• Journal of Life Science
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• v.20 no.8
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• pp.1193-1200
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• 2010

The cDNA cloning and developmental profiles of the mRNA for A. pernyi arylphorin was determined. The complete A. pernyi arylphorin cDNA sequence comprised 2,234 bp (without the poly $A^+$ tail), including an open reading frame of 2,112 bp beginning with a methionine ATG at bp34. The A. pernyi arylphorin contained 704 amino acids which are highly enriched in aromatic amino acids, phenylalanine and tyrosine. The calculated molecular mass of the A. pernyi arylphorin from the ORF was 83,439 Da. The deduced amino acid sequence of A. pernyi arylphorin showed 78, 71, 62 and 64% identity with those of H. cecropia, M. sexta $\alpha$ subunit, M. sexta $\beta$ subunit and B. mori storage protein. In Northern blot analysis, the A. pernyi arylphorin mRNA only in the fat body of the 5th instar larvae was responsible for gene expression of the protein, and the synthetic activity of the mRNA was detected strongly in the early larvae, but not in the middle or late-stage larvae. In addition, a very weak signal in mRNA activity was detected in pupal stages, but this was considered to be inactive mRNA after reviewing the results of the labeling experiment of this protein.

• Journal of Microbiology
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• v.39 no.1
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• pp.31-36
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• 2001

A cDNA clone encoding the ribosomal protein S20 has been isolated from the Schizosaccharomyces pombe cDNA library by colony hybridization. The insert contained in the original plasmid pYJ10 was transferred intro shuttle vector pRS316 generate plasmid pYJll. The dDNA insert of plasmid pYJll, contains 484 nucleotides and encodes a protein of 118 amino acids with a calculated mass of 13,544 daltons. The deduced amino acid sequence of S. pombe ribosomal protein S20 is very homologous with fruit fly, rat, and budding yeast counterparts. It is also homologous with Xenopus S22 ribosomal protein. S. pombe ribosomal protein S20 appears to be relatively hydruphobic except the C-terminal region. The 728 bp upstream region of the S20 gene was amplified from chromosomal DNA and transferred into the BamHI/EcoRI site of the promoterles $\beta$-galactosidase gene of the vector YEp357R, which resulted in fusion plasmid pYS20. The synthesis of $\beta$-galactosidase from the fusion plasmid appeared to be the highest in the mid-exponential phase. The S. pombe cells with the fusion plasmid grown at 35$\^{C}$ gave lower $\beta$-galactosidase activity than the cells grown at 30$\^{C}$. Computer analysis showed the consensus sequence CAGTCACA in the upstream regions of various ribosomal protein genes in S. pombe, which would be involved in the coordinated expression of small ribosomal proteins.

• KSBB Journal
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• v.10 no.1
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• pp.89-96
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• 1995

An Avabidofsis thaliana gene encoding phosphoribosyl anthranilate transferase is shown to be the gene that is defective in blue fluorescent trp 1 mutant plants. This gene, named PAT1, coding region is homologous to those for the phosphoribosyl anthranilate transferase from many microorganisms. This is due to a defect in tryptophan biosynthesis that leads to an accumulation of anthranilate, a fluorescent intermediate in the tryptophan pathway. PAT1 is a single-copy gene that complements all of the visible phenotypes of the different trp1 mutants. Experiments to determine the regulation of the PAT1 gene are in progress. The wild-type PAT1 promoter and several promoter deletions of PAT1 gene have been transformed into Arabidopsis tryptophan mutants. These constructs might identify promoter elements that control this patterns. We have isolated the homozygous lines in T3 seeds and analysed the protein levels using PAT antibody and PAT protein level increased two fold in pHSl07. Finally, the potential of using PAT1 as a selectable marker or visible reporter of gene expression is being explored.

• Plant Biotechnology Reports
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• v.4 no.3
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• pp.201-211
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• 2010

We present a highly effective T-DNA inserted gene screening method as part of a reverse genetics model system using the Chinese cabbage (Brassica rapa L. spp. pekinensis). Three-step two-dimensional (2D) matrix strategies are potentially accurate and useful for the identification of specific T-DNA inserted mutants from a large population. To construct our Chinese cabbage model, we utilized a forward genetics screening approach for the abnormal phenotypes that were obtained from transgenic plants of Brassica rapa generated with Agrobacteria tumefaciens containing the pRCV2 vector. From one transgenic plant with an abnormal phenotype, we observed that the st1 gene (which is related to senescence-associated process proteins) contained a T-DNA fragment, and that its expression level was decreased. This T-DNA insert was then used as a control to construct an effective screening pool. As a result, the optimum template concentration was found to be 0.1-1 ng in our PCR strategy. For other conditions, positive changes to the Gibbs free energy prevented the formation of oligo dimers and hairpin loop structures, and autosegment extension gave better results for long fragment amplification. Using this effective reverse genetics screening method, only 23 PCR reactions were necessary to select a target gene from a pool of 100 individual DNAs. Finally, we also confirmed that the sequence we obtained from the above method was identical to the flanking sequence isolated by rescue cloning.

• Journal of Applied Biological Chemistry
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• v.53 no.4
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• pp.189-194
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• 2010

We confirmed the hypo-osmotic shock strengths and duration, different type of vectors, and subcelluar localization to identify the optimum analysis condition of plant aquaporin activity in Xenopus ooctye using Arabidopsis thaliana AtPIP2-1 gene. Six minutes and 1/5ND buffer hypoosmotic shock treatment was the best condition to show the maximum swelling of Xenopus oocytes where AtPIP2-1 was expressed using pcDNA3.1 vector. AtPIP2-1 protein was expressed more efficiently in pGEMHE vector which has 5' and 3' UTR (untranslation region) of Xenopus ${\beta}$-GLOBIN gene in multiple cloning site than in pcDNA3.1 vector. Also green fluorescence of GFP fused to AtPIP2-1 was detected onto oocyte plasmamembrane where is the proper subcellular localization target of AtPIP2-1.

• Journal of Life Science
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• v.19 no.11
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• pp.1522-1528
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• 2009

A marine bacterium was isolated from brown seaweeds for its ability to degrade alginate. Analysis of 16S ribosomal DNA sequence revealed that the strain belongs to Streptomyces like strain ALG-5 which was reported previously. New alginate lyase gene of Streptomyces sp. M3 was cloned by using PCR with the specific primers designed from homologous nucleotide sequences. The consensus sequences of N-terminal YXRSELREM and C-terminal YFKAGXYXQ were conserved in the M3 alginate lyase amino acid sequences. The homology model for the M3 alginate lyase showed a characteristic structure of $\beta$-jelly roll fold main domain like alyPG from Corynebacterium sp. ALY-1. The homogenate of the recombinant E. coli with the alginate lyase gene showed more degrading activity for polyguluronate block than polymannuronate block. The results from the multiple alignments and the homology modeling elucidated in the M3 alginate lyase can be classified into family PL-7.

• Korean Journal of Weed Science
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• v.15 no.3
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• pp.206-213
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• 1995

Glyphosate(N-[phosphonomethyl]glycine) applied to the assimilate-exporting leaves(i.e. third old leaf) of tomato(Lycopersicon esculentum Mil var. Moneymaker). Herbicide binding protein, QB protein(D1), has been immunoblotted using the antibodies raised against the hybrid-protein expressed by a part of spinach psb A gene cloned in frame with the 3'end of lac Z gene to allow expression of the ${\beta}$-galactosidase(EC 3.21.23) in Escherichia coli. Glyphosate has an effect on a turnover of D1 within photosystem II of thylakoid membrane. The dysfunction of D1 protein within light harvesting complex(LHC-II) seems to be a pleiotropic effect of glyphosate.

• Korean Journal of Microbiology
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• v.36 no.2
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• pp.103-108
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• 2000

We have cloned and analyzed cDNA coding for non-virion (NV) protein of the m V - P R T The NV gene contained 336 bp open readmg frame and encoded a protein of 11 1 amino acids with a molecular weight of 13.2 kDa. The deduced amino acid sequence of NV of IHNVPRT was found to be 90-95% identical to those of foreign isolates of IHNV. These results indicate that NV gene of the MNV is highly conserved among &ifferent strains of THNV Northern blot analyses revealed that the levels of NV gene expression were strongly elevated after 20 h post-infection. In order to identify the role of NV in the replication of MNV in fish cells, IHNVinfected cells were treated with antisense oligonucleotides. While IHNV-PRT exposed to glycoprotein (G) antisense oligonucleotide showed severely reduced growth, the growth of virus exposed to NV antisense oligonucleotide was not affected by NV antisense oligonucleotide, which suggests that NV is not essential for replication of IHNV in fish cells.

• Animal cells and systems
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• v.6 no.4
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• pp.317-322
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• 2002

To improve conventional yeast one-hybrid screening, we have developed an efficient mammalian one-hybrid system that allows rapid isolation of com-plementary DNAs which are able to induce human p14$^{ARF}$. tumor suppressor gene. A 1.5 kb promoter region of p14$^{ARF}$ was fused to EGFP to generate ARF promoter-EGFP reporter vector. This reporter plasmid was stably trans-fected into NIH3T3 cells for generation of reporter cell line. When the reporter cell line was infected with E2F-1 together with excess amounts of empty vector, the cells that received the positive modulator were readily identifiable by green fluorescence using FACS. The GFP-positive cells were cloned directly from the cultured cells and expanded in bulk culture. The genomic DNAs from GFP-positive cells were prepared and the CDNA insert in integrated retroviral genome was recovered by PCR using primers annealing to the retroviral vector sequences flanking the insert-cloning site. This system should be useful for efficient screening of expression CDNA libraries in mammalian cells to identify novel upstream regulators for spe-cific genes by one-hybrid interaction.ion.

• BMB Reports
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• v.44 no.2
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• pp.123-128
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• 2011

Leucine-rich repeat containing protein 10 (LRRC10) is characterized as a cardiac-specific gene, suggesting a role in heart development and disease. A severe cardiac morphogenic defect in zebrafish morphants was recently reported but a contradictory result was found in mice, suggesting a more complicated molecular mechanism exists during mouse embryonic development. To elucidate how LRRC10 is regulated, we analyzed the 5'enhancer region approximately 3 kilo bases (kb) upstream of the Lrrc10 start site using luciferase reporter gene assays. Our characterization of the Lrrc10 promoter indicates it possesses complicated cis-and trans-acting elements. We show that GATA4 and MEF2C could both increase transcriptional activity of Lrrc10 promoter individually but that they do not act synergistically, suggesting that there exists a more complex regulation pattern. Surprisingly, knockout of Gata4 and Mef2c binding sites in the 5’enhancer region (-2,894/-2,889) didn't change the transcriptional activity of the Lrrc10 promoter and the likely GATA4 binding site identified was located in a region only 100 base pair (bp) upstream of the promoter. Our data provides insight into the molecular regulation of Lrrc10 expression, which probably also contributes to its tissue-specific expression.