• Journal of Photoscience
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• 제9권2호
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• pp.267-268
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• 2002

A vertebrate retina is an organ belonging to the central nerve system (CNS), and is usually difficult to regenerate except at an embryonic stage in life. However, certain species of urodele amphibians, such as newts and salamanders, possess the ability to regenerate a functional retina from retinal pigment epithelial (RPE) cells even as adults. After surgical removal of neural retinas from adult newt eyes, the remaining RPE cells lose their pigment granules, transdifferentiate into retinal progenitor cells, which further differentiate into various retinal neurons, and then finally reform a functional neural network. To understand the molecular mechanisms of CNS regeneration, we attempted to investigate the genes expressing in regenerating newt retina. mRNAs were isolated from regenerating retinas at 18-19 days after the surgical removal of the normal retina, and a cDNA library (regenerating retinal cDNA library) were constructed. Our EST analysis of 112 clones in the regenerating cDNA library revealed that about 70% clones are closely related to the genes previously identified. About 40% clones are housekeeping genes, and about 15% clones encode proteins related to the regulation of gene expression and to the proliferation of the cells. Sequences similar to neural retina- and RPE-specific genes were not detected at all. These results led us to suppose that the regenerating retinal cells are in a state considerably different from those of neither neural retina nor RPE cells.

• BMB Reports
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• 제40권4호
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• pp.501-510
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• 2007

Sex-related genes expressed in vitellogenic ovaries of the giant tiger shrimp, Penaeus monodon, were identified by an EST approach. A total of 1051 clones were unidirectionally sequenced from the 5 terminus. Nucleotide sequences of 743 EST (70.7%) significantly matched known genes previously deposited in the GenBank (E-value <$10^{-4}$) whereas 308 ESTs (29.3%) were regarded as newly unidentified transcripts (E-value >$10^{-4}$). A total of 559 transcripts (87 contigs and 472 singletons) were obtained. Thrombospondin (TSP) and peritrophin (79 and 87 clones accounting for 7.5 and 8.3% of clones sequenced, respectively) predominated among characterized transcripts. everal full length transcripts (e.g. cyclophilin, profillin and thioredoxin peroxidase) were also isolated. A gene homologue encoding chromobox protein (PMCBX, ORF of 567 nucleotides encoding a protein of 188 amino acids) which is recognized as a new member of the HP1 family was identified. Expression patterns of 14 of 25 sex-related gene homologues in ovaries and testes of P. monodon broodstock were examined by RT-PCR. Female sterile and ovarian lipoprotein receptor homologues were only expressed in ovaries whereas the remaining transcripts except disulfide isomerase related P5 precursor and adenine nucleotide translocator 2 were higher expressed in ovaries than testes of P. monodon broodstock. A homologue of ubiquitin specific proteinase 9, X chromosome (Usp9X) revealed a preferential expression level in ovaries than testes of broodstock-sized P. monodon (N = 13 and 11, P<0.05) but was only expressed in ovaries of 4-month-old shrimp (N = 5 for each sex).

• Molecular & Cellular Toxicology
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• 제1권1호
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• pp.17-25
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• 2005

There are some critical drawbacks in the use of biomarkers for a global assessment of the toxicological impacts many chemicals and environmental pollutants have, primarily due to an individual biomarker's specificity for an explicit chemical or toxicant. In other words, the biomarker-based assessment methodology used to analyze toxicological effects lacks a high-throughput capability. Therefore, eco-toxicogenomics, or the study of toxicogenomics with organisms present within a given environmental locale, has recently been introduced with the advent of the so-called "-omics" era, which began with the creation of microarray technologies. Fish are comparable with humans in their toxicological responses and thus data from toxicogenomic studies performed with fish could be applied, with appropriate tools and implementation protocols, to the evaluation of environments where human or animal health is of concern. At present, there have been very active research streams for developing expression sequence tag (EST) databases (DBs) for zebra fish and rainbow trout. Even though few reports involve toxicogenomic studies with fish, a few groups have successfully fabricated and used cDNA microarrays or oligo DNA chips when studying the toxicological impacts of hypoxia or some toxicants with fish. Furthermore, it is strongly believed that this technology can also be implemented with non-model fish. With the standardization of DNA microarray technologies and ample progress in bioinformatics and proteomic technologies, data obtained from DNA microarray technologies offer not only multiple biomarker assays or an analysis of gene expression profiles, but also a means of elucidating gene networking, gene-gene relations, chemical-gene interactions, and chemical-chemical relationships. Accordingly, the ultimate target of eco-toxicogenomics should be to predict and map the pathways of stress propagation within an organism and to analyze stress networking.

• Journal of Ginseng Research
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• 제44권2호
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• pp.321-331
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• 2020

Background: The patatin-related phospholipase AIII family (pPLAIIIs) genes alter cell elongation and cell wall composition in Arabidopsis and rice plant, suggesting diverse commercial purposes of the economically important medicinal ginseng plant. Herein, we show the functional characterization of a ginseng pPLAIII gene for the first time and discuss its potential applications. Methods: pPLAIIIs were identified from ginseng expressed sequence tag clones and further confirmed by search against ginseng database and polymerase chain reaction. A clone showing the highest homology with pPLAIIIβ was shown to be overexpressed in Arabidopsis using Agrobacterium. Quantitative polymerase chain reaction was performed to analyze ginseng pPLAIIIβ expression. Phenotypes were observed using a low-vacuum scanning electron microscope. Lignin was stained using phloroglucinol and quantified using acetyl bromide. Results: The PgpPLAIIIβ transcripts were observed in all organs of 2-year-old ginseng. Overexpression of ginseng pPLAIIIβ (PgpPLAIIIβ-OE) in Arabidopsis resulted in small and stunted plants. It shortened the trichomes and decreased trichome number, indicating defects in cell polarity. Furthermore, OE lines exhibited enlarged seeds with less number per silique. The YUCCA9 gene was downregulated in the OE lines, which is reported to be associated with lignification. Accordingly, lignin was stained less in the OE lines, and the expression of two transcription factors related to lignin biosynthesis was also decreased significantly. Conclusion: Overexpression of pPLAIIIβ retarded cell elongation in all the tested organs except seeds, which were longer and thicker than those of the controls. Shorter root length is related to auxinresponsive genes, and its stunted phenotype showed decreased lignin content.

• 한국미생물·생명공학회지
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• 제44권3호
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• pp.317-323
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• 2016

본 연구에서는 C. fermentati SI가 생산하는 isoflavone 배당체 가수분해 효소를 클로닝하여 염기 서열을 밝힌 뒤 P. pastoris X-33에 형질전환하여 재조합 효소의 과발현을 시켰고, 또한 재조합 isoflavone 가수분해 효소의 효소학적 특성을 조사하였다. 재조합 isoflavone 가수분해 효소의 분자량은 약 50.4 kDa이었으며, Meyerozyma guilliermondii ATCC 6260의 exo-1,3-β-glucanase와 96%로 가장 높은 homology를 나타내었다. exo-1,3-β-glucanase의 ORF는 pPICZA 벡터로 클로닝 후 P. pastoris X-33으로 형질전환을 하였으며, His6-tag을 이용하여 효소를 정제하였다. 정제된 효소는 citrate phosphate buffer pH 4.5에서 최적 활성을 나타내었으며, 효소의 최적 활성 온도는 40℃로 나타났다. 40℃이상에서는 효소의 활성이 급격하게 감소함을 확인 하였으며, pH 안정성을 조사한 결과 비교적 넓은 범위인 4−8 사이에서 80%이상의 활성을 유지하였다. 따라서, 재조합 효소의 과발현을 통해 isoflavone aglycone의 효율적인 생산에 이용할 수 있을 것으로 사료된다.

• 대한소아치과학회지
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• 제33권3호
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• pp.401-410
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• 2006

치아 우식은 구강 내 미생물에 의해 치아 석회 조직의 일부가 용해되고 파괴되는 감염성 질환이다. 치아 우식의 원인균은 Streptococcus mutans와 같은 Mutans streptococci로 알려져 있고, 이 미생물이 치면에 접착하여 군집을 형성하는 능력이 균의 독성에 중요한 역할을 한다. S. mutans가 치면의 타액성 피막에 부착하는 데에는 AgI/II와 같은 세포표면의 섬유성 단백질을 매개로 한다. 그러므로, AgI/II는 S. mutans GS-5에 대한 백신 개발에 적절한 목표가 된다. 본 실험은 S. mutans GS-5로부터 AgI/II 유전자를 복제하고 염기서열분석을 하였다. 복제된 AgI/II와 앞서 보고된 S. mutans GS-5의 해당 부위의 280개의 핵산은 완벽하게 일치하였다. 복제된 유전자를 두 부위로 절단하여 형질전환을 통해 재조합 단백질인 AgI/IImr을 얻었고, 정제된 재조합 단백질을 쥐에게 주입하여 다클론 항체를 얻었다. 추출된 다클론 항체는 AgI/IImr항원단백질에 반응하였고, 대조군으로 쓰인 단백질에는 반응하지 않았다.

• 한국작물학회지
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• 제55권2호
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• pp.151-158
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• 2010

지금까지 양구슬냉이의 유전정보는 거의 연구되지 않았으므로 우리는 양구슬냉이의 잎으로부터 cDNA library를 제작하고 발현유전자의 종류와 기능별 분류를 조사하였다. 그 결과를 요약하면 다음과 같다. 1. cDNA library에서 1334개 의 클론들을 얻었고 삽입된 단편들의 염기서열의 평균길이는 736bp였다. 우리는 1269개의 high-quality expressed sequence tags (ESTs) 서열을 얻었다. 이러한 EST의 클러스터 분석결과 고유 염기서열(unigene)을 가진 유전자의 수는 851개를 나타냈다. 2. Unigene 476개는 GeneBank에 기능이 알려진 유전자들과 고도의 상동성을 나타내었다. 다른 375개의 unigene들은 기능이 알려지지 않은 것들이었다. 나머지 63개는 NCBI데이터베이스에 어떤 유전자와도 상동성을 보이지 않았고 이러한 유전자들은 아마도 양구슬냉이의 잎에서 발현되는 새로운 유전자일 것으로 보인다. 3. 데이터베이스에서 상동성을 나타낸 EST들을 기능별 주석에 따라서 17개의 카테고리로 분류하였다. 대표적으로 가장 분포도가 높은 카테고리는 결합 기능 또는 보조인자 요구의 단백질(27%), 대사(11%), 세포 소기관 위치(11%), 세포수송과 수송기관 그리고 수송 경로(7%), 에너지(6%), 대사와 단백질 기능의 조절(6%) 등이 있다. 이러한 우리의 연구 결과는 양구슬냉이의 유용한 유전적 자원과 전반적인 mRNA 발현 정보를 제공해 줌으로써 대체 에너지 작물로 떠오르는 양구슬냉이의 다양한 분자적 연구에 기여할 것으로 사료된다.

• 생명과학회지
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• 제17권9호통권89호
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• pp.1197-1203
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• 2007

세균인 Paenibacillus sp. DG-22의 유전체 DNA library가 제조되었으며, ${\beta}-xylosidase-$양성 클론이 형광기질인 $4-methylumbelliferyl-{\beta}-D-xylopyranoside$ $({\beta}MUX)$를 사용하여 확인되었다. 이 클론으로부터 재조합 플라스미드가 분리되었고 삽입된 4.3-kb 크기 DNA의 염기서열이 결정되었다. ${beta}-xylosidase$ 유전자는 분자량이 78.710 dal-ton이고 pI가 5.0인 701개의 아미노산을 암호화하는 2,106 염기쌍의 열린해독틀(ORF)로 구성되어있었다. xylA 유전자산물의 추론된 아미노산 서열은 과(family) 52에 속하는 클리코실 가수분해효소로 분류된 ${beta}-xylosidase$들과 상당한 유사성을 가지고 있었다. 이 xylA 유전자에 6개의 히스티딘-꼬리표를 붙이기 위해 pQE60 발현벡터에 다시 클로닝하였다. 재조합 ${beta}-xylosidase$ $(XylA-H_6)$가 열처리와 고정화금속친화성 크로마토그래피(IMAC)에 의해 순수하게 정제되었다. $XylA-H_6$ 효소의 최적 pH와 온도는 각각 pH 5.5-6.0과 $60^{\circ}C$이었다.

• Applied Biological Chemistry
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• 제38권5호
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• pp.382-386
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• 1995

감자의 단백질 분해효소 억제제-II(PI-II)단백질은 카이모트립신 저해부위와 트립신 저해부위로 나뉘어 있는데 PI-II 유전자중의 하나인 PI-IIT 유전자의 염기서열에서 비롯된 아미노산 서열이 폴리펩타이드 카이모트립신 저해제(PCI) 단백질의 아미노산 서열과 84%의 높은 동질성을 가지고 있으므로 감자의 단백질 분해효소 억제제유전자 집단 (family) 중의 하나인 PCI와 homology가 있는 유전자단편을 클로닝하기 위하여 이미 클론된 PI-IIT 유전자로부터 PCR을 행하여 얻어진 DNA 단편을 백터에 클로닝하고 염기서열을 결정하였다. 염기서열을 확인한 유전자 단편을 박테리오파아지 T7 promoter와 terminator를 갖고있는 플라스미드 pET3a에 옮겨 대장균 BL2l(DE3)에서 발현시켰던바 IPTG의 부가에 따라 유기되는 것을 확인 하였으나 발현수준은 기대했던것에 미치지 못하였다.

• Asian-Australasian Journal of Animal Sciences
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• 제22권11호
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• pp.1555-1565
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• 2009

Anaerobic rumen fungi have been regarded as good genetic resources for enzyme production which might be useful for feed supplements, bio-energy production, bio-remediation and other industrial purposes. In this study, an expressed sequence tag (EST) library of the rumen anaerobic fungus Neocallimastix frontalis was constructed and functional genes from the EST library were analyzed to elucidate carbohydrate metabolism of anaerobic fungi. From 10,080 acquired clones, 9,569 clones with average size of 628 bp were selected for analysis. After the assembling process, 1,410 contigs were assembled and 1,369 sequences remained as singletons. 1,192 sequences were matched with proteins in the public data base with known function and 693 of them were matched with proteins isolated from fungi. One hundred and fifty four sequences were classified as genes related with biological process and 328 sequences were classified as genes related with cellular components. Most of the enzymes in the pathway of glucose metabolism were successfully isolated via construction of 10,080 ESTs. Four kinds of hemi-cellulase were isolated such as mannanase, xylose isomerase, xylan esterase, and xylanase. Five $\beta$-glucosidases with at least three different conserved domain structures were isolated. Ten cellulases with at least five different conserved domain structures were isolated. This is the first solid data supporting the expression of a multiple enzyme system in the fungus N. frontalis for polysaccharide hydrolysis.