• Journal of Microbiology
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• 제43권2호
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• pp.209-212
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• 2005

The purpose of this study was to develop species-specific PCR primers for use in the identification and detection of Actinobacillus actinomycetemcomitans. These primers target variable regions of the 168 ribosomal RNA coding gene (rDNA). We assessed the specificity of the primers against 9 A. actinomycetemcomitans strains and 11 strains (3 species) of the Haemophilus genus. Primer sensitivity was determined by testing serial dilutions of the purified genomic DNAs of A. actinomycetemcomitans ATCC$ 33384^$T Our obtained data revealed that we had obtained species-specific amplicons for all of the tested A. actinomycetemcomitans strains, and that none of these amplicons occurred in any of the other species. Our PCR protocol proved able to detect as little as 4 fg of A. actinomycetemcomitans chromosomal DNA. Our findings suggest that these PCR primers are incredibly sensitive, and should prove suitable for application in epidemiological studies, as well as the diagnosis and monitoring of periodontal pathogens after treatment for periodontitis.

• Fisheries and Aquatic Sciences
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• 제6권3호
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• pp.116-124
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• 2003

Isolation and cloning of seven-band grouper (Epinephelus septemfasciatus) growth hormone cDNA from pituitary gland revealed an open reading frame of 612 bp coding for a pre-growth hormone of 204 amino acids with a 17 amino acid putative signal peptide. Deduced amino acid sequence showed that there was one possible N-glycosylation site at $Asn^{l84}$ and four cysteine residues $(Cys^{52},\;Cys^{160},\;Cys^{177},\;Cys^{185})$ on t e same positions as in some other species where they were involved in the stabilization of the tertiary structure. The seven-band grouper growth hormone (sbgGH) presented a $99.5\%$ amino acid sequence identity with the growth hormone of Epinephelus coioides and contained the conserved hormone domain region. Comparison of growth hormone sequences from evolutionarily diverse species revealed 25 amino acid residues conserved in jawless fishes to modern mammals. It also revealed an evolutionary trend to retain the same polypeptide sequence even in the distantly related animals while allowing alterations to occur in polypeptides of the closely related species. In order to create a recombinant system to produce high levels of the growth hormone, it was expressed in Escherichia coli (BL21) cells. The gel analysis revealed theoretically expected molecular weights for both mature and pre-sbgGHs.

• Molecules and Cells
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• 제28권4호
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• pp.341-345
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• 2009

MiRNAs are non-coding RNAs that play a role in the regulation of major processes. The inhibition of miRNAs using antisense oligonucleotides (ASOs) is a unique and effective technique for the characterization and subsequent therapeutic targeting of miRNA function. Recent advances in ASO chemistry have been used to increase both the resistance to nucleases and the target affinity and specificity of these ASOs. Peptide nucleic acids (PNAs) are artificial oligonucleotides constructed on a peptide-like backbone. PNAs have a stronger affinity and greater specificity to DNA or RNA than natural nucleic acids and are resistant to nucleases, which is an essential characteristic for a miRNA inhibitor that will be exposed to serum and cellular nucleases. For increasing cell penetration, PNAs were conjugated with cell penetrating peptides (CPPs) at N-terminal. Among the tested CPPs, Tat-modified peptide-conjugated PNAs have most effective function for miRNA inhibition. PNA-based ASO was more effective miRNA inhibitor than other DNA-based ASOs and did not show cytotoxicity at concentration up to 1,000 nM. The effects of PNA-based ASOs were shown to persist for 9 days. Also, PNA-based ASOs showed considerable stability at storage temperature. These results suggest that PNA-based ASOs are more effective ASOs of miRNA than DNA-based ASOs and PNA-based ASO technology, compared with other technologies used to inhibit miRNA activity can be an effective tool for investigating miRNA functions.

• Journal of Microbiology and Biotechnology
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• 제22권2호
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• pp.159-165
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• 2012

The rhizobacterial composition varies according to the soil properties. To test if the effect of herbicides on the rhizobacterial communities of genetically modified NK603 glyphosate-tolerant maize varies according to different soil locations, a comparison was made between the effects of glyphosate (Roundup Plus), a post-emergence applied herbicide, and a pre-emergence applied herbicide (GTZ) versus untreated soil. The potential effect was monitored by direct amplification, cloning, and sequencing of the soil DNA encoding 16S rRNA, and high-throughput DNA pyrosequencing of the bacterial DNA coding for the 16S rRNA hypervariable V6 region. The results obtained using three different methods to analyze the herbicide effect on the rhizobacterial communities of genetically modified NK603 maize were comparable to those previously obtained when glyphosate-tolerant maize was grown in soil with different characteristics. Both herbicides decreased the bacterial diversity in the rhizosphere, with Actinobacteria being the taxonomic group most affected. The results suggest that both herbicides affected the structure of the maize rhizobacterial community, but glyphosate was environmentally less aggressive.

• 한국응용곤충학회지
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• 제36권1호
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• pp.30-36
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• 1997

미국과 캐나다는 한국에 분포하는 잎응애속(Tetranychus)중 범세계적 분포종인 점박이응애(T. urticae Koch)를 제외한 벚나무응애(Tetranychus vienensis Zacher), 차응애(T. kanzawai Kishida), 뽕나무응애(T. truncatus Ehara)를 검역대항으로 하고 있다. 잎응애속 응애들은 암컷성충으로 월동휴면에 들어가는데 기존의 수컷생색기의 형태를 위주로 한 동정방법으로는 이 휴면태의 암컷을 정확히 동정하기 어렵다. 월동을 위해 사과 과실 꼭지부에 우발적으로 부착할 가능성이 있는 것으로 우려되는 잎응애속 응애들의 월동휴면태에 대한 신속, 정확한 동정법이 수출검역현장에서 절실히 요구되는 실정이다. 사과의 주요해충인 점박이응애와 과수원 주변에서 발견되는 벚나무응애, 뽕나무응애, 차응애의 미토콘드리아 DNA(mtDNA)내 cytochrome oxidase subunit I(CO-I) 유전자를 PCR로 증폭하고 증폭된 DNA의 종간 변이를 이용하여 발육영기나 암수에 관계없이 동정할 수 있는 방법을 찾는 연구를 수행하였다. 세쌍의 primer에 의해 미토콘드리아 DNA의 CO-I 유전자 일부(680 bp)를 중복되게 증폭하였고 증폭된 유전자는 제한효소 AluI, DdeI, Sau3A 대하여 응애종간 특이적 인식부위를 가지고 있었다. 제한효소에 의해 절단되는 특이적 DNA 단편은 Tetranychus 응애류를 동정하는데 유용한 표식인자로 사용될 수 있을 것이다. 아울러 증폭한 CO-I 유전자내의 제한효소 인식부위에 대한 이들 4종 응애의 유전자지도를 작성하였다.

• 한국약용작물학회지
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• 제14권3호
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• pp.178-182
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• 2006

둥굴레 유전자원의 유연관계를 위한 기초 자료를 얻고자 둥굴레속 식물 수집종 10종에서 18S ribosomal RNA를 암호화하는 18S rDNA 영역의 염기서열을 결정하고 그 특성을 분석한 결과를 요약하면 다음과 같다. 둥굴레속 10종의 18S rDNA 영역 전체의 길이는 $913{\sim}914bp$로 비슷하였으나, 총 8개 지점에서 염기의 치환 및 결실에 의한 변이가 발생하였다. 전위는 $T{\rightarrow}C$전위가 4개 지점에서 발생하였고, $A{\rightarrow}G$ 전위가 1개 지점에서 발생하였으며, 전좌는 $C{\rightarrow}A$ 전좌가 1개 지점에서 발생하여 전위가 전좌보다 5배 만큼 발생하였다. 결실은 2개 지점에서 발생하였다. 18S rDNA의 염기의 조성은 adenine $23.09{\sim}23.33%$, guanine $23.33{\sim}23.52%$, thymine $25.60{\sim}25.85%$, cytosine $27.38{\sim}27.79%$로 pyrimidine계가 purine계보다 많았다. 18S rDNA의 A + T 함량은 $48.80{\sim}49.18%$로 평균 48.99%였고, G+C 함량은 $50.82{\sim}51.20%$로 평균51.01%였다. 다중 정렬에 의해 염기서열을 비교한 결과 $99.7{\sim}100%$ 일치하여 종간에 차이가 적었다.

• Archives of Pharmacal Research
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• 제20권5호
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• pp.465-470
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• 1997

The human cDNA clone UDPGTh2, encoding a liver UDP-glucuronosyltransferase (UDPGT), was isolated from a .gamma.gt 11 cDNA library by hybridization to mouse transferase cDNA clone, UDPGTm1. The two clones had 74% nlicleotide sequence identities in the coding region. UDPGTh2 encoded a 529 amino acid protein with an amino terminus membrane-insertion signal peptide and a carboxyl terminus membrane-spanning region. In order to establish substrate specificity, the clone was inserted into the pSVL vector (pUDPGTh2) and expressed in COS 1 cells. Sixty potential substrates were tested using cells transfected with pUDPGTh2. The order of relative substrate activity was as follows: 4-hydroxyestrone > estriol >2-hydroxyestriol > 4-hydroxyestradiol > $6{\alpha}$-hydroxyestradiol >$5{\alpha}$-androstane-$3{\alpha}$, $11{\beta}$, $17{\beta}$-triol=5${\beta}$-androstane-$3{\alpha}$ ${\beta}$, $17{\beta}$-triol. There were only trace amounts of gulcuronidation of 2-hydroxyestradiol and 2-hydroxyestrone, and in contrast to other cloned transferase, no gulcuronidation of either the primary estrogens and androgens (estrone, $17{\beta}$estradiol/testosterone, androsterone) or any of the exogenous substrates tested was detected. A lineweaver-Burk plot of the effect of 4-hydroxystrone concentration on the velocity of glucuronidation showed an apparent Km of $13{\mu}M$. The unique specificity of this transferase might play an important role in regulating the level and activity of these potent and active estrogen metabolites.

• 한국어병학회지
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• 제14권2호
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• pp.59-64
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• 2001

두툽상어의 간조직 cDNA library의 EST를 통해 중금속의 세포내 농도 조절과 환경으로부터 흡수한 유해 중금속의 해독작용 등의 기능을 수행하는 MT 유전자를 cloning하였다. 염기서열 분석 결과 두툽상어의 MT 유전자는 204bp의 coding 영역과 182bp의 3'UTR 영역으로 구성되어 있었으며, 종결코돈 이후 162bp의 polyadenylatin 신호서열과 이로부터 15bp 이후의 poly(A)서열 등이 확인되었다. 염기서열로부터 유추한 68개의 아미노산 서열에는 다른 척추동물에서와 같이 Cys 잔기가 전체의 29.4%(20/68)로 풍부하였으며, 아미노산 서열수준에서 포유류와는 43~54%, 어류들과는 41~45%의 상동성을 나타냈었다. 특히 20개의 Cys은 어류와 18개가 다른 척추동물과는 19개가 잘 보존되어 있었다. 두툽상어 MT는 특이하게 모든 척추동물에서 잘 보존된 $\beta$-domain 끝 쪽의 9번째 Cys앞에 5개의 아미노산을 더 갖고 있었으며, 경골어류 MT의 특징인 4번째 위치의 gap이 없고, 18번째 Cys의 위치가 어류와 달리 다른 척추동물들과 같았다. 또한 C 말단의 아미노산 잔기가 다른 생물체와는 모두 다른 Ser을 갖는 특징을 나타내었다. 이와 같은 두툽상어 MT유전자의 특징들은 연골어류의 분자적 진화과정을 알 수 있는 분자 표지유전자로 이용할 수 있을 것이다.

• Asian Pacific Journal of Cancer Prevention
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• 제16권17호
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• pp.7935-7941
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• 2015

The aim of this study was to implement massive parallel sequencing (MPS) technology in clinical genetics testing. We developed and tested an amplicon-based method for resequencing the BRCA1 and BRCA2 genes on an Illumina MiSeq to identify disease-causing mutations in patients with hereditary breast or ovarian cancer (HBOC). The coding regions of BRCA1 and BRCA2 were resequenced in 96 HBOC patient DNA samples obtained from different sample types: peripheral blood leukocytes, whole blood drops dried on paper, and buccal wash epithelia. A total of 16 random DNA samples were characterized using standard Sanger sequencing and applied to optimize the variant calling process and evaluate the accuracy of the MPS-method. The best bioinformatics workflow included the filtration of variants using GATK with the following cut-offs: variant frequency >14%, coverage ($>25{\times}$) and presence in both the forward and reverse reads. The MPS method had 100% sensitivity and 94.4% specificity. Similar accuracy levels were achieved for DNA obtained from the different sample types. The workflow presented herein requires low amounts of DNA samples (170 ng) and is cost-effective due to the elimination of DNA and PCR product normalization steps.

• BMB Reports
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• 제35권3호
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• pp.273-282
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• 2002

Sp3 is a bifunctional transcription factor that has been reported to stimulate or repress the transcription of numerous genes. Although the size of Sp3 mRNA is 4.0kb, the size of the known Sp3 cDNA sequence is 3.6kb. Thus, Sp3 functional studies have been performed with an artificially introduced start codon, and thus an amino-terminus that differs from the wild-type. Ideally, full-length cDNA expression vectors with the appropriate start codon should be utilized for these studies. Using 5'rapid amplification of cDNA ends, a full-length Sp3 cDNA clone was generated and the sequence verified in nine cell lines. No AUG initiation codon was present. However, stop codons were present in all three frames 5' to the known coding sequence. In vitro translation of this full-length cDNA clone produced the expected three isoforms-one at 100 kDa and two in the mid 60 kDa range. Electrophoretic mobility shift assays showed that the protein products had the ability to bind to the Sp1/3 consensus sequence. In vitro studies, using our Sp3 clone and site directed mutagenesis, identified the translation initiation site for the larger isoform as AUA. AUA has not been previously described as an endogenous initiation codon in eukaryotes.