• Applied Biological Chemistry
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• 제30권4호
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• pp.293-299
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• 1987

Bradyrhizobium japonicum ROKS 26 균주를 glycine및 Iysozyme을 처리하여 spheroplast를 형성시킨 후 Agrobacterium tumefaciens에서 분리한 Ti plasmid를 분리하여 형질전환 시키고 octopine 영양 요구성에 의하여 transformants를 분리하였다. 그 결과 Ti plasmid가 도입된 transformants의 형질전환율은 $1{\times}10^{-7}$ 정도 되었다. 얻어진 transformant의 plasmid를 조사한 결과 도입된 Ti plasmid 존재가 확인되었으며, 2차원 전기영동법으로 균체 단백질을 분석한 결과 수용세포인 Bredyrhizobium japonicum ROKS 26과 transformant간의 단백질 구성 차이도 화인되었다. 또한 transformant의 근류 형성력을 조사한 결과 본래의 근류의근류형성력을 가지고 있었으나, crown gall형성에 있어서는 Agrobacterium tumefaciens 15955에 의한 gall보다는 크기에 있어 차이가 있었다.

• 대한수의학회지
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• 제57권2호
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• pp.117-126
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• 2017

Bovine mastitis is an important microbial disease in the dairy industry. We investigated the frequencies of bacterial pathogens in 62 farms and pathogen antibiotic resistance from mastitis samples (n = 748). We tested the antimicrobial activity of chicken and duck egg white and lysozyme purified from chicken egg white. Moreover, we compared the microbiomes of normal and mastitic raw milk obtained by 16S rRNA gene pyrosequencing and culture methods. The results showed that the frequencies of Gram-positive pathogens (Enterococcus faecalis 37% and Staphylococcus aureus 36%) were higher than that of a Gram-negative pathogen (Escherichia coli 15%). Resistance frequencies to ampicillin and norfloxacin were lowest in Staphylococcus aureus (21%), Enterococcus faecalis (23%), and Escherichia coli (33%), and the antimicrobial activity of chicken egg white was higher than those of lysozyme and duck egg white. Pyrosequencing results revealed clear differences between the microbiomes of mastitic and normal raw milk samples and revealed a slightly similar, but clearly different, composition of pathogens compared to that from the culture method. Thus, pyrosequencing may be useful for elucidating changes in microbiomes during mastitis progression and treatment. A chicken egg white and antibiotic combination may help with mastitis treatment; however, further studies are needed.

• Journal of Microbiology and Biotechnology
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• 제17권7호
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• pp.1162-1168
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• 2007

Although widely used as a host for recombinant protein production, Escherichia coli is unsuitable for massive screening of recombinant clones, owing to its poor secretion of proteins. A vector system containing T4 holin and T7 lysozyme genes under the control of the ptsG promoter derivative that is inducible in the absence of glucose was developed for programmed cell lysis of E. coli. Because E. coli harboring the vector grows well in the presence of glucose, but is lysed upon glucose exhaustion, the activity of the foreign gene expressed in E. coli can be monitored easily without an additional step for cell disruption after the foreign gene is expressed sufficiently with an appropriate concentration of glucose. The effectiveness of the vector was demonstrated by efficient screening of the amylase gene from a Bacillus subtilis genomic library. This vector system is expected to provide a more efficient and economic screening of bioactive products from DNA libraries in large quantities.

• Journal of Microbiology and Biotechnology
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• 제18권5호
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• pp.946-951
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• 2008

The opd gene, encoding organophosphorus hydrolase (OPH) from Flavobacterium sp. capable of degrading a wide range of organophosphate pesticides, was surface- and intracellular-expressed in Synechococcus PCC7942, a prime example of photoautotrophic cyanobacteria. OPH was displayed on the cyanobacterial cell surface using the truncated ice nucleation protein as an anchoring motif. A minor fraction of OPH was displayed onto the outermost surface of cyanobacterial cells, as verified by immunostaining visualized under confocal laser scanning microscopy and OPH activity analysis; however, a substantial fraction of OPH was buried in the cell wall, as demonstrated by proteinase K and lysozyme treatments. The cyanobacterial outer membrane acts as a substrate (paraoxon) diffusion barrier affecting whole-cell biodegradation efficiency. After freeze-thaw treatment, permeabilized whole cells with intracellular-expressed OPH exhibited 14-fold higher bioconversion efficiency ($V_{max}/K_m$) than that of cells with surface-expressed OPH. As cyanobacteria have simple growth requirements and are inexpensive to maintain, expression of OPH in cyanobacteria may lead to the development of a low-cost and low-maintenance biocatalyst that is useful for detoxification of organophosphate pesticides.

• Molecules and Cells
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• 제41권4호
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• pp.290-300
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• 2018

Using an in vitro model of intestinal organoids derived from intestinal crypts, we examined effects of indole-3-carbinol (I3C), a phytochemical that has anticancer and aryl hydrocarbon receptor (AhR)-activating abilities and thus is sold as a dietary supplement, on the development of intestinal organoids and investigated the underlying mechanisms. I3C inhibited the in vitro development of mouse intestinal organoids. Addition of ${\alpha}$-naphthoflavone, an AhR antagonist or AhR siRNA transfection, suppressed I3C function, suggesting that I3C-mediated interference with organoid development is AhR-dependent. I3C increased the expression of Muc2 and lysozyme, lineage-specific genes for goblet cells and Paneth cells, respectively, but inhibits the expression of IAP, a marker gene for enterocytes. In the intestines of mice treated with I3C, the number of goblet cells was reduced, but the number of Paneth cells and the depth and length of crypts and villi were not changed. I3C increased the level of active nonphosphorylated ${\beta}$-catenin, but suppressed the Notch signal. As a result, expression of Hes1, a Notch target gene and a transcriptional repressor that plays a key role in enterocyte differentiation, was reduced, whereas expression of Math1, involved in the differentiation of secretory lineages, was increased. These results provide direct evidence for the role of AhR in the regulation of the development of intestinal stem cells and indicate that such regulation is likely mediated by regulation of Wnt and Notch signals.

• BMB Reports
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• 제39권1호
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• pp.26-36
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• 2006

Differential Display PCR technique (DD-PCR) was used for the analysis of altered gene expression in hemocytes of Vibrio harveyi-infected Penaeus monodon. Forty-four combinations of arbitrary and oligo(dT) primers were used to screen for differentially expressed genes. A total of 79 differentially expressed bands could be identified from 33 primer combinations. These included 48 bands (61%) whose expression level increased and 31 bands (39%) decreased after V. harveyi challenge. Subsequently, forty-eight differential display fragments were successfully reamplified and cloned. A total of 267 clones were randomly selected and sequenced. The sequence analysis showed that 85 (31%) out of 267 clones were matched with sequences in the GenBank database which represented 24 different genes with known functions. Among the known genes, glucose transporter 1, interferon-related developmental regulator 1, lysozyme, profilin, SERPINB3, were selected for further confirmation of their differentially expression patterns by real-time PCR. The results showed increasing in expression level of the selected genes in shrimp hemocytes after microbial challenge suggesting the involvement of such genes in bacterial response in shrimp. The anti-lipopolysaccharide factor type 3 (ALFPm3) gene, previously reported in P. monodon (Supungul et al., 2002) was found among the up-regulated genes but diversity due to amino acid changes was observed. Increase in ALFPm3 transcripts upon V. harveyi injection is in accordance with that found in the previous study.

• Journal of Microbiology and Biotechnology
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• 제28권9호
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• pp.1433-1442
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• 2018

To identify and quantify the effects of a combination of dietary $1{\times}10^8CFU/g$ Lactococcus lactis subsp. lactis I2 ($LI_2$) and 0.1% ${\beta}$-glucooligosaccharides (BGO) on the growth and immunity of olive flounder (Paralichthys olivaceus), a feeding experiment was conducted. Flounder ($14{\pm}0.5g$) were divided into two groups and fed control and synbiotic feeds for 8 weeks. Investigations were carried out on growth and feed utilization, innate immunity, serum biochemical parameters, intestinal lactic acid bacterial (LAB) viability, microvillus length, and changes in the expression levels of genes encoding pro-inflammatory cytokines (tumor necrosis factor $[TNF]-{\alpha}$, interleukin $[IL]-1{\beta}$, and IL-6). Results demonstrated the synbiotic diet had significantly better (p < 0.05) responses in terms of weight gain and specific growth rate, three innate immune parameters (respiratory burst, serum lysozyme, and superoxide dismutase), intestinal LAB viability, and the relative $TNF-{\alpha}$ expression level (p < 0.05). Moreover, after challenge with Streptococcus iniae ($1{\times}10^8CFU/ml$), the synbiotically fed group exhibited significantly higher (p < 0.05) protection against streptococcosis, validating the observed changes in immune parameters and induction of the cytokine-encoding gene. Therefore, according to the results of the present study, synbiotic feed ($LI_2+BGO$) increased growth, modulated innate immune parameters and protected olive flounder against streptococcosis.

• Animal cells and systems
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• 제12권1호
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• pp.15-21
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• 2008

Nitric oxide(NO) has been known to play important roles in numerous physiologic processes including neurotransmission, vasorelaxation, and cellular apoptosis. Using a mouse cDNA gene chip, we examined expression patterns and time course of NO-dependent genes in mouse macrophage RAW264.7 cells. Genes shown to be upregulated more than two fold or at least at two serial time points were further selected and validated by RT-PCR. Finally, 81 selected genes were classified by function as signaling, apoptosis, inflammation, transcription, translation, ionic homeostasis and metabolism. Among those, genes related with signaling, apoptosis and inflammation, such as guanylate cyclase 1, soluble, alpha3(Gucy1a3); protein kinase C, alpha($Pkc{\alpha}$); lymphocyte protein tyrosine kinase(Lck); BCL2/adenovirus E1B 19 kDa-interacting protein(Bnip3); apoptotic protease activating factor 1(Apaf1); X-linked inhibitor of apoptosis(Xiap); cyclin G1(Ccng1); chemokine(C-C motif) ligand 4(Ccl4); B cell translocation gene 2, anti-proliferative(Btg2); lysozyme 2(Lyz2); secreted phosphoprotein 1(Spp1); heme oxygenase(decycling) 1(Hmox1); CD14 antigen(Cd14); and granulin(Grn) may play important roles in NO-dependent responses in murine macrophages.

• Journal of Plant Biotechnology
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• 제1권1호
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• pp.46-55
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• 1999

The expressed sequence tags(ESTs) from immature seed of rice, Oryza sativa cv Milyang 23, were partially sequenced and analyzed by homology. As of 1998, the partial sequences of about 6,600 cDNA clones were analyzed from normal and normalized immature seed cDNA libraries. About 2,200 ESTs were putatively identified by BLASTX deduced amino acid sequence homology analysis. About 20% of them were putatively identified as storage proteins. Also the clones were highly homologous to genes involved particularly in starch biosynthesis, glycolysis, signal transduction and defenses. Compared to 35% of redundancy in the ESTs of normal cDNA library, that from the substracted library was 15%. The Korea Rice Genome Network is maintained to provide the updated information of sequences, their homologies and sequence alignments of ESTs. For the stable expression of transgene in rice, diverse vectors were developed for overexpression, targeting and gene dosage effect with transit peptides (Tp) and matrix attachment region (MAR) sequence from chicken lysozyme locus. The rice calli were transformed via Agrobacterium tumefaciens LBA4404(pSB1) with the triparental mating technique and selected by herbicide resistance. The green fluorescent protein(GFP) gene in expression vector under the control of rbcS promoter-Tp was overexpressed upto 10 % of the total soluble protein. In addition, the Tp-sGFP fusion protein was properly processed during translocation into chloroplast. The expression of sGFP in the presence of MAR sequences was analyzed with Northern and immunoblot analysis. All the lines in which sGFP transgene with MAR sequence, showed position independent and copy number-dependent expression, while the lines without MAR showed the varied level of expression with the integration site. Thus the MAR sequence significantly reduced the variation in transgene expression between independent transformants.

• Genomics & Informatics
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• 제4권3호
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• pp.97-102
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• 2006

In acute leukemia patients, several successful methods of expression profiling have been used for various purposes, i.e., to identify new disease class, to select a therapeutic target, or to predict chemo-sensitivity and clinical outcome. In the present study, we tested the peripheral blood of 47 acute leukemia patients in an attempt to identify differentially expressed genes in AML and ALL using a Korean-made 10K oligo-nucleotide microarray. Methods: Total RNA was prepared from peripheral blood and amplified for microarray experimentation. SAM (significant analysis of microarray) and PAM (prediction analysis of microarray) were used to select significant genes. The selected genes were tested for in a test group, independently of the training group. Results: We identified 345 differentially expressed genes that differentiated AML and ALL patients (FWER<0.05). Genes were selected using the training group (n=35) and tested for in the test group (n=12). Both training group and test group discriminated AML and ALL patients accurately. Genes that showed relatively high expression in AML patients were deoxynucleotidyl transferase, pre-B lymphocyte gene 3, B-cell linker, CD9 antigen, lymphoid enhancer-binding factor 1, CD79B antigen, and early B-cell factor. Genes highly expressed in ALL patients were annexin A 1, amyloid beta (A4) precursor protein, amyloid beta (A4) precursor-like protein 2, cathepsin C, lysozyme (renal amyloidosis), myeloperoxidase, and hematopoietic prostaglandin D2 synthase. Conclusion: This study provided genome wide molecular signatures of Korean acute leukemia patients, which clearly identify AML and ALL. Given with other reported signatures, these molecular signatures provide a means of achieving a molecular diagnosis in Korean acute leukemia patents.