• Horticultural Science & Technology
• /
• v.29 no.2
• /
• pp.116-122
• /
• 2011

In this study, we report the generation and analysis of 1,392 expressed sequence tags (ESTs) from Korean Stewartia (Stewartia koreana Nakai). A cDNA library was generated from the young leaf tissue and a total of 1,392 cDNA were partially sequenced. EST and unigene sequence quality were determined by computational filtering, manual review, and BLAST analyses. Finally, 1,301 ESTs were acquired after the removal of the vector sequence and filtering over a minimum length 100 nucleotides. A total of 893 unigene, consisting of 150 contigs and 743 singletons, was identified after assembling. Also, we identified 95 new microsatellite-containing sequences from the unigenes and classified the structure according to their repeat unit. According to homology search with BLASTX against the NCBI database, 65% of ESTs were homologous with known function and 11.6% of ESTs were matched with putative or unknown function. The remaining 23.2% of ESTs showed no significant similarity to any protein sequences found in the public database. Annotation based searches against multiple databases including wine grape and populus sequences helped to identify putative functions of ESTs and unigenes. Gene ontology (GO) classification showed that the most abundant GO terms were transport, nucleotide binding, plastid, in terms biological process, molecular function and cellular component, respectively. The sequence data will be used to characterize potential roles of new genes in Stewartia and provided for the useful tools as a genetic resource.

• Korean Journal of Clinical Laboratory Science
• /
• v.50 no.2
• /
• pp.100-109
• /
• 2018

The emergence and dissemination of multidrug-resistant (MDR) Acinetobacter baumannii isolates have been reported worldwide, with most of these possessing the ability to form biofilms. Biofilm formation is an important virulence factor associated with the resistance to disinfection and desiccation. This study examined the genetic basis of antimicrobial resistance mechanisms of biofilm-forming A. baumannii clinical isolates. Imaging and quantification of biofilms were performed by a crystal violet assay and 46 biofilm-forming A. baumannii isolates were selected. Subsequently, 16 isolates belonging to different clones were identified using REP-PCR, and detection of the antimicrobial determinants in the isolates was carried out. The 16 isolates included 9 non-MDR and 7 MDR isolates. The mean biomass $OD_{560}$ values of the non-MDR (0.96) and MDR (1.05) isolates differed but this difference was not significant. In this study, most biofilm-forming MDR A. baumannii isolates contained various antimicrobial resistance determinants ($bla_{OXA-23}$, armA, and mutations of gyrA and parC). On the other hand, most biofilm-forming non-MDR A. baumannii isolates did not contain antimicrobial resistance determinants. These results suggest that there is little correlation between the biofilm-forming ability and antimicrobial susceptibility in A. baumannii isolates. In addition, the emergence of MDR A. baumannii clinical isolates is generally caused by mutations of the genes associated with antimicrobial resistance and/or the acquisition of various antimicrobial resistance determinants.

• Korean Journal of Food Science and Technology
• /
• v.34 no.2
• /
• pp.311-318
• /
• 2002

A new bacteriocin producing lactic acid bacteria having antagonistic activity against Lactobacillus plantarum, was isolated from Kimchi. It was identified as Leuconostoc mesenteroides, and designated as Leuconostoc mesenteroides B7. The bacteriocin from Leuconostoc mesenteroides B7 named as bacteriocin B7 was stable in the pH range $2.5{\sim}9.5$. Bacteriocin B7 was active over a wide temperature range from $4^{\circ}C$ to $120^{\circ}C$. It was inactivated by proteinase K, trypsin, ${\alpha}-chymotrypsin$, and protease treatments indicating its proteinous nature. Tricine-SDS-PAGE of the purified bacteriocin B7 showed the presence of a single band, having a molecular mass of about 3,500 dalton. Mixed culture of the producer and the indicator, Lb. plantarum KFRI 464 or Lb. delbruekii KFRI 347, increased production of bacteriocin B7. This result suggested the presence of bacteriocin inducing factor in the indicator strain. The inducing factor was localized in cell debris and intracellular faction of the indicator cell, Lb. plantarum KFRI 464. Treatment of the inducing factor with proteinase K destroyed inducing activity. This result strongly suggested that the inducing factor is a protein.

• Asian-Australasian Journal of Animal Sciences
• /
• v.31 no.8
• /
• pp.1366-1372
• /
• 2018

Objective: A disintegrin and metallopeptidase with thrombospondin motifs type 8 (ADAMTS8) is crucial for diverse physiological processes, such as inflammation, tissue morphogenesis, and tumorigenesis. The chicken ADAMTS8 (chADAMTS8) gene was differentially expressed in the kidney following exposure to different calcium concentrations, suggesting a pathological role of this protein in metabolic diseases. We aimed to examine the molecular characteristics of chADAMTS8 and analyze the gene-expression differences in response to toll-like receptor 3 (TLR3) stimulation. Methods: The ADAMTS8 mRNA and amino acid sequences of various species (chicken, duck, cow, mouse, rat, human, chimpanzee, pig, and horse) were retrieved from the Ensembl database and subjected to bioinformatics analyses. Reverse-transcription polymerase chain reaction (RT-PCR) and quantitative PCR (qPCR) experiments were performed with various chicken tissues and the chicken fibroblast DF-1 cell line, which was stimulated with polyinosinic-polycytidylic acid (poly[I:C]; a TLR3 ligand). Results: The chADAMTS8 gene was predicted to contain three thrombospondin type 1 (TSP1) domains, whose amino acid sequences shared homology among the different species, whereas sequences outside the TSP1 domains (especially the amino-terminal region) were very dif­ferent. Phylogenetic analysis revealed that chADAMTS8 is evolutionarily clustered in the same clade with that of the duck. chADAMTS8 mRNA was broadly expressed in chicken tissues, and the expression was significantly up-regulated in the DF-1 cells in response to poly(I:C) stimulation (p<0.05). These results showed that chADAMTS8 may be a target gene for TLR3 signaling. Conclusion: In this report, the genetic information of chADAMTS8 gene, its expression in chicken tissues, and chicken DF-1 cells under the stimulation of TLR3 were shown. The result suggests that chADAMTS8 expression may be induced by viral infection and correlated with TLR3-mediated signaling pathway. Further study of the function of chADAMTS8 during TLR3-dependent inflammation (which represents RNA viral infection) is needed and it will also be important to examine the molecular mechanisms during different regulation, depending on innate immune receptor activation.

• Korean Journal of Poultry Science
• /
• v.38 no.2
• /
• pp.89-96
• /
• 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.

• Molecules and Cells
• /
• v.20 no.3
• /
• pp.385-391
• /
• 2005

As a first step towards identifying genes involving in the signal transduction pathways mediating rice blast resistance, we isolated 3 mutants lines that showed enhanced susceptibility to rice blast KJ105 (91-033) from a T-DNA insertion library of the japonica rice cultivar, Hwayeong. Since none of the susceptible phenotypes co-segregated with the T-DNA insertion we adapted a map-based cloning strategy to isolate the gene(s) responsible for the enhanced susceptibility of the Hwayeong mutants. A genetic mapping population was produced by crossing the resistant wild type Hwayeong with the susceptible cultivar, Nagdong. Chi-square analysis of the $F_2$ segregating population indicated that resistance in Hwayeong was controlled by a single major gene that we tentatively named Pi-hy. Randomly selected susceptible plants in the $F_2$ population were used to build an initial map of Pi-hy. The SSLP marker RM2265 on chromosome 2 was closely linked to resistance. High resolution mapping using 105 $F_2$ plants revealed that the resistance gene was tightly linked, or identical, to Pib, a resistance gene with a nucleotide binding sequence and leucine-rich repeats (NB-LRR) previously isolated. Sequence analysis of the Pib locus amplified from three susceptible mutants revealed lesions within this gene, demonstrating that the Pi-hy gene is Pib. The Pib mutations in 1D-22-10-13, 1D-54-16-8, and 1C-143-16-1 were, respectively, a missense mutation in the conserved NB domain 3, a nonsense mutation in the 5th LRR, and a nonsense mutation in the C terminus following the LRRs that causes a small deletion of the C terminus. These findings provide evidence that NB domain 3 and the C terminus are required for full activity of the plant R gene. They also suggest that alterations of the resistance gene can cause major differences in pathogen specificity by affecting interactions with an avirulence factor.

• Food Science of Animal Resources
• /
• v.40 no.5
• /
• pp.689-698
• /
• 2020

The aim of study was to scrutinize the physicochemical and protein profile of milk obtained from local Pakistani breeds of milch animals such as Nilli-Ravi buffalo, Sahiwal cow, Kajli sheep, Beetal goat and Brela camel. Physicochemical analysis unveiled maximum number of total solids and protein found in sheep and minimum in camel. Buffalo milk contains the highest level of fat (7.45%) while camel milk contains minimum (1.94%). Ash was found maximum in buffalo (0.81%) and sheep (0.80%) while minimum in cow's milk (0.71%). Casein and whey proteins were separated by subjecting milk to isoelectric pH and then analyzed through sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE). The results showed heterogeneity among these species. Different fractions including α_S1, α_S2, κ-casein, β-casein and β-lactoglobulen (β-Lg) were identified and quantitatively compared in all milk samples. Additionally, this electrophoretic method after examining the number and strength of different protein bands (α_S1, α_S2, β-CN, α-LAC, BSA, and β-Lg, etc.), was helpful to understand the properties of milk for different processing purposes and could be successfully applied in dairy industry. Results revealed that camel milk was best suitable for producing allergen free milk protein products. Furthermore, based on the variability of milk proteins, it is suggested to clarify the phylogenetic relationships between different cattle breeds and to gather the necessary data to preserve the genetic fund and biodiversity of the local breeds. Thus, the study of milk protein from different breed and species has a wide range of scope in producing diverse protein based dairy products like cheese.

• Applied Biological Chemistry
• /
• v.42 no.1
• /
• pp.12-19
• /
• 1999

To improve biopolymer productivity and properties of Bacillus strains, protoplast fusion was performed between biopolymer producing Bacillus subtilis K-1 and lactose utilizing Bacillus coagulans. The results were as follows; Protoplasts mixed in fusion fluid containing 33% PEG 6000, 1% PVP and 10 mM $CaCl_2$ were reacted for 5 min at $37^{\circ}C$ and then centrifused protoplasts were directly overlaid on the selective media containing $100\;{\mu}g/ml$ antibiotics and incubated for 3 days. At this conditions, the frequency of protoplast fusion was generally in the range of $4.6{\times}10^{-5}\;to\;1.8{\times}10^{-7}$ in ratio. Segregation ratio was observed between 1 to 6% indicating genetic stability of all the fusants. Fusants growth were also observed on the media contained amino acid and antibiotics as required marked materials. DNA contents of the selected fusants were 1.6 to 2.7 times more than that of parent strains. With observation by TEM microscopy, spherical protoplasts were first released from the swollen parental cells and then contracted to fuse in the process of fusion. And fused cells were observed representative vesicle. Originally, the parental cells were observed as in the morphology of thick-walled and double membrane-surrounded rod shape with TEM microscopy.

• Journal of Microbiology and Biotechnology
• /
• v.28 no.6
• /
• pp.997-1006
• /
• 2018

As shown during the 2009 pandemic H1N1 (A(H1N1)pdm09) outbreak, egg-based influenza vaccine production technology is insufficient to meet global demands during an influenza pandemic. Therefore, there is a need to adapt cell culture-derived vaccine technology using suspended cell lines for more rapid and larger-scale vaccine production. In this study, we attempted to generate a high-growth influenza vaccine strain in MDCK cells using an A/Puerto/8/1934 (H1N1) vaccine seed strain. Following 48 serial passages with four rounds of virus plaque purification in MDCK cells, we were able to select several MDCK-adapted plaques that could grow over $10^8PFU/ml$. Genetic characterization revealed that these viruses mainly had amino acid substitutions in internal genes and exhibited higher polymerase activities. By using a series of Rg viruses, we demonstrated the essential residues of each gene and identified a set of high-growth strains in MDCK cells ($PB1_{D153N}$, $M1_{A137T}$, and $NS1_{N176S}$). In addition, we confirmed that in the context of the high-growth A/PR/8/34 backbone, A/California/7/2009 (H1N1), A/Perth/16/2009 (H3N2), and A/environment/Korea/deltaW150/2006 (H5N1) also showed significantly enhanced growth properties (more than $10^7PFU/ml$) in both attached- and suspended-MDCK cells compared with each representative virus and the original PR8 vaccine strain. Taken together, this study demonstrates the feasibility of a cell culture-derived approach to produce seed viruses for influenza vaccines that are cheap and can be grown promptly and vigorously as a substitute for egg-based vaccines. Thus, our results suggest that MDCK cell-based vaccine production is a feasible option for producing large-scale vaccines in case of pandemic outbreaks.

• Korean Journal of Microbiology
• /
• v.28 no.1
• /
• pp.55-64
• /
• 1990

Cell volume and DNA contents of the fusants were similar to those of parents. Genetic stability of the fusants was increased when they were cultured on minimal medium (MM) rather than on complete medium (CM), and the fusants were stabilized by subculturing 7 generations each 7 day on MM agar. The finally selected fusants after being cultured for 6 months on CM were stable without segregation. The fusants could also form nuclein and ascospores, and show red and pink colors by the test of TTC colorization. Assimilability and fermentability of carbon sources of the fusants were similarto those of parents. The tolerance of KCl, NaCl, sodium propionate and cycloheximide showed the traits of one strain of parents. When the fusants were cultured for 72 hr and 60 hr in the medium containing 20% glucose and sucrose, respectively, the yield of ethanol for FWKS 260 was reached to 9.6 v/v% and 9.8v/v%, respectively. The sensitive strain Kyokai 7 was found to be killed entirely after cultivation of 48 hr by the killer toxin from the fusants. The recipient S 29 and Kyokai 7 were found to have neither L nor M dsRNA plasmid. However, K 52 and fusants had both L and M dsRNA plasmid of 4.7 kb and 2.5 kb, respectively. The curants treated by heat and cycloheximide did not contain M dsRNA plasmid, but had large amounts of L dsRNA plasmid of those of killer yeasts.