positions 226 (H3 numbering) and 228 of HA, suggesting avian-type receptor-binding specificity. None of the Korean H7 isolates had the amino acid substitutions E627K in PB2 and I368V in PB1, which are critical for efficient replication in human cells. The Korean H7 isolates showed no deletions in the NA stalk region and in NS. These results suggest that the Korean H7 isolates from wild birds are different from the H7N9 influenza viruses isolated in China in 2013, which are capable of infecting humans.

Identification and characterization of laccase genes in the Flammulina velutipes var. lupinicola genome (Flammulina velutipes var. lupinicola의 유전체 정보기반 laccase 유전자 동정 및 특성 규명)

  • Yu, Hye-Won;Park, Young-Jin
    • Journal of Mushroom
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    • v.19 no.4
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    • pp.285-293
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    • 2021
  • The purpose of this study was to identify and characterize the laccase genes of Flammulina velutipes var. lupinicola. Five laccase genes (g1934, g1937, g2415, g2539, g5858) were selected based on the copper binding site and signal peptide analysis results using the laccase gene selected from the F. velutipes var. lupinicola genome. The size of the laccase genes of F. velutipes var. lupinicola were 1,488 bp~1,662 bp. As a result of cDNA sequence analysis, 14 to 17 introns were identified in the laccase genes. The cleavage site predicted as the signal peptide of the laccase gene was found to be located between 20 bp and 34 bp from the N-terminus. In addition, separation and purification were performed to characterize the F. velutipes var. lupinicola laccases, and the optimal activity of the separated and purified proteins were analyzed by pH, temperature and time. Five bands with laccase activity were found from zymogram analysis. The optimal pH of the reaction was 5.5, the optimal temperature was found to be 40℃. Therefore, characterization of the laccase genes identified in this study should help in better understanding the biomass decomposition of F. velutipes var. lupinicola.

Molecular Orbital Anaysis of Water Activation on TiO2(110) Surface (TiO2(110) 표면에 흡착된 물분자의 결합 활성화에 관한 MO 연구)

  • Kang, Dae-Bok;
    • Journal of the Korean Chemical Society
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    • v.46 no.3
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    • pp.179-186
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    • 2002
  • A molecular orbital analysis based on the extended Huckel calculations has been carried out to study the OH bond activation of water on the $TiO_2$(110) surface. $H_2O$ binds with its axis perpendicular to the surfac on top of the five-coordinate $Ti^{4+}$ atom via its $3a_1$ orbital. In this bonding situation, the two-coordinated bridging $O^{2-}$ atom ($O_b$, basic site) on $TiO_2$(110) is too distant from an H atom of water to form hydrogen-bondig interactions with water that facilitate O-H bond cleavage. It has been elucidated that the O-H bond is appreciably weakened when the water molecule is tilted to give a hydrogen bond with the $O_b$ atom. This mechanism includes mutual transfer of electron density from the $3a_1$ orbital of the water molecule to the $Ti^{4+} 3d_{z2}$ orbital and from the $O_b$ P orbitals to the $2b_1$ of the adsorbed water molecule This should result in lengthening of the O-H bond in the surface complex and the subsequent dissociation into the fragments OH and H.

Characterizations of Restriction Endonuclease EagBI from Enterobacter agglomerans CBNU45 (Enterobacter agglomerans CBNU45로부터 분리된 제한효소 EagBI 의 특성)

  • Choe, Yeong-Ju;Kim, Seong-Jae;Hwang, Hye-Yeon;Im, Jeong-Bin;Kim, Yeong-Chang
    • Korean Journal of Microbiology
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    • v.32 no.1
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    • pp.91-95
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    • 1994
  • EagBI is a type II restriction endonuclease from Enterobacter agglomerans strain CBNU45 isolated from soil. EagBI was partially purified by DEAE-cellulose, phosphocellulose P11 and hydroxylapatite column chromatography. EagBI recognizes and cleaves the sequence 5'-CGAT${\downarrow}$CG-3' and generates 2-base 3'-protruding cohesive ends. The optimal reaction conditions of EagBI are 10 mM Tris-HCl (pH 7.8), 6-10 mM $MgCl_2$, at 37 ${\circ}C$. The enzyme is maximally active in the absence of NaCl, able to cleave both $dam^-$ and $dam^+$ DNAs, and sensitive to heat treatment (at 65 ${\circ}C$ for 10 min). Therefore, although EagBI is an isoschizomer of PvuI, it is more useful than PvuI in respect of the NaCl requirement and heat-stability.

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Surveillance of Newcastle Disease Virus in Chicken Slaughterhouses (닭 도축장에서의 뉴캣슬병 바이러스 오염 실태 조사)

  • Choi, Kang-Seuk;Lee, Eun-Kyoung;Jeon, Woo-Jin;Kwon, Jun-Hun;Lee, Jin-Hwa;Sung, Haan-Woo
    • Korean Journal of Poultry Science
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    • v.38 no.2
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    • pp.97-104
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    • 2011
  • We conducted a 10-month (March to October 2009) surveillance of Newcastle disease virus (NDV) in 13 slaughterhouses in Korea. NDV was isolated in 13.0%, 13.3%, 16.0%, and 10.8% of chicken farms, transport vehicles, hang rooms, and chilling water, respectively. Of NDV isolates from slaughterhouses, 37% were isolated in July. All NDV isolates were determined to be lentogenic viruses by RT-PCR-based pathotyping, and all NDV isolates had the $^{112}GKQGR/L^{117}$ motif at the cleavage site of the F protein. Phylogenetic analysis based on the hypervariable region of the F protein gene classified all 25 NDV isolates examined into genotype I within class II. Of these, 24 were clustered together with the NDV V4 strain, while the remaining isolate was placed in the cluster belonging to the NDV Ulster 2C strain. Our results indicate that lentogenic NDV was a high-frequency contaminant in the serial process of ranging live birds to slaughtering at slaughterhouses.

Identification of a Causal Pathogen of Watermelon Powdery Mildew in Korea and Development of a Genetic Linkage Marker for Resistance in Watermelon (Citrullus lanatus)

  • Han, Bal-Kum;Rhee, Sun-Ju;Jang, Yoon Jeong;Sim, Tae Yong;Kim, Yong-Jae;Park, Tae-Sung;Lee, Gung Pyo
    • Horticultural Science & Technology
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    • v.34 no.6
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    • pp.912-923
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    • 2016
  • Watermelon production is often limited by powdery mildew in areas with a large daily temperature range. Development of resistant watermelon cultivars can protect against powdery mildew; however, little is known about the characteristics of its causal agents. Here, we identified the genus and race of a causal pathogen of powdery mildew in Ansung province of South Korea, and developed molecular markers for the generation of resistant watermelon cultivars. The causal pathogen was determined to be Podosphaera xanthii based on multiple sequence alignments of internal transcribed spacers (ITS) of rDNA. The physiological race was identified as 1W, and the Ansung isolate was named P. xanthii 1W-AN. Following inoculation with the identified P. xanthii 1W-AN, we found inheritance of the resistant gene fitting a single dominant Mendelian model in a segregated population ('SBA' ${\times}$ PI 254744). To develop molecular markers linked to fungus-resistant loci, random amplified polymorphic DNA (RAPD) was accomplished between DNA pooled from eight near-isogenic lines (NILs; $BC_4F_6$), originated from PI 254744 and susceptible 'SBB' watermelon. After sequencing bands from RAPD were identified in all eight NILs and PI254744, 42 sequence-characterized amplifiedregion (SCAR) markers were developed. Overall, 107 $F_2$ plants derived from $BC_4F_6$ NIL-1 ${\times}$ 'SBB' were tested, and one SCAR marker was selected. Sequence comparison between the SCAR marker and the reference watermelon genome identified three Nco I restriction enzyme sites harboring a single nucleotide polymorphism, and codominant cleavage-amplified polymorphic site markers were subsequently developed. A CAPS marker was converted to a high-resolution melt (HRM) marker, which can discriminate C/T SNP (254PMR-HRM3). The 254PMR-HRM3 marker was evaluated in 138 $F_{2:3}$ plants of a segregating population ('SBA' ${\times}$ PI254744) and was presumed to be 4.3 cM from the resistance locus. These results could ensure P. xanthii 1W-AN resistance in watermelon germplasm and aid watermelon cultivar development in marker-assist breeding programs.

Differentiation of Korean isolates of Entamoeba histolytica from Entamoeba dispar (우리 나라에서 분리한 이질아메바(Entamoeba histoItica)와 동형아메바(Entamoebn dispar)의 감별)

  • 최성준;이미정
    • Parasites, Hosts and Diseases
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    • v.34 no.1
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    • pp.15-20
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    • 1996
  • Cysts of Entamoebn histoIMtica are still found from humans in Korea, but notall of the cysts are known as pathogenic. The non-pathogenic strain is regarded as a differenL species, E. nispnr. In this study, Korean isolates of conventional E. histolvticn were subjected for the differentiation by polymerase chain reaction (PCR) and restriction fragment length polymorphism (RFLP) analysis. Human stools were screened by routine microscopic examination, and cyst or trophozoite positive stools were inoculated into Robinson media. The cultivated trophozoites were prepared for DNA extraction, and the DNAs were used for PCR with common primers of Pl gene. The PCR products were divested with 3 restriction enzymes and RFLP was observed. Also anti-sense primers containing the cleavage site of each restriction eWe were designed for differentiation only by PCR. The PCR products of Korean isolates 59,512, YS-6, and YS-27 were spliced by Taq I and Xmnl but not byAccl, and the isolates S1, S3, S11, S15, S16, S17, S20, YS- l7, and YS-44 were spliced by Acc I but not by Taq I and Xmn I. These RFLP pattern correlated well with PCR products by the species specific primers. The findings confirm that the Korean isolates 59,512, YS-6, and YS-27 are E. histolwtico and others are E. dispar. In Korea, most of the asymptomatic cyst carriers are infected by E. dispar, not by E. histolytica. Key words: Entcmoebc histolytica, Entcmoebn dispar Korean isolates, polymerase chain reaction (PCR), restriction fragment length polymorphism (RFLP)

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Molecular Biological Characterization of the First Newcastle Disease Virus Isolated in Mongolia (몽골에서 최초로 분리된 뉴캣슬병 바이러스의 분자생물학적 특성)

  • Choi, Kang-Seuk;Lee, Eun-Kyoung;Jeon, Woo-Jin;Batchuulon, D.;Sodnomdarjaa, R.;Park, Mi-Ja;Yoo, Ye-Nah;Kwon, Jun-Hun
    • Korean Journal of Poultry Science
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    • v.38 no.2
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    • pp.89-96
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    • 2011
  • The outbreak of Newcastle disease occurred for the first time at a commercial chicken farm near Ulaanbaatar, Mongolia in August 2010. Newcastle disease virus (NDV) obtained from infected chickens in Mongolia was characterized by biological and molecular biological approches. Mongolian NDV isolate killed all of chicken embryos within 60 h in the mean death time assay, indicating virulent for chicken. A genomic region of 695 nts between nts 1055 of the M gene and 508 of the F gene was amplified by RT-PCR and sequenced. The deduced amino acid sequence of the F protein cleavage site was $^{112}RRQKRF^{117}$, which is a typical sequence of velogenic strains of NDV and is agreement with the result of the MDT assay. The sequence of the partial F gene (nts 47 to 435) was used for genotyping by phylogenetic analysis. The phylogenetic analysis showed that the Mongolian isolate was of genotype VII within class II of NDV. Further phylogenetic analysis on the genotype VII strains revealed that the isolates placed in a genetic sublineage of VIId and most closely related with velogenic strains of NDV circulating in Far-east Asian region especially China, suggesting the introduction of velogenic NDV into Mongolia from neighboring countries.

Novel reassortant 2.3.4.4B H5N6 highly pathogenic avian influenza viruses circulating among wild, domestic birds in Xinjiang, Northwest China

  • Zhang, Qian;Mei, Xindi;Zhang, Cheng;Li, Juan;Chang, Nana;Aji, Dilihuma;Shi, Weifeng;Bi, Yuhai;Ma, Zhenghai
    • Journal of Veterinary Science
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    • v.22 no.4
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    • pp.43.1-43.10
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    • 2021
  • Background: The H5 avian influenza viruses (AIVs) of clade 2.3.4.4 circulate in wild and domestic birds worldwide. In 2017, nine strains of H5N6 AIVs were isolated from aquatic poultry in Xinjiang, Northwest China. Objectives: This study aimed to analyze the origin, reassortment, and mutations of the AIV isolates. Methods: AIVs were isolated from oropharyngeal and cloacal swabs of poultry. Identification was accomplished by inoculating isolates into embryonated chicken eggs and performing hemagglutination tests and reverse transcription polymerase chain reaction (RT-PCR). The viral genomes were amplified with RT-PCR and then sequenced. The sequence alignment, phylogenetic, and molecular characteristic analyses were performed by using bioinformatic software. Results: Nine isolates originated from the same ancestor. The viral HA gene belonged to clade 2.3.4.4B, while the NA gene had a close phylogenetic relationship with the 2.3.4.4C H5N6 highly pathogenic avian influenza viruses (HPAIVs) isolated from shoveler ducks in Ningxia in 2015. The NP gene was grouped into an independent subcluster within the 2.3.4.4B H5N8 AIVs, and the remaining six genes all had close phylogenetic relationships with the 2.3.4.4B H5N8 HPAIVs isolated from the wild birds in China, Egypt, Uganda, Cameroon, and India in 2016-2017, Multiple basic amino acid residues associated with HPAIVs were located adjacent to the cleavage site of the HA protein. The nine isolates comprised reassortant 2.3.4.4B HPAIVs originating from 2.3.4.4B H5N8 and 2.3.4.4C H5N6 viruses in wild birds. Conclusions: These results suggest that the Northern Tianshan Mountain wetlands in Xinjiang may have a key role in AIVs disseminating from Central China to the Eurasian continent and East African.

Effect of Implant Types and Bone Resorption on the Fatigue Life and Fracture Characteristics of Dental Implants (임플란트 형태와 골흡수가 임플란트 피로 수명 및 파절 특성에 미치는 효과에 관한 연구)

  • Won, Ho-Yeon;Choi, Yu-Sung;Cho, In-Ho
    • Journal of Dental Rehabilitation and Applied Science
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    • v.26 no.2
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    • pp.121-143
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    • 2010
  • To investigate the effect of implant types and bone resorption on the fracture characteristics. 4 types of Osstem$^{(R)}$Implant were chosen and classified into external parallel, internal parallel, external taper, internal taper groups. Finite elements analysis was conducted with ANSYS Multi Physics software. Fatigue fracture test was performed by connecting the mold to the dynamic load fatigue testing machine with maximum load of 600N and minimum load of 60N. The entire fatigue test was performed with frequency of 14Hz and fractured specimens were observed with Hitachi S-3000 H scanning electron microscope. The results were as follows: 1. In the fatigue test of 2 mm exposed implants group, Tapered type and external connected type had higher fatigue life. 2. In the fatigue test of 4 mm exposed implants group, Parallel type and external connected types had higher fatigue life. 3. The fracture patterns of all 4 mm exposed implant system appeared transversely near the dead space of the fixture. With a exposing level of 2 mm, all internally connected implant systems were fractured transversely at the platform of fixture facing the abutment. but externally connected ones were fractured at the fillet of abutment body and hexa of fixture or near the dead space of the fixture. 4. Many fatigue striations were observed near the crack initiation and propagation sites. The cleavage with facet or dimple fractures appeared at the final fracture sites. 5. Effective stress of buccal site with compressive stress is higher than that of lingual site with tensile stress, and effective stress acting on the fixture is higher than that of the abutment screw. Also, maximum effective stress acting on the parallel type fixtures is higher. It is careful to use the internal type implant system in posterior area.