• BMB Reports
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• 제39권3호
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• pp.297-303
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• 2006

As lipolytic enzymes, GDSL lipases play an important role in plant growth and development. In order to identify their functions and roles, the full-length cDNA of a GDSL lipase gene, designated BnLIP2, was isolated from Brassica napus L. BnLIP2 was 1,300 bp long, with 1,122 bp open reading frame (ORF) encoding 373 amino acid residues. Sequence analysis indicated that BnLIP2 belonged to GDSL family. Southern blot analysis indicated that BnLIP2 belonged to a small gene family in rapeseed genome. RT-PCR analysis revealed that BnLIP2 was a tissue-specific expressing gene during reproductive growth and strongly expressed during seed germination. BnLIP2 expression could not be detected until three days after germination, and it subsequently became stronger. The transcript of this gene was deficient in root of seedlings growing at different stages. When juvenile seedlings were treated by methyl jasmonate (MeJ), salicylic acid (SA) and naphthalene acetic acid (NAA), BnLIP2 expression could not be induced in root. Our study implicates that BnLIP2 probably plays an important role in rapeseed germination, morphogenesis, flowering, but independent of root growth and development.

• BMB Reports
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• 제31권6호
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• pp.578-584
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• 1998

In a previous paper (Kim et al., 1996a), the immediate 5' -flanking region and coding region of the human UDP-N -acetylglucosamine:-D-mannoside-1,4-Nacetylglucosaminyltransferase III (N-acetylglucosaminyitransferase- III; GnT-III) gene was reported, isolated and analyzed. Herein, we report on amplification of a new 5' -noncoding region of the GnT-III mRNA by single-strand ligation to single-stranded cDNA-PCR (5' -RACE PCR) using poly(A)+ RNA isolated from human fetal liver cells. A cDNA clone was obtained with 5' sequences (96 bp) that diverged seven nucleotides upstream from the ATG (+1) start codon. A concensus splice junction sequence, TCTCCCGCAG, was found immediately 5' to the position where the sequences of the cDNA diverged. The result suggested the presence of an intron in the 5' -noncoding region and that the cDNA was an incompletely reversetranscribed cDNA product derived from an mRNA containing a new noncoding exon. When mRNA expression of GnT-III in various human tissues and cancer cell lines was examined, Northern blot analysis indicated high expression levels of GnT-III in human fetal kidney and brain tissues, as well as for a number of leukemia and lymphoma cancer cell lines. Promoter activities of the 5' -flanking regions of exon 1 and the new noncoding region were measured in a human hepatoma cell line, HepG2, by luciferase assays. The 5'-flanking region of exon 1 was the most active, whilst that of exon 2 was inactive.

• Asian-Australasian Journal of Animal Sciences
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• 제23권5호
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• pp.640-648
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• 2010

Insulin-responsive glucose transporter 4 (GLUT4) is a member of the glucose transporter family and mainly presents in skeletal muscle and adipose tissue. To clarify the molecular structure of porcine GLUT4, RACE was used to clone its cDNA. Several cDNA clones corresponding to different regions of GLUT4 were obtained by amplifying reverse-transcriptase products of total RNA extracted from Landrace porcine skeletal muscles. Nucleotide sequence analysis of the cDNA clones revealed that porcine GLUT4 cDNA was composed of 2,491 base pairs with a coding region of 509 amino acids. The deduced amino acid sequence was over 90% identical to human, rabbit and cattle GLUT4. The tissue distribution of GLUT4 was also examined by Real-time RT-PCR. The mRNA expression abundance of GLUT4 was heart>liver, skeletal muscle and brain>lung, kidney and intestine. The developmental expression of GLUT4 and insulin receptor (IR) was also examined by Real-time RT-PCR using total RNA extracted from longissimus dorsi (LM), semimembranosus (SM), and semitendinosus (SD) muscle of Landrace at the age of 1, 7, 30, 60 and 90 d. It was shown that there was significant difference in the mRNA expression level of GLUT4 in skeletal muscles of Landrace at different ages (p<0.05). The mRNA expression level of IR also showed significant difference at different ages (p<0.05). The developmental change in the mRNA expression abundance of GLUT4 was similar to that in IR, and both showed a higher level at birth and 30 d than at other ages. However, there was no significant tissue difference in the mRNA expression of GLUT4 or IR (p>0.05). These results showed that the nucleotide sequence of the cDNA clones was highly identical with human, rabbit and cattle GLUT4 and the developmental change of GLUT4 mRNA in skeletal muscles was similar to that of IR, suggesting that porcine GLUT4 might be an insulin-responsive glucose transporter. Moreover, the tissue distribution of GLUT4 mRNA showed that GLUT4 might be an important nutritional transporter in porcine skeletal muscles.

• 원예과학기술지
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• 제35권3호
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• pp.333-343
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• 2017

Bloomless cucumber fruits are commercially produced by grafting onto the pumpkin stocks (Cucurbita moschata) to restricted silicon ($SiO_2$) absorption. Inhibition of silicon absorption in bloomless stocks is conferred by a mutant allele of the CmLsi1 homologous to Lsi1 in rice. In this study, we characterized the Lsi1 homologs in pumpkin (C. moschata) and its cold-tolerant wild relative C. ficifolia ('Heukjong') in order to develop a DNA marker for selecting a bloomless trait and to establish the molecular basis for breeding bloomless stock cultivars of C. ficifolia. A Cleaved amplified polymorphic sequence (CAPS) marker (CM1-CAPS) was designed based on a non-sysnonymous single nucleotide polymorphism (SNP, C>T) of the CmLsi1 mutant-type allele, and its applicability for Marker-assisted selection (MAS) was confirmed by evaluating three bloom and five bloomless pumpkin stock cultivars. Quantitative RT-PCR of the CmLsi1 for these stock cultivers implied that expression level of the CmLsi1 gene does not appear to be associated with the bloom/bloomless trait and may differ depending on plant species and tissues. A full length cDNA of the Lsi1 homolog [named CfLsi1($B^+$)] of 'Heukjong' (C. ficifolia), was cloned and sequence comparison between CmLsi1($B^+$) and CfLsi1($B^+$) revealed that there exists total 24 SNPs, of which three were non-synonymous. Phylogenetic analysis of CfLsi1($B^+$) and Lsi1 homologs further revealed that CfLsi1($B^+$) is closesly related to Nodulin 26-like intrinsic proteins (NIPs) and most similar to CpNIP1 of C. pepo than C. moschata.

• 원예과학기술지
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• 제28권4호
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• pp.648-655
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• 2010

본 연구에서는 들잔디와 돌연변이체에서 Glyphosate 내성 관련 유전자인 EPSPS를 코딩하는 cDNA를 분리하여, 시퀸스 비교분석과 발현 양상 차이 등에 관하여 조사하였다. 5'/3' RACE를 통하여 밝혀진 EPSPS의 cDNA는 각각 1176bp의 open reading frame으로 이루어져 있으며 391개의 아미노산을 코딩하고 있었다. 이는 보고된 다른 EPSPS 유전자들과 높은 유사성을 가지고 있다. Genomic southern 결과 들잔디 내에 EPSPS 유전자는 단일copy로 존재하였다. Wild type과 제초제 내성 변이체의 EPSPS 유전자는 6개의 아미노산 시퀀스의 차이를 보였으며 기존에 보고된 EPSPS active site에서 시퀀스의 차이를 나타냈다. 한편 glyphosate의 독성기작의 하나인 EPSPS 효소활성 저해 작용을 알아본 결과, 내성 개체에서 높은(1.5배 이상) EPSPS 활성을 확인할 수 있었다. Northern 분석과 RT-PCR로 제초제 처리 시간에 따른 EPSPS의 발현량을 살펴본 결과, 제초제 처리 후 시간이 경과할수록 발현량이 증가하다가 7일 이후에는 감소하였고, wild type에 비해 glyphosate 내성 선발개체에서 전체적인 발현량이 높음을 알 수 있었다. 본 연구 결과 선발된 glyphosate 내성 돌연변이체는 높은 EPSPS 효소활성 증가 및 EPSPS active site의 아미노산 시퀀스 변화를 통해 원할하고 지속적인 방향족 아미노산의 합성이 가능한 것으로 보여졌다. 본 실험을 통해 얻어진 glyphosate 내성 잔디 돌연변이체와 방법은 앞으로 유용한 제초제 저항성 돌연변이 품종개발 연구에 활용될 수 있을 것으로 기대된다.

• Asian-Australasian Journal of Animal Sciences
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• 제21권8호
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• pp.1134-1142
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• 2008

Cationic amino acid transporter $b^{0,+}AT$ (HGMW-approved gene symbol SLC7A9, solute carrier family 7, member 9) plays a crucial role in amino acid nutrition. In the present study, we describe the cloning and sequencing of porcine $b^{0,+}AT$. Based on the sequence of porcine $b^{0,+}AT$ deposited in the NCBI (National Center for Biotechnological Information), we identified a putative porcine homologue. Using rapid amplification of cDNA ends (RACE), the full-length cDNA encoding porcine $b^{0,+}AT$ was isolated. The porcine $b^{0,+}AT$ cDNA was 1,680 bp long, encoding a 487 amino acid trans-membrane protein. The predicted amino acid sequence was found to have 88.9% and 87.1% identity with human and mouse $b^{0,+}AT$, respectively. Real-time RT-PCR indicated porcine $b^{0,+}AT$ transcripts expressed in heart, kidney, muscle and small intestine. The small intestine had the highest $b^{0,+}AT$ mRNA abundance while the muscle had the lowest (p<0.05). Along the longitudinal axis, the ileum had the highest $b^{0,+}AT$ mRNA abundance while the colon had the lowest (p<0.05). The $b^{0,+}AT$ mRNA level was highest on day 7 and 90 in the duodenum (p<0.05). It increased from day 1 to day 26 in the jejunum (p>0.05) and had the highest abundance on day 60 (p<0.05). There was, however, no difference between day 1, 7, 26, 30, 90 and 150 (p>0.05). The strongest $b^{0,+}AT$ expression appeared on day 7 in the ileum before weaning, and then decreased till day 30 but rose gradually again from day 60 to 150 (p<0.05).

• BMB Reports
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• 제39권2호
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• pp.158-166
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• 2006

In many organisms, trehalose acts as protective metabolite against harsh environmental stresses, such as freezing, drought, nutrient starvation, heat and salt. Herein a cDNA (designated as GbTPS, GenBank Accession Number AY884150) encoding a trehalose-6-phosphate synthase homologue was isolated and characterized from the living fossil plant, Ginkgo biloba, which is highly tolerant to drought and cold. GbTPS encoded an 868-amino-acid polypeptide with a predicted isoelectric point of 5.83 and molecular mass of 97.9 kD. Amino acid sequence alignment revealed that GbTPS shared high identity with class II trehalose-6-phosphate synthase homologues (67% identical to AtTPS7), but had only 17% and 23% of identity with OstA from Escherichia coli and ScTPS1 from S. cerevisiae, respectively. DNA gel blot analysis indicated that GbTPS belonged to a small multi-gene family. The expression analysis by RT-PCR showed that GbTPS expressed in a tissue-specific manner in G biloba and might involve in leaf development. GbTPS was also found to be induced by a variety of stresses including cold, salt, drought and mannitol.

• BMB Reports
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• 제41권2호
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• pp.112-118
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• 2008

Camptothecin is an anti-cancer monoterpene indole alkaloid. The gene encoding 1-hydroxy-2-methyl-2-(E)-butenyl 4-diphosphate reductase (designated as CaHDR), the last catalytic enzyme of the MEP pathway for terpenoid biosynthesis, was isolated from camptothecin-producing Camptotheca acuminata. The full-length cDNA of CaHDR was 1686 bp encoding 459 amino acids. Comparison of the cDNA and genomic DNA of CaHDR revealed that there was no intron in genomic CaHDR. Southern blot analysis indicated that CaHDR belonged to a low-copy gene family. RT-PCR analysis revealed that CaHDR expressed constitutively in all tested plant organs with the highest expression level in flowers, and the expression of CaHDR could be induced by 100 ${\mu}M$ methyl-jasmonate (MeJA), but not by 100 mg/L salicylic acid (SA) in the callus of C. acuminata. The complementation of CaHDR in Escherichia coli ispH mutant MG1655 demonstrated its function.

• Journal of Periodontal and Implant Science
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• 제28권4호
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• pp.659-678
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• 1998

본 연구에서는 한국인 성인성 치주염의 관련세균 중에서 구강 스피로헤타의 분포를 조사하기 위하여 배양하지 않고도 구강 스피로헤타를 분리할 수 있는 16S rRNA에 의거한 올리고뉴클레오타이드 소식자를 사용하였다. 성인성 치주염 환자 29명을 대상으로 한 사람당 6mm 이상의 탐침 깊이를 보이는 부위 4곳(실험군)과 3mm 이하의 탐침 깊이를 보이는 건강한 부위 1곳(대조 1군), 건강한 치주조직을 가진 학생 20명을 대상으로 한 사람당 5부위로부터 치은연하 치태를 채취한 뒤(대조 2군) 중합효소연쇄반응과 점상블롯보합결합법을 시행하였다. 소식자로는 구강 스피로헤타의보편 소식자 및 현재 배양이 되는 구강 스피로헤타중에서 T. denticola, T. pectinovorum, T. socranskii, T. vincentii, T. maltophilum에 대한 종 특이 소식자 TDEN, TPEC, TSOC, TVIN, TMAL과 현재 배양이 되지않은 구강스피로헤타중에서 I-VII군에 대한 군 특이 소식자 TRE I-TRE VII을 이용하여 다음과 같은 결론을 얻었다. 1. 위상차 현미경으로 본 결과는 실험군, 대조 1군에서 각기 91.37%, 14.28%의 구강스피로헤타가 관찰되었으며 대조 2군에서는 관찰되지 않았다. 2. 보편 소식자를 사용한 경우는 실험군, 대조 1군, 대조 2군에서 각기 98.27%, 46.42%, 22.0%의 구강 스피로헤타가 관찰되었다. 3. 특이 소식자를 사용한 경우는 실험군, 대조 1군, 대조 2군에서 각기 95.68%, 35.71%, 19.0%의 구강 스피로헤타가 관찰되었다. 4. 종 특이 소식자를 사용한 경우는 T. socranskii가 가장 많이 관찰되었으며 (81.89%), 그다음이 T. maltophilum(50.0%), T. vincentii(36.20%), T. denticola(13.79%)순이었고, 군 특이 소식자를 사용한 경우는 TREIV(85.34%), TRE II(77.58%), TREI(56.89%), TRE III(25.86%), TREVI(5.17%), TRE V(2.58%) 순이었다. 5. T. vincentii는 치주염에 이환된 부위에서만 관찰되었고, 건강한 부위에서는 관찰되지 않았다. 6. T. pectinovorum과 VII군에 속하는 구강스피로헤타는 어느 표본에서도 관찰되지 않았다. 이상의 결과에서 16S rRNA에 의거한 올리고뉴클레오타이드 소식자로 구강 스피로헤타의 성인성 치주염과의 연관성과 분리되지 않은 구강 스피로헤타를 확인하였으며, 인종 및 치주염의 형태에 따른 구강 스피로헤타의 분포 차이, T. vincentii의 병원성, 치료 전후의 구강 스피로헤타의 분포 변화등의 보다 세분화된 연구가 필요하다고 생각된다.