• International Journal of Industrial Entomology and Biomaterials
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• 제5권1호
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• pp.73-77
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• 2002

The Sec61 trimeric complex ($\alpha$,$\beta$, and ${\gamma}$ subunits) is one of the Sec-complex responsible for post-translational protein translocation across the endoplasmic reticulum membrane in diverse organisms. In this study, a cDNA encoding the Sec61p ${\gamma}$ subunit homologue was isolated from the cDNA library of the mole cricket, Gryllotalpa orientalis. Sequence analysis of a 442-bp cDNA clone showed it to contain an open reading frame of 68 amino acid residues consisted of 204-bp. The homologues of the gene were found in the GenBank database in a diverse organism including insect, mammals, fungi, and plants. The deduced amino acid sequence of Sec61p ${\gamma}$ subunit homologue of the mole cricket showed the highest homology to the gene of the singly known insect, Drosophila melanogester (93% identity), and the least homology to that of the baker's yeast, Saccharomyces cerevisiae (37.2%). Phylogenetic analysis also confirmed a close relationship between the insect Sec61p ${\gamma}$ subunit homologues of G. orientalis and D. melanogester. Hydropathy analysis of the cricket mole and published other data suggested that the hydrophobic segment close to C-terminus is predicted to be the putative membrane anchor, Multiple alignment of the Sec61p ${\gamma}$ subunit homologue among several organisms showed the presence of several conserved domains including the conserved proline at position 28.

• Journal of Microbiology and Biotechnology
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• 제6권6호
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• pp.474-481
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• 1996

Since the commercially available rabbit anti-cyclin D3, generated from c-terminal 16 amino acid residues which are common to human and murine cyclin D3, is highly cross-reactive with many other cellular proteins of mouse, a new rabbit polyclonal anti-cyclin D3 has been raised by using murine cyclin D3 protein expressed at a high level in Escherichia coli as the immunogen. To express murine cyclin D3 protein in E. coli, the cyclin D3 cDNA fragment encoding c-terminal 236 amino acid residues obtained by polymerase chain reaction (PCR) was inserted into the NcoI/BamHI site of protein expression vector, pET 3d. Molecular mass of the cyclin D3 overexpressed in the presence of IPTG (Isopropyl $\beta$-D-thiogalactopyranoside) was approximately 26 kDa as calculated from the reading frame on the DNA sequence, and the protein was insoluble and mainly localized in the inclusion bodies that could be easily purified from the other cellular soluble proteins. When renaturation was performed following denaturation of the insoluble cyclin D3 protein in the inclusion bodies using guanidine hydrochloride, 4.4 mg of soluble form of cyclin D3 protein was produced from the transformant cultured in 100ml of LB media under the optimum conditions. Four-hundred micrograms of the soluble form of cyclin D3 protein was used for each immunization of a rabbit. When the antiserum obtained 2 weeks after tertiary immunization was applied to Western blot analysis, it was able to detect 33 kDa cyclin D3 protein in both murine lymphoma cell line BW5147.G.1.4 and human Jurkat T cells at 3,000-fold dilution with higher specificity to murine cyclin D3, demonstrating that the new rabbit polyclonal anti-murine cyclin D3 generated against c-terminal 236 amino acid residues more specifically recognizes murine cyclin D3 protein than does the commercially available rabbit polyclonal antibody raised against c-terminal 16 amino acids residues.

• Journal of Microbiology and Biotechnology
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• 제18권7호
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• pp.1235-1244
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• 2008

Indole-3-acetic acid (IAA) is produced commonly by plants and many bacteria, however, little is known about the genetic basis involving the key enzymes of IAA biosynthetic pathways from Bacillus spp. IAA intermediates from the Gram-positive spore-forming bacterium Paenibacillus polymyxa E681 were investigated, which showed the existence of only an indole-3-pyruvic acid (IPA) pathway for IAA biosynthesis from the bacterium. Four open reading frames (ORFs) encoding indole-3-pyruvate decarboxylase-like proteins and putative indole-3-pyruvate decarboxylase (IPDC), a key enzyme in the IPA synthetic pathway, were found on the genome sequence database of P. polymyxa and cloned in Escherichia coli DH5$\alpha$. One of the ORFs, PP2_01257, was assigned as probable indole-3-pyruvate decarboxylase. The ORF consisted of 1,743 nucleotides encoding 581 amino acids with a deduced molecular mass of 63,380 Da. Alignment studies of the deduced amino acid sequence of the ORF with known IPDC sequences revealed conservation of several amino acids in PP2_01257, essential for substrate and cofactor binding. Recombinant protein, gene product of the ORF PP2_01257 from P. polymyxa E681, was expressed in E. coli BL21 (DE3) as a glutathione S-transferase (GST)-fusion protein and purified to homogeneity using affinity chromatography. The molecular mass of the purified enzyme showed about 63 kDa, corresponding closely to the expected molecular mass of IPDC. The indole-3-pyruvate decarboxylase activity of the recombinant protein, detected by HPLC, using IPA substrate in the enzyme reaction confirmed the identity and functionality of the enzyme IPDC from the E681 strain.

• Journal of Microbiology
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• 제43권5호
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• pp.406-410
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• 2005

In contrast to Saccharomyces cerevisiae, little is known about the kinesin-like protein (KLP) in Candida albicans. The motor domain of kinesin, or KLP, contains a subregion, which is well conserved from yeast to humans. A similarity search, with the murine ubiquitous kinesin heavy chain region as a query, revealed 6 contigs that contain putative KLPs in the genome of C. albicans. Of these, the length of an open reading (ORF) of 375 amino acids, temporarily designated CaKAR3, was noticeably short compared with the closely related S. cerevisiae KAR3 (ScKAR3) of 729 amino acids. This finding prompted us to isolate a ${\lambda}$ genomic clone containing the complete CaKAR3 ORF, and here the complete sequence of CaKAR3 is reported. CaKAR3 is a C-terminus motor protein, of 687 amino acids, encoded by a non-disrupting gene. When compared with ScKAR3, the amino terminal region of 112 amino acids was unique, with the middle part of the 306 amino acids exhibiting $25\%$ identity and $44\%$ similarity, while the remaining C-terminal motor domain exhibited $64\%$ identity and $78\%$ similarity, and have been submitted to GeneBank under the accession number AY182242.

• Plant Biotechnology Reports
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• 제3권2호
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• pp.147-155
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• 2009

Oryza grandiglumis Chitinase IVa (OgChitIVa) cDNA encoding a class IV chitinase was cloned from wild rice (Oryza grandiglumis). OgChitIVa cDNA contains an open reading frame of 867 nucleotides encoding 288 amino acid residues with a predicted molecular weight of 30.4 kDa and isoelectric point of 8.48. Deduced amino acid sequences of OgChitIVa include the signal peptide and chitin-binding domain in the N-terminal domain and conserved catalytic domain. OgChitIVa showed significant similarity at the amino acid level with related monocotyledonous rice and maize chitinase, but low similarity with dicotyledoneous chitinase. Southern blot analysis showed that OgChitIVa genes are present as two copies in the wild rice genome. It was shown that RNA expression of OgChitIVa was induced by defense/stress signaling chemicals, such as jasmonic acid, salicylic acid, and ethephon or cantharidin and endothall or wounding, and yeast extract. It was demonstrated that overexpression of OgChitIVa in Arabidopsis resulted in mild resistance against the fungal pathogen, Botrytis cinerea, by lowering disease rate and necrosis size. RT-PCR analysis showed that PR-1 and PR-2 RNA expression was induced in the transgenic lines. Here, we suggest that a novel OgChitIVa gene may play a role in signal transduction process in defense response against B. cinerea in plants.

• 대한지역사회영양학회지
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• 제15권4호
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• pp.513-524
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• 2010

This study was designed to examine nutrition label use, self-efficacy, snacking and eating behaviors of middle school students, and to investigate if these characteristics were different by nutrition label use. A cross-sectional survey was conducted to 348 middle school students in Kyunggi, Korea. About a third of subjects read nutrition labels when they purchased snacks/packaged foods. Most nutrition label users were interested in reading information on calories, fat and trans-fat. Self-efficacy of eating/selecting snacks or general nutrition behavior was moderate (mean score: 44.4 out of 60), with significantly higher score in nutrition label users compared to nonusers (p < 0.001). Nutrition label users felt more confident in 9 items out of 15 items of self-efficacy, such as "taking fruits instead of cookies/candy for snack" (p < 0.001), "choosing milk instead of soft drink" (p < 0.01), "not having snacks after dinner" and "avoiding processed foods for snacks" (p < 0.05). Subjects had snacks 1.3 times a day, and nutrition label nonusers consumed snacks more frequently than the counterparts (p < 0.01). About 55% of nutrition label users and 64.7% of nonusers mainly purchased snacks for themselves (p < 0.05). Commonly purchased snacks by adolescents were ice cream, cookies/chips, breads and ramen. Major considerations in purchasing snacks were taste (46.9%) and price (34.6%). In selecting snacks, the influence of friends and parents was greater than the other sources. Based on eating frequency of snacks, nutrition label users were more likely to consume healthy snacks, such as fruit juices, vegetables, milk, yogurt, and potato/sweet potato than nonusers (p < 0.05). Eating behaviors measured by 15 items scored 33.6 out of 45. Nutrition label users showed better eating behaviors, such as "eating meals slowly", "eating foods cooked with plant oil", and "eating out less frequently" (p < 0.05). Study results showed that majority of adolescents did not read nutrition labels, selected snacks for themselves and had somewhat unhealthy foods for snacks. This study also showed the differences in self-efficacy, snacking and eating behaviors between nutrition label users and nonusers. In nutrition education, it is necessary to stress the importance and skills for reading nutrition labels. It is also needed to help adolescents to select healthy snacks and have desirable eating behaviors, as well as increasing self-efficacy.

• KSBB Journal
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• 제21권2호
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• pp.144-150
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• 2006

본 연구는 메타게놈 유전자 특성과 E. coli에서 정상적으로 발현되는 유전자 특성을 생물정보학 기법으로 비교 분석하고 그 결과를 메타게놈 선별 연구에 활용하고자 하는데 그 목적을 두었다. 이를 위하여 메타게놈 유래의 URF 와 숙주세포로 이용되는 E. coli이 ORF에 대한 염기구조, 발현되는 단백질의 크기 및 분자량, 아미노산의 구성 및 코돈사용은 물론 전사와 번역에 관여하는 프로모터 부위와 리보솜 결합부위의 보존서열 특성을 비교 분석하였다. 메타게놈과 E. coli가 합성하는 단백질의 크기와 분자량은 매우 비슷한 경향을 보였으나, 아미노산의 조성비, G+C 함량 및 코돈사용에서는 매우 다른 경향을 나타내었다. 특히 전사와 번역에 직접적으로 관여하는 프로모터와 RBS 영역에서의 DNA 보존서열이 상당부분 부합되지 않아 E. coli에서 메타게놈의 발현율이 현저히 낮을 것으로 예측할 수 있었다. RBS와 같이 유전자 발현에 필수적인 조절인자가 메타게놈과 E. coli에서 큰 차이를 나타내는 문제점은 메타게놈으로부터 유용한 유전자원을 탐색하는 연구에서 심도있게 개선하여야 할 사항이다. 부분적으로는 라이브러리 구축에 사용되는 벡터 및 숙주의 개량을 통하여 위의 문제를 극복할 수도 있을 것이다.

• 한국작물학회지
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• 제51권5호
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• pp.386-395
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• 2006

목표 수량과 단백질함량을 얻기 위한 질소 수비처방을 위해서는 유수형성기 전후 생체정보의 정확한 진단뿐만 아니라 유수형성기 이후 작물의 질소 축적 및 이에 따른 수량 및 미립 단백질 함량 반응이 정량화 되어야 한다. 본 연구에서는 유수분화기 생육 및 질소영양상태를 잘 대표하는 RVI green과 현재 널리 이용되고 있는 SPAD값의 유수분화기와 유수분화기 1주일전의 측정치 및 유수분화기부터 수확기까지 즉 생식생장기 지상부 질소 축적량(PNup)을 변수로 하는 수량 및 단백질함량 예측 중회귀 모델과 PNup 예측 회귀모델을 작성하여 이들의 수비 처방에의 이용 가능성을 검토하였다. 1. 유수분화기 및 유수분화기 1주일전의 RVIgreen과 SPAD값, 그리고 PNup을 이용하여 얻은 수량과 단백질함량의 중회귀모형은 어느 경우에나 모델의 결정계수($R_{2}$)가 0.9 이상으로 매우 높았다 2. 수량을 최대로 하는 생식생장기 질소흡수량(PNup)은 유수형성기 전후 RVIgreen이 증가할수록 감소하는 경향을 보였는데 본 연구의 유수형성기 전후 RVIgreen 범위로 보면 $9{\sim}13.5kg/10a$ 으로 추정되었다. 또한 PNup은 유수형 성기 전후 SPAD값과는 무관하게 $10{\sim}11kg/10a$ 범위로 나타났다. 3. 미립의 단백질함량을 7% 이하로 하는 유수형성기 질소흡수량은 유수형성기 전후 RVIgreen과 SPAD값이 증가할수록 감소하는 경향으로 어느 경우에나 $6{\sim}8kg/10a$로 추정되어 최대수량을 위한 생식생장기 질소흡수량 $9{\sim}13.5kg/10a$ 보다 크게 낮았다. 따라서 고품질 쌀 생산을 위한 수비 처방을 위해서는 수량보다도 단백질함량을 기준으로 하여 처방하여야 할 것으로 판단되었다. 4. 본 실험결과 수비질소의 회수율은 $53{\sim}83%$의 변이를 보였는데, 생식생장기 생육량이 많을 수록 회수율이 증가하는 경향이었으며, 수비 시용량이 증가함에 따라서 감소하였다. 생식생장기 천연질소공급량은 $3{\sim}4kg/10a$ 범위였으며 유수분화기 생육량이 많을 경우 증가하는 경향이었다 수비 질소시비량 및 유수분화기 생육 및 질소 영양 지표들을 예측변수로하는 PNup 예측모델을 작성하였으며 이 모델들은 적합도가 매우 높았다. 5. 영양생장기 생육 및 질소영양 상태의 비파괴적 측정치를 이용하여 목표 수량과 단백질함량에 달할 수 있도록 수비질소 시용량을 결정할 수 있을 것으로 판단되었다. 그러나 여기서 제시한 모델들이 광범위한 조건에서 이용될 수 있기 위해서는 보다 다양한 품종, 토양, 기상 조건에서 모델의 검증과 보완이 되어야 할 것으로 판단된다.

• 생명과학회지
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• 제19권5호
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• pp.566-572
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• 2009

S. marcescens KCTC 2172로부터 유전자 은행을 작성하여 재조합 클론 pDH3를 얻었으며, pDH3 유래의 서브클론을 작성하였다. 플라스미드 pPH4의 전염기서열 5,137 bp 영역을 결정한 결과 3개의 ORF가 있음을 확인하였다. 이들은 pst 오페론의 pstC, pstA, 및 pstB, 세 유전자를 동일 전사방향으로 코드하고 있었다. 타 세균의 유전자와 비교한 결과 S. marcescens의 pst 오페론은 pstS와 phoU가 결손되어 있다. 조절영역에는 CRP 결합영역과 pho box 서열이 존재하였다. 보고된 유전자와 상동성 조사결과, PstC 단백질은 Yersinia sp., Vibrio sp. 및 Pseudomonas sp.와는 49, 37, 33%의 상동성을, PstA 단백질은 Yersinia sp., Vibrio sp. 및 Pseudomonas sp.와 64, 51, 47%의 상동성을, PstB 단백질은 Methanocaldococcus sp., E. coli 및 Mycoplasma sp.와 60, 50, 48%의 상동성을 나타내었다. Pst 유전자들은 조절영역의 cAMP-CRP 복합체에 의해 in vivo에서 양성적으로 발현됨을 확인하였다. Pst 오페론을 포함하는 플라스미드를 도입한 대장균은 인산운송에 관여하는 능력을 확인하였다.

• Journal of Microbiology and Biotechnology
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• 제18권3호
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• pp.404-409
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• 2008

The brown-rot basidiomycete Fomitopsis palustris is known to degrade crystalline cellulose (Avicel) and produce three major cellulases, exoglucanases, endoglucanases, and ${\beta}$-glucosidases. A gene encoding endoglucanase, designated as cel12, was cloned from total RNA prepared from F. palustris grown at the expense of Avicel. The gene encoding Cel12 has an open reading frame of 732 bp, encoding a putative protein of 244 amino acid residues with a putative signal peptide residing at the first 18 amino acid residues of the N-terminus of the protein. Sequence analysis of Cel12 identified three consensus regions, which are highly conserved among fungal cellulases belonging to GH family 12. However, a cellulose-binding domain was not found in Cel12, like other GH family 12 fungal cellulases. Northern blot analysis showed a dramatic increase of cel12 mRNA levels in F. palustris cells cultivated on Avicel from the early to late stages of growth and the maintenance of a high level of expression in the late stage, suggesting that Cel12 takes a significant part in endoglucanase activity throughout the growth of F. palustris. Adventitious expression of cel12 in the yeast Pichia pastoris successfully produced the recombinant protein that exhibited endoglucanase activity with carboxymethyl cellulose, but not with crystalline cellulose, suggesting that the enzyme is not a processive endoglucanase unlike two other endoglucanases previously identified in F. palustris.