• 한국미생물·생명공학회지
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• 제17권3호
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• pp.178-182
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• 1989

고온, 호알칼리성 Bacillus K-17 균주에서 한가지 xylanase 유전자를 pBR322를 벡터로 이용하여 클로닝시켰다. Xylan을 함유하는 LB 한천배지에서 분해환을 형성하는 대장균 형질전환주에서 재조합 플라스미드 pAXl13을 분리하였으며, 본 pAXl13 은 pBR322와 고온, 호알칼리성 Bacillus K-17균주 염색체 DNA의 4.3Kb HindIII 절편으로 구성되어 있었다. Biotin으로 표식된 pAXl13을 probe로 하여 상동성시험을 하여 본 결과, pAXl13에 존재하는 4.3Kb Hind III 절편은 고온, 호알칼리성 Bacillus K-17 균주 유래임을 확인하였다. pAXl13 을 가지는 E. coli 균주가 생성하는 xylanase는 균체외에 존재하였으며 그 효소학적 성질은 고온, 호알칼리성 Bacillus K-17 균주의 xylanase I 과 II중에서 xylanase I과 동일하였다.

• 생명과학회지
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• 제21권12호
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• pp.1784-1788
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• 2011

전형적인 마이크로 RNA는 주로 해당 마이크로RNA의 호스트 유전자와 동시에 발현하는 형상을 보인다. 마이크로RNA miR-7b와 그 호스트 유전자인 FICT는 유전자 발현 조절부위인 프로모터를 함께 공유할 것으로 추정되며, 이는 이 유전자들의 뇌 특이적인 발현 양상에 기여할 것으로 추정된다. 바이오인포메틱 방법을 이용하여 사람과 마우스의 miR-7혹은 miR-7b의 프로모터 부위가 상호 유사성을 가짐을 확인하였고, 이 부위에 다양한 전자조절 부위가 있는 것을 확인 하였다. 또한 이 가설을 증명하기 위하여 형광발현 리포터 유전자 시스템을 사용하여 형광발현 벡터에 마이크로 RNA miR-7b와 그 호스트 유전자인 FICT의 5' 전부위를 클로링하여 프로모터의 활성정도를 다양한 세포주에서 확인하였다. 이 결과를 통하여 마이크로 RNA와 그 호스트 유전자인 FICT의 프로모터에는 네거티브 유전자 조절인자를 포함하는 것을 확인 할 수 있었다.

• The Plant Pathology Journal
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• 제15권5호
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• pp.270-279
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• 1999

cDNAs complementary to the 3'-terminal regions of two potyvirus genomes were cloned and sequenced. The clone G7 contains one open reading frame (ORF) of 1,338 nucleotides and a 3' untranslated region (3'-UTR) of 403 nucleotides at the 3'-end excluding the 3'end poly(A) tail. The putative viral coat protein (CP) shows 55%-92% amino acid sequence homology to those of Allium potyviruses. The genome size of the virus was analyzed to be about 9.0 kb by Northern blot analysis. Five cDNA clones were screened out using GPV2 oligonucleotide as a probe. One of these clones, DEA72, which has a longest cDNA insert, contains one ORF of 1,459 nucleotides and a 3'-UTR of 590 nucleotides at the 3'-end excluding the 3'-end poly(A) tail. The putative viral CP shows 57%-88% amino acid sequence homologies to those of Allium potyviruses. The genome size of the virus was analyzed to be about 9.6 kb by Northern blot analysis. The results of immunoblot and Northern blot analyses suggest that almost all of the tested garlic plants showing mosaic or streak symptoms are infected with DEA72-potyvirus in variable degrees but rarely infected with G7-potyvirus in variable degrees but rarely infected with DEA72-potyvirus in variable degrees but rarely infected with G7-potyvirus. Immunoelectron microscopy using anti-DEA72 CP antibody shows that this potyvirus is about 750 nm long and flexuous rod shaped.

• 한국양식학회지
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• 제17권1호
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• pp.1-7
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• 2004

A rainbow trout uncoupling protein 3 (UCP3) cDNA clone, encoding a 310 amino acid protein, was cloned and sequenced from a liver cDNA library. Two different splice variants designated UCP3-vl and UCP3-v2, were identified through liver cDNA library screening using rainbow trout UCP3 cDNA clone as a probe. UCP3-vl has 3 insertions in the UCP3 cDNA: the first insertion (133 bp), the second (141 bp), and the third (370 bp) were located 126 bp, 334 bp and 532 bp downstream from the start codon, respectively. UCP3-v2 contained a single insertion, identical in sequence and location to the second insertion of UCP3-vl. UCP3, a mitochondrial protein, functions to modulate the efficiency of oxidative phosphorylation. UCP3 has been detected from heart, testis, spinal cord, eye, retina, colon, muscle, brown adipose tissue and white adipose tissue in mammalian animals. Human and rodent UCP3s are highly expressed in skeletal muscle and brown adipose tissue, while they show weak expression of UCP3 in heart and white adipose tissue. In contrast to mammalian studies, RT-PCR and Southern blot analysis of the rainbow trout demonstrated that UCP3 is strongly expressed in liver and heart. UCP3, UCP3-vl, and UCP3-v2 all contain an Ava III short interspersed element (SINE), located in the 3'untraslated region (UTR). PCR using primers from the Ava III SINE and the UCP3 3'UTR region indicates that the UCP3 cDNA is structurally conserved among salmonids and that these primers may be useful for salmonid species genotyping.

• 대한수의학회지
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• 제36권1호
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• pp.109-118
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• 1996

The Salmonella rfb gene encoding for the biosynthesis of the oligosaccharide-repeating units of the O-antigenic determinants was cloned and sequenced. A set of nucleotide primers(a forward and reverse) was selected to target a defined region of the guanosine diphospho-mannose(GDP-Man) pyrophosphorylase synthase gene : rfbM of Salmonella C serogroup. The primer set was used to develop a PCR-based rapid and specific detection system for Salmonella C1 serogroup. Amplification bands of predicted size(1,422bp) were generated from 11 different Salmonella C1 isolates. The bands were verified to be specific for the C1 serogroup by Southern blot analysis using reference homologous DNA specificity was further confirmed by the lack of reactivity with heterologous DNA derived from non-salmonella members of the family enterobacteriaeceae. A specificity of 100% was deduced along with a very high sensitivity shown by a detection limit of 1fg of a purified DNA template. The isolated DNA sequence was found to be 99.8% homologous to S montevideo but the related primers amplified with the predicted band sizes with all the Salmonella C1 serogroups tested. It is concluded that the PCR protocol based on the rfbM gene from S cholerasuis is optimal fast and specific for the detection of Salmonella C1 serogroup and also the corresponding probe is suitable for rapid detection of all Salmonella C1 serogroup DNA tested. This technology should facilitate the identification of contaminated pig products and for any other products contaminated with the Salmonalla C1 serogroup. The immediate impact of this developed method will be in the area of food safety of pig products with the potential prospect for adaptation to other food inspection technologies.

• 식물조직배양학회지
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• 제27권6호
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• pp.423-428
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• 2000

In 1997 when cloned sheep Dolly and soon after Polly were born, it had become head-line news because in the former the nucleus that gave rise to the lamb came from cells of six-year-old adult sheep and in the latter case a foreign gene was inserted into the donor nucleus to make the cloned sheep produce human protein, factor IX, in e milk. In the last few years, once the realm of science fiction, cloned mammals especially in livestock have become almost commonplace. What the press accounts often fail to convey, however, is that behind every success lie hundreds of failures. Many of the nuclear-transferred egg cells fail to undergo normal cell divisions. Even when an embryo does successfully implant in the womb, pregnancy often ends in miscarriage. A significant fraction of the animals that are born die shortly after birth and some of those that survived have serious developmental abnormalities. Efficiency remains at less than one % out of some hundred attempts to clone an animal. These facts show that something is fundamentally wrong and enormous hurdles must be overcome before cloning becomes practical. Cloning researchers now tent to put aside their effort to create live animals in order to probe the fundamental questions on cell biology including stem cells, the questions of whether the hereditary material in the nucleus of each cell remains intact throughout development, and how transferred nucleus is reprogrammed exactly like the zygotic nucleus. Stem cells are defined as those cells which can divide to produce a daughter cell like themselves (self-renewal) as well as a daughter cell that will give rise to specific differentiated cells (cell-differentiation). Multicellular organisms are formed from a single totipotent stem cell commonly called fertilized egg or zygote. As this cell and its progeny undergo cell divisions the potency of the stem cells in each tissue and organ become gradually restricted in the order of totipotent, pluripotent, and multipotent. The differentiation potential of multipotent stem cells in each tissue has been thought to be limited to cell lineages present in the organ from which they were derived. Recent studies, however, revealed that multipotent stem cells derived from adult tissues have much wider differentiation potential than was previously thought. These cells can differentiate into developmentally unrelated cell types, such as nerve stem cell into blood cells or muscle stem cell into brain cells. Neural stem cells isolated from the adult forebrain were recently shown to be capable of repopulating the hematopoietic system and produce blood cells in irradiated condition. In plants although the term$\boxDr$ stem cell$\boxUl$is not used, some cells in the second layer of tunica at the apical meristem of shoot, some nucellar cells surrounding the embryo sac, and initial cells of adventive buds are considered to be equivalent to the totipotent stem cells of mammals. The telomere ends of linear eukaryotic chromosomes cannot be replicated because the RNA primer at the end of a completed lagging strand cannot be replaced with DNA, causing 5' end gap. A chromosome would be shortened by the length of RNA primer with every cycle of DNA replication and cell division. Essential genes located near the ends of chromosomes would inevitably be deleted by end-shortening, thereby killing the descendants of the original cells. Telomeric DNA has an unusual sequence consisting of up to 1,000 or more tandem repeat of a simple sequence. For example, chromosome of mammal including human has the repeating telomeric sequence of TTAGGG and that of higher plant is TTTAGGG. This non-genic tandem repeat prevents the death of cell despite the continued shortening of chromosome length. In contrast with the somatic cells germ line cells have the mechanism to fill-up the 5' end gap of telomere, thus maintaining the original length of chromosome. Cem line cells exhibit active enzyme telomerase which functions to maintain the stable length of telomere. Some of the cloned animals are reported prematurely getting old. It has to be ascertained whether the multipotent stem cells in the tissues of adult mammals have the original telomeres or shortened telomeres.

• Applied Biological Chemistry
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• 제38권1호
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• pp.49-54
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• 1995

한국 마늘에 감염된 바이러스의 종류와 병 발생 메카니즘을 구명하기 위하여, 마늘 바이러스 cDNA clone들을 분리하였다. 24개 cDNA clone들의 부분적인 염기 서열을 결정하였고, 이 중 poly(A) tail을 가진 5개 clone들의 염기 서열을 결정하였다. 이를 이미 알려진 다른 식물 바이러스와 비교했을 때, clone V9은 일차구조가 carlavirus와 유사성을 보이므로 GLV cDNA clone으로 여겨진다. Northern blot 결과로부터 GLV genome의 크기는 8.5 knt이고, poly(A) tail을 가지고 있다는 것을 알 수 있었다. clone V9의 3' 말단부분에는 바이러스 복제과정에서 cis-acting element로 작용한다고 여겨지는 hexanucleotide motif(5'-ACCUAA)가 존재한다. 또한 carlavirus의 껍질 단백질 subgenomic RNA의 5' 말단에 보존되어 있는 5'-TTAGGT도 나타난다. 이들은 모두 carlavirus의 특징들이다. 껍질 단백질 유전자를 pRSET-A 발현 벡터에 재조합하고, E. coli BL21에서 발현시켰다. 발현된 껍질 단백질을 $Ni^{2+}$ NTA affinity chromatography에 의해 정제하였다. 껍질 단백질을 토끼에 주사하여 항체를 만든 후, immunoblot을 한 결과 GLV 껍질 단백질에 해당하는 24 kDa polypeptide가 인지되었다. 또한 다양한 마늘 품종에 대해서 immunoblot을 한 결과, GLV 껍질 단백질의 크기와 GLV의 감염정도가 마늘 품종에 따라서 차이가 있다는 것을 알 수 있었다.

• Parasites, Hosts and Diseases
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• 제36권3호
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• pp.207-212
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• 1998

질편모충 7개 분리주 (국내분리주 UT8, KT6, UT-Kim 및 fr-Lee, 외국 분리주 CDC85, IR78 및 NYH286주)의 유전학적 차이점을 관찰하고자 Southemhybridization을 하였다. 탐침 (probe) 은 질편모충 DNA에 있는 반복적인 염기서열을 기초로 하여 337 bp의 탐침을 제작하였다. 질편모충 각 분리주를 클로닝하고 각각의 원충을 따로 배양하여 DNA를 분리하여 제한효소를 처리한 후 전기영동하고 Southemhybridization을 하였다. 질편모충 분리주에 관계엄이 11개 내외의 븐회이 관찰되었다. 콜로니가 2개 형성된 KT8, IR78 및 KT-Lee 분리주에서는 클로닝하기 전의 분리주와 클로닝하여 형성된 콜로니를 배양한 질편모충에서 같은 bandpattem이 관찰되었다. 사용된 모든 질편모충을 bandpattem에 따라 3군으로 나눌 수 있었는데, KT8 분리주는 국내 분리주인 KT6, KT-Kim 분리주와 같은 band pattern을 보여 1 kb, 1,2 kb, 1.6 kb, 1.9 kb, 2.3 kb, 2.7 kb, 3.2 kb, 3.4 kb, 3.8 kb, 4.9 kb 및 6.0 kb의 11개의 공통 분획을 보였다. 외국분리주로 metronidazole에 내성인 IR78 분리주는 국내 분리주인 fr-Lee 분리주와 같은 분획을 보여 1 kb, 1.2 kb, 1.6 kb, 1.8 kb, 2.1 kb, 2.5 kb, 2.7 kb, 2.9 kb, 3.4 kb, 5.0 kb 및 6.0 kb의 분획을 보였으며 IR78과 같이 약제 내성이 있다고 알려진 CDC85의 경우 IR78, KT-Lee 분리주와 비슷한 분획을 보였으나 2.9 kb가 없고 3.2 kb의 분획이 관찰되었다. 세번째 군에 해당되는 NYH286주는 12개의 분획을 보였는데 IR78, KT-Lee 분리주와 유사한 분획을 보였으나 그 차이점 은 6.0 kb 대신 6.2 kb를, 2. 1 kb 대신 2.0 kb와 2.2 kb를 나타냈다. 이상의 결과로 질편모충 여러 분리주의 유전학적 다양성이 관찰되었다.

• International Journal of Industrial Entomology and Biomaterials
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• 제8권1호
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• pp.95-99
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• 2004

Synthetic oligodeoxynucleotides (ODNs) containing unmethylated CpG dinucleotides in particular base contexts are known to induce immunity in vertebrate cells. In insect, however, it was recent to find out that ODNs induces insect immunity as other immune inducer such as lipopolysaccharide. However, the finding was solely based on one lepidopteran insect, Bombyx mori, and the expression of insect immunity was neither dependent on numbers of CpG repeats nor methylation of CpG repeats within ODNs. Instead, foreignness of DNA has been suggested to be a key factor governing induction of antibacterial peptide. In this study, we expanded our previous understanding to the potentiality of ODNs as an immune inducer for antifungal peptide in Galleria mellonella and B. mori. To do this, a defensin-type antifungal peptide gene, reported from G. mellonella was cloned and partially sequenced from G. mellonella and B. mori successfully and utilized as a probe in the Northern blot analysis. We found out that ODNs also work as an immune inducer for antifungal peptide in the fat body and midgut of G. mellonella and B. mori larvae. Also, induction pattern of antifungal peptide was irrelevant to the numbers of CpG repeats within ODNs as previously reported on the induction pattern of antibacterial peptides.

• Journal of Microbiology and Biotechnology
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• 제16권1호
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• pp.77-83
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• 2006

A gene encoding a $\gamma-butyrolactone$ autoregulator receptor was cloned from Saccharopolyspora erythraea, and the biochemical characteristics, including the autoregulator specificity, were determined with the purified recombinant protein. Using primers designed for the conserved amino acid sequence of Streptomyces $\gamma-butyrolactone$ autoregulator receptors, a 120 bp S. erythraea DNA fragment was obtained by PCR. Southern and colony hybridization with the 120 bp fragment as a probe allowed to select a genomic clone of S. erythraea, pESG, harboring a 3.2 kb SacI fragment. Nucleotide sequencing analysis revealed a 615 bp open reading frame (ORF), showing moderate homology (identity, $31-34\%$; similarity, $45-47\%$) with the $\gamma-butyrolactone$ autoregulator receptors from Streptomyces sp., and this ORF was named seaR (Saccharopolyspora erythraea autoregulator receptor). The seaR/pET-3d plasmid was constructed to overexpress the recombinant SeaR protein (rSeaR) in Escherichia coli, and the rSeaR protein was purified to homogeneity by DEAE-Sephacel column chromatography, followed by DEAE-ion-exchange HPLC. The molecular mass of the purified rSeaR protein was 52 kDa by HPLC gel-filtration chromatography and 27 kDa by SDS-polyacrylamide gel electrophoresis, indicating that the rSeaR protein is present as a dimer. A binding assay with tritium-labeled autoregulators revealed that rSeaR has clear binding activity with a VB-C-type autoregulator as the most effective ligand, demonstrating for the first time that the erythromycin producer S. erythraea possesses a gene for the $\gamma-butyrolactone$autoregulator receptor.