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Molecular cloning, expression and characterization of a squalene synthase gene from grain amaranth (Amaranthus cruentus L.)

  • Park, Young-Jun;Nemoto, Kazuhiro;Matsushima, Kenichi;Um, Han-Yong;Choi, Jung-Hoon;Oh, Chan-sung;Nishikawa, Tomotaro
    • Proceedings of the Korean Society of Crop Science Conference
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    • 2017.06a
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    • pp.89-89
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    • 2017
  • A gene encoding squalene synthase from grain amaranth was cloned and characterized. The full-length cDNA was 1805-bp long and contained a 1248-bp open reading frame encoding a protein of 416 amino acids with a molecular mass of 47.6 kDa. Southern blot analysis revealed that the A. cruentus genome contained a single copy of the gene. Comparison of the cDNA and genomic sequences indicated that the amaranth SQS gene had 12 introns and 13 exons. All of the exons contributed to the coding sequence. The predicted amino acid sequence of the SQS cDNA shared high homology with those of SQSs from several other plants. It contained conserved six domains that are believed to represent crucial regions of the active site. We conducted qRT-PCR analyses to examine the expression pattern of the SQS gene in seeds at different developmental stages and in several tissues. The amaranth SQS gene was low levels of SQS transcripts at the initial stage of seed development, but the levels increased rapidly at the mid-late developmental stages before declining at the late developmental stage. These findings showed that the amaranth SQS is a late-expressed gene that is rapidly expressed at the mid-late stage of seed development. In addition, we observed that the SQS mRNA levels in stems and roots increased rapidly during the four- to six-leaf stage of development. Therefore, our results showed that the expression levels of SQS in stem and root tissues are significantly higher than those in leaf tissues. In present study provides useful information about the molecular characterization of the SQS clone isolated from grain amaranth. Finally, a basic understanding of these characteristics will contribute to further studies on the amaranth SQS.

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Isolation and Characterization of a Gene Encoding Hexokinase from Loquat (Eriobotrya japonica Lindl.)

  • Qin, Qiaoping;Zhang, Lanlan;Xu, Kai;Jiang, Li;Cheng, Longjun;Xu, Chuanmei;Cui, Yongyi
    • Horticultural Science & Technology
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    • v.30 no.3
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    • pp.243-249
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    • 2012
  • Hexokinase is the first enzyme in the hexose assimilation pathway; it acts as a sensor for plant sugar responses, and it is also important in determining the fruit sugar levels. The full-length cDNA of a hexokinase gene was isolated from loquat through reverse transcription polymerase chain reaction (RT-PCR) and rapid amplification of cDNA ends, which was designated as EjHXK1. EjHXK1 is 1,839 bp long and contains an entire open reading frame encoding 497 amino acids. The predicted protein of EjHXK1 shares 72%-81% similarity with other plant hexokinases. Phylogeny analysis indicated that EjHXK1 is closely related to maize and rice hexokinases. Transient expression of the 35S: EjHXK1-GFP fusion protein was observed on the cell membrane and cytoplasm. Real-time RT-PCR indicated that EjHXK1 is expressed in loquat leaves, stems, flowers, and fruits. EjHXK1 transcripts were higher during early fruit development, but decreases before maturation, which is consistent with hexokinase enzyme activity during fruit development and conducive for hexose accumulation in mature fruits. These results imply that EjHXK1 may play important roles in the regulation of sugar flux during fruit ripening.

Complete Genome Sequences of the Genomic RNA of Soybean mosaic virus Strains G7B and G5

  • Kim, Kook-Hyung;Lim, Won-Seok;Kim, Yul-Ho
    • The Plant Pathology Journal
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    • v.19 no.3
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    • pp.171-176
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    • 2003
  • The complete nucleotide sequences of the genomic RNAs of Soybean mosaic virus strains GS (SMV-G5) and G7H (SMV-G7H) were determined and compared with sequences of other SMV strains. Each viral RNA was determined to be 9588 nucleotides in length excluding the poly (A) tail and contained an open reading frame to encode a polyprotein subsequently processed into up to ten proteins by proteolytic cleavage. Com-parison of the amino acid sequences with those of other SMV strains showed high percentage of amino acid sequence homology with the same genome organization. The nucleotide and the deduced amino acid sequences between SMV-G5 and SMV-G7H were greater than 99% identity. When compared with those of other SMV strains in a phylogenetic analysis of the nucleotide and deduced amino acid sequences, they formed a distinct virus clade showing over 97% amino acid identity, but were more distantly related to the other potyvirus (44.1-69.6% identity). Interestingly, SMV G7H strain caused a severe mosaic or necrosis symptom in soybean cultivars including Jinpum-1, Jinpum-2, and Sodam, whereas, no symptom was observed in SMV-G5 inoculation. Complete nucleotide sequences of these strains will give clues for determining symptom determinant(s) in future research.

Effect of Cross Beams on Live Load Distribution in Rolled H-beam Bridges (압연형강(H형강) 거더교의 가로보가 활하중 횡분배에 미치는 영향)

  • Yoon, Dong Yong;Eun, Sung Woon
    • Journal of Korean Society of Steel Construction
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    • v.18 no.5
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    • pp.535-542
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    • 2006
  • In this study, the effects of cross beams on the lateral distribution of live loads in composite rolled H-beam girder bridges, were investigated through three-dimensional finite element analysis. The parameters considered in this study were the inertial moment ratio between the main girder and the cross beam, the presence of the cross beam, and the number of cross beams. The live load lateral distribution factors were investigated through finite element analysis and the customary grid method. The results show that there was no difference between the bridge models with and without a cross beam. The cross beam of the beam and frame types also showed almost the same live load lateral distribution factors. However, the finite element analysis showed that the concrete slab deck plays a major role in the lateral distribution of a live load, and consequently, the effect of the cross beam is not so insignificant that it can be neglected.

Characteristics of Gene Structure of Bovine Ghrelin and Influence of Aging on Plasma Ghrelin

  • Kita, K.;Harada, K.;Nagao, K.;Yokota, H.
    • Asian-Australasian Journal of Animal Sciences
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    • v.18 no.5
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    • pp.723-727
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    • 2005
  • Ghrelin is a novel growth-hormone-releasing acylated peptide, which has been purified and identified in rat stomach. In the present study, the full-length sequence of bovine ghrelin cDNA was cloned by RT-PCR. The bovine ghrelin cDNA sequence derived in the present study included a 348 bp open reading frame and a 137 bp 3'UTR. The putative amino acid sequence of bovine prepro-ghrelin consisted of 116 amino acids, which contained the 27-amino acid ghrelin. The sequence analysis of the bovine ghrelin gene revealed that an intron existed between Gln$^{13}$ and Arg$^{14}$ of ghrelin. This exon-intron boundary matched the GT-AG rule of the splicing mechanism. Compared with rats, which have two tandem CAG sequences in the 3'end of intron, bovine ghrelin genome has only one CAG sequence. Therefore, although rats can produce 28 amino acid-ghrelin and 27 amino acid-des-Gln$^{14}$-ghrelin by alternative splicing, ruminant species, including bovines, might be able to produce only one type of ghrelin peptide, des-Gln$^{14}$-ghrelin. The influence of aging on plasma ghrelin concentration was also examined. Plasma ghrelin concentration increased after birth to approximately 600 days of age, and then remained constant.

Characterization of Heterologously Expressed Acetyl Xylan Esterase1 Isolated from the Anaerobic Rumen Fungus Neocallimastix frontalis PMA02

  • Kwon, Mi;Song, Jaeyong;Park, Hong-Seog;Park, Hyunjin;Chang, Jongsoo
    • Asian-Australasian Journal of Animal Sciences
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    • v.29 no.11
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    • pp.1576-1584
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    • 2016
  • Acetyl xylan esterase (AXE), which hydrolyzes the ester linkages of the naturally acetylated xylan and thus known to have an important role for hemicellulose degradation, was isolated from the anaerobic rumen fungus Neocallimastix frontatlis PMA02, heterologously expressed in Escherichi coli (E.coli) and characterized. The full-length cDNA encoding NfAXE1 was 1,494 bp, of which 978 bp constituted an open reading frame. The estimated molecular weight of NfAXE1 was 36.5 kDa with 326 amino acid residues, and the calculated isoelectric point was 4.54. The secondary protein structure was predicted to consist of nine ${\alpha}$-helixes and 12 ${\beta}$-strands. The enzyme expressed in E.coli had the highest activity at $40^{\circ}C$ and pH 8. The purified recombinant NfAXE1 had a specific activity of 100.1 U/mg when p-nitrophenyl acetate (p-NA) was used as a substrate at $40^{\circ}C$, optimum temperature. The amount of liberated acetic acids were the highest and the lowest when p-NA and acetylated birchwood xylan were used as substrates, respectively. The amount of xylose released from acetylated birchwod xylan was increased by 1.4 fold when NfAXE1 was mixed with xylanase in a reaction cocktail, implying a synergistic effect of NfAXE1 with xylanase on hemicellulose degradation.

Experimental seismic behaviour of L-CFST column to H-beam connections

  • Zhang, Wang;Chen, Zhihua;Xiong, Qingqing;Zhou, Ting;Rong, Xian;Du, Yansheng
    • Steel and Composite Structures
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    • v.26 no.6
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    • pp.793-808
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    • 2018
  • In this study, the seismic performance of the connections between L-shaped columns composed of concrete-filled steel tubes (L-CFST columns) and H-beams used in high-rise steel frame structures was investigated. Seven full-scale specimens were tested under quasi-static cyclic loading. The variables studied in the tests included the joint type, the axial compression ratio, the presence of concrete, the width-to-thickness ratio and the internal extension length of the side plates. The hysteretic response, strength degradation, stiffness degradation, ductility, plastic rotation capacity, energy dissipation capacity and the strain distribution were evaluated at different load cycles. The test results indicated that both the corner and exterior joint specimens failed due to local buckling and crack within the beam flange adjacent to the end of the side plates. However, the failure modes of the interior joint specimens primarily included local buckling and crack at the end plates and curved corners of the beam flange. A design method was proposed for the flexural capacity of the end plate connection in the interior joint. Good agreement was observed between the theoretical and test results of both the yield and ultimate flexural capacity of the end plate connection.

Molecular Cloning and Nucleotide Sequence of Connexin 35 cDNA in the Ovary from the Sweetfish, Plecoglossus altivelis (은어, Plecoglossus altivelis 난소에서 발현하는 Connexin 35 cDNA의 해석)

  • Choi, Cheol-Young;Chang, Young-Jin
    • Korean Journal of Fisheries and Aquatic Sciences
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    • v.33 no.6
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    • pp.565-571
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    • 2000
  • Mixed primers based on the high sequence homology of selected regions of known connexins (Cxs) was used for PCR reaction. A full-length connexin cDNA of sweetfish (Plecoglossus altivelis) was cloned by rapid amplification of cDNA 5'and (5'RACE) and 3'RACE method. When compared to other known Cx sequences, homology of sweetfish Cx cDNA to Atlantic croaker, Mycropogonias undulatus Cx32.7, bovine, Bos taurus Cx44 and Atlantic croaker Cx32.2 were $63.8{\%},\;61.6{\%}\;and\;56.7{\%}$, respectively. This cDNA encoded 308 amino acids (35,028 dalton) and named as sweetfish Cx35. Hydropathicity analysis of predicted amino acid sequences indicated that sweetfish Cx35 have four major hydrophobic regions and four major hydrophilic regions, suggesting its topology is similar to that of known Cxs. The presence of a tfical Cx consensus sequences were identified in each of the extracellular loops (first loop and second loop).

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COMPARISON OF IMAGE REFORMATION USING PERSONAL COMPUTER WITH CT SCAN RECONSTRUCTION (CT 스캔 영상재구성과 개인용 컴퓨터를 이용한 영상 재형성과의 비교에 관한 연구)

  • Jung Gi-Hun;Kim Eun-Kyung;Kim Sang-Joon
    • Journal of Korean Academy of Oral and Maxillofacial Radiology
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    • v.24 no.2
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    • pp.361-368
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    • 1994
  • Radiographic planning is needed for implant placement in order to determine implant length, jaw bone volume, anatomical stucture and so on. Radiographic examination includes conventional radiography, conventional tomography and CT scan. The most accurate mesurement can be obtained from CT scan. For the cross-sectional view of mandible, CT scan reconstruction is generally needed. But the cross-sectional view of mandible can be reformed by personal computer. This study was performed to examine the clinical usefulness of reformed image using personal computer in comparison with CT scan reconstructed image. CT axial slices of 4 mandibles of 4 volunteers were used. Digital imaging system was composed of Macintosh Ⅱ ci computer, high resolution Sony XC-77 CCD camera, Quick Capture frame grabber board and 'NIH Image' program. Seven reconstructed cross-sectional images within CT machine(CT group) were obtained. And seven reformed cross-sectional images(PC group) after digitization of CT axial slices into the personal computer were obtained. PC group was compared with CT group in the objective and subjective aspects. The results were as follow: 1. Measurement of mandibular height & width in both group showed insignificant difference(P>0.05). 2. Subjective assessment of the mandibular canal in both group showed insignificant difference(P>0.05). 3. Image reformation using personal computer could provide panoramic view, which could not be obtained in CT scan reconstruction.

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Molecular Cloning, Characterization and Expression Analysis of an ILF2 Homologue from Tetraodon nigroviridis

  • Wang, Hui-Ju;Shao, Jian-Zhong;Xiang, Li-Xin;Shen, Jia
    • BMB Reports
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    • v.39 no.6
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    • pp.686-695
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    • 2006
  • Interleukin-2 enhancer binding factor 2 (ILF2) was reported to regulate transcription of interleukin-2 (IL-2), a central cytokine in the regulation of T-cell responses. This property of ILF2 was well characterized in human and mammals, but little is known in bony fish. In this paper, an ILF2 homologue was cloned and well characterized from Tetraodon nigrovirid is for the further investigation of the function of ILF2 in bony fish. The full-length Tetraodon ILF2 cDNA was 1380 bp in size and contained an open reading frame (ORF) of 1164 bp that translates into a 387 amino-acid peptide with a molecular weight of 42.9 kDa, a 5' untranslated region (UTR) of 57 bp, and a 3' UTR of 159 bp containing a poly A tail. The deduced peptide of Tetraodon ILF2 shared an overall identity of 58%~93% with other known ILF2 sequences, and contained two N-glycosylation sites, two N-myristoylation sites, one RGD cell attachment sequence, six protein kinase C phosphorylation sites, one amino-terminal RGG-rich single-stranded RNA-binding domain, and a DZF zinc-finger nucleic acid binding domain, most of which were highly conserved through species compared. Constitutive expression of Tetraodon ILF2 was observed in all tissues examined, including gill, gut, head kidney, spleen, liver, brain and heart. The highest expression was detected in heart, followed by liver, head kidney and brain. Stimulation with LPS did not significantly alter the expression of Tetraodon ILF2. Gene organization analysis showed that the Tetraodon ILF2 gene have fifteen exons, one more than other known ILF2 genes in human and mouse. Genes up- and down-stream from the Tetraodon ILF2 were Rpa12, Peroxin-11b, Smad4, Snapap and Txnip homologue, which were different from that in human and mouse.