• 한국발생생물학회:학술대회논문집
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• 한국발생생물학회 2003년도 전기 한국발생생물학회 제16차 학술대회논문집
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• pp.21-23
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• 2003

Mechanisms regulate the arrest and growth of the resting primordial follicles are very poorly understood. To elucidate genes involved in the early folliculogenesis, we conducted suppression subtractive hybridization using mRNA from day1 and day5 ovaries and selected weel for further analysis, since it was most frequent gene in the day1-subtracted cDNA library (1). Expression of weel and correlated components of the cell cycle machinery, such as cdc2, cyclin B1, cdc25C, and phosphorylated cdc2 was evaluated by immunohistochemistry. In primordial follicles, expression of weel, cdcw, and cyclin B1 was cytoplasmic in oocytes, but phosphorylated cdc2 was weakly expressed in oocytes. While cdc25C expression was in ovarian somatic and in some theca cells. None of components was expressed in the pre-granulosa cells of the primordial follicles, while weel weakly, and cdc2 and cyclin B1 was strongly expressed in the granulosa cells of the growing follicles. Results from the present study suggest that 1) the mejotic arrest of the oocytes may not due to of cell cycle machinery, and 2) the weel may arrest meiosis by sequestering cdc2 and cyclin B1 in the cytoplasm by protein-protein interactions and/or by inhibitory phosphorylation.

• 식물조직배양학회지
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• 제28권5호
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• pp.255-261
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• 2001

VVTL1은 포도 과육에서 특이적으로 다량 발현되는, PR5 계열의 thaumatin과 높은 상동성을 나타내는 단백질로서, 품종에 따라 염기서열의 차이를 나타낸다고 알려져 있다. 현재까지 포도의 VVTL1에 대해서 몇몇 연구가 진행되었지만, 우리나라에서 가장 많이 재배되는 품종인 캠벨에서는 전혀 이루어지지 않았다. 본 연구에서는 캠벨 품종으로부터 VVTL1-homolog 유전자의 게놈 DNA를 분리하여, 염기서열을 분석하였다. VVTL1-homolog 유전자는 일반적으로 PR5 유전자의 구조와 동일한 구조인, intron이 없는 하나의 exon으로만 구성되어 있었다. 염기서열로부터 추론된 VVTL1-homolog 단백질의 아미노산 서열은 VVTL1을 비롯한 다른 품종의 포도에서 분리된 TLP와는 달리 염기성의 등전점을 가지고 있었다. Primer extension 분석을 통해 전사개시 부위를 결정하였고, promoter영역을 포함하는 5'upstream 지역에 전사에 중요한 TATA box (4개)와 CAAT box (1개)가 존재하였으나, 이들의 위치와 수는 다른 PR5 유전자의 promoter와는 다랐다. 이러한 연구결과는 VVTL1-homolog 유전자의 발현이 과육 성숙과정동안 abscisic acid와 스트레스 또는 자극에 의해 발현이 유도되고 있음을 제시해준다. 포도 과육특이발현 promoter인 VVTL1-homolog 유전자의 promoter 분리는, 유전자의 도입에 의해 유용형질을 과육에 나타내는 포도품종을 개발하고자 할 때 효율적으로 사용될 수 있을 것으로 사료된다.

• Applied Biological Chemistry
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• 제31권3호
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• pp.274-283
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• 1988

Aqualysin I is an alkaline serine protease which is secretet into the culture medium by Thermus aquaticus YT-1, an extreme thermophile. Aqualysin I was purified, and its partial amino acid sequence was determined. The gene encoding aqualysin I was cloned into E. coli using synthetic oligodeoxyribonucleotides as hybridization probes. The nucleotide sequence of the cloned DNA was determined. The primary structure of aqualysin I, deduced from the nucleotide sequenc, agreed with the determid amino acid sequences, including the $NH_2-$ and COOH terminal sequence of the tryptides derived from aqualysin I. Aqualysin I comprised 281 amino acid residues and its molecular mass was determined to be 28350. On alignment of the whole amino acid sequence, aqualysin I showed high sequence homology with the subtilisin type serine protease, and 43% identity with proteinase K, 37-30% with subtilisins and 34% with thermitase. Extremely high sequence identity was observed in the regions containing the active-site residues, corresponding to Asp32, His64 and Ser221 of subtilisin BPN'. Aqualysin I contains two disulfide bonds, Cys67-Cys99 and Cys163-Cys194, and these disulfide bonds seem to contribute to the heat stability of the enzyme. The determined positions of the twe disulfide bonds of aqualysin I agreed with those predicted previously on the basis of computer graphics of the crystallographic data for subtilisin BPN'. Therefore, these findings sugests that the three-dimensional structure of aqualysin I is similar to that of subtilisin BPN' Aqualysin I is produced as a lage precursor, which contains $NH_2-$ and COOH- terminal portions besides the mature protease sequence.

• 대한수의학회지
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• 제39권1호
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• pp.45-54
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• 1999

In the present study we have analyzed the characteristics and distribution of the mu-opioid receptor(MOR) by raising anti-peptide antisera to the C-terminal peptide of MOR. The antisera against MOR was produced in New Zealand White rabbit against 15 residue corresponding to amino acids, 384-398 of the cloned rat MOR. The antigenic peptide was synthesized using an Applied Biosystems 432 solid-phase peptide synthesizer. The specificity and identification of the antisera were tested by analysis of transfected cells, epitope mapping and immunohistochemical method. COS-7 cells electroporated with MOR cDNA were used to evaluate the characteristics and subcellular distribution of MOR. MOR immunoreactivity was prodominent in the plasmalemma and subcellular compartments such as endoplasmic reticulum, Golgi apparatus and vesicle like structure. Furthermore, both tissue sections and transfected cell lines could be immunostained with these antisera and the immunoreactivity was abolished when anti-MOR sera were preincubated with the peptide against which they were raised. Based on epitope mapping analysis, all antisera appeared to have a similar epitope, which was determined to be within the last amino acid, 391-398. Moreover, immunohistochemistry showed that MOR immunoreactivity was observed in many brain areas including cerebral cortex, striatum, hippocampus, locus coeruleus and the superficial laminae of the dorsal horn. These stained spinal cord and brain areas showed the mirrored pattern observed in auto radiographic studies of mu-opioid binding as well as a pattern similar to that seen by is situ hybridization for MOR. Thus, several lines of evidence support the conclusion that the antisera produced in the present study most likely recognize mu-opioid receptor. These results suggest that MOR antisera may be utilized as useful tool to analyze the physiological and pharmacological studies for mu-opioid receptor in the future.

• Journal of Microbiology
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• 제34권2호
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• pp.144-150
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• 1996

During growth of the genetically engineered E. coli CU103 in different media, extracellular DNAs released from the cells were studied. The extracellular DNAs released in the medium were concentrated by an thanol precipitation method and then quantified by a fluorescence method using Hoechst 33258. The released extracellular DNAs were also examined by gel electrophoresis and identified by Southern hybridization for the cloned pcbCD genes. The chromosomal DNAs and recombinant plasmid containing the cloned genes were observed to be released in an exponential growth phase. In Luria-Bertani (LB) broth and MM2-GLUCOSE, 210 and 69 ng/ml of DNAs were detected, respectively, after 3-4 days incubation at $30^{\circ}C$ and at pH 7.0. But the released DNAs were measured to be about 10-15 ng/ml in filtered river water (FW) and Tris-EDTA (TE). The at both $15^{\circ}C$ and $4^{\circ}C$, but the released DNAs were more easily degraded at the higher temperature. The extracellular DNAs were produced about 2 times more at pH 7.0 than at both pH 5.0 and pH 9.0 in MM2-glucose medium at $30^{\circ}C$. Therefore, the extracellular DNAs were found to be released actively from the cells during growth in liquid media.

• Mycobiology
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• 제34권4호
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• pp.209-213
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• 2006

Agrocybe cylindracea, an edible mushroom belonging to Bolbitiaceae, Agaricales, is widely used as invaluable medicinal material in the oriental countries. This study was initiated to find the genes expressed during the fruiting body formation of A. cylindracea. The cDNAs expressed differentially during fruiting body morphogenesis of A. cylindracea were isolated through subtractive hybridization between vegetative mycelia and fruiting bodies. The cDNAs expressed in the fruiting body morphogenesis of A. cylindracea were cloned and twenty genes were identified. Eleven were homologous to genes of known functions, three were homologous to genes in other organism without any function known. Six were completely novel genes specific to A. cylindracea so far examined. Some genes with known functions were a pleurotolysin, a self-assembling poreforming cytolysins; Aa-Pril and Pir2p, specifically induced genes during fruiting initiation of other mushroom, Agrocybe aegerita; an amino acid permease; a cytochrome P450; a MADS-box gene; a peptidylprolyl isomerase; and a serine proteinase. For other clones, no clear function was annotated so far. We believe the first report of the differentially expressed genes in fruiting process of A. cylindracea will be great helps for further research.

• 생명과학회지
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• 제14권4호
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• pp.644-649
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• 2004

GA 생합성에 결정적 역할을 하는 GA 3$\beta$-hydroxylase를 pIG121-Hm 벡터에 GUS유전자를 빼고 antisense로 클로닝하여 이를 동진벼에 도입한 결과 17개체의 절간신장이 억제된 형질전환한 식물체를 얻을 수 있었다. 일반 재배 동진벼를 대조군으로 하여 비교하였을때 antisense GA 3$\beta$-hydroxylase 유전자가 형질 도입된 식물체의 획득형질은 평균적으로 대조군에 비해 절간 신장의 억제가 확인되었다. 절간신장의 억제가 보인 개체의 엽육조직을 co취하여 Southen blot hybridization분석 결과 3개의 line에서 모두 single copy로 도입된 것으로 나타났다. 이로써$T_o$ 식물체 내에 antisense GA 3$\beta$-hydroxylase 유전자를 내포하고 있는 것으로 확인되었다. 이것은 antisense GA 3$\beta$-hydroxylase 유전자가 생체내에서 직접 또는 간접적으로 GA 3$\beta$-hydroxylase유전자의 발현에 관여한 것이라 사료되어진다.

• 한국응용곤충학회지
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• 제51권4호
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• pp.377-382
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• 2012

경기지역에서 담배가루이의 발생 및 약제반응을 조사하였다. 담배가루이는 13개 시군에서 발생되었으며, 유전자(16S rRNA, MtCOI) 분석결과, 고양시 일부지역은 biotype B와 Q가 혼재되었으나, 그 밖의 지역에서 2005부터 국내 발생이 보고되었던 biotype Q로 나타났다. 채집곤충 및 기주식물의 total DNA에 대해 PCR을 통한 염기서열 분석과 southern hybridization 분석 결과 모든 지역에서 Tomato Yellow Leaf Curl Virus(TYLCV)는 검출되지 않았다. 5개 지역에서 채집한 6 개체군의 담배가루이를 실내사육하면서 유묘검정법으로 성충에 대한 살충제 감수성을 검정한 결과, 지역별로 다소 차이는 있었지만 네오니코티노이드계 약제의 경우 biotype B에게 높은 살충활성을 나타낸 반면 biotype Q에게는 상대적으로 낮은 살충활성을 나타내었다.

• Journal of Microbiology
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• 제43권4호
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• pp.325-330
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• 2005

Rhodococcus sp. strain YU6 was isolated from soil for the ability to grow on o-xylene as the sole carbon and energy source. Unlike most other o-xylene-degrading bacteria, YU6 is able to grow on p-xylene. Numerous growth substrate range experiments, in addition to the ring-cleavage enzyme assay data, suggest that YU6 initially metabolizes 0- and p-xylene by direct aromatic ring oxidation. This leads to the formation of dimethylcatechols, which was further degraded largely through meta-cleavage path-way. The gene encoding meta-cleavage dioxygenase enzyme was PCR cloned from genomic YU6 DNA using previously known gene sequence data from the o-xylene-degrading Rhodococcus sp. strain DK17. Subsequent sequencing of the 918-bp PCR product revealed a $98\%$ identity to the gene, encoding meth-ylcatechol 2,3-dioxygenase from DK17. PFGE analysis followed by Southern hybridization with the catechol 2,3-dioxygenase gene demonstrated that the gene is located on an approximately 560-kb megaplasmid, designated pJY J1

• Molecules and Cells
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• 제42권10호
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• pp.711-720
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• 2019

Sink strength optimizes sucrose import, which is fundamental to support developing seed grains and increase crop yields, including those of rice (Oryza sativa). In this regard, little is known about the function of vacuolar invertase (VIN) in controlling sink strength and thereby seed size. Here, in rice we analyzed mutants of two VINs, OsVIN1 and OsVIN2, to examine their role during seed development. In a phenotypic analysis of the T-DNA insertion mutants, only the OsVIN2 mutant osvin2-1 exhibited reduced seed size and grain weight. Scanning electron microscopy analysis revealed that the small seed grains of osvin2-1 can be attributed to a reduction in spikelet size. A significant decrease in VIN activity and hexose level in the osvin2-1 spikelets interfered with spikelet growth. In addition, significant reduction in starch and increase in sucrose, which are characteristic features of reduced turnover and flux of sucrose due to impaired sink strength, were evident in the pre-storage stage of osvin2-1 developing grains. In situ hybridization analysis found that expression of OsVIN2 was predominant in the endocarp of developing grains. A genetically complemented line with a native genomic clone of OsVIN2 rescued reduced VIN activity and seed size. Two additional mutants, osvin2-2 and osvin2-3 generated by the CRISPR/Cas9 method, exhibited phenotypes similar to those of osvin2-1 in spikelet and seed size, VIN activity, and sugar metabolites. These results clearly demonstrate an important role of OsVIN2 as sink strength modulator that is critical for the maintenance of sucrose flux into developing seed grains.