Earticle

다양한 포유동물의 미토콘드리아 유전자들에서 선택적 개시코돈(AIC)들이 보고되었다. 본 연구는 돼지 5 품종에서 mtDNA ND2 유전자의 AIC 양상을 분석하였다. 두 종류의 AIC 서열들이 발견되었고, 품종 집단간에서 각기 다른 출현빈도를 보였다. ND2의 methionine codon으로 Large White와 Landrace는 각각 전 개체에서 ATA와 ATT 서열로 확인되었다. 다른 3 품종(Berkshire, Duroc, Hampshire)의 경우는 두 서열이 모두 발생하였으며, 91.9, 21.3, 60.0%의 빈도로 ATA를 보였다. 기존의 연구들에 의하면 중국재래돼지들이 모두 개시코돈 ATA를 갖는다고 보고되어 있다. 미토콘드리아 ND2 단백질의 합성과정에서 AIC가 어떤 영향을 미치는지는 설명할 수 없으나, 본 연구에서 나타난 AIC 다형성과 품종특이적인 분포양상은 돼지의 육종에 있어 모계추적을 위한 분자적 표지인자로서 이용될 수 있을 것을 사료된다.

Alternative initiation codon(AIC) has been reported in the mitochondrial genes in various mammalian species. We investigated the AIC of mitochondrial NADH dehydrogenase 2 gene(mtDNA ND2) in five pig breeds. Two kinds of initiation codons(ATA/ATT) showing different frequencies among tested pig breeds were used. While all Large White pigs had ATA as an initiator methionine codon, all Landrace pigs had ATT. The other breeds(Berkshire, Duroc and Hampshire) had both initiation codons with the ATA frequencies, 91.9, 21.3 and 60.0%, respectively. In the previous reports, all Chinese indigenous pig breeds were identified to have unique initiation codon ATA. Although the effect of AIC on the translation of mtDNA ND2 has not been studied in this study, AIC patterns in mtDNA ND2 will contribute to the maternity test using molecular markers in pig breeding.

본 논문에서는 유전정보 DNA 표준 코드인 [UC;AG]를 64괘로 분석했다. 인간의 유전정보를 담고 있는 DNA는 인산과 당에 A(아데닌)‧C(시토신)‧G(구아닌)‧T(티민) 등 네 종류의 염기가 30억쌍 이어 붙여진 형태이다. DNA 표준 코드를 64괘로 나타내고, 이 코드를 Kronecker product를 이용하여 164행렬로 나타냈다. 이 164 행렬은 이중나선의 중복성을 가지고 있으며, 이 중복성을 제거하면 RNA코드 44 행렬을 얻는다. 164행렬은 Kronecker product를 이용하여 소 행렬로 분해되었다. DNA 이중나선을 행렬로 표시하고 유전정보 괘 배열 코드를 분석하였으며 그 예를 예 5, 6에 나타냈다.

In this paper, we analyze the [UC;AG] code which is genetic information standard DNA code, with 64 trigram. DNA which contains human genetic information, is a shape of adding three billion pairs of four bases which are A(adenine), C(cytosine), G(guanine) and T(thymine) to phosphoric acid and glucose. We present standard DNA code to 64 trigram which is 644 matrix with Kronecker product. This 644 matrix has double helix duplex property, and we can get the 44 matrix RNA code by removing the duplex of it. We present the DNA double helix to matrices and analysis the trigram array code of genetic information and the examples of it are presented in example 5, 6.

제주지역에 산재된 錠木올레와 드러누워 있는 세계 최대 피라미드인 墓의 神門올레를 조사하고 제주인의 풍속 Protocol을 현대 과학과 접목시키는 Link를 구성했다. 3개의 이승 정낭과 2개의 墓의 저승 神門은 올레길 공간체로 연 결되어 있다. 그 공간체에는 삶과 죽음사이의 相生과 相剋이 공존하는 相補性(complementarity) 원리가 제주 文化에 숨어있다. 對와 待이다. 즉 반대되는 것은 서로 보완적이다. (Contraria Sunt Complementa 라틴어) 서로 대립하면서도 서로 의존하는 관계로 서로가 서로를 품은 관계를 뜻한다. 정낭은 통신 원리를 사용될 뿐 아니라 인체의 DNA Codon 에 기본 원리으로 사용된다.

We investigate the God gate olleh of the largest tomb, pyramid, in the world which is scattered in Jeju Island and construct link which is connecting Jeju people’s custom to modern science. The three sacred gates and the two tombs are connected to the olegil space. In this space, the principle of complementarity in which coexistence exist between life and death is hidden in Jeju culture. It is a question and wait. Contrarily, the opposite is complementary. (Contraria Sunt Complementa Latin) This refers to the relationship of each other in relation to one another and in a mutually dependent relationship. Seminal vesicles are used as basic logic in DNA codon of human body as well as communication principle.

Codon optimization is a technology which changes the codon usage to highly express a target gene or protein in host. To express a gene derived from marine bacteria, codon optimization was used in this study. Using SDS-PAGE, we confirmed the expression level of target protein. The band meaning expression of codon optimized gene was only observed in SDS-PAGE. Also we measured a enzyme activity using analysis of NADH consumption per minute in extract sample of recombinant. The recombinant having codon optimized gene had the highest enzyme activity. Observing the gene having the highest activity in Escherichia coli, we have studied the confirmed genes of marine bacteria to obtain specific chemicals in E. coli by fermentation.

Xylose reductase (XR) is a key enzyme in xylitol production, catalyzing the reduction of D-xylose to xylitol. However, most XRs from yeasts and fungi have higher activity for L-arabinose than D-xylose and this broad substrate acceptance causes formation of byproduct, arabitol. Neurospora crassa XR(NcXR) is known to have high overall catalytic efficiency and higher activity toward D-xylose than L-arabinose significantly. C. tropicalis and N. crassa have different patterns in codon usage and three codons, CCC(P), CGC(R) and CTC(L) are particularly expected to be problems in functional expression of NcXR. Consequently, all codons of NcXR were changed into preferred codons in C. tropicalis. A promoter region of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was isolated from the genomic DNA of C. tropicalis ATCC 20913 to construct a constitutive expression cassette for this strain. The codon-optimized NcXR(NXRG) gene was placed under the control of GAPDH promoter and integrated into the genome of C. tropicalis N43 which is xyl2-disrupted and xyl1-partially disrupted mutant. Expression of NXRG was confirmed by determining enzyme activity in cells grown on a medium containing glucose as a carbon source. The resulting recombinant yeast, C. tropicalis NG4, showed higher XR activity (1287mU/mg of proteins) than that of the parental strain (26mU/mg of proteins) and 3.6 times higher substrate specificity for D-xylose than L-arabinose.

Drug development research requires a large amount of target proteins. Screening of drug target often requires 13C- and 15N- labeled protein, and higher protein expression has great advantages for obtaining isotope-labeled proteins. Among many of the proteins, glycosyltransferases(GTs) are attractive biocatalysts in producing a series of important bioactive natural products. In case of kanamycin, kanF gene encodes the first glycosyltransferase which acts both as glucosyltransferase and N-acetyl-glucosaminyl-transferase in kanamycin biosynthetic pathway of Streptomyces kanamyceticus. The recombinant expression of kanF gene in E. coli BL21 (DE3), BL21pLysS and BL21-CodonPlus® (DE3)-RP under T7 promoter-based system showed the expression in insoluble rather than soluble form. Further analysis of the codons revealed that this gene includes number of rare codons which are difficult to be translated in E. coli. Due to variations in codon usage between E. coli and Streptomyces, with high free energy of secondary structure of mRNA, KanF was not expressed under various condition tested. Thus, using codon optimized gene based on codon usage of E. coli has helped to overcome this problem and led to soluble expression of protein. Thus obtained KanF was used for making in vitro reaction for glycosylation of 2-DOS and the product was confirmed to be 2′ -Deamino-2′-hydroxyparomamine by high resolution Q-TOF mass analysis.

Many researches have been focused on the development of cellulases having high catalytic efficiency on crystalline cellulose and thermo-stability at elevated temperature to obtain fermentable sugars for bio-ethanol source from the most abundant plant biomass. Previously, we produced a cellulase of Clostridium thermocellum (C-cellulase) whose structure has been elucidated in recombinant Escherichia coli BL21 displaying a high soluble expression and a superior activity at 50~55 oC. However, the stability of C-cellulase decreased rapidly above 55 oC. In this study, we investigate the two strategies for improving C-cellulase; the first is to obtain a new cellulase which can be evolved to more efficient cellulase by protein engineering and the second is to increase the thermo-stability of C-cellulase at a higher temperature by fusing it to maltose-binding protein (MBP), glutathionestransferase (GST) and ferritin (Fn), respectively. We searched a new cellulase-like protein from Clostridium sp.L2-50 which shows identity with C-cellulase, resulting in a high soluble expression in E. coli. The PCR fragment of C-cellulase was cloned into the C-terminus of MBP gene using pMAL-2x, producing MBP-C-cellulase or the C-terminus of GST gene using pET 42b+ vector, producing GST-C-cellulase. The gene of C-cellulase was fused to N terminus of ferritin gene which was already cloned in pETDuet vector, producing C-cellulase-Fn.

Proteins composed of twenty amino acids play important biological roles in a cell and their biological activities have been employed in many academic and industrial fields. Recently, great efforts have been devoted to expanding the genetic code for the in vivo biological incorporation of non-canonical amino acids into proteins. This methodology will be useful to manipulate the protein with non natural amino acids that possess unique side chains, which could be used for probing protein function and structure. Here, we report the global incorporation of 3, 4-dihydroxy L-phenylalanine (L-DOPA) into green fluorescent protein (GFPdopa) which altered the spectral and functional properties of the protein. The successful incorporation L-DOPA further facilitates the selective chemical cross-linking of protein-polysaccharide interaction in vitro. GFPdopa protein were oxidized upon sodium periodate (NaIO4) which lead to the formation an orthoquinone intermediate that is subsequently attacked by nucleophilic side chains of amine group interacting polysaccharide, through either Michael addition or Schiff base formation. In general, we have developed an efficient bioconjugants by selective covalent cross linking using genetically encoded unnatural amino acid. Developing such a bioconjugants having lot of potential application in tissue engineering, regenerative medicine and developing a novel drug carrier. Finally, our current approach could be a valuable and efficient addition tool to the protein engineering field as well as for developing of protein-polysaccharide based therapeutics for mankind

The purpose of this study is to find out how to use emotional activity rate in sentence. The aim is to estimate the Emotion-Codon status of emotion-DNA wiggling as well as the sequence of codons in which amino acids of human body are spread. It is to use as if the amino acids are translated from the DNA of the human body, and the emotions that cooperate with each other are diversified and refined. Through these attempts, we anticipate the change of emotions by literary works and present the direction of the synapse working and the direction of the encoding. Therefore, this study analyzed the emotional activity rate of Rated Emotion-Codon, which works as Rated Sijo. At this time, the emotional change in the human body, in which the sentence is more clearly pronounced, is predicted and detected. This result shows how emotions can activate the human body. This state of activation is an indicator of how much the sentence pushes the physiological action of the human body. It is judged that Codon is constructed precisely for the activation of the rated Sijo additive definition by this index. Our result is evidenced by the fact that three sentences of Rated Sijo show an array with the same rating as the Rated Codon's, The first sentence fuses with the sentiment between the second sentence and the third sentence of Rated Sijo. The emotional symbols of the first sentence’s space, which are fused with emotions flowing between the second and third sentence of Rated Sijo, are "A, U, J, L", which are rated-structured in the first base of the Codon table. The emotional symbols of the second sentence’s space, which are fused with emotions flowing between the first and third sentence of Rated Sijo, are "A, U, J, L", which are rated-structured in the second base of the Codon table. The emotional symbols of the third sentence’s space, which are fused with emotions flowing between the first and second sentence of Rated Sijo, are "A, U, J, L", which are rated-structured in the third base of the Codon table. The Rated Sijo, which is resiliently and precisely assigned to the human body's Rated Emotion-Codon, will require a rich experience in our Rated Life.

Free fatty acids (FFAs) are attractive intermediates for producing a wide range of high-energy-density biofuels such as biodiesel. Acyl-acyl carrier protein (ACP) thioesterase (acyl-ACP TE) from Streptococcus pyogenes MGAS10270, was codon-optimized and expressed in Escherichia coli K-12 MG1655 and Escherichia coli K-12 W3110. By utilizing codon-optimized S. pyogenes acyl-ACP TE to improve the total FFAs and to tailor the composition of FFAs, high-specificity production of saturated fatty acids (C12, C14) and unsaturated fatty acids (C18:1 C18:2) were achieved in recombinants. E. coli SGJS41 and SGJS46 (codon-optimized acyl-ACP TE of S. pyogenes) demonstrated the highest intracellular total FFAs content; in particular, the content of C12 and C14 FFAs was about 3–5 fold, and the content of C18:1 and C18:2 FFAs content about 8–42 fold higher than that in the control E. coli and E. coli JES1017 (original acyl-ACP TE of S. pyogenes).

Amino acyl tRNA synthetase (AARS) is an ATP dependent enzyme helps in charging of tRNA with cognate amino acid during protein synthesis. The accuracy of the protein translation is maintained by the proof reading activity of AARS by preventing charging of tRNA with non cognate amino acids (Unnatural amino acids). Recently researchers identified a methodology in which the endogenous tRNA synthetases can recognize and incorporate the close structural analogs of the cognate amino acid containing novel functional groups in the absence of the natural substrate called as codon reassignment. Currently, more than 15 methionine analogs were incorporated into protein in response to the sense codon with the help of methionine auxotrophic cell which provide novel application such as metabolic labeling. In this study, we have performed molecular docking analysis to predict the binding mode of the methionine analogs with the active site residues of Escherichia coli methionyl tRNA synthetase and we identified the key residues responsible for the recognition of these methionine analogs. Here, we clearly demonstrate the substrate selectivity of these methionine analogs with methionyl tRNA synthetase and offer the clear structural knowledge about the active site which will provide knowledge for rational designing of methionyl tRNA synthetase.

Translational efficiency in Escherichia coli is known to be strongly influenced by the secondary structure around the ribosome-binding site and the initiation codon in the translational-initiation region of the mRNA. Several quantitative studies have reported that translational efficiency is attributable to effects on ribosome accessibility predominantly caused by the secondary structure surrounding the ribosome-binding site. However, the influence of mRNA secondary structure around regions downstream of the initiation codon on translational efficiency after ribosome-binding step has not been quantitatively studied. Here, we quantitatively analyzed the relationship between secondary structure of mRNA surrounding the region downstream of the initiation codon, referred to as the downstream region (DR), and protein expression levels. Modified hairpin structures containing the initiation codon were constructed by site-directed mutagenesis, and their effects on expression were analyzed in vivo. The minimal folding free energy (dG) of a local hairpin structure was found to be linearly correlated with the relative expression level over a range of 0.3- to 1.2-fold that of the wild-type structure. These results demonstrate that expression can be quantitatively controlled by changing the stability of the secondary structure surrounding the DR.

본 연구에서는 컴퓨터 기반 예측 프로그램을 활용하여 알라닌 아미노산이 반복적으로 포함된 항결빙 단백질 (antifreeze proteins, AFPs)의 효율적인 생산 시스템을 개발하고자 하였다. 이를 위해 대장균 숙주 세포의 유전자 코돈 과 GC 염기서열 함량 비율을 최적화하는 전략을 적용하였다. 먼저, 다양한 유전자 및 단백질 데이터베이스를 분석하여 여러 종류의 AFP 유전자 중 AFP01과 AFP02를 선정한 후, OptimumGene™ 코돈 최적화 분석을 수행하여 숙주 세포 에 적합하도록 코돈 선호도 및 GC 함량을 조정하였다. 최적화 결과, AFP01 유전자의 코돈 적응 지수(Codon Adaptation Index, CAI)는 0.7에서 0.83으로 개선되었으며, GC 함량은 72.6%에서 65.4%로 조정되었다. 또한, AFP02 유전자의 CAI는 0.64에서 0.84로 증가하였고, GC 함량은 최적화 전 67.5%에서 최적화 후 68.2%로 변경되었 다. 이러한 최적화된 유전자 서열을 기반으로 인공적으로 synAFP01과 synAFP02 유전자를 합성하고, 이를 발현 벡터 시스템에 삽입하여 대장균에서의 단백질 발현을 유도하였다. 그 결과, 두 종류의 synAFPs가 높은 수준으로 생산됨이 분자 수준에서 확인되었다. 본 연구에서 개발된 방법을 통해 합성된 AFPs는 식품 및 의약 산업과 같은 결빙 방지가 필요한 다양한 분야에서 활용될 수 있을 것으로 기대된다.

This study aimed to develop an efficient production system for antifreeze proteins (AFPs) containing repetitive alanine residues by optimizing the codon usage and GC content ratio of Escherichia coli host cell genes using a computer-based predictive program. To achieve this, various gene and protein databases were analyzed to select two AFP genes (AFP01 and AFP02). These genes were then subjected to OptimumGene™ codon optimization analysis to adjust codon preference and GC content according to the host cell requirements. As a result, the codon adaptation index (CAI) of the AFP01 gene was improved from 0.7 to 0.83, and its GC content was modified from 72.6% to 65.4%. Similarly, the CAI of the AFP02 gene was increased from 0.64 to 0.84, and its GC content was adjusted from 67.5% to 68.2%. Based on these optimized gene sequences, synAFP01 and synAFP02 genes were artificially synthesized and inserted into an expression vector system to induce protein expression in E. coli. The results confirmed that both synAFPs were produced at high levels at the molecular level. The AFPs synthesized using this method are expected to have broad applications in industries requiring antifreeze properties, such as the food and pharmaceutical sectors.

목적 : p53 다형성과 자궁내막증과의 연관성을 파악하고자 하였다. 연구 방법 : 복강경술 또는 개복술로 자궁내막증으로 진단된 환자 271명과 복강경술 또는 개복술에서 자궁내막증이 없는 것으로 확인된 대조군 219명을 대상으로 하여 p53 유전자 codon 72 다형성을 조사하였다. 결과 : p53 유전자 codon 72 다형성의 유전자형 분포는 자궁내막증 환자군과 대조군 사이에 유의한 차이는 없었다 (p=0.086). 반면 stage I-II인 경한 자궁내막증군에서 p53 유전자 codon 72 유전자형 분포는 대조군과 유의한 차이를 보였다 (p=0.043). 그러나 stage III-IV 자궁내막증군은 대조군과 p53 codon 72 유전자형의 분포에서 차이가 없었다. Pro/Pro 유전자형의 경우 Arg/Arg 유전자형과 비교시 stage I-II 자궁내막증 발생 위험도가 2.75로 유의하게 증가하였다. 결론 : 한국 여성에서 p53유전자의 codon 72 다형성은 경증의 자궁내막증 발생과 연관이 있었다.

Objective : To explore the association of p53 codon 72 polymorphism with endometriosis. Methods : Two hundred seventy-one women with surgically or histologically diagnosed edometriosis of stage I-IV, and 219 patients with no evidence of endometriosis by laparoscopy or laparotomy served as control. Allele frequencies and genotype distribution of p53 polymorphisms (arginine homozygosity, heterozygosity, and proline homozygosity) in affected women and controls were evaluated. Results : The genotype distributions of p53 codon 72 polymorphisms did not differ significantly between endometriosis group and control group (p=0.086). However, the genotype distributions of p53 codon 72 polymorphisms differ significantly between stage I-II endometriosis group and control group (p=0.043). Proline homozygotes had higher risk for stage I-II endometriosis compared to arginine homozygotes (odds ratio=2.75, p=0.013). Conclusion : These results suggest that proline homozygote of p53 codon 72 polymorphism is associated with the risk of minimal or mild stage of endometriosis in the Korean population.

Background: Ras genes are thought to play an important role in human cancer since they have been found to be activated frequently in several types of tumors including breast cancer, where the overall incidence of K-RAS oncogene activation is 0-10%. Evaluation of K-RAS gene not only for mutational frequency but also for mutation types in this downstream signaling gene pathway is necessary to determine the mechanisms of action. The present study was conducted to test the hypothesis that K-RAS activation is involved in breast cancer risk of south Indian population. Materials and Methods: A total of 70 paired pathologically confirmed tumor and non-tumor tissues from the same breast cancer patients were analysed for most common K-RAS mutations of codon 12,13 and 61 by polymerase chain reaction followed by restriction digestion and direct nucleotide sequencing method. Results: We found that a high rate of homozygous and heterozygous mutations of codon 12, but not codon 13 and 61, may influence the invasive ductal carcinoma of breast risk in this study. Conclusions: Our study indicated that only codon 12 may be involved in initiating breast carcinogenesis in India.

Background: We investigated the association between polymorphisms in the $p53$ tumor suppressor gene and breast cancer risk in women especially in the Southern part of India. Methods: Genotyping was performed for 50 breast cancer women and 50 controls to determine the status of $p53$ exon 4 codon 72 polymorphism and exon 7 codon 249 mutation and their possible role in breast cancer risk. Results: Frequency of Arg/Arg at codon 72 was 18% in controls and 28% in patients, Arg/Pro frequency was 56% and 66%, Pro/Pro genotype was 8% in controls and 8% in patients. No significance was observed for breast cancer risk with either Arg/Arg or Pro/Pro genotype in codon 72 polymorphism. Similarly, mutation analysis of exon 7 codon 249 revealed that 72% of breast cancer patients have mutation, which is not statistically significant. However, there is a strong association between increase in exon 7 codon 249 mutation and exposure to pollution. Conclusion: The results suggested that there is no risk for exon 4 with Arg/Arg or Pro/Pro polymorphisms in the $p53$ gene and there is no strong correlation between breast cancer patients and mutation in exon 7 codon 249 in South Indian women.

The enzyme encoded by the MGMT gene is involved in the repair of alkylated lesions formed in DNA by carcinogenic nitrosamines. Since dietary items consumed by the Kashmiri population contain high concentrations of these agents, it is biologically plausible that MGMT polymorphic variants may be associated with their risk of esophageal cancer. The present study was performed to assess whether non-synonymous SNPS at codon Leu84Phe and codon Ileu143Val of the MGMT gene, close to the active site of the protein, might be linked to predisposition of Kashmiris to esophageal cancer. Genotyping was carried out by polymerase chain reaction-restriction fragment length polymorphism on 92 cases and 77 healthy controls. Codon 84 and codon 143 SNPs of the MGMT gene were not associated with any increase in risk. While the frequency of the Phe allele at codon 84 in cases was (0.16), slightly higher than controls (0.12), the difference was not statistically significant. Similarly, the frequency of Valine allele in cases at codon 143 (0.08) and controls (0.09) was nearly equal. Moreover, no significant association of MGMT genotypes with the clinicopatholgic variables of esophageal cancer patients was observed. In conclusion, MGMT variants at codon 84 and codon143 may not be involved in the susceptibility of the Kashmiri population to esophageal cancer.