• 한국생물정보학회:학술대회논문집
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• 한국생물정보시스템생물학회 2000년도 International Symposium on Bioinformatics
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• pp.21-22
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• 2000

Expressed sequence tags (EFTs) are the partial segments of cDNA produced from 5 or 3 single-pass sequencing of cDNA clones, error-prone and generated in highly redundant sets. Advancement and expansion of Genomics made biologists to generate huge amount of ESTs from variety of organisms-human, microorganisms as well as plants, and the cumulated number of ESTs is over 5.3 million, As the EST data being accumulate more rapidly, it becomes bigger that the needs of the EST analysis tools for extraction of biological meaning from EST data. Among the several needs of EST analyses, the extraction of protein sequence or functional motifs from ESTs are important for the identification of their function in vivo. To accomplish that purpose the precise and accurate identification of the region where the coding sequences (CDSs) is a crucial problem to solve primarily, and it will be helpful to extract and detect of genuine CD5s and protein motifs from EST collections. Although several public tools are available for EST analysis, there is not any one to accomplish the object. Furthermore, they are not targeted to the plant ESTs but human or microorganism. Thus, to correspond the urgent needs of collaborators deals with plant ESTs and to establish the analysis system to be used as general-purpose public software we constructed the pipelined-EST analysis system by integration of public software components. The software we used are as follows - Phred/Cross-match for the quality control and vector screening, NCBI Blast for the similarity searching, ICATools for the EST clustering, Phrap for EST contig assembly, and BLOCKS/Prosite for protein motif searching. The sample data set used for the construction and verification of this system was 1,386 ESTs from human intrathymic T-cells that verified using UniGene and Nr database of NCBI. The approach for the extraction of CDSs from sample data set was carried out by comparison between sample data and protein sequences/motif database, determining matched protein sequences/motifs that agree with our defined parameters, and extracting the regions that shows similarities. In recent future, in addition to these components, it is supposed to be also integrated into our system and served that the software for the peptide mass spectrometry fingerprint analysis, one of the proteomics fields. This pipelined-EST analysis system will extend our knowledge on the plant ESTs and proteins by identification of unknown-genes.

• Asian-Australasian Journal of Animal Sciences
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• 제23권5호
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• pp.640-648
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• 2010

Insulin-responsive glucose transporter 4 (GLUT4) is a member of the glucose transporter family and mainly presents in skeletal muscle and adipose tissue. To clarify the molecular structure of porcine GLUT4, RACE was used to clone its cDNA. Several cDNA clones corresponding to different regions of GLUT4 were obtained by amplifying reverse-transcriptase products of total RNA extracted from Landrace porcine skeletal muscles. Nucleotide sequence analysis of the cDNA clones revealed that porcine GLUT4 cDNA was composed of 2,491 base pairs with a coding region of 509 amino acids. The deduced amino acid sequence was over 90% identical to human, rabbit and cattle GLUT4. The tissue distribution of GLUT4 was also examined by Real-time RT-PCR. The mRNA expression abundance of GLUT4 was heart>liver, skeletal muscle and brain>lung, kidney and intestine. The developmental expression of GLUT4 and insulin receptor (IR) was also examined by Real-time RT-PCR using total RNA extracted from longissimus dorsi (LM), semimembranosus (SM), and semitendinosus (SD) muscle of Landrace at the age of 1, 7, 30, 60 and 90 d. It was shown that there was significant difference in the mRNA expression level of GLUT4 in skeletal muscles of Landrace at different ages (p<0.05). The mRNA expression level of IR also showed significant difference at different ages (p<0.05). The developmental change in the mRNA expression abundance of GLUT4 was similar to that in IR, and both showed a higher level at birth and 30 d than at other ages. However, there was no significant tissue difference in the mRNA expression of GLUT4 or IR (p>0.05). These results showed that the nucleotide sequence of the cDNA clones was highly identical with human, rabbit and cattle GLUT4 and the developmental change of GLUT4 mRNA in skeletal muscles was similar to that of IR, suggesting that porcine GLUT4 might be an insulin-responsive glucose transporter. Moreover, the tissue distribution of GLUT4 mRNA showed that GLUT4 might be an important nutritional transporter in porcine skeletal muscles.

• Molecules and Cells
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• 제25권4호
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• pp.467-478
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• 2008

Simple sequence repeats (SSRs) and interSSR (ISSR) marker systems were used in this study to reveal genetic changes induced by artificial selection for short/long larval duration in the tropical strain Nistari of the silkworm Bombyx mori. Artificial selection separated longer larval duration (LLD) ($29.428{\pm}0.723days$) and shorter larval duration (SLD) ($22.573{\pm}0.839days$) lines from a base, inbred population of Nistari (larval span of $23.143{\pm}0.35days$). SSR polymorphism was observed between the LLD and SLD lines at one microsatellite locus, Bmsat106 ($CA_7$) and at two loci of 1074 bp and 823 bp generated with the ISSR primer UBC873. Each of these loci was present only in the LLD line. The loci segregated in the third generation of selection and were fixed in opposite directions. In the $F_2$ generation of the $LLD{\times}SLD$ lines, the alleles of Bmsat106 and $UBC873_{1074bp}$ segregated in a 1:1 ratio and the loci were present only in the LLD individuals. $UBC873_{823bp}$ was homozygous. Single factor ANOVA showed a significant association between the segregating loci and longer larval duration. Together, the two alleles contributed to an 18% increase in larval duration. The nucleotide sequences of the $UBC873_{1074bp}$ and $UBC873_{823bp}$ loci had 67% A/T content and consisted of direct, reverse, complementary and palindromic repeats. The repeats appeared to be "nested" (59%) in larger repeats or as clustered elements adjacent to other repeats. Of 203 microsatellites identified, dinucleotides (67.8%) predominated and were rich in A/T and T/A motifs. The sequences of the $UBC873_{1074bp}$ and $UBC873_{823bp}$ loci showed similarity (E = 0.0) to contigs located in Scaffold 010774 and Scaffold 000139, respectively, of the B. mori genome. BLASTN analysis of the $UBC873_{1074bp}$ sequence showed significant homology of (nt.) 45-122 with upstream region of three exons from Bombyx. The complete sequence of this locus showed ~49% nucleotide conservation with transposon 412 of Drosophila melanogaster and the Ikirara insertions of Anopheles gambiae. The A + T richness and lack of coding potential of these small loci, and their absence in the SLD line, reflect the active process of genetic change associated with the switch to short larval duration as an adaptation to the tropics.

• Journal of Plant Biotechnology
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• 제37권1호
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• pp.102-109
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• 2010

Fructan은 식물이 저온에 노출 되었을 때 다양한 조직에 축적됨으로써 여러 스트레스에 저항을 나타내는 것으로 알려져 있다. 본 연구에서는 fructan 생합성 경로에 관여하는 효소인 1-sst와 1-fft 유전자를 돼지감자 구근으로 부터 분리하였다. 분리한 1-sst와 1-fft 유전자는 Ti-plasmid vector인 KJG V-B2 vector에 35S promoter에 의해 발현할 수 있도록 형질전환용 벡터를 구축하였다. Agrobacterium tumefaciens법에 의해 1-sst와 1-fft 유전자의 형질전환 벼를 육성하였고, 1-sst, 1-fft 및 HPT 유전자 특이적인 primer를 사용하여 PCR 분석한 결과 유전자가 벼의 callus 게놈내에 안정적으로 삽입되었음을 확인하였다. 또한 Southern 및 RT-PCR 분석에서도 같은 결과를 얻었다. 형질전환 벼의 후대에서도 안정적으로 유전자가 발현되는 homo 계통을 선발하였고 이를 이용해 1-sst와 1-fft 유전자의 삽입이 확인된 형질전환 벼에서 유전자의 발현양상을 알아보기 위해 RT-PCR 및 Real-Time PCR를 수행한 결과 형질전환 벼에서 1-sst와 1-fft 유전자 모두 안정적으로 발현되고 있음을 확인하였다. 또한 1-sst와 1-fft 유전자가 삽입된 형질전환 벼를 이용한 기능 분석 연구를 통해 식물체가 저온에 노출되었을 때 1-sst와 1-fft의 작용에 의해 fructan 생합성량이 증가됨을 알 수 있었다. 따라서 본 연구를 통해 얻어진 fructan 생합성 관련 유전자가 삽입된 형질전환 벼는 탄수화물대사 및 저온, 건조 등의 환경 stress에 대한 내성에 대해 좋은 육종 소재로 이용 가능할 것으로 사료된다.

• 한국자원식물학회지
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• 제19권5호
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• pp.628-633
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• 2006

태백제비꽃군내 식물들은 동소적으로 생육하면서 단엽에서 장상복엽까지 연속적인 중간형을 나타내어 분류학적 어려움이 있다. 본 연구의 목적은 태백제비꽃, 단풍제비꽃, 남산제비꽃 그리고 각 분류군 사이의 중간형을 잎의 형태에 따라 5 집단으로 구분하고 각 집단별 대표적인 개체를 선별하여 ITS DNA 염기서열을 분석하고 분류학적 어려움을 해결하는데 있다. 정렬된 ITS1, ITS2 그리고 5.8S 지역의 염기서열은 702 bp로 나타났다. 5.8S 지역은 163 bp로 조사된 모든 개체에서 변이가 없었으며, ITS1과 ITS2는 일부 변이가 있어 분산분석, 염기 서열 분기 조사 그리고 분계분석에 이용하였다. 분산분석 결과 조사된 잎 형태별 개체들 간에 차이가 없는 것으로 나타났다. 염기서열 분기 조사 결과, 군외군으로 설정한 낚시제비꽃과 노랑제비꽃의 경우 Kimura 2-parameter distance에서 절대치가 0.05보다 훨씬 높게 나타나서 뚜렷한 차이가 확인되었다. 그러나 군내군 5 개체는 절대치가 모두 매우 낮게 나타나서 염기서열 분기는 종 수준이 아닌 종 이하의 수준으로 판단되었다. 분계분석에서 군외군으로 설정한 2 종은 기저 분계조를 형성하였다. 군내군은 하나의 분계조를 형성하였지만, 부트스트랩이 50% 이하로 나타나 계통학적 의미는 적은 것으로 사료된다.

• Molecules and Cells
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• 제19권1호
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• pp.114-123
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• 2005

Nitropyrene, the predominant nitropolycyclic hydrocarbon found in diesel exhaust, is a mutagenic and tumorigenic environmental pollutant that requires metabolic activation via nitroreduction and ring oxidation. In order to determine the role of ring oxidation in the mutagenicity of 1-nitropyrene, its oxidative metabolites, 1-nitropyrene 4,5-oxide and 1-nitropyrene 9,10-oxide, were synthesized and their mutation spectra were determined in the coding region of hprt gene of CHO cells by a PCR amplification of reverse-transcribed hprt mRNA, followed by a DNA sequence analysis. A comparison of the two metabolites for mutation frequencies showed that 1-nitropyrene 9,10-oxide was 2-times higher than 1-nitropyrene 4,5-oxide. The mutation spectrum for 1-nitropyrene 4,5-oxide was base substitutions (33/49), one base deletions (11/49) and exon deletions (5/49). In the case of 1-nitropyrene 9,10-oxide, base substitutions (27/50), one base deletions (15/50), and exon deletions (8/50) were observed. Base substitutions were distributed randomly throughout the hprt gene. The majority of the base substitutions in mutant from 1-nitropyrene 4,5-oxide treated cells were $A{\rightarrow}G$ transition (15/33) and $G{\rightarrow}A$ transition (8/33). The predominant base substitution, $A{\rightarrow}G$ transition (11/27) and $G{\rightarrow}A$ transition (8/27), were also observed in mutant from 1-nitropyrene 9,10-oxide treated cells. The mutation at the site of adenine and guanine was consistent with the previous results, where the sites of DNA adduct formed by these compounds were predominant at the sites of purines. A comparison of the mutational patterns between 1-nitropyrene 4,5-oxide and 1-nitropyrene 9,10-oxide showed that there were no significant differences in the overall mutational spectrum. These results indicate that each oxidative metabolite exhibits an equal contribution to the mutagenicity of 1-nitropyrene, and ring oxidation of 1-nitropyrene is an important metabolic pathway to the formation of significant lethal DNA lesions.

• Journal of Genetic Medicine
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• 제8권1호
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• pp.53-57
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• 2011

목 적: 윌슨병은 간조직에 구리의 과도한 침착으로 발병하는 상염색체 열성 유전질환이다. 지금까지 ATP7B 유전자가 유일한 원인유전자로 알려져 왔다. 그러나, 약 15%의 환자에서는 ATP7B 유전자 돌연변이가 발견되지 않는다. 본 연구는 ATP7B 유전자의 돌연변이가 발견되지 않은 윌슨병 환자를 대상으로 새로운 원인 유전자를 발견하기 위하여 시행되었다. 대상 및 방법: ATP7B 돌연변이가 발견되지 않은 12명의 윌슨병 환자를 대상으로 ATP7B 와 상호작용을 하는 것으로 알려진 ATOX1, COMMD1, GLRX, DCTN4와 ZBTB16 유전자의 전사부위와 엑손-인트론 경계부위의 염기서열을 분석하였다. 결 과: DCTN4 유전자의 12번 엑손에 존재하는 c.1084A>G(p.Thr362Ala)를 포함하는 3가지의 변이가 환자에서 발견되었다. in silico 분석을 통해 3가지 변이 중 c.1084A>G가 유일하게 단백질 기능 변화를 일으킬 것으로 예측되었다. 176명의 윌슨병 환자와 414명의 정상인을 대상으로 이 변이의 빈도를 조사한 결과, 정상인보다 윌슨병 환자에서 더 높은 빈도를 나타내었다(상대비, odds ratio [OR]=3.14, 95% 신뢰도=1.36-7.22, P=0.0094). 결 론: 본 연구의 결과는 ATP7B 와 상호작용하는 DCTN4 유전자의 c.1084A>G (p.Thr362Ala) 다형성이 윌슨병의 발병과 연관이 있음을 시사한다.

• Journal of Microbiology
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• 제44권3호
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• pp.301-310
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• 2006

Two pathways of ammonium assimilation and glutamate biosynthesis have been identified in microorganisms. One pathway involves the NADP-linked glutamate dehydrogenase, which catalyzes the amination of 2-oxoglutarate to form glutamate. An alternative pathway involves the combined activities of glutamine synthetase, which aminates glutamate to form glutamine, and glutamate synthase, which transfers the amide group of glutamine to 2-oxoglutarate to yield two molecules of glutamate. We have cloned the large subunit of the glutamate synthase (GOGAT) from Salmonella typhimurium by screening the expression of GOGAT and complementing the gene in E. coli GOGAT large subunit-deficient mutants. Three positive clones (named pUC19C12, pUC19C13 and pUC19C15) contained identical Sau3AI fragments, as determined by restriction mapping and Southern hybridization, and expressed GOGAT efficiently and constitutively using its own promoter in the heterologous host. The coding region expressed in Escherichia coli was about 170 kDa on SDS-PAGE. This gene spans 4,732 bases, contains an open reading frame of 4,458 nucleotides, and encodes a mature protein of 1,486 amino acid residues (Mr =166,208). The EMN-binding domain of GOGAT contains 12 glycine residues, and the 3Fe-4S cluster has 3 cysteine residues. The comparison of the translated amino acid sequence of the Salmonella GOGAT with sequences from other bacteria such as Escherichia coli, Salmonella enterica, Shigella flexneri, Yersinia pestis, Vibrio vulnificus and Pseudomonas aeruginosa shows sequence identity between 87 and 95%.

• 한국동물학회지
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• 제38권4호
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• pp.550-556
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• 1995

c-myc proto-oncogene은 여러 세포들의 분화와 형질전화에 뿐만 아니라 정상세포의 분열조절에도 관여한다고 알려져왔다. 특히 생쥐의 초기배아에서 c-myc mRNA가 발현되고 antisense c-myc oligomer의 미세주입에 의해 배발생이 억제된다는 연구결과는 c-myc이 초기배아의 발생 및 분열에 관여하는 것을 시사한다. 그러나 최근까지 초기배아에 존재하는 c-myc promoter의 기능적 활성화에 관한 연구는 미진하였다. 이를 위하여, c-myc promoter와 대장균의 lacZ 유전자를 결합시킨 두 종류의 vector(pcmyc-Gall, pcmyc-Ga12)를 만들어 수정란의 전핵에 미세주입한 후, 배 발생에 따른 c-myc promoter의 활성화를 lacZ 유전자의 산물인 $\beta$-galactosidase 에 의한 X-gal 염색으로 조사하였다. 미세주입된 초기 배아는 2세포기 배아를 포함하는 여러 발생단계에서 $\beta$-galactosidase 의 활성을 보였다. 이는 c-myc 유전자가 배아의 게놈유전자로부터 발현되며, 또한 궁극적으로 초기 배아의 발생과정에 중요한 역할을 하고 있음을 시사하고 있다.

• 생명과학회지
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• 제13권3호
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• pp.291-297
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• 2003

인간 내생 레트로바이러스 HERV-W는 다발성 경화증 환자로부터 탐지된 MSRV와 연루되어 있다. 인간 태아의 뇌로부터 유래된 cDNA library를 이용하여 PCR법으로 2개의 HERV-W 패밀리(HWP-FB10과 HWP-FB12)를 동정하고 분석하였다. 그들은 HERV-W (accession no. AF009668)와 89%의 염기서열의 유사성을 보였다. Pol 유전자를 아미노산의 서열로 분석해 본 결과 점돌연변이 또는 삽입/결실로 말미암아 frameshift 및 종결코돈을 나타내었다. 유전자정보의 데이터베이스를 이용하여 HERV-W 패밀리간의 분자계통분류도를 작성해 본 결과 HWP-FB10은 인간의 염색체 7q21-22로부터 유래된 AC000064와 매우 가깝게 관련되어 있음을 시사하였다. 이들의 새로운 HERV-W pol 패밀리가 이웃하는 어떤 유전자와 상호 연결되어 있으며, 어떠한 기능을 수행하는지에 대한 전망에 대해 토의하였다.