• Archives of Pharmacal Research
• /
• v.20 no.5
• /
• pp.465-470
• /
• 1997

The human cDNA clone UDPGTh2, encoding a liver UDP-glucuronosyltransferase (UDPGT), was isolated from a .gamma.gt 11 cDNA library by hybridization to mouse transferase cDNA clone, UDPGTm1. The two clones had 74% nlicleotide sequence identities in the coding region. UDPGTh2 encoded a 529 amino acid protein with an amino terminus membrane-insertion signal peptide and a carboxyl terminus membrane-spanning region. In order to establish substrate specificity, the clone was inserted into the pSVL vector (pUDPGTh2) and expressed in COS 1 cells. Sixty potential substrates were tested using cells transfected with pUDPGTh2. The order of relative substrate activity was as follows: 4-hydroxyestrone > estriol >2-hydroxyestriol > 4-hydroxyestradiol > $6{\alpha}$-hydroxyestradiol >$5{\alpha}$-androstane-$3{\alpha}$, $11{\beta}$, $17{\beta}$-triol=5${\beta}$-androstane-$3{\alpha}$ ${\beta}$, $17{\beta}$-triol. There were only trace amounts of gulcuronidation of 2-hydroxyestradiol and 2-hydroxyestrone, and in contrast to other cloned transferase, no gulcuronidation of either the primary estrogens and androgens (estrone, $17{\beta}$estradiol/testosterone, androsterone) or any of the exogenous substrates tested was detected. A lineweaver-Burk plot of the effect of 4-hydroxystrone concentration on the velocity of glucuronidation showed an apparent Km of $13{\mu}M$. The unique specificity of this transferase might play an important role in regulating the level and activity of these potent and active estrogen metabolites.

• BMB Reports
• /
• v.35 no.3
• /
• pp.273-282
• /
• 2002

Sp3 is a bifunctional transcription factor that has been reported to stimulate or repress the transcription of numerous genes. Although the size of Sp3 mRNA is 4.0kb, the size of the known Sp3 cDNA sequence is 3.6kb. Thus, Sp3 functional studies have been performed with an artificially introduced start codon, and thus an amino-terminus that differs from the wild-type. Ideally, full-length cDNA expression vectors with the appropriate start codon should be utilized for these studies. Using 5'rapid amplification of cDNA ends, a full-length Sp3 cDNA clone was generated and the sequence verified in nine cell lines. No AUG initiation codon was present. However, stop codons were present in all three frames 5' to the known coding sequence. In vitro translation of this full-length cDNA clone produced the expected three isoforms-one at 100 kDa and two in the mid 60 kDa range. Electrophoretic mobility shift assays showed that the protein products had the ability to bind to the Sp1/3 consensus sequence. In vitro studies, using our Sp3 clone and site directed mutagenesis, identified the translation initiation site for the larger isoform as AUA. AUA has not been previously described as an endogenous initiation codon in eukaryotes.

• Applied Biological Chemistry
• /
• v.37 no.3
• /
• pp.148-153
• /
• 1994

Rice black-streaked dwarf virus (RBSDV), a member of the plant reoviridae fijivirus group, causes a serious damage for rice production in Korea. To characterize the RBSDV genome, virus particles were produced by feeding of planthopper (Laodelphax striatellus F.) carring RBSDV to maize plants for 2 days. In $30{\sim}40$ days after feeding, the viral particles were purified from the infected maize roots by using $10{\sim}40%$ sucrose gradient centrifugation. After treatment of 10% SDS to remove the viral coat proteins, ten viral double-stranded RNAs were resolved in agrose gel electrophoresis. Total dsRNA was then used to synthesize cDNA by reverse transcriptase and a cDNA library was constructed in the ${\lambda}gt11$ vector. The phages that contain RBSDV cDNA fragments were selected by hybridizing with the random-primed probe prepared from RBSDV dsRNAs. After subcloning of several cDNA fragments into the pUC19 plasmid vector, one clone (pRV3) was chosen for sequencing. The pRV3 clone was shown to be located on the RBSDV genome fragment No.3 by RNA gel-blot analysis. Sequence analysis of the clone revealed that the pRV3 contains two partial open reading frames.

• Journal of Life Science
• /
• v.9 no.2
• /
• pp.72-75
• /
• 1999

Using RT-PCR, a cDNA encoding tomato (Lycopersicon esculentum) ACC oxidase has partially been cloned, sequenced and identified. The nucleotide suquence of the clone was in the coding region and shared about 80% of homology iwht the other ACC oxidase genes of tomato, and 70∼84% with those of other plants such as Oryza sativa, Nicotiana tabacum and Helianthus annuus. In the wounded tomato leaves, this nucleotide transcripts were accumulated rapidly and declined slowly thereafter. These results suggested that the predicted clone might be another member of tomato ACC oxidase gene family.

• The Korean Journal of Zoology
• /
• v.37 no.2
• /
• pp.240-248
• /
• 1994

골격근 세포는 미분화 단핵 근원세포로부터 신장과 융합을 거쳐 다핵 횡문근섬유로 분화되어 가며 동시에 근특이 유전자의 발현이 선택적으로 일어난다. 본 연구에서는 계배 배양 근원세포의 분화동안 유전자 발현 조절 양상에 대한 연구를 위해, 계배 근원세포를 72시간 배양한 근섬유로부터 CDNA 라이브러리를 제작하였다. 이 cDNA 라이브러리를 미분화 단핵 근원세포(배양 36시간)와 분화된 다핵 근섬유(배양 72시간)의 poly(A)+ RNA 주형에서 합성된 [32P〕cDNA를 Probe로 사용한 differential plaque hybridization 방법으로 스크리닝하였다 분화된 다핵 근섬유 CDNA probe에 강한게 흔성화되는 CDNA clone을 선별하여 클로닝하였다. 선별한 CDNA clone 들 중 하나는 약 1.3 Kb 크기의 삽입절편을 갖고 있는 것으로 나타났고, 이 CDNA를 probe로 사용하여 northern blotting 한 결과, 이 CDNA엑 대한 유전자는 미분화 단핵 근원세포에서 분화된 다핵 근섬유로 분화가 진행됨에 따라 유전자 산물인 RNA 양이 증가되는 것으로 나타났다 또한 이 1.3 Kb CDNA에 대한 RNA의 크기는 약 2 7 Kb로 확인되었다.

• International Journal of Industrial Entomology and Biomaterials
• /
• v.3 no.2
• /
• pp.163-168
• /
• 2001

We isolated and sequenced a cDNA clone corresponding to apolipophorin-III (apoLp-III) from the fall webworm, Hyphantria cunea. The cDNA for apoLp-III codes fer a 187-residue protein (561 bp) with a predicted molecular mass of 20 kDa. The calculated isoelectric point is 8.76. Multiple alignment analysis of the amino acid sequence revealed that H. cunea apoLp-III is most similar to that of Spodoptera litura (71.5% identity), followed by that of Manduca sexta (69.7% identity). They share five amphipathic $\alpha$-helices that are proposed to play a critical role in the binding of apoLp-III to lipophorin.

• Korean journal of applied entomology
• /
• v.35 no.4
• /
• pp.321-325
• /
• 1996

To isolate a novel gene for antibacterial peptide, an inducible clone(BmInc8) was selected by differential screening strategy from Bombyx mori cDNA library prepared from lavae injected with Escherichia coli. This clone contained a cDNA insert of 564 nucleotides and encoded 59 amino acids with an apparent molecular mass of 6.3 kDa. The cDNA sequence of BmInc8 had 61.2% identity compared to that of the bactericidin from Manduca sexta and also the deduced amino acids sequences from this insert had 65% identity compared to that of the cecropin D peptide Hyalophora cecropia. The transient expression assay of this insert using prokaryotic expression vector system revealed that the expressed peptide displayed the antibacterial activity. The cDNA sequence was deposited in GenBank under the accession number U30289.

• Korean Journal of Plant Resources
• /
• v.18 no.2
• /
• pp.240-245
• /
• 2005

A cDNA clone homologous to glutathione S-transferase gene was isolated and characterized from Codonopsis lanceolata(ClGST). The ClGST is 761 nucleotides long and has an open reading frame of 522 bp with a deduced amino acid sequence of 173 residues. The ClGST shows meaning homology to A. thaliana(AAC63629) $71\%$, C. chinense(CAI51314) $73\%$, E. esula(AAE65767) $75\%$, H. muticus(CAA55039) $70\%$, N. plumbaginifolia(CAA96431) $77\%$, S. commersonii(AAB65163).

• Applied Biological Chemistry
• /
• v.38 no.1
• /
• pp.55-62
• /
• 1995

To understand the molecular structure of Korean garlic viruses, cDNA cloning of virus genomic RNA was attempted. Virus particles were isolated from virus-infected garlic leaves and a cDNA library was constructed from garlic virus RNA. One of these clones, S81, selected by random sequencing has been identified as a member of potexvirus group other than potyvirus and carlavirus. The clone is 873 bp long contains most of the coat protein (CP) coding region and 3'-noncoding region including poly(A) tail. A putative polyadenylation signal sequence (AAUAAA) and the hexanucleotide motif (ACUUAA), a replicational cis-acting element conserved in the 3'-noncoding region of potexvirus RNAs are noticed. The clone S81 shows about 30-40% identity in both nucleotide and amino acid sequences with CPs of potexviruses. The genome size of the virus was analysed to be 7.46 knt by Northern blot analysis, which was longer than those of other potexviruses. The open reading frame encoding CP was expressed as a fusion protein (S81CP) in Escherichia coli and the recombinant protein was purified by immobilized metal binding affinity chromatography. Polyclonal antibody was raised against S81CP in rabbit to examine the occurrence of garlic potexvirus in Korean garlic plants by immunoblot analysis. Two virus protein bands of Mr 27,000 and 29,000 from garlic leaf extract of various cultivars reacted with the antibody. It was shown that Mr 27,000 band might not be a degradation product of Mr 29,000 band, suggesting that two types of potexvirus different in size of coat protein could exist in Korean garlic plants.

• Journal of Ginseng Research
• /
• v.19 no.1
• /
• pp.56-61
• /
• 1995

Molecular cloning and restriction mapping on ATPase $\alpha$-subunit gene (atpA) were carried out to obtain genomic information concerned with the gene structure and organization in Korean ginseng mitochondria. Two different clones containing the homologous sequence of atpA gene were selected from SalI and PstI libraries of mitochondrial DNA (mtDNA) of Korean ginseng. The sizes of mtDNA fragments inserted in SalI and PstI clones were 3.4 kb and 13 kb, respectively. Southern blot analysis with [$^{32}P$] labelled Oenothera atPA gene probe showed that atpA gene sequence was located in 2.0 kb XkaI fragment in PstI clone and in 1.7 kb XbaI fragment in SalI clone. A partial sequening ascertained that the SalI clone included about 1.2 kb fragment from SalI restriction site to C-terminal sequence of this gene but about 0.3 kb N-terminal sequence of open reading frame was abscent. The PstI fragment was enough large to cover the full sequence of atpA gene. The same restriction pattern of the overlapped region suggests that both clones include the same fragment of atiA locus. Data of Southern blot analysis and partial nucleotide sequencing suggested that mtDNA of Korean ginseng has a single copy of atpA gene. Key words ATPase a-subunit, mitochondrial DNA, Panax ginseng.