• 한국어병학회지
• /
• 제6권2호
• /
• pp.93-101
• /
• 1993

효모의 RNAI유전자는 RNA processing에 관여 하는지 혹은 RNA transport에 관여 하는지 아직까지 유전자의 기능이 정확히 알려져 있지 않은 실정이다. 효모의 RNAI 유전자의 기능을 파악하기 위한 방법으로 본 연구에서는 rna1-1 mutant gene을 cloning하여 이에 대한 DNA sequence를 조사함으로써 RNAI 유전자와 rna1-1 유전자의 차이점을 이해하고자 하였다. rna1-1 marker를 갖는 yeast strain(R49)로 부터 genomic DNA를 추출하여 이를 BglII로 절단하여 genomic southern blotting을 행한 결과 wild type의 경우와 동일하게 3.4 kb에서 hybridization되는 signal을 얻었으며, RNAl 및 rna1-1이 yeast genome내에 single site로 존재함을 보여 주는 결과를 얻었다. mutant strain으로 부터 얻은 3.4 kb의 BglI fragment를 pUC19의 BamHI site에 subcloning하여 transformant들을 얻었고, wild type RNAl 유전자를 probe로 하여 rna1-1 mutant 유전자를 cloning할 수 있었다. pUC19에 cloning된 RNA1유전자 및 rna1-1유전자로부터 다양한 Ba131유도체를 얻어 이들에 대한 염기 서열을 비교한 결과 transcription initation site에서부터 down stream쪽으로 17 아미노산위치에 TCC가 TTC로 대치되어 있었으며 그 결과 serine이 phenylalanine으로 변환되는 결과를 얻었다. Wild type RNAI gene의 5'-region에는 3군데의 TATA-like sequence가 true TATA box인지 확인하기 위하여 Bal3I deletion에 의해 -103nt까지 deletion된 유도체를 얻었으며 ${\Delta}RNAI$, rna1-1, 81-2-6 clone이 rna1-1 allele와 complementation한지 확인하였으나 ${\Delta}RNAI$은 TS-complementation을 하지 못하였다. 따라서 현재까지 TATA-box라고 알려진 부분은 promoter로 작용하지 못함을 확인하였다.

• Journal of Microbiology
• /
• 제39권1호
• /
• pp.37-41
• /
• 2001

The fission yeast Schizosaccharomyces pombe contains two distinct superoxide dismutase (SOD) activities, one in the cytosol encoded by the $sod2^{+}$ gene and the other in mitochondria. The $sod2^{+}$ gene encoding putative mitochondrial manganese superoxide dismutase (MnSOD) was isolated from the S. pombe genomic library using a PCR fragment as the probe. The nucleotide sequence of the $sod2^{+}$ gene and its flanking region (4051 bp HindIII fragment) was determined. An intron of 123 nt in size was predicted and confirmed by sequencing the cDNA following reverse transcription PCR. The predicted Sod2p consists of 218 amino acid residues with a molecular mass of 24,346 Da. The deduced amino acid sequence showed a high degree of homology with other MnSODs, especially in the metal binding residues at the active site and their relative positions. The transcriptional start site was mapped by primer extension at 231 at upstream from the ATG codon. A putative TATA box(TATAAAA) was located 58 nt upstream from the transcriptional start site and putative polyadenylation sites were located at 1000, 1062, and 1074 nt downstream from the ATG start codon.

• Journal of Microbiology
• /
• 제35권4호
• /
• pp.309-314
• /
• 1997

The manganese-containing superoxide dismutase (MnSOD) is a major component of the cellular defence mechanisms against the toxic effects of the superoxide radical. Within the framework of studies on oxidative stress=responsible enzymes in the Candida sp., the gene encoding the MnSOD was isolated and examined in this study. A specific primer was designed based on conserved regions of MnSOD sequences from other organisms, and was used to isolate the gene by PCR on reverse-transcribed Candida poly($A^{+}$) RNA. The PCR product was used to screen a Candida genomic lambda library and the nucleotide wequence of positive clone was determined. The deduced primary sequence encodes a 25kDa protein which has the conserved residues for enzyme activity and metal binding. The 28 N-terminal amino acids encoded by the Candida cDNA comprise a putatice mitochondrial transit peptide. Potential regulatory elements were identified in the 5' flanking sequences. Northern blot analysis showed that the transcription of the MnSOD gene is induced 5-to 10-fold in response to mercury, cadmium ions and hydrogen peroxide.

• Asian-Australasian Journal of Animal Sciences
• /
• 제21권5호
• /
• pp.624-627
• /
• 2008

An enrichment library of GATA-repeats from genomic DNA was constructed in this study to isolate and characterize microsatellite loci in Tsaiya duck (Anas platyrhynchos). Thirty-three microsatellite markers were developed and used to detect polymorphisms in 30 Tsaiya ducks. A total of 177 alleles were observed and all loci except APT022 were polymorphic. The number of alleles ranged from 2 to 9 with an average of 5.5 per microsatellite locus. The observed and expected heterozygosity of these polymorphic markers ranged from 0.07 to 0.93 with an average number of 0.60 and 0.10 to 0.86 with an average number of 0.61, respectively. Among the polymorphic markers, the observed heterozygosities of 23 loci were higher than 0.50 (69.70%). The polymorphism information content (PIC) in the 32 loci ranged from 0.09 to 0.83 with an average of 0.57. Seven of the 33 duck microsatellite loci had orthologs in the chicken genome, but only APT004 had a similar core repeat to chickens. These microsatellite markers will be useful in constructing a genetic linkage map for the duck and a comparative mapping with the chicken can also provide a valuable tool for studies related to biodiversity and population genetics in this duck species.

• Journal of Microbiology and Biotechnology
• /
• 제18권3호
• /
• pp.397-403
• /
• 2008

Nikkomycins are a group of peptidyl nucleoside antibiotics with potent fungicidal, insecticidal, and acaricidal activities. sanN was cloned from the partial genomic library of Streptomyces ansochromogenes 7100. Gene disruption and complementation analysis demonstrated that sanN is essential for nikkomycin biosynthesis in S. ansochromogenes. Primer extension assay indicated that sanN is transcribed from two promoters (sanN-P1 and sanN-P2), and sanN-P2 plays a more important role in nikkomycin biosynthesis. Purified recombinant SanN acts as a dehydrogenase to convert benzoate-CoA to benzaldehyde in a random-order mechanism in vitro, with respective $K_{cat}/K_m$$ values of $3.8mM^{-1}s^{-1}\;and\;12.0mM^{-1}s^{-1}$ toward benzoate-CoA and NADH, suggesting that SanN catalyzes the formation of picolinaldehyde during biosynthesis of nikkomycin X and Z components in the wild-type stain. These data would facilitate us to understand the biosynthetic pathway of nikkomycins and to consider the combinatorial synthesis of novel antibiotic derivatives.

• Asian Pacific Journal of Cancer Prevention
• /
• 제15권11호
• /
• pp.4423-4428
• /
• 2014

A novel monoclonal antibody (mAb), known as AC10364, was identified from an antibody library generated by immunization of mice with human carcinoma cells. The mAb recognized proteins in lysates from multiple carcinoma cell lines. Cell cytotoxicity assays showed that AC10364 significantly inhibited cell growth and induced apoptosis in multiple carcinoma cell lines, including Bel/fu, KATO-III and A2780. Compared with mAb AC10364 or chemotherapeutic drugs alone, the combination of mAb AC10364 with chemotherapeutic drugs demonstrated enhanced growth inhibitory effects on carcinoma cells. These results suggest that mAb AC10364 is a promising candidate for cancer therapy.

• 한국미생물·생명공학회지
• /
• 제44권2호
• /
• pp.140-144
• /
• 2016

사람의 분변으로부터 분리한 Bacteroides stericoris HJ-15로부터 chondroitinase ABC 유전자를 클로닝하였다. 클로닝한 chondroitinase ABC 유전자는 3,090 bp, 1,029 아미노산으로 구성되어 있었다. B. stercoris chondroitinase ABC 유전자는 이미 보고된 chondroitinase ABC 유전자들과 호몰로지가 없었으나, 아미노산서열에서는 82% 호몰로지를 보였다. T7 promoter를 가진 pET-26b+ expression vector에 클로닝한 chondroitinase ABC 유전자를 Escherichia coli BL21 (DE3)에서 발현하여 정제한 재조합 chondroitinase ABC는 chondroitin sulfate A, B 및 C를 모두 분해하였다.

• 한국생물공학회:학술대회논문집
• /
• 한국생물공학회 2001년도 추계학술발표대회
• /
• pp.652-655
• /
• 2001

The comparative study of enzymes that catalyze a similar reactions but have different substrate spectrum and/or stereospecificity is a powerful approach to understanding the reaction mechanism between the relative enzymes, and it was also an useful tool to cloning the related enzyme, without the typical cloning from DNA library of genomic pools. For this purpose, we conducted an approach that the comparison at the molecular and protein level of esterases, from various sources including a previously identified (S)-stereospecific esterase of Pseudomonas sp. ES1. As expected, we found an esterase family genes that shared a high similarity at the protein and genetic level in the identical genus Pseudomonad. The striking structural and biochemical identity strongly suggested the family genes to be an identical one. We, hence, aligned the family genes and designated a degenerated primer for PCR-cloning using six Pseudomonas strains as templates. As a result, a recombinant esterase from Pseudomonas fluorescens KCTC 1767 was cloned and high-level expressed with high selectivity to (R,S)-ketoprofen ethyl ester. The enzyme exhibited a high ester-hydrolyzing activity to (S)-ketoprofen but did not hydrolyzed the opposite stereoisomer. Further characteristics were discussed in our presentation.

• 한국식물병리학회:학술대회논문집
• /
• 한국식물병리학회 2003년도 정기총회 및 추계학술발표회
• /
• pp.71.2-71
• /
• 2003

Ralstonia solancearum infects many solanaceous plants, however race 3 infects only potato and tomato weakly. To identify genes responsible for race specificity of R. solanacearum, we mobilized genomic library of LSD2029 (race 3) into LSD341 (race 1) and inoculated 1,000 transconjugants into hot pepper. One transconjugant that did not induce wilt symptom in hot pepper was isolated. We found that a cosmid clone, pRSl, conferred avirulence to LSD341. By deletion and mutational analyses of pRSl, we found the 0.9-kb PstI/Hindlll fragment carries avirulence functions. We sequenced the fragment and identified one possible open reading frame, a rsal gene, possibly encoding 110 amino acids. The rsal was preceded with a plant-inducible promoter (PIP) box, indicating that the gene might be regulated by HrpB. Interestingly, the promoter region of the rsal homolog in the strain GM11000 (race 1) did not have the PIP box. Rsal did not show any significant homologies with proteins in the database, indicating th e protein is different from the previously reported avirulence proteins. When we mutated the rsal gene by marker-exchange in LSD2029, the mutant was less virulent in potato.

• Journal of Microbiology and Biotechnology
• /
• 제3권1호
• /
• pp.6-11
• /
• 1993

An autonomously replicating sequence (Kf-ARS1) of Kluyveromyces fragilis was cloned from the genomic library which was constructed using pHN134 as a cloning vector to make a new host-vector system for the production of heterologous protein from K. fragilis as a host. The cloning vector pHN134 was composed of $Km^r, Ap^r$ and multiple cloning site in LacZ . A clone carrying Kf-ARS1 was isolated and the recombinant plasmid was designated as pIKD102. The cloned fragment was 2.3 kb (EcoRI/EcoRI) in length. Subcloning experiment showed that the region for ARS activity was 1.5 kb (SalI/EcoRI) fragment. It was shown that the Kf-ARS1 was active in Saccharomyces cerevisiae and Kluyveromyces fragilis.