• BMB Reports
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• v.32 no.4
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• pp.409-413
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• 1999

The cDNA encoding CMP-NeuAc:lactosylceramide ${\alpha}2$,3-sialyltransferase (GM3 synthase) was isolated from a human fetal brain cDNA library using sequence information obtained from amino acid sequences found in the conserved regions of the previously-cloned mouse GM3 synthase (mST3Gal V) and human sialyltransferases. The cDNA sequence included an open reading frame coding for 362 amino acids, and the primary structure of this enzyme predicted all the structural features characteristic of other sialyltransferases, including a type II membrane protein topology and both sialylmotifs. Comparative analysis of this cDNA with mST3Gal V showed 85% and 86% identity of the nucleotide and amino acid residues, respectively. The expression of this gene is highly restricted in both human fetal and adult tissues.

• Journal of Life Science
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• v.21 no.3
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• pp.459-463
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• 2011

The genome of the rhesus monkey has diverged as an average sequence identity of ~93%. The rhesus monkey has been widely used as a non-human primate in the field of biomedical and evolutional research. Insertion of transposable elements (TEs) induced several events such as transcriptional diversity and different expression in host genes. In this study, 112 transcripts were identified from a full-length cDNA library of brain tissues of the rhesus monkey. One transcript (R54) showed a different expression pattern between human and rhesus monkey tissues. This phenomenon can be an explanation that R54 transcript was acquired by splicing a donor site derived from exonization of the L2A element. Therefore, integration of TEs during primate radiation could contribute to transcriptional diversity and gene regulation.

• Journal of Aquaculture
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• v.20 no.4
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• pp.248-254
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• 2007

Representative cDNA libraries were constructed from various tissue sources of Hemibarbus mylodon, an endangered freshwater fish species in Korea, for the mining of expressed sequence tags (ESTs). Randomized and non-normalized EST analysis was performed with 7 unidirectional cDNA libraries generated from brain, intestine, kidney, liver, muscle, ovary or testis. Of 3,383 ESTs in total, the number of singleton was 2,029, and 333 contigs containing 1,354 ESTs were assembled (percent of unigene = 70.0%). Abundantly expressed gene transcripts and broad clustering of putative gene function were tissue-specific in general, and the redundancy was also variable among those libraries. Over half of H. mylodon ESTs were matched with orthologues from other teleosts among which zebrafish gene sequences were the most frequent in those matches. This initial setting of EST libraries achieved in the present study would be a fundamental basis for the banking of gene resources from this endangered fish species.

• Biomedical Science Letters
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• v.13 no.4
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• pp.305-311
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• 2007

cAMP-dependent protein kinase A (PKA) is the best-characterized protein kinases and has served as a model of the structure and regulation of cAMP-binding protein as well as of protein kinases. To determine the function of PKA in development, we employed the yeast two-hybrid system to screen for catalytic subunit of PKA $(C\alpha)$ interacting partners in a cDNA library from mouse embryo. A Testis-brain RNA-binding protein (TB-RBP), specifically bound to $C\alpha$. This interaction was verified by several biochemical analysis. Our findings indicate that $C\alpha$ can modulate nucleic acid binding proteins of TB-RBP and provide insights into the diverse role of PKA.

• BMB Reports
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• v.30 no.2
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• pp.95-100
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• 1997

Two kinds of cDNA encoding mouse $Gal{\beta}$1,3(4)GlcNAc ${\alpha}$2,3-sialyltransferase (mST3Gal III) and $Gal{\beta}$1,4(3)GlcNAc ${\alpha}$2,3-sialyltransferase (mST3Gal IV) were isolated from mouse brain cDNA library by means of a PCR-based approach. The cDNA sequences included an open reading frame coding for proteins of 374 and 333 amino acids, respectively, and the primary structure of these enzymes suggested a putative domain structure consisting of four regions, like that in other glycosyltransferases. The deduced amino acid sequences of mST3GaI III and IV showed a 98% and 89% identity with rat ST3GaI III and human ST3Gal IV, respectively. Northern analysis indicated that the expression of mST3Gal III mRNA was abundant in heart, liver and adult brain, while that of mST3GaI IV mRNA was detected in all tissues tested except for testis, but the level was the highest in liver. Soluble forms of mST3GaI III and IV transiently expressed in COS cells exhibited enzyme activity toward acceptor substrates containing the terminal either $Gal{\beta}$1,3GlcNAc or $Gal{\beta}$1,4GlcNAc sequences. The substrate preferences of both enzymes were stronger for tetrasaccharides than for disaccharides.

• Animal cells and systems
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• v.13 no.3
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• pp.283-288
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• 2009

The ubiquitous plasma membrane $Na^+/H^+$ exchanger 1 (NHE1) is rapidly activated in response to various extracellular stimuli and maintains normal cytoplasmic pH. Yeast two-hybrid screening was used in order to identify proteins interacting with NHE1 using its cytoplasmic domain as a bait from HeLa cDNA library. One of the interacting cDNA clones was human Lactate dehydrogenase B (LDHB). In vitro translated LDHB was pulled down together with GST-NHE1.cd protein in the GST pull down assay, confirming the interaction in vitro. LDHB antibody immunoprecipitated endogenous LDHB together with NHE1 from H9c2 cells, validating cellular interaction between NHE1 and LDHB. Subsequent analysis revealed that the overexpression of LDHB increased intracellular PH, implying opening of the NHE1 transporter. Moreover, overexpression of LDHB activated caspase 3 and induced cell death, consistent with the expected phenotype of hyper-activation of NHE1. Collectively, our data indicate that LDHB modulates NHE1 activity via physical interaction.

• BMB Reports
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• v.29 no.2
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• pp.146-150
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• 1996

A 2.1 kb cDNA clone for rat transketolase was isolated from rat liver ${\lambda}gt11$ cDNA library and its sequence was determined. The predicted rat transketolase (655 amino acids with $M_r$ 71,186) is highly similar (92%) to that of the human enzyme except that it contains an extra 32 amino acids at its N-terminus. Although it is less similar (<27%) to transketolases from non-mammalian species, the functional motifs such as the catalytic sites and thiamine binding domain are well conserved in the rat enzyme. Southern blot analysis of genomic DNA verified that transketolase appears to be derived from a single gene. Immunoblot and Northern blot analyses suggested that hepatic transketolase was activated pretranslationally by a 2.1-fold while little change was observed in brain enzyme, indicating a tissue-specific pretranslational activation during postnatal development.

• Proceedings of the Zoological Society Korea Conference
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• 2001.04a
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• pp.58.2-58
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• 2001

No Abstract, See Full Text

• Microbiology and Biotechnology Letters
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• v.32 no.4
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• pp.297-306
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• 2004

Heat shock proteins (HSPs) were induced in cells in the thermal stress, and the HSP90 family is one of the major classes of HSPs. Gene encoding HSPs have been characterized from various mammals and piscine. We have cloned and sequenced the HSP90 cDNA from a brain cDNA library constructed from flounder (Paralichthys oliThe result of sequence analysis shows it to be the HSP90~. The nucleotide sequence of the HSP90$\beta$ was composed of 2791 long, encoding 726 amino acid residues. The flounder hsp90$\beta$ gene showed very high sequence homology with hsp90f3 of European sea bass (96.6%), zebrafish (92.9%), Atlantic salmon (92.0%) and human (89.5%). We also constructed a phylogenetic tree based on HSP90 amino acid sequences from vertebrate species. Gene-specific primers were selected and used in RT-PCR reactions to measure the basal hsp90$\beta$ mRNA. The hsp90f3 gene is constitutively expressed at a fairly high level in all examined tissues (brain, liver, kidney, muscle, and spleen). In order to express protein of flounder hsp90$\beta$ in E. coli, we used the His-tagged pETvector. Then, the expression of flounder HSP90$\beta$ was confirmed by Western blot analysis.

• Journal of Microbiology and Biotechnology
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• v.15 no.2
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• pp.321-329
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• 2005

Ornithine decarboxylase (ODC) antizyme is a key regulatory protein in the control of cellular polyamines. We have isolated two distinct ODC antizyme cDNA clones (AZS and AZL) from a flounder (Paralichthys olivaceus) brain cDNA library. Their sequences revealed that both clones required translational frameshifting for expression. Taking + 1 frameshifting into account, AZS and AZL products were 221 and 218 amino acid residues long, respectively, and shared $83.3\%$ amino acid sequence identity. Comparison of the structure and nucleotide sequence of the antizyme genes showed that the genes were highly conserved in flounder, zebrafish, mouse, and human. A phylogenetic tree was constructed, based on the antizyme amino acid sequences from various species. The presence of the two types of antizyme mRNA species in brain, kidney, liver, and embryo was confirmed by using the reverse transcription­polymerase chain reaction (RT-PCR) and Northern blot analysis. Recombinant proteins of flounder ODC antizymes, containing His-Nus-S tag at the amino-terminus, were overexpressed as His-AZL and His-AZS fusion proteins in Escherichia coli BL21 (DE3) pLys by using the pET­44a(+) expression vector.