• Genomics & Informatics
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• 제16권4호
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• pp.28.1-28.6
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• 2018

Due to the increasing interest in synonymous codons, several codon bias-related terms were introduced. As one measure of them, the tRNA adaptation index (tAI) was invented about a decade ago. The tAI is a measure of translational efficiency for a gene and is calculated based on the abundance of intracellular tRNA and the binding strength between a codon and a tRNA. The index has been widely used in various fields of molecular evolution, genetics, and pharmacology. Afterwards, an improved version of the index, named specific tRNA adaptation index (stAI), was developed by adapting tRNA copy numbers in species. Although a subsequently developed webserver (stAIcalc) provided tools that calculated stAI values, it was not available to access pre-calculated values. In addition to about 100 species in stAIcalc, we calculated stAI values for whole coding sequences in 148 species. To enable easy access to this index, we constructed a novel web database, named STADIUM (Species-specific tRNA adaptive index compendium). STADIUM provides not only the stAI value of each gene but also statistics based on pathway-based classification. The database is expected to help researchers who have interests in codon optimality and the role of synonymous codons. STADIUM is freely available at http://stadium.pmrc.re.kr.

• 미생물학회지
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• 제45권2호
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• pp.215-221
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• 2009

대장균에서 비천연 아미노산을 단백질 생합성시 특정 위치에 삽입하는 방법의 하나로 amber suppressor tRNA와 여기에 비천연 아미노산을 특이적으로 결합할 수 있는 변형된 aminoacyl-tRNA synthetase 쌍을 이용한다. 이러한 기술의 개발을 위해 필요한 여러 요소 중 하나는 이러한 시스템이 대장균에서 얼마나 잘 작동하는지를 확인할 수 있는 in vivo 보고시스템을 설정하는 것이다. 본 논문에서는 $\beta$-galactosidase 유전자의 N-말단에 amber 코돈을 삽입한 보고유전자를 제작하였으며, 이를 대장균(DH10B)의 chromosomal DNA에 삽입하여 DH10B(Tn:lacZam) 균주를 개발하였다. Genomic PCR과 Southern blot 분석을 통하여 lacZ amber 유전자가 대장균의 염색체에 삽입된 것을 확인하였으며, DH10B(Tn:lacZam)은 amber suppression을 유도할 수 있는 벡터가 형질 전환될 경우만 $\beta$-galactosidase 활성을 나타냈다. DH10B(Tn:lacZam)에 효모균의 amber suppressor $tRNA^{Tyr}$와 Tyrosyl-tRNA synthetase 쌍을 동시에 발현하는 벡터를 형질전환하였을 때, amber suppression에 의해서 $\beta$-galactosidase 활성이 나타났다. 하지만 이 활성은 대장균의 amber suppressor $tRNA^{Gln}$를 발현하는 pSupE2를 형질전환하였을 때와 비교 하여 매우 낮은 $\beta$-galactosidase 활성을 나타냈다. 이러한 결과는 DH10B(Tn:lacZam) 균주가 $\beta$-galactosidase 활성을 통하여 정성 및 정량적으로 in vivo amber suppression 활성을 비교 분석할 수 있는 특성을 가졌음을 나타낸다.

• Journal of Microbiology and Biotechnology
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• 제18권1호
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• pp.23-27
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• 2008

Self-splicing group I introns in tRNA anticodon loops have been found in diverse groups of bacteria. In this work, we identified $tRNA^{fMet}$ group I introns in six strains of marine Synechococcus elongatus. Introns with sizes around 280 bp were consistently obtained in all the strains tested. In a phylogenetic analysis using the nucleotide sequence determined in this study with other cyanobacterial $tRNA^{fMet}$ and $tRNA^{Leu}$ intron sequences, the Synechococcus sequence was grouped together with the sequences from other unicellular cyanobacterial strains. Interestingly, the phylogenetic tree inferred from the intronic sequences clearly separates the different tRNA introns, suggesting that each family has its own evolutionary history.

• 미생물학회지
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• 제50권1호
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• pp.1-7
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• 2014

생명체에서 비천연 아미노산을 단백질의 특정 위치에 삽입하는 방법으로 orthogonal suppressor tRNA와 여기에 비천연 아미노산을 특이적으로 결합시킬 수 있는 유전자 변형된 aminoacyl-tRNA synthetase (ARS)가 활용되고 있다. 이 기술개발을 위해서는 돌연변이를 유발한 ARS library로부터 비천연 아미노산만을 특이적으로 결합시킬 수 있는 변형된 ARS를 탐색하기 위한 선별시스템이 필요하다. 본 논문에서는 대장균에서 작용하는 2단계로 구성된 새로운 선별시스템을 개발하였다. 먼저 양성선별 시스템은 27번 잔기를 amber 코돈으로 치환한 Chloramphenicol acetyl transferase 유전자로 구성되어 있으며, 이유전자의 amber suppression에 의해 chloramphenicol 배지에서 생존함에 따라 활성을 나타내는 ARS를 최고 $9.0{\times}10^5$배로 농축할 수 있었다. 반면 음성선별 시스템은 대장균의 Topoisomerase II의 기능을 억제하는 단백질을 암호화하는 control of cell death B (ccdB) 유전자의 N-말단 앞에 3개의 amber 코돈을 삽입하여 제작하였다. 이 음성선별 시스템을 가진 대장균에 orthogonal pair인 Saccharomyces cerevisiae tyrosyl-tRNA synthetase (Scc TyrRS)와 amber suppressor tRNA를 형질전환하면 amber suppression으로 CcdB가 발현되어 대장균의 성장이 억제되는 것을 확인하였으며, 천연 아미노산에 대한 특이성을 가진 ARS를 효과적으로 제거하는 것을 관찰하였다. 따라서, 양성선별 및 음성선별 시스템을 순차적으로 거침으로써 무작위적으로 아미노산에 대한 특이성을 변형시킨 ARS 라이브러리로부터 비천연 아미노산을 suppressor tRNA에 특이적으로 결합하는 유전자 변형 ARS를 탐색하는데 유용하게 사용될 수 있을 것이다.

• Journal of Microbiology and Biotechnology
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• 제22권2호
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• pp.190-198
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• 2012

RNA interference (RNAi) is rapidly becoming a valuable tool in biological studies, as it allows the selective and transient knockdown of protein expression. The short-interfering RNAs (siRNAs) transiently silence gene expression. By contrast, the expressed short-hairpin RNAs induce long-term, stable knockdown of their target gene. Trichoplusia ni (T. ni) cells are widely used for mammalian cell-derived glycoprotein expression using the baculovirus system. However, a suitable shRNA expression system has not been developed yet. We investigated the potency of shRNA-mediated gene expression inhibition using human and Drosophila U6 promoters in T. ni cells. Luciferase, EGFP, and ${\beta}$-N-acetylglucosaminidase (GlcNAcase) were employed as targets to investigate knockdown of specific genes in T. ni cells. Introduction of the shRNA expression vector under the control of human U6 or Drosophila U6 promoter into T. ni cells exhibited the reduced level of luciferase, EGFP, and ${\beta}$-N-acetylglucosaminidase compared with that of untransfected cells. The shRNA was expressed and processed to siRNA in our vector-transfected T. ni cells. GlcNAcase mRNA levels were down-regulated in T. ni cells transfected with shRNA vectors-targeted GlcNAcase as compared with the control vector-treated cells. It implied that our shRNA expression vectors using human and Drosophila U6 promoters were applied in T. ni cells for the specific gene knockdown.

• Applied Biological Chemistry
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• 제45권4호
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• pp.190-194
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• 2002

Bacillus subtilis의 glutamyl-tRNA synthetase(GluRS)는 대장균에서 발현될 때 숙주세포의 $tRNA_1^{Gln}$에 glutamate를 잘못 아실화하여 독성을 나타내는 것으로 추정되고 있다. 이러한 B. subtilis GluRS를 대장균에서 과발현 시키기 위하여 B. subtilis 168 균주의 chromosomal DNA에서 GluRS의 유전자(gltX)를 PCR을 이용하여 증폭하고 T7 promoter에 의해 발현이 조절되는 pET11a expression vector에 클로닝하였다. 이 재조합된 pEBER plasmid DNA로 T7 RNA polymerase를 갖는 대장균 NovaBlue(DE3)에 형질전환하였다. 형질전환된 대장균에 IPTG를 처리하여 과량 생성된 GluRS 단백질은 ammonium sulfate 분별침전 후 EPLC를 이용한 Source Q column anion exchange chromatography, Superdex 200 column gel filtration, Mono Q column anion exchange chromatography로 정제하였다. 정제된 B. subtilis의 GluRS 분자량은 약 55 kDa이었으며 효소의 활성도는 조효소액에 비해 18배로 증가하였다.

• Journal of Plant Biology
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• 제26권1호
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• pp.1-6
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• 1983

In order to investigate the effects of GA3 on RNA biosynthesis, the amounts of rRNA and tRNA in germinating maize seeds were measured. The amount of rRNA in the endospermless seedlings was remarkably increased by GA3 tretment after 48 h of germination, but no effect was observed after 12 h of germination. While the amout of rRNA in 0.5 cm shoots in length was decreased by GA3 treatment, both of the amounts of rRNA and tRNA were increased in 1~1.5 cm shoots. According to the above mentioned results, it may be suggested that RNA biosynthesis is affected by GA3 treatment, and that GA3 participates in the biosynthesis of rRNA rather than tRNA in germinating maize seeds.

• Bulletin of the Korean Chemical Society
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• 제26권6호
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• pp.921-924
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• 2005

It is known that metallothionein (MT) plays a role in the scavenging of free radicals, which is produced under various stress conditions. MT may function as an antioxidant that protects against oxidative damage of DNA, protein, and lipid induced by superoxide anion, hydrogen peroxide, hydroxyl radical, nitric oxide, and peroxynitrite. This study was undertaken to test the hypothesis that MT also protects from RNA damage induced by peroxynitrite, an important reactive nitrogen species that causes a diversity of pathological processes. A cell-free system was used. RNA damage was detected by the mobility of $tRNA^{Phe}$ in electrophoresis. Cleavages on tRNA were not induced by 3-morpholinosydnomine, which produces peroxynitrite directly. MT induced tRNA damage which was site specific.

• Parasites, Hosts and Diseases
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• 제51권4호
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• pp.401-412
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• 2013

Because of an increased number of Acanthamoeba keratitis (AK) along with associated disease burdens, medical professionals have become more aware of this pathogen in recent years. In this study, by analyzing both the nuclear 18S small subunit ribosomal RNA (18S rRNA) and mitochondrial 16S rRNA gene loci, 27 clinical Acanthamoeba strains that caused AK in Japan were classified into 3 genotypes, T3 (3 strains), T4 (23 strains), and T5 (one strain). Most haplotypes were identical to the reference haplotypes reported from all over the world, and thus no specificity of the haplotype distribution in Japan was found. The T4 sub-genotype analysis using the 16S rRNA gene locus also revealed a clear subconformation within the T4 cluster, and lead to the recognition of a new sub-genotype T4i, in addition to the previously reported sub-genotypes T4a-T4h. Furthermore, 9 out of 23 strains in the T4 genotype were identified to a specific haplotype (AF479533), which seems to be a causal haplotype of AK. While heterozygous nuclear haplotypes were observed from 2 strains, the mitochondrial haplotypes were homozygous as T4 genotype in the both strains, and suggested a possibility of nuclear hybridization (mating reproduction) between different strains in Acanthamoeba. The nuclear 18S rRNA gene and mitochondrial 16S rRNA gene loci of Acanthamoeba spp. possess different unique characteristics usable for the genotyping analyses, and those specific features could contribute to the establishment of molecular taxonomy for the species complex of Acanthamoeba.

• 자연과학논문집
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• 제14권2호
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• pp.83-91
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• 2004

박테리오 파이지 K11 lysozyme은 최근에 우리 실험실에서 클로닝 되었으며, 숙주균주의 세포벽을 분해하는 고유의 lysozyme활성과 박테리오 파아지 K11 RNA 중합효소의 전사반응을 억제하는 활성을 가지고 있는 것으로 확인되었다. 이미 잘 연구된 박테리오 파아지 T7 lysozyme의 경우 클로닝되고 분리 정제된 T7 lysozyme 단백질의 3차 구조 및 T7 RNA 중합효소와의 결합양상에 대하여 밝혀졌다. 따라서 우리 실험실에서는 K11 lysozyme과 K11 RNA 중합효소와의 결합 정도 및 그 특성을 파악할 목적으로 yeast two hybrid 시스템을 통하여 K11 RNA 중합효소와 K11 lysozyme의 단백질-단백질 상호작용을 알아보고자 하였다. LexA 시스템을 이용하여 LexA DNA 결합 부위를 갖고 있는 pLexA에 K11 lysozyme 유전자를 삽입하여 prey로 하였따. 활성 부위로는 B42 융합 단백질을 만드는 pJG4-5에 K11 RNA 중합효소의 결합은 생체 밖에서 reporter 유전자인 lacZ와 leu2의 발현으로 확인되었으며 이들의 결합정도와 결합부위에 대한 연구들은 진행중에 있다.