• 대한치과교정학회지
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• 제33권3호
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• pp.185-197
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• 2003

비증후군성 구순구개열을 발생시키는 주요유전자로 추측이 되는 OFC1 유전자(위치 염색체 6p24.3)의 한국인에서 나타나는 특성을 연구하였다. 3대에 걸쳐서 처음으로 비증후군성 구순구개열이 나타난 40 명의 환자(남자 20명, 여자 20명, 평균 나이 : 14.2세)와 3대에 걸쳐서 비증후군성 구순구개열을 포함한 어떤 선천성 기형도 나타나지 않았던 정상 성인 40명 (남자 20명, 여자 20명, 평균 나이 : 25.6세)을 연구 대상으로 하였다. 중합효소 연쇄 반응법을 이용하여 OFC1 유전자를 분리 증폭한 후, 염기 서열 분석을 통해서 대립유전자형을 밝히고, BLAST 와 Pedant-Pro 데이터베이스를 이용하여 단백질의 상동성 검색을 수행하였으며, 그 결과는 다음과 같다. 1. OFC1 유전자는 'CA' 연쇄반복서열을 가진 극소위성 표지자로 밝혀졌다. 2. 환자군과 대조군의 OFC1 유전자의 특별한 차이는 발견되지 않았다. 3. 한국인에서 나타난 'CA' 연쇄반복서열의 형태는 'ABI linkage map 2'의 TA(CA)11TA(CA)10과는 달리, TA(CA)n의 형태를 띄었으며, 연쇄반복의 수는 17회에서 26회로 다양하게 나타났다. 4. 'CA' 연쇄반복서열의 횟수에 따라서, 9가지의 대립유전자형이 발견되었으며, 나타나는 빈도는 환자군과 대조군에서 유사하였다. 5. 'ABI linkage map 2'의 'CA' 연쇄반복서열 사이의 염기서열 T가 한국인에서는 C로 치환되어 있었지만, ORF예측을 하였을 때 예상되는 아미노산의 배열 차이는 관찰되지 않았다. 6. 한국인 OFC1 유전자의 염기서열로 예측되는 단백질을 알아보기 위하여 BLAST 검색을 한 결과, Telomerase reverse transcriptase(TERT, locus 5p15.33, NCBI Genome Annotation ; NT023089)와 Nucleotide binding protein 2(NBP2, locus 17q22, NCBI Genome Annotation; NT010783)가 유사한 구조를 가지는 단백질로 밝혀졌다. 7. Pedant-Pro 데이터베이스로 단백질 구조의 상동성 검색을 한 결과, OFC1 유전자는 적어도 하나의 transmembrane region과 non-gloular region을 가지는 구조로 밝혀졌다.

• Parasites, Hosts and Diseases
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• 제55권4호
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• pp.367-374
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• 2017

Despite the broad distribution of leishmaniasis among Iranians and animals across the country, little is known about the genetic characteristics of the causative agents. Applying both HSP70 PCR-RFLP and sequence analyses, this study aimed to evaluate the genetic diversity and phylogenetic relationships among Leishmania spp. isolated from Iranian endemic foci and available reference strains. A total of 36 Leishmania isolates from almost all districts across the country were genetically analyzed for the HSP70 gene using both PCR-RFLP and sequence analysis. The original HSP70 gene sequences were aligned along with homologous Leishmania sequences retrieved from NCBI, and subjected to the phylogenetic analysis. Basic parameters of genetic diversity were also estimated. The HSP70 PCR-RFLP presented 3 different electrophoretic patterns, with no further intraspecific variation, corresponding to 3 Leishmania species available in the country, L. tropica, L. major, and L. infantum. Phylogenetic analyses presented 5 major clades, corresponding to 5 species complexes. Iranian lineages, including L. major, L. tropica, and L. infantum, were distributed among 3 complexes L. major, L. tropica, and L. donovani. However, within the L. major and L. donovani species complexes, the HSP70 phylogeny was not able to distinguish clearly between the L. major and L. turanica isolates, and between the L. infantum, L. donovani, and L. chagasi isolates, respectively. Our results indicated that both HSP70 PCR-RFLP and sequence analyses are medically applicable tools for identification of Leishmania species in Iranian patients. However, the reduced genetic diversity of the target gene makes it inevitable that its phylogeny only resolves the major groups, namely, the species complexes.

• Parasites, Hosts and Diseases
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• 제55권2호
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• pp.149-158
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• 2017

Variant surface antigens (VSAs) encoded by pir families are considered to be the key proteins used by many Plasmodium spp. to escape the host immune system by antigenic variation. This attribute of VSAs is a critical issue in the development of a novel vaccine. In this regard, a population genetic study of vir genes from Plasmodium vivax was performed in the Republic of Korea (ROK). Eighty-five venous blood samples and 4 of the vir genes, namely vir 27, vir 21, vir 12, and vir 4, were selected for study. The number of segregating sites (S), number of haplotypes (H), haplotype diversity (Hd), DNA diversity (${\pi}$ and ${\Theta}_w$), and Tajima's D test value were conducted. Phylogenetic trees of each gene were constructed. The vir 21 (S=143, H=22, Hd=0.827) was the most genetically diverse gene, and the vir 4 (S=6, H=4, Hd=0.556) was the opposite one. Tajima's D values for vir 27 (1.08530, P>0.1), vir 12 (2.89007, P<0.01), and vir 21 (0.40782, P>0.1) were positive, and that of vir 4 (-1.32162, P>0.1) was negative. All phylogenetic trees showed 2 clades with no particular branching according to the geographical differences and cluster. This study is the first survey on the vir genes in ROK, providing information on the genetic level. The sample sequences from vir 4 showed a clear difference to the Sal-1 reference gene sequence, whereas they were very similar to those from Indian isolates.

• Asian Pacific Journal of Cancer Prevention
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• 제17권7호
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• pp.3309-3315
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• 2016

Helicobacter pylori as the second most common cause of gastric cancer in the world infects approximately half of the developed countries population and 80% of the population living in developing countries. Integrons as genetic reservoirs play major roles in dissemination of antimicrobial resistance genes. To the best of our knowledge, this is the first study to report carriage of class 1 and 2 integrons and associated gene cassettes in H. pylori isolates from Iran. This cross-sectional study was conducted in Tehran among 110 patients with H. pylori infection. Antimicrobial susceptibility testing (AST) for H. pylori strains were assessed by the micro broth dilution method. Class 1 and 2 integrons were detected using PCR. In order to determine gene cassettes, amplified fragments were subjected to DNA sequencing of both amplicon strands. The prevalence of resistance to clarithromycin, metronidazole, clarithromycin, tetracycline, amoxicillin, rifampin, and levofloxacin were 68.2% (n=75), 25.5% (n=28), 24.5% (n=27), 19.1% (n=21), 18.2% (n=20) and 16.4% (n=18), respectively. Frequency of multidrug resistance among H. pylori isolates was 12.7%. Class 2 integron was detected in 50 (45.5%) and class 1 integron in 10 (9.1%) H. pylori isolates. The most predominant gene cassette arrays in class 2 integron-bearing H. pylori were included sat-era-aadA1, dfrA1-sat2-aadA1, blaoxa2 and, aadB whereas common gene cassette arrays in class 1 integron were aadB-aadA1-cmlA6, aacA4, blaoxa2, and catB3. The high frequency of class 2 integron and multidrug resistance in the present study should be considered as a warning for clinicians that continuous surveillance is necessary to prevent the further spread of resistant isolates.

• 한국해양학회지:바다
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• 제5권2호
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• pp.157-168
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• 2000

우리나라 동해안에 서식하는 새치성게(Strongylocentrotus intermedius)는 둥근성게과(Strongylocentrotidae)에 속하는 냉수성 해양 무척추동물이다. 둥근성게과에는 현재 9종의 성게가 속해 있으나, 아직 종간의 분류 기준, 계통 분류학적 유연관계, 진화과정 등이 잘 밝혀져 있지 않다. 본 연구는 유전자 염기서열이라는 분자형질을 이용하여 새치성게의 종 분류기준을 확립하고 이 종의 계통진화 및 분화 시기를 파악하고자 수행되었다. 이를 위하여 변화율이 빠르고 모계로만 유전되는 특성을 가진 미토콘드리아의 한 유전자인 cytochrome c oxidase subunit I(COI)을 분석하였다. 새치성게의 생식소에서 DNA를 추출하고 중합효소연쇄반응으로 COI 유전자 단편을 선택적으로 증폭하였으며, 클로닝과 시퀀싱 과정을 거쳐 COI 유전자의 단편 1077개 염기쌍 순서(염기서열)를 확정하였다. 이 염기서열과 유전자 데이터베이스(GenBank)에 들어있는 다른 성게 및 해삼, 불가사리의 유전자를 비교하고 그 분자 계통수를 작성함으로써 새치성게의 진화과정을 분석하였다. COI 유전자 계통수는 새치성게가 태평양 동쪽 연안에 서식하는 S. purpuratus와 계통적으로 자매군(sister species)의 관계에 있음을 보였다. 두 종의 분화 시기는 계통수 상 분지의 길이와 화석연대를 고려하여 산출했을 때 지구 온도의 변동이 심했던 약 890만년 전으로 추정되었다. 태평양의 동안과 서안으로 분리된 두 종의 현재 분포와 종분화 시기의 지구 환경조건은 두 종간의 분화가 환경변화에 따른 개체군의 지리적 분리(vicariance)에 의한 것임을 시사해 준다. 한편, 새치성게의 COI 유전자염기서열은 이 종을 대표하는 분자형질로서 둥근성게과의 성게들을 서로 구분할 수 있는 종분류의 기준이 될 것이다.

• 대한수의학회지
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• 제39권3호
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• pp.523-530
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• 1999

Sal enteritidis thin fimbriae, SEF14, were found to be restricted to the predominantly poultry-associated members of the Salmonella serogroup D1 that are considered as the important pathogens in poultry industry. SefA together with sefB and sefC encode the proteins involved in SEF14 biosynthesis. In order to develop the rapid and specific detection methods for Salmonella serogroup D1, a PCR technique for the amplification of sefA gene was established, and its specificity and sensitivity were investigated with various microorganisms. The bacterial genomic DNA was extracted by colony-picking and rapid boiled-lysate technique. In comparison of Sef I and Sef II primers used in the PCR, Sef I primer for sefA gene of 513bp showed higher specificity than that of Sef II. The established PCR was as sensitive as to detect 1pg of Sal enteritidis DNA. When 73 strains in 28 genera including the reference strains and the field isolates of various Salmonella serotypes, Bacillus subtilis, Bordetella bronchisepdca, E coli, Listeria spp., Micrococcus luteus, Rhodococcus equi, Staphylococcus spp., Streptococcus spp., Vibrio parahemolyticus, Yersinia spp. were studied, the established PCR yielded specifically positive results with only Salmonella serogroup D1. The results suggested that the PCR for sefA gene could be a potential candidate among the specific detection methods for Salmonella serogroup D1.

• Clinical and Experimental Pediatrics
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• 제53권3호
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• pp.397-407
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• 2010

목 적 : F9 유전자는 혈우병B의 병인 유전자이다. 기존의 RFLP를 이용한 연관분석은 정보제공율이 55.6%에 불과하였다. 직접 염기서열 분석법은 98%의 돌연변이를 진단할 수 있지만, 고가의 비용이 든다. 본 연구는 F9 유전자를 대상으로 돌연변이의 선별검사로써 mPCR-CSGE를 사용하고, 이후 특정 유전자 부위만을 염기서열 분석하여 mPCR-CSGE의 유용성을 확인하기 위해 고안되었다. 방 법 : 연구대상은 비혈연 관계인 27명의 혈우병B 환자였다. 직접염기서열 분석법은 독립된 다른 기관에서 시행하였고, mPCR-CSGE 선별 후 염기서열 분석법은 본 연구자의 기관에서 시행되었다. 직접 염기서열 분석법의 결과가 참고치가 되어 mPCR-CSGE 선별 후 염기서열 분석법을 정확성, 경제성, 신속성, 편이성 측면에서 비교하였다. 두가지 방법으로 진단이 되지 않는 환자에게는 MLPA를 이용하여 돌연변이를 발견하였다. 결 과 : 직접 염기서열 분석법으로 26명(96.3%)의 환자에서 돌연변이를 확인할 수 있었다. mPCR-CSGE 선별 후 염기서열 분석법으로는 23명(85.2%)의 환자에서 돌연변이를 찾아낼 수 있었다. 1명의 환자는 MLPA로써 돌연변이를 확인할 수 있었다. 27명의 환자에게 21개의 독립적인 돌연변이가 있었다. mPCR-CSGE 선별 후 염기서열 분석법은 직접 염기서열 분석법에 비해 비용은 55.7%로 줄일 수 있었으나, 실험 단계는 더욱 복잡하였고, 시간도 하루가 더 걸렸으며, 세심한 실험상의 주의가 필요하였다. 결 론 : mPCR-CSGE 선별 후 염기서열 분석법은 85.2%의 높은 돌연변이 선별력을 보이고, 직접 염기서열 분석법의 57.7%의 비용만 소모하였으나, 실험과정에 세한 주의가 요구되었으며, 노동집약적이고, 실험 시간도 하루가 더 소요되었다.

• 대한수의학회지
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• 제45권1호
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• pp.45-53
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• 2005

GroE that is a heat shock protein composed of GroEL and GroES is known as an immunodominant target of both the humoral and cellular immune responses in bovine brucellosis. This study was carried out to characterize groE gene encoding heat shock proteins of B. abortus isolated in Korea and to evaluate the immunogenicity of the GroE protein expressed in E. coli system. In PCR the specific signals with the size of 2,077 bp were detected in five strains isolated from the mammary lymphnodes of the dairy cattle that were serologically positive and the reference strains. In comparison of the sequences of nucleotides and amino acids among the strains, GroES showed 100% identity in both sequences. GroEL was evaluated 99.0~99.9% in nucleotides and 98.0~100% homology in amino acids. The groE gene including groES and groEL was inserted into pET29a vector and constructed pET29a-GroE recombinant plasmids. The inserted groE was confirmed by digestion with Nco1 and EcoR1 endonucleases and nucleotide sequencing. E. coli BL (DE3) was transformed with pET29a-GroE, named as E. coli BL (DE3)/pET29a-GroE. In SDS-PAGE, it was evident that the recombinant plasmid effectively expressed the polypeptides for GroES (10 kDa) and GroEL (60 kDa) in 0.5, 1 and 2 hours after IPTG induction. The immuno-reactivity of the expressed proteins were proved in mouse inoculation and Western blot analysis.

• Molecules and Cells
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• 제40권2호
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• pp.100-108
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• 2017

Cathepsin F, which is encoded by CTSF, is a cysteine proteinase ubiquitously expressed in several tissues. In a previous study, novel transcripts of the CTSF gene were identified in the crab-eating monkey deriving from the integration of an Alu element-AluYRa1. The occurrence of AluYRa1-derived alternative transcripts and the mechanism of exonization events in the CTSF gene of human, rhesus monkey, and crabeating monkey were investigated using PCR and reverse transcription PCR on the genomic DNA and cDNA isolated from several tissues. Results demonstrated that AluYRa1 was only integrated into the genome of Macaca species and this lineage-specific integration led to exonization events by producing a conserved 3' splice site. Six transcript variants (V1-V6) were generated by alternative splicing (AS) events, including intron retention and alternative 5' splice sites in the 5' and 3' flanking regions of CTSF_AluYRa1. Among them, V3-V5 transcripts were ubiquitously expressed in all tissues of rhesus monkey and crab-eating monkey, whereas AluYRa1-exonized V1 was dominantly expressed in the testis of the crab-eating monkey, and V2 was only expressed in the testis of the two monkeys. These five transcript variants also had different amino acid sequences in the C-terminal region of CTSF, as compared to reference sequences. Thus, species-specific Alu-derived exonization by lineage-specific integration of Alu elements and AS events seems to have played an important role during primate evolution by producing transcript variants and gene diversification.

• 한국동물위생학회지
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• 제44권2호
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• pp.73-83
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• 2021

Although many swine farms continuously vaccinated to sow to prevent Porcine epidemic diarrhea(PED), PED has occurred annually in swine herds in Jeonbuk province, Korea. In the present study, the small intestine and feces samples from 17 farms where severe watery diarrhea and death of newborn piglets occurred in 2019 were collected, amplified by RT-PCR and determined the complete nucleotide sequences of the spike (S) glycoprotein genes of nine Jeonbuk PEDV isolates. The spike (S) glycoprotein is an important determinant for molecular characterization and genetic relationship of PEDV. These nine complete S gene isolates were compared with other PEDV reference strains to identify the molecular diversity, phylogenetic relationships and antigenicity analysis. 9 field strains share 98.5~100% homologies with each other at the nucleotide sequence level and 97.3~100% homologies with each other at the amino acid level. The nine Jeonbuk PEDV isolates were classified into G2b group including a genetic specific signal, S-indels (insertion and deletion of S gene). In addition, comparisons the neutralizing epitopes of S gene between 9 field strains and domestic vaccine strains of Korea mutated 12-15 amino acids with SM-98-1 (G1a group) and mutated 0-3 amino acids with QIAP1401 (G2b group). Therefore, the development of G2b-based live vaccines will have to be expedited to ensure effective prevention of endemic PED in Korea. In addition, we will need to be prepared with periodic updates of preventive vaccines based on the PEDV variants for the re-emergence of a virulent strain.