• 한국응용약물학회:학술대회논문집
• /
• 한국응용약물학회 2001년도 추계학술대회 및 정기총회
• /
• pp.102-102
• /
• 2001

Taurine (2-ethaneaminosulfonic acid) is one of the major intracellular ${\beta}$ -amino acids in mammals and is required for a number of biological processes including membrane stabilization, osmoregulation, antioxidation, detoxification, modulation of calcium flux and neurornodulation. The taurine transporter (TAUT) which contains 12 hydrophobic membrane-spanning domains has been cloned from dog kidney, rat brain, mouse brain, human thyroid, placenta and retina. In this study, The TAUT cDNA from the human intestinal epithelial cell, HT-29 was cloned and sequenced. Reverse-transcription polymerase chain reaction (RT-PCR) was performed to amplify partial cDNA encoding human intestinal TAUT. The coding region of the PCR product was 732 bp long. The primers were designed to encode highly conserved amino acid sequences near the transmembrane domains III (IPYFIFLF) and Ⅵ (KYKYNSYR) both in human and mouse. The TAUT cDNA amplified was ligated into the pGEX 4T-1 expression vector. The resulting sequence of human intestinal TAUT cDNA (Accession number of NCBI Genebank is AF346763) was identical to the sequences of the TAUTs previously determined in the human placenta and retina except 3 base pairs from that of the reported human thyroid. TAUT specific antibodies were generated to use them as biological tools in the studies of the biological role of TAUT. Peptides of 149-162 amino acid residue (14 amino acids) of the TAUT were synthesized. The synthetic peptide used in this study was LFQSFQKELPWAHC. This region was chosen not only to avoid putative glycosylation sites but also to exclude regions of known homology with GABA transporters in the extracellular hydrophilic domains. The synthetic peptide, TAUT-1 was conjugated with carrier protein, kehole lympet hemocyanin (KLH) to use as an antigen. When used for immunization on a rabbit to produce polyclonal antiserum, the conjugates elicited high -titered specific anti-TAUT-1 antibodies, which reacted well with the ovalbumin (OVA) conjugated peptides in ELISA. The KLH-conjugated peptide was also used as immunizing antigen in BALB/c mice to produce TAUT specific monoclonal antibodies. From the culture supernatant of the hybridoma, the specificity of anti-TAUT-1 monoclonal antibodies was confirmed by ELISA. Further applications of more tools in TAUT expression analysis will be performed such as western blotting and flow cytometry.

• BMB Reports
• /
• 제34권4호
• /
• pp.334-341
• /
• 2001

Even though three isotypes of thioredoxins (-f, -m and -h types) have been identified in a variety of plant cells, there are only a few reports on thioredoxin-h that were recently identified. In this study, a cDNA encoding a h-type of thioredoxin was isolated from a cDNA library of Chinese cabbage, and named here CTrx-h. An open reading frame of the gene contained a polypeptide of 133 amino acids with a conserved active center, WCGPC, which appeared in all of the thioredoxin proteins. A deduced amino acid sequence of the CTrx-h showed the highest sequence identity with those of Arabidopsis thioredoxin-h2 (75.2%) and thioredoxin-h5 (46.6%) proteins, but it shared a low sequence homology to other isotypes of plant thioredoxinm and thioredoxin-f. The CTrx-h protein that is expressed in E. coli represented not only an insulin reduction activity, but also electron transferring activity from NADPH to thioredoxin-dependent peroxidase. A genomic Southern blot analysis using the cDNA insert of CTrx-h revealed that the gene consisted of a small multigene family in Chinese cabbage genome. On the contrary to other thioredoxin-h proteins that were widely distributed in most tissues of the plant, the CTrx-h was predominantly expressed in flowers. The expression was very low in other tissues. The data of the Northern blot analysis suggests that the CTrx-h may have other functions in flower development or differentiation, in addition to its defensive role.

• 한국발생생물학회지:발생과생식
• /
• 제13권4호
• /
• pp.249-256
• /
• 2009

구조적으로 estrogen 수용체(estrogen receptor, ER)와 유사한 estrogen receptor-related receptor(ERR)는 포유동물에서 배발생 후기에 외배엽 형성과 관련되어 있다고 알려진 고아핵수용체(orphan nuclear receptor)이다. ERR은 ER과 DNA binding domain의 보존성은 유사하지만, ligand 결합 및 전사 활성은 다르다. 포유동물의 ERR에 관한 연구에 비하여 해양 무척추동물의 ERR에 대한 기능 연구는 매우 부족하다. 본 연구에서는 우리나라 동해안에 주로 서식하는 둥근성게(Strongylocentrotus nudus) ERR의 초기 발생기 유전자 발현 변화와 전사 활성 기능을 조사하기 위해 먼저 polymerase chain reaction(PCR)을 이용하여 cDNA를 동정하였다. 둥근성게 ERR은 S. purpuratus와 91%의 높은 상동성을 보였으며, 계통수 분석을 통해 무척추동물 ERR의 clade에 포함되는 것을 알았다. 둥근성게 배발생 시기에 ERR 유전자 발현을 알아보기 위하여 real-time PCR을 실시한 결과, 4~64세포기와 유생기에 mRNA level이 높은 경향을 나타내었다. 또한 호르몬 및 co-regulator에 의한 둥근성게 ERR의 전사 활성을 조사한 결과, 호르몬에 의한 특이성은 확인되지 않았지만, peroxisome proliferator activated receptor-(PPAR) $\gamma$ coactivator $1\alpha$(PGC-$1\alpha$)가 둥근성게 ERR의 coactivator임을 입증할 수 있었다. 이 연구 결과는 향후 새로운 ligand 발굴과 coregulator와 의 상호작용 연구에 도움이 될 것으로 사료된다.

• 생명과학회지
• /
• 제24권12호
• /
• pp.1291-1300
• /
• 2014

본 연구에서는 참전복(Haliotis discus hannai)의 대용량 염기서열 분석을 통해 peroxiredoxin (Prx) 2 유전자의 full length의 cDNA를 동정하였다. 참전복 Prx 2 유전자의 총 길이는 1,052 bp로 597 bp의 open reading frame는 총 199개의 아미노산을 코딩하고 있으며 분자량은 22 kDa, 등전점은 7.58로 예측되었다. 참전복 Prx 2 아미노산서 열에는 typical 2-Cys Prx의 특징을 갖는 motif와 효소활성에 중요한 cysteine잔기가 매우 보존되어 있었다. 참전복 Prx 2 아미노산 서열은 다른 종의 Prx 2와 64~99% 유사하였고, 특히 패류의 Prx 2와 가장 유사성이 높았다. 참전복 Prx 2 유전자의 mRNA는 관찰된 모든 조직에서 발현하고 있었으며, 특히 외투막, 아가미, 족부, 간췌장, 소화관에서 높은 발현이 확인되었다. 참전복의 Prx 2는 비브리오균으로 감염시킨 전복의 혈구세포에서 감염 후 1시간 째 발현이 증가했다가 서서히 감소하였고, 간췌장 조직에서는 감염 6시간 경과 후 발현 정도가 최고로 증가했다가 감소하였다. 따라서 typical 2-Cys Prx의 특징을 잘 보존되어 있는 참전복 Prx 2는 병원균 감염에 따른 산화스트레스 조절에 관여하는 인자로 중요한 역할을 할 것으로 예상된다.

• Journal of Microbiology
• /
• 제39권1호
• /
• pp.37-41
• /
• 2001

The fission yeast Schizosaccharomyces pombe contains two distinct superoxide dismutase (SOD) activities, one in the cytosol encoded by the $sod2^{+}$ gene and the other in mitochondria. The $sod2^{+}$ gene encoding putative mitochondrial manganese superoxide dismutase (MnSOD) was isolated from the S. pombe genomic library using a PCR fragment as the probe. The nucleotide sequence of the $sod2^{+}$ gene and its flanking region (4051 bp HindIII fragment) was determined. An intron of 123 nt in size was predicted and confirmed by sequencing the cDNA following reverse transcription PCR. The predicted Sod2p consists of 218 amino acid residues with a molecular mass of 24,346 Da. The deduced amino acid sequence showed a high degree of homology with other MnSODs, especially in the metal binding residues at the active site and their relative positions. The transcriptional start site was mapped by primer extension at 231 at upstream from the ATG codon. A putative TATA box(TATAAAA) was located 58 nt upstream from the transcriptional start site and putative polyadenylation sites were located at 1000, 1062, and 1074 nt downstream from the ATG start codon.

• Journal of Microbiology and Biotechnology
• /
• 제26권1호
• /
• pp.66-71
• /
• 2016

PikD is a widely known pathway-specific regulator for controlling pikromycin production in Streptomyces venezuelae ATCC 15439, which is a representative of the large ATP-binding regulator of the LuxR family (LAL) in Streptomyces sp. RapH and FkbN also belong to the LAL family of transcriptional regulators, which show greatest homology with the ATP-binding motif and helix-turn-helix DNA-binding motif of PikD. Overexpression of pikD and heterologous expression of rapH and fkbN led to enhanced production of pikromycin by approximately 1.8-, 1.6-, and 1.6-fold in S. venezuelae, respectively. Cross-complementation of rapH and fkbN in the pikD deletion mutant (ΔpikD) restored pikromycin and derived macrolactone production. Overall, these results show that heterologous expression of rapH and fkbN leads to the overproduction of pikromycin and its congeners from the pikromycin biosynthetic pathway in S. venezuelae, and they have the same functionality as the pathwayspecific transcriptional activator for the pikromycin biosynthetic pathway in the ΔpikD strain. These results also show extensive "cross-communication" between pathway-specific regulators of streptomycetes and suggest revision of the current paradigm for pathwayspecific versus global regulation of secondary metabolism in Streptomyces species.

• 한국식물병리학회:학술대회논문집
• /
• 한국식물병리학회 2003년도 정기총회 및 추계학술발표회
• /
• pp.114.2-115
• /
• 2003

Rice stripe virus causes severe damage to rice in Korea, Japan and China. RSV is a type member of the tenuivirus group and transmitted by the small brown planthopper, Laodeiphax striatellus, in a persistant manner. Until now, occurrence of RSV is limited in of southern part of Korea. But recently occurrence of RSV is increasing and spreading in central part of Korea including Chungcheong and Kyonggi province. So we analyzed recent occurrence trend of RSV which is increased and cloned and sequenced coat protein gene for isolating of RSV strain. Infected rice of each species(Ilpumbyeo, Sindongjinbyeo, Keumobyeo-2, Dongjinbyeo, Jongnambyeo, Samcheonbyeo, etc.)is collected, we extracted total RNA from infected leaves and detected RSV viral RNA by reverse transcription(RT)-PCR using specific primer of coat protein gene. The result of RT-PCR, we observed specific band. We already cloned cDNA from the band, is analyzing sequence variety and homology of each species.

• 한국식물병리학회지
• /
• 제11권3호
• /
• pp.271-277
• /
• 1995

The RNA nucleotide sequences of the 3/-noncoding regions (3'-NCRs) of two Korean strains of turnip mosaic virus (TuMV), Ca and cqs, have been determined from their cDNA clones that encompassed the 3'-terminal regions of the viral genomic RNAs. The 3'-NCRs of both strains were 209 nucleotides long, terminated with GAC residues and poly (A) tails. The potential polyadenylational signal motif, UAUGU, was located 140 nucleotides upstream from the poly (A) tail in each of the virus. A highly conserved hexanucleotide sequence [A G U G A/U G/C], which was common in the 3'-NCRs of the potyvirus RNAs, was also found at the regions of 119 bases upstream from the 3'-end. Comparison of the 3'-NCRs of the two Korean isolates with those of four strains from Canada, China and Japan showed significantly identical genotypes (94.3∼99.5%). The secondary structure of three loops with long stems was found within the 3'-NCRs by sequence analysis. The substituted bases in the region among the six TuMV strains did not alter their secondary structures. Length of the 3'-NCRs of the know 11 potyviral RNAs and TuMV RNAs was different from one another and their nucleotide sequences showed 55.7% to 24.0% of homology. The 3'-NCR, therefore, is considered to be useful for phylogenetic studies in potyviruses.

• Biotechnology and Bioprocess Engineering:BBE
• /
• 제10권5호
• /
• pp.432-443
• /
• 2005

Microarray technology has contributed Significantly to the understanding of bacterial genetics and transcriptional regulation. One neglected aspect of this technology has been optimization of microarray-generated signals and quality of generated information. Full genome microarrays were developed for Clostridium acetobutylicum through spotting of PCR products that were designed with minimal homology with all other genes within the genome. Using statistical analyses it is demonstrated that Signal quality is significantly improved by increasing the hybridization volume. possibly increasing the effective number of transcripts available to bind to a given spot, while changes in labeled probe amounts were found to be less sensitive to improving signal quality. In addition to Q-RT-PCR, array validation was tested by examining the transcriptional program of a mutant (M5) strain lacking the pSOL1 178-gene megaplasmid relative to the wildtype (WT) strain. Under optimal conditions, it is demonstrated that the fraction of false positive genes is 1% when considering differentially expressed genes and 7% when considering all genes with signal above background. To enhance genomic-scale understanding of organismal physiology, using data from these microarrays we estimated that $40{\sim}55%$ of the C. acetobutylicum genome is expressed at any time during batch culture, similar to estimates made for Bacillus subtilis.

• Molecules and Cells
• /
• 제23권1호
• /
• pp.100-107
• /
• 2007

The moss Physcomitrella patens has two life cycles, filamentous protonema and leafy gametophore. A modified from of suppression subtractive hybridization (SSH), mirror orientation selection (MOS), was applied to screen genes differentially expressed in the P. patens protonema. Using reverse Northern blot analysis, differentially expressed clones were identified. The identified genes were involved mainly in metal binding and detoxification. One of these genes was an AP2 (APETALA2) domain-containing protein (PpACP1), which was highly up-regulated in the protonema. Alignment with other AP2/EREBPs (Ethylene Responsive Element Binding Proteins) revealed significant sequence homology of the deduced amino acid sequence in the AP2/EREBP DNA binding domain. Northern analysis under various stress conditions showed that PpACP1 was induced by ethephon, cadmium, copper, ABA, IAA, and cold. In addition, it was highly expressed in suspension-cultured protonema. We suggest that PpACP1 is involved in responses to metals, and that suspension culture enhance the expression of genes responding to metals.