• Journal of Plant Biotechnology
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• 제38권1호
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• pp.77-86
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• 2011

Alcohol dehydrogenase (E.C.1.1.1.1) is an enzyme present in higher plants involved in the anaerobic fermentation pathway that catalyzes the reduction of pyruvate to ethanol, resulting in continuous $NAD^+$ regeneration. It also plays an important role in many plant developments including tolerance to anoxia condition. Here, a cDNA clone encoding alcohol dehydrogenase (ADH) was isolated from Chinese cabbage (Brassica rapa) seedlings. The gene named Bradh1 had a total length of 1,326 bp that contains a single open reading frame of 1,140 bp. The predicted protein consists of 379 amino acid residues with a calculated molecular mass of 41.17 kDa. Expression pattern analysis revealed a tissue-specific expressing gene in different tissues and strongly expressed in the shoot, roots and seeds of Chinese cabbage. Agrobacterium transformation of full-length cDNA Bradh1 into rice Gopumbyeo showed high efficiency. Furthermore, induction of ADH in transgenic rice enhanced tolerance to anaerobiosis stresses and elevated mRNA transcripts. The overexpression of Bradh1 in rice increases germination under anaerobiosis stresses, implying the possibility of developing new varieties suited for direct seeding or flood-prone rice field.

• 한국자원식물학회지
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• 제23권6호
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• pp.526-534
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• 2010

Genomes of clusters of related eukaryotes are now being sequenced at an increasing rate. In this paper, we developed an accurate, low-cost method for annotation of gene prediction and exon-intron structure. The gene prediction was adapted for delta 1-pyrroline-5-carboxylate-synthetase (p5cs) gene from China wild-type of the halophytic Leymus chinensis (Trin.), naturally adapted to highly-alkali soils. Due to complex adaptive mechanisms in halophytes, more attentions are being paid on the regulatory elements of stress adaptation in halophytes. P5CS encodes delta 1-pyrroline-5-carboxylate-synthetase, a key regulatory enzyme involved in the biosynthesis of proline, that has direct correlation with proline accumulation in vivo and positive relationship with stress tolerance. Using analysis of reverse transcription-polymerase chain reaction (RT-PCR) and PCR, and direct sequencing, 1076 base pairs (bp) of cDNA in length and 2396 bp of genomic DNA in length were obtained from direct sequencing results. Through gene prediction and exon-intron structure verification, the full-length of cDNA sequence was divided into eight parts, with seven parts of intron insertion. The average lengths of determinated coding regions and non-coding regions were 154.17 bp and 188.57 bp, respectively. Nearly all splice sites displayed GT as the donor sites at the 5' end of intron region, and 71.43% displayed AG as the acceptor sites at the 3' end of intron region. We conclude that this method is a cost-effective way for obtaining an experimentally verified genome annotation.

• Genomics & Informatics
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• 제3권3호
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• pp.86-93
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• 2005

Human brain EST data provide important clues for our understanding of the molecular biology associated with the function of the normal brain and the molecular pathophysiology with brain disorders. To systematically and efficiently study the function and disorders of the human brain, 45,773 human brain ESTs were collected from 27 human brain cDNA libraries, which were constructed from normal brains and brain disorders such as brain tumors, Parkinson's disease (PO) and epilepsy. An analysis of 45,773 human brain ESTs using our EST analysis pipeline resulted in 38,396 high-quality ESTs and 35,906 ESTs, which were coalesced into 8,246 unique gene clusters, showing a significant similarity to known genes in the human RefSeq, human mRNAs and UniGene database. In addition, among 8,246 gene clusters, 4,287 genes ($52\%$) were found to contain full-length cONA clones. To facilitate the extraction of useful information in collected these human brain ESTs, we developed a user-friendly interface system, the Korea Brain Unigene Database (KBUD). The KBUD web interface allows access to our human brain data through three major search modes, the BioCarta pathway, keywords and BLAST searches. Each result when viewed in KBUD offers comprehensive information concerning the analyzed human brain ESTs provided by our data as well as data linked to various other publiC databases. The user-friendly developed KBUD, the first world-wide web interface for human brain EST data with ESTs of human brain disorders as well as normal brains, will be a helpful system for developing a better understanding of the underlying mechanisms of the normal brain well as brain disorders. The KBUD system is freely accessible at http://kugi.kribb.re.kr/KU/cgi -bin/brain. pI.

• Molecules and Cells
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• 제28권6호
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• pp.529-536
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• 2009

Genome sequencing of the pig is being accelerated because of its importance as an evolutionary and biomedical model animal as well as a major livestock animal. However, information on expressed porcine genes is insufficient to allow annotation and use of the genomic information. A series of expressed sequence tags of 5' ends of five full-length enriched cDNA libraries (SUSFLECKs) were functionally characterized. SUSFLECKs were constructed from porcine abdominal fat, induced fat cells, loin muscle, liver, and pituitary gland, and were composed of non-normalized and normalized libraries. A total of 55,658 ESTs that were sequenced once from the 5′ ends of clones were produced and assembled into 17,684 unique sequences with 7,736 contigs and 9,948 singletons. In Gene Ontology analysis, two significant biological process leaf nodes were found: gluconeogenesis and translation elongation. In functional domain analysis based on the Pfam database, the beta transducin repeat domain of WD40 protein was the most frequently occurring domain. Twelve genes, including SLC25A6, EEF1G, EEF1A1, COX1, ACTA1, SLA, and ANXA2, were significantly more abundant in fat tissues than in loin muscle, liver, and pituitary gland in the SUSFLECKs. These characteristics of SUSFLECKs determined by EST analysis can provide important insight to discover the functional pathways in gene networks and to expand our understanding of energy metabolism in the pig.

• Journal of Nutrition and Health
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• 제48권5호
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• pp.390-397
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• 2015

본 연구에서는 Sprague-Dawley 종 랫트 수컷을 대조군, EtOH군, Fe군, EtOH + Fe군으로 나누어, 알코올과 철분을 액상 사료로 8주간 공급한 후, 간 조직과 간 세포 mtDNA의 손상 정도를 알아보았다. EtOH + Fe군은 대조군, EtOH군, Fe군의 다른 세 군에 비해 혈청 ALT와 혈청 AST 수치가 가장 유의적으로 높았으며, 간 조직 검사의 결과에서도 다수의 지방구, 염증성 세포 침입 및 조직의 괴사가 관찰되는 등 가장 심한 간 손상이 확인되었다. DNA 손상 여부를 긴 영역 PCR을 사용하여 분석한 결과, 만성적인 알코올과 철분에 의한 노출은 간 세포의 mtDNA 손상을 유발하는 것으로 나타났으며, 핵 DNA에는 영향을 미치지 않았다. 또한 미토콘드리아의 호흡에 관여하는 Cox1과 Nd4 유전자 발현 정도를 real-time PCR으로 분석한 결과, 알코올 또는 철분은 간 세포의 Cox1 mRNA와 Nd4 mRNA 수준을 유의적으로 낮추는 것으로 나타났다. 이상의 결과는 만성 알코올 또는 과잉의 철분에 의한 간 손상에 mtDNA 손상 및 미토콘드리아 기능 저하가 관여함을 제시한다.

• Molecules and Cells
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• 제37권7호
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• pp.526-531
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• 2014

Human PARP family consists of 17 members of which PARP-1 is a prominent member and plays a key role in DNA repair pathways. It has an N-terminal DNA-binding domain (DBD) encompassing the nuclear localisation signal (NLS), central automodification domain and C-terminal catalytic domain. PARP-1 accounts for majority of poly-(ADP-ribose) polymer synthesis that upon binding to numerous proteins including PARP itself modulates their activity. Reduced PARP-1 activity in ageing human samples and its deficiency leading to telomere shortening has been reported. Hence for cell survival, maintenance of genomic integrity and longevity presence of intact PARP-1 in the nucleus is paramount. Although localisation of full-length and truncated PARP-1 in PARP-1 proficient cells is well documented, subcellular distribution of PARP-1 fragments in the absence of endogenous PARP-1 is not known. Here we report the differential localisation of PARP-1 Nterminal fragment encompassing NLS in PARP-$1^{+/+}$ and PARP-$1^{-/-}$ mouse embryo fibroblasts by live imaging of cells transiently expressing EGFP tagged fragment. In PARP-$1^{+/+}$ cells the fragment localises to the nuclei presenting a granular pattern. Furthermore, it is densely packaged in the midsections of the nucleus. In contrast, the fragment localises exclusively to the cytoplasm in PARP-$1^{-/-}$ cells. Flourescence intensity analysis further confirmed this observation indicating that the N-terminal fragment requires endogenous PARP-1 for its nuclear transport. Our study illustrates the trafficking role of PARP-1 independently of its enzymatic activity and highlights the possibility that full-length PARP-1 may play a key role in the nuclear transport of its siblings and other molecules.

• 한국발생생물학회지:발생과생식
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• 제21권3호
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• pp.237-247
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• 2017

Mammalian spermatogenesis occurs in a precise and coordinated manner in the seminiferous tubules. One of the attempts to understand the detailed biological process during mammalian spermatogenesis at the molecular level has been to identify the testis specific genes followed by study of the testicular expression pattern of the genes. From the subtracted cDNA library of rat testis prepared using representational difference analysis (RDA) method, a complimentary DNA clone encoding type III member of a DnaJ family protein, DnaJC18, was cloned (GenBank Accession No. DQ158861). The full-length DnaJC18 cDNA has the longest open reading frame of 357 amino acids. Tissue and developmental Northern blot analysis revealed that the DnaJC18 gene was expressed specifically in testis and began to express from postnatal week 4 testis, respectively. In situ hybridization studies showed that DnaJC18 mRNA was expressed only during the maturation stages of late pachytene, round and elongated spermatids of adult rat testis. Western blot analysis with DnaJC18 antibody revealed that 41.2 kDa DnaJC18 protein was detected only in adult testis. Immunohistochemistry study further confirmed that DnaJC18 protein, was expressed in developing germ cells and the result was in concert with the in situ hybridization result. Confocal microscopy with GFP tagged DnaJC18 protein revealed that it was localized in the cytoplasm of cells. Taken together, these results suggested that testis specific DnaJC18, a member of the type III DnaJ protein family, might play a role during germ cell maturation in adult rat testis.

• Parasites, Hosts and Diseases
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• 제44권3호
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• pp.251-254
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• 2006

Monoclonal antibody (mAb) Tg786 against Toxoplasma gondii has been found to detect a 42-kDa rhoptry protein (ROP6) which showed protease activity and host cell binding characteristics after secretion. Using the mAb, a colony containing a 3'-UTR was probed in a T. gondii cDNA expression library. A full length cDNA sequence of the rhoptry protein was completed after 5'-RACE, which consisted of 1,908 bp with a 1,443 bp ORF. The deduced amino acid sequence of ROP6 consisted of a polypeptide of 480 amino acids without significant homology to any other known proteins. This sequence contains an amino terminal stop transfer sequence downstream of a short neutral sequence, hydrophilic middle sequence, and hydrophobic carboxy terminus. It is suggested that the ROP6 is inserted into the rhoptry membrane with both N- and C-termini.

• The Plant Pathology Journal
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• 제37권2호
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• pp.162-172
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• 2021

Soybean mosaic virus (SMV) is the predominant viral pathogen that affects the yield and quality of soybean. The natural host range for SMV is very narrow, and generally limited to Leguminosae. However, we found that SMV can naturally infect Pinellia ternata and Atractylodes macrocephala. In order to clarify the molecular mechanisms underlying the cross-family infection of SMV, we used double-stranded RNA extraction, rapid amplification of cDNA ends polymerase chain reaction and Gibson assembly techniques to carry out SMV full-length genome amplification from susceptible soybeans and constructed an infectious cDNA clone for SMV. The genome of the SMV Shanxi isolate (SMV-SX) consists of 9,587 nt and encodes a polyprotein consisting of 3,067 aa. SMV-SX and SMV-XFQ008 had the highest nucleotide and amino acid sequence identities of 97.03% and 98.50%, respectively. A phylogenetic tree indicated that SMV-SX and SMV-XFQ018 were clustered together, sharing the closest relationship. We then constructed a pSMV-SX infectious cDNA clone by Gibson assembly technology and used this clone to inoculate soybean and Ailanthus altissima; the symptoms of these hosts were similar to those caused by the virus isolated from natural infected plant tissue. This method of construction not only makes up for the time-consuming and laborious defect of traditional methods used to construct infectious cDNA clones, but also avoids the toxicity of the Potyvirus special sequence to Escherichia coli, thus providing a useful cloning strategy for the construction of infectious cDNA clones for other viruses and laying down a foundation for the further investigation of SMV cross-family infection mechanisms.

• 한국약용작물학회지
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• 제13권5호
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• pp.293-297
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• 2005

A thoredoxin (CTRX) gene was cloned and characterized from a full length cDNA library prepared from taproot of three-year old Codonopsis lanceolata. A CTRX was 666 nucleotides long and has an open reading frame of 372 bp with 124 amino acid residues (pI = 4.92). The deduced amino acid sequence of the CTRX matched to the previously reported plant thioredoxin h genes. The deduced amino acid sequence of CTRX exhibited the similarity of 33-67% among previously registered thioredoxin genes. The expression of CTRX in leaves of Codonopsis lanceolata was increased by wounding and 1 mM $H_2O_2$, but decreased by 0.1 mM cadmium.