• Journal of Microbiology and Biotechnology
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• 제28권7호
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• pp.1133-1140
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• 2018

Pseudomonas fluorescens KLR101 was found to be capable of producing polyhydroxyalkanoate (PHA) using various sugars and fatty acids with carbon numbers ranging from 2 to 6. The PHA granules consisted mainly of a poly(3-hydroxybutyrate) homopolymer and/or poly(3-hydroxybutyrate-co-3-hydroxyvalerate) copolymer. Genomic DNA of P. fluorescens was fractionated and cloned into a lambda library, in which a 5.8-kb fragment that hybridized to a heterologous phaC probe from Ralstonia eutropha was identified. In vivo expression in Klebsiella aerogenes KC2671 (pUMS), restriction mapping, Southern hybridization experiments, and sequencing data revealed that PHA biosynthesis by P. fluorescens relied upon a polypeptide encoded by a 1,683-bp non-operonal ORF, which was preceded by a possible -24/-12 promoter and highly similar to DNA sequences of a gene encoding PHA synthase in the genus Pseudomonas. In vivo expression of the putative PHA synthase gene ($phaC_{Pf}$) in a recombinant Escherichia coli strain was investigated by using glucose and decanoate as substrates. E. coli (${phaC_{Pf}}^+$, pUMS) grown in medium containing glucose accumulated PHA granules consisting mainly of 3-hydroxybutyrate, whereas only a trace amount of 3-hydroxydecanoate was detected from an E. coli fadR mutant (${phaC_{Pf}}^+$) grown in medium containing decanoate. In vitro enzymatic assessment experiments showed that 3-hydroxybutyryl-CoA was efficiently used as a substrate of purified $PhaC_{Pf}$, suggesting that the putative PHA synthase of P. fluorescens utilizes mainly short-chain-length PHA precursors as a substrate.

• BMB Reports
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• 제40권4호
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• pp.501-510
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• 2007

Sex-related genes expressed in vitellogenic ovaries of the giant tiger shrimp, Penaeus monodon, were identified by an EST approach. A total of 1051 clones were unidirectionally sequenced from the 5 terminus. Nucleotide sequences of 743 EST (70.7%) significantly matched known genes previously deposited in the GenBank (E-value <$10^{-4}$) whereas 308 ESTs (29.3%) were regarded as newly unidentified transcripts (E-value >$10^{-4}$). A total of 559 transcripts (87 contigs and 472 singletons) were obtained. Thrombospondin (TSP) and peritrophin (79 and 87 clones accounting for 7.5 and 8.3% of clones sequenced, respectively) predominated among characterized transcripts. everal full length transcripts (e.g. cyclophilin, profillin and thioredoxin peroxidase) were also isolated. A gene homologue encoding chromobox protein (PMCBX, ORF of 567 nucleotides encoding a protein of 188 amino acids) which is recognized as a new member of the HP1 family was identified. Expression patterns of 14 of 25 sex-related gene homologues in ovaries and testes of P. monodon broodstock were examined by RT-PCR. Female sterile and ovarian lipoprotein receptor homologues were only expressed in ovaries whereas the remaining transcripts except disulfide isomerase related P5 precursor and adenine nucleotide translocator 2 were higher expressed in ovaries than testes of P. monodon broodstock. A homologue of ubiquitin specific proteinase 9, X chromosome (Usp9X) revealed a preferential expression level in ovaries than testes of broodstock-sized P. monodon (N = 13 and 11, P<0.05) but was only expressed in ovaries of 4-month-old shrimp (N = 5 for each sex).

• 한국자원식물학회지
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• 제17권3호
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• pp.239-247
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• 2004

더덕 뿌리에서 유래한 EST cDNA library로부터 cyclophilin 유전자와 높은 상동성을 나타내 는 full clone cDNA를 얻었다. 더덕의 cyclophilin, ClCyP1은 747 bp의 cDNA로 174개의 아미노산을 코딩하는 525 bp의 ORF를 가지고 있다. ClCyP1의 아미노산 서열을 분석한 결과, 기존에 보고된 식물들의 cytosolic cyclophilin들과 높은 상동성을 나타내었으며, 여러 식물의 cytosolic CyP의 공통적인 특징인 7개의 아미노산 잔기(KSGKPLH)를 가지고 있었다. RT-PCR분석 결과, 더덕의 ClCyP1은 식물의 조직 전체에서 발현되며, 저온, 고온, 그리고 염 스트레스에 대해 그 발현량이 증가하였다.

• 한국수산과학회지
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• 제57권1호
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• pp.41-52
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• 2024

Serotransferrin cDNA from the Siberian sturgeon Acipenser baerii was isolated and its expression patterns during early life intervals were characterized. It contained an ORF encoding a 708-aa-long polypeptide, including a 19-aa signal peptide. Bioinfomatic analysis and 3D modeling indicated a typical bi-lobal structure with conserved iron-coordinating residues. During embryonic development, the potential transition of maternally provisioned transcripts to zygotically de novo transcribed ones occurred around blastula stage. The transferrin mRNA levels peaked at stages responsible for pronephros, heart and erythropoietic component differentiation. After hatching, the transferrin mRNA expression gradually increased at early ontogenic phases (0 to 3 DPH) corresponding to the periods in which prolarvae exhibited increased blood circulation and liver differentiation. The expression decreased at subsequent stages in which prolarvae exhibited benthic movement. The tissue distribution assay indicated liver-predominant expression at fingerling stage. From the microinjection-based challenge with Aeromonas hydrophila at day-0 and day-7, the transcriptional response was modulated toward upregulation, in which the amounts induced at 6, 12 and 24 HPI were greater in prolarvae injected at day-7 than at day-0. Therefore, transferrin plays important roles in both early development and host protective responses to pathogens in the Siberian sturgeon.

• 한국미생물·생명공학회지
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• 제33권2호
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• pp.96-105
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• 2005

공시균인 S. longwoodensis IFO 14251의 autoregulators 및 receptor gene 탐색의 일환으로 기존의 Streptomyces속 receptor gene의 공통배열을 primer로 이용하여 PCR을 수행하였다. 예상되는 100 bp 크기의 단편을 pUC19 vector에 ligation하여 E. coli $DH5{\alpha}$에 transformation한 후, plasmid를 분리하여 BamHI을 처리하여 $2\%$ agarose gel에 전기영동한 결과, pUC19 외에 receptor gene PCR product가 100 bp 위치에 존재하는 것을 확인하였다. 형질전환된 plasmid로 PCR을 수행한 후, 염기배열을 결정하여 분석한 결과, Streptomyces sp. 유래의 receptor gene의 일부분임이 확인되었다. 따라서 S. longwoodensis IFO 14251에는 lysocellin 생산에 관여한다고 추정되는 autoregulator receptor protein을 코드하는 유전자가 존재할 것으로 예상되어 100 bp의 PCR product를 probe로 이용하여 Southern 및 colony hybridization을 통하여 4.4 kb의 SphI 단편을 가지는 plasmid(pSLT)를 제작하였고 이를 sequencing한 결과, Streptomyces 속 유래의 autoregulator receptor 단백질을 코드하는 유전자와 3개의 open reading frame(651 bp)을 확인하여 sltR이라 명명하였다. 유전자 해석 결과, 기존의 autoregulator receptor proteins과 비교시 $35{\sim}46\%$의 homology를 나타내었다. 재조합 단백질의 발현을 위해, sltR/pET-l7b plasmid를 제작하고 E. coli BL21(DE3)/pLysS을 host cell로 이용하여 재조합 단백질을 발현시켰다. SltR 재조합 단백질의 정제는 DEAE-Sephacel column chromatography와 DEAE-5PW chromatography(HPLC)를 통해 수행하였다. Gel filtration chromatography(HPLC, 55 kDa)와 SDS-PAGE(28 kDa)를 통해 분자량을 확인한 결과, 재조합 단백질은 dimer형태로 존재하는 것으로 확인되었다. 또한, 결합활성에 있어 A-factor type의 autoregulator에 대해 가장 강한 결합활성을 나타내었다.

• 미생물학회지
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• 제52권1호
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• pp.110-115
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• 2016

남극 해양세균 Pseudoalteromonas sp. PAMC 21150에서 분리한 소형 플라스미드(small plasmid, pDK4)의 크기는 3,480bp이고 G+C 함량은 41.64%이며, 3개의 open reading frames(ORFs)을 포함하고 있다. 3개의 ORF는 replication initiation protein (RepA), conjugative mobilization protein (Mob), 그리고 기능이 밝혀지지 않은 단백질을 코팅하고 있다. PCR 반응으로 증폭한 pDK4를 Escherichia coli high-copy pUC19 클로닝 벡터에 삽입하여 fusion vector (pDOC153)를 제작하였고, pDOC 153에 chloramphenicol 저항성 유전자를 삽입하여 ampicillin/chloramphenicol 저항성 Pseudoalteromonas - Escherichia coli 셔틀 벡터(shuttle vector; 7,216 bp 크기; pDOC155)를 제작하였다. 북극 해양세균 P. issachenkonii PAMC 22718이 보유한 2개의 유전자(TonB-dependent receptor gene, chi22718_IV, and exochitinase gene, chi22718_III)를 pDOC155에 삽입하여 두 개의 pDOC155 변형체(pDOC158, pDOC165)를 제작하였다. pDOC158 혹은 pDOC165을 이용하여 triparental mating 방법에 의해 플리스미드 미보유 해양세균인 Pseudoalteromonas sp. PAMC 22137를 형질전환하였다. PCR을 이용한 유전자 증폭실험을 통해서, pDOC158와 pDOC165에 삽입된 유전자들은 Pseudoalteromonas sp. PAMC 22137와 E. coli $DH5{\alpha}$ 내에서 안정적으로 유지되는 것을 확인하였다. 위의 결과는 셔틀 벡터 pDOC155는 Pseudoalteromonas spp. 유래 유전자들을 다른 Pseudoalteromonas spp. 세포 안으로 전달할 수 있는 새로운 유전자 전달시스템으로 이용될 수 있음을 보여주었다.

• 한국어병학회지
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• 제33권1호
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• pp.77-81
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• 2020

This study was performed for screening yellow head virus(YHV) complex in 252 including 235 white leg shrimps (Litopenaeus vannamei) and 17 oriental shrimp (Fenneropenaeus chinensis) collected from 18 farms located in southwestern province of Korea. The virus complex was detected by nested reverse-transcriptase polymerase chain reaction (RT-PCR) assay. In the assay, amplicons were resulted in RNAs exracted from 38 shrimps (21 white leg shrimps and 17 oriental shrimps) obtained from 7 farms. In phylogenetic analysis using sequences of ORF1b gene, all 38 sequences obtained in this study formed an independent lineage with YHV-8 genotype firstly isolated in China, belonged to an YHV-8 clade.

• Journal of Microbiology and Biotechnology
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• 제20권3호
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• pp.579-586
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• 2010

A genomic DNA fragment encoding a putative maltooligosyltrehalose synthase (NfMTS) for trehalose biosynthesis was cloned by the degenerate primer-PCR from cyanobacterium Nostoc flagelliforme. The ORF of NfMTS was 2,799 bp in length and encoded 933 amino acid residues constituting a 106.6 kDa protein. The deduced amino acid sequence of NfMTS contained 4 regions highly conserved for MTSs. By expression of NfMTS in E. coli, it was demonstrated that the recombinant protein catalyzed the conversion of maltohexaose to maltooligosyl trehalose. The $K_m$ of the recombinant enzyme for maltohexaose was 1.87 mM and the optimal temperature and pH of the recombinant enzyme was at $50^{\circ}C$ and 7.0, respectively. The expression of MTS of N. flagelliforme was upregulated, and both trehalose and sucrose contents increased significantly in N. flagelliforme during drought stress. However, trehalose accumulated in small quantities (about 0.36 mg/g DW), whereas sucrose accumulated in high quantities (about 0.90 mg/g DW), indicating both trehalose and sucrose were involved in dehydration stress response in N. flagelliforme and sucrose might act as a chemical chaperone rather than trehalose did during dehydration stress.

• Journal of Periodontal and Implant Science
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• 제31권4호
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• pp.787-801
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• 2001

The molecular mechanisms control the function of PDL(periodonta1 ligament) cells and/or fibroblasts remain unclear. PDLsl7, PDL-specific gene, had previousely　identified the cDNA for a novel protein from cultured　PDL fibroblasts using subtraction hybridization between gingival fibroblasts and　PDL fibroblasts. The purpose　of this study was to determine the regulation by growth factors and cytokines on　PDLsl7 gene expression in　cultured human periodontal ligament cells and observe the immunohistochemical　localization of PDLsl7 protein in various tissues of mouse. Primary PDL fibroblasts isolated by scraping the root of the extracted human mandibular third molars. The cells were incubated with various concentration of human recombinant $IL-1{\beta}$, PDGF-BB and TGF\;${\beta}$ for 48h nd 2 weeks. At each time point total RNA was extracted and the levels of transcription ere assessed by reverse transcription-polymerase chain reaction (RT-PCR assay). polyclonal antiserum raised against PDLsl7 peptides, CLSVSYNRSYQINE and SEAVHETDLHDGC, were made, and stained the tooth, periodontium, developing bone, bone marrow and mid-palatal suture of the mouse. The results were as follows. 1. PDLsl7 mRNA levels were increased in response to PDGF (10ng/ml) and $TGF\;{\beta}$(20ng/ml) after treatment of the $IL-1{\beta}$, PDGF-BB and $TGF{\beta}$for 48 h. 2. PDLsl7 was up-regulated only by $TGF{\beta}$(20 ng/ml) after treatment of the $IL-1{\beta}$, PDGF-BB and $TGF\;{\beta}$ for 2 weeks and unchanged by the other stimulants. 3. PDLsl7 was a novel protein coding the 142 amino acid peptides in the ORF and the nucleotide sequences of the obtained cDNA from RT-PCR was exactly same as the nucleotides of the database. 4. Immunohistochemical analysis showed that PDLsl7 is preferentially expressed in the PDL, differentiating osteoblast-like cells and stromal cells of the bone marrow in the adult mouse. 5. The expression of PDLsl7 protein was barely detectable in gingival fibroblasts, hematopoetic cells of the bone marrow and mature osteocytes of the alveolar bone. These results suggest that PDLsl7 might upregulated by PDGF-BB or $TGF{\beta}$ and acts at the initial stage of differentiation when the undifferentiated mesenchymal cells in the bone marrow and PDL differentiate into multiple cell types. However, more research needs to be performed to gain a better understanding of the exact function of PDLsl7 during the differentiation of bone marrow mesenchymal and PDL cells.

• Journal of Microbiology and Biotechnology
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• 제18권8호
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• pp.1445-1452
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• 2008

An open reading frame (ORF) encoding a putative epoxide hydrolase (EHase) was identified by analyzing the genome sequence of Sphingophyxis alaskensis. The EHase gene (seh) was cloned and expressed in E. coli. To facilitate purification, the gene was fused in-frame to 6$\times$ histidine at the C-terminus. The recombinant EHase (rSEH) was highly soluble and could be purified to apparent homogeneity by one step of metal affinity chromatography. The purified SEH displayed hydrolyzing activities toward various epoxides such as styrene oxide, glycidyl phenyl ether, epoxyhexane, epoxybutane, epichlorohydrin, and epifluorohydrin. The optimum activity toward styrene oxide was observed at pH 6.5 and $35^{\circ}C$. The purified SEH showed a cold-adapted property, displaying more than 40% of activity at low temperature of $10^{\circ}C$ compared with the optimum activity. Despite the catalytic efficiency, the purified SEH did not hydrolyze various epoxides enantioselectively. $K_m$ and $k_{cat}$ of SEH toward (R)-styrene oxide were calculated as 4$\pm$0.3 mM and 7.42$s^{-1}$ respectively, whereas $K_m$ and $k_{cat}$ of SEH toward (S)-styrene oxide were 5.25$\pm$0.3 mM and 10.08$s^{-1}$ respectively.