• Journal of Life Science
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• v.10 no.5
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• pp.533-541
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• 2000

Compared to mammal including human, many bioreactive genes that regulate various biological events has not been cloned and characterized yet in fishes, especially shark, Scyliorhinus torazame. In orther to isolate genes that regulate physiological processes in cartilaginors fishes, we performed reverse transcription-polymerase chain reaction (RT-PCR) using the RNA of cartilage tissues of Scyliofhinus torazame. The cloned partial genes were 86%, 80%, 73%, 84%, 75%, 79% identical to $\alpha$- actin, 90-kDa heat-shock protein, methyle-neterahydrofolate dehydrogenase-methenyltertrahydrofolate cyclohudrolase-formyltetrahydrofolate synthetase, ubiquitin, glutamine synthetase and connective tissue growth factor genes of human, respectively. They also have similar nucleotide sequence homologues with those of another species. These partial bioreactive genes elucidated in this study may support to studies of phylogenetic analysis based on evolutionary relationships between shark and other species.

• Journal of Photoscience
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• v.4 no.3
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• pp.141-145
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• 1997

Polymerase chain reaction(PCR) was conducted with a pea cDNA library using two primers synthesized from homology analysis of amino acid sequences for animal and plant cytosolic FBPases. A PCR product with 650 bp long was cloned into pGEM-T vector and sequenced. The deduced amino acid sequence of the cDNA fragment was 98, 91, and 85% homologous with those of cytosolic FBPases from spinach, sugarbeet, and sugarcane, respectively. It was 51% homologous with amino acid sequence of FBPase from pea chloroplasts. Northern blot analysis was proceeded with the cDNA clone resulting that 1.2 kb transcript was highly expressed in light-grown pea leaves but almost not expressed in dark-grown etiolated pea seedlings. When peas grown in the light for 10 days were transferred to darkness, the transcript was gradually decreased with dark treatment, indicating that the expression of the enzyme was induced by continuous white light but suppressed by dark treatment. Pea cytosolic FBPase was highly expressed in leaves with trace amounts in stems. but almost not expressed in roots.

• Journal of the Korean Society of Tobacco Science
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• v.18 no.2
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• pp.101-106
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• 1996

Tobacco mosaic virus (TMV) tomato strain was isolated from tomato "Seo-Kwang" in Korea. The virion was purified by density gradient centrifugation, and total viral RNA was isolated from the purified particles. Coat protein (CP) cDNA of the virus was synthesized by RT-PCR, and the purified cDNA fragment was subcloned to pBluescript II SK-. The analysis of nucleotide sequence showed that this cDNA was 693 nucleotides long from the insert of clone p1571 and p1572 which contain complete codons of the viral coat protein gene (474 nucleotides) and 3' untranslated region. The nucleotides of coat protein encoding cDNA of the strain were 6 nucleotides less than that of TMV common strain isolated from tobacco plant in Korea. The CP gene showed 70% maximum homology with that of the common strain in the nucleotide level and 86% maximum homology in amino acid level.cid level.

• Applied Biological Chemistry
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• v.40 no.6
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• pp.495-500
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• 1997

To isolate a gene for cyclodextrin glycosyltransferase (CGTase) from alkalophilic Bacillus sp. E1, polymerase chain reaction (PCR) amplification was carried out. Direct molecular cloning of 1.2 kbp fragment and partial nucleotide sequence analysis of the PCR amplified clone, pH12, showed close homology with CGTases from Bacillus species. To investigate the genomic structure of the gene, Southern blot analysis of genomic DNA was carried out with the clone pH12 as a molecular probe. It showed that 5.3 kbp XbaI fragment was hybridized with the probe pH12. To isolate a genomic clone, genomic DNA library was constructed and a genomic clone for CGTase, pCGTE1, was isolated. Nucleotide sequence analysis of the clone pCGTE1 revealed that BCGTE1 contained 2,109 bp open reading frame encoding a polypeptide of 703 amino acids and showed over 94.3% amino acid sequence homology with CGTase of ${\beta}-cyclodextrin$ producer, Bacillus sp. KC201.(Received October 7, 1997; accepted October 20, 1997)

• Journal of Life Science
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• v.9 no.2
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• pp.9-14
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• 1999

Pseudomonas iodinum IFO 3558 adenosine deaminase(ADA) gene was cloned by the polymerase chain reaction and deduced the amino acid sequence of the enzyme. DNA sequence homology of Pseudomonas iodinum IFO 3558 ADA gene was compared to those of E. coli, human and mouse ADA genes. Unambiguous sequence from both strands of pM21 was obtained for the region believed to encode ADA. The sequence included a 804-nucleotide open reading frame, bounded on one end by sense primer and on the other end by two antisense primer. This open reading frame encodes a protein of 268 amino acids having a molecular weight of 29,448. The deduced amino acid sequence shows considerable similarity to those of E. coli, mouse and human ADA. Pseudomonas iodinum IFO 3558 nucleotide sequence shows 98.5% homology with that of the E. coli ADA sequence and 51.7% homology with that of the mouse ADA sequence and 52.5% homology with that of the human ADA sequence. The ADA protein sequence of Pseudomonas iodinum IFO 3558 shows 96.9% homology with that of the E. coli and 40.7% homology with that of the mouse and 41.8% homology with that of the human. The distance between two of the conserved elements, TVHAGE and SL(1)NTDDP has veen exactly conserved at 76 amino acids for all four ADAs. Two of the four conserved sequence elements shared among the four ADAs are also present in the yeast, rat, human (M), and Human(L) AMP deaminase. The SLSTDDP sequence differs only in the conservative substitution of a serine for an asparagine. A conserved cysteine with conserved spacing between these two regions is also found. Thus, sequence analysis of four ADAs and four AMP deaminases revealed the presence of a highly conserved sequence motif, SLN(S)TDDP, a conserved dipeptide, HA, and a conserved cysteine residue.

• Journal of Microbiology and Biotechnology
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• v.14 no.5
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• pp.1022-1030
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• 2004

The gene encoding Aquifex pyrophilus (Apy) DNA polymerase was cloned and sequenced. The Apy DNA polymerase gene consists of 1,725 bp coding for a protein with 574 amino acid residues. The deduced amino acid sequence of Apy DNA. polymerase showed a high sequence homology to Escherichia coli DNA polymerase I-like DNA polymerases. It was deduced by amino acid sequence alignment that Apy DNA polymerase, like the Klenow fragment, has only the two domains, the $3'{\rightarrow}5'$ exonuclease domain and the $5'{\rightarrow}3'$ polymerase domain, containing the characteristic motifs. The Apy DNA polymerase gene was expressed under the control of T7lac promoter on the expression vector pET-22b(+) in E. coli. The expressed enzyme was purified by heat treatment, and Cibacron blue 3GA and $UNO^{TM}$ Q column chromatographies. The optimum pH of the purified enzyme was 7.5, and the optimal concentrations of KCl and $Mg^{2+}$ were 20 mM and 3 mM, respectively. Apy DNA polymerase contained a double strand-dependent $3'{\rightarrow}5'$ proofreading exonuclease activity, but lacked any detectable $5'{\rightarrow}3'$ exonuclease activity, which is consistent with its amino acid sequence. The somewhat lower thermostability of Apy DNA polymerase than the growth temperature of A. pyrophilus was analyzed by the comparison of amino acid composition and pressure effect.

• Korean Journal of Microbiology
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• v.30 no.5
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• pp.397-402
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• 1992

The cDNA of RNA segment coding for VP7 of human rotavirus isolated from patient's stool at Seoul area was synthesized, amplified by polymerase chain reaction, field in with Klenow fragment of DNA polymerase I and cloned into pUC19. The cDNA sequence was determined and compared with that of VP7 coding RNA segments of group A rotaviruses isolates in foreign country. Over 90% sequence homology was found with serotyppe I sepcific WA1 and RE9 strains. Comparative analysis of the deduced amino acid sequences within the two variable regions (amino acid residue 87 through 101 and 208 through 221) with WA1 and RE9 strains also showed high degree of sequence similarity with each other.

• Animal cells and systems
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• v.11 no.2
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• pp.175-182
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• 2007

The lysozyme II gene of cabbage butterfly Artogeia rapae was cloned from fat body of the larvae injected with E. coli and its nucleotide sequence was determined by the RACE-PCR. It has an open reading frame of 414 bp nucleotides corresponding to 138 amino acids including a signal sequence of 18 amino acids. The estimated molecular weight and the isoelectric point of the lysozyme II without the signal peptide were 13,649.38 Da and 9.11, respectively. The A. rapae lysozyme II (ARL II) showed the highest identity (81%) in the amino acid sequence to Manduca sexta lysozyme among other lepidopteran species. The two catalytic residues ($Glu^{32}$ and $Asp^{50}$) and the eight Cys residue motifs, which are highly conserved among other c-type lysozymes in invertebrates and vertebrates, are also completely conserved. A phylogenetic analysis based on amino acid sequences indicated that the ARL II was more closely related to M. sexta, Hyphantria cunea, Heliothis virescens, and Trichoplusia ni lysozymes. The ARL II gene was expressed in Spodoptera frugiperda 21 insect cells and the recombinant ARL II (rARL II) was purified from cell-conditioned media by cation exchange column chromatography and reverse phase FPLC. The purified rARL II was able to form a clear zone in lysoplate assay against Micrococcus luteus. The lytic activity was estimated to be 511.41 U/mg, 1.53 times higher than that of the chicken lysozyme. The optimum temperature for the lytic activity of the rARL II was $50^{\circ}C$, the temperature dependency of the absolute lytic activity of rARL II was higher than that of the chicken lysozyme at low temperatures under $65^{\circ}C$.

• International Journal of Industrial Entomology and Biomaterials
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• v.3 no.2
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• pp.121-126
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• 2001

A cDNA encoding a cathepsin D homologue was cloned from a cDNA library of the mulberry longicorn beetle, Apriona germari. Sequence analysis of the cDNA encoding the cathepsin D homologue of A. germari revealed that the 1,158 bp cDNA has an open reading frame of 386 amino acid residues. The deduced protein sequence of the A. germari cathepsin D homologue shows high homology with cathepsin D in insects, Aedes aegypti (68.2% amino acid similarity) and Drosophila melanogaster (67.2% amino acid similarity). Two aspartic residues and six cystein residues in the A. germari cathepsin D homologue are present at identical locations in all of the other catepsins D. Unlike cathepsins D in two insect species, A. gemari cathepsin D homologue appears to have two putative glycosylation sites, rather than one. Phylogenetic analysis revealed the A. germari cathepsin D homologue is more closely related to insect cathepsins D than to the other animal cathepsins D. Northern blot analysis suggests that A. germari cathepsin D homologue gene is expressed in most if not all, body tissues.

• Proceedings of the Zoological Society Korea Conference
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• 1999.10b
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• pp.263.2-263
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• 1999

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