• Journal of Plant Biology
• /
• v.39 no.2
• /
• pp.85-92
• /
• 1996

Small GTP-binding proteins are divided into three major group: Ras, Rho and Ypt/Rab. They have the conserved regions designed G1 to G5 that are critical in GDP/GTP exchange, GTP-induced conformational change and GTP hydrolysis. We isolated and characterized genomic DNA or cDNAfragments encoding G1 to G3 domains of small GTP-binding protein Rab and Rho from several plant species using two different PCR-based cloning strategies. Seven rab DNA fragments were isolated from 4 different plants, mung-bean, tobacco, rice and pepper using two degenerate primers corresponding to the GTP-binding domain G1 and G3 in small GTP-binding proteins. The amino acid sequences among these rab DNA fragments and other known small GTP-binding proteins shows that they belong to the Ypt/Rab family. Six rho DNA fragments were isolated from 5 different plants, mung-bean, rice, Arabidopsis, Allium and Gonyaulax using the nested PCR method that involves four degenerate primers corresponding to the GTP-binding domain G1, G3 and G4. The rho DNA fragments cloned show more than 90% homology to each other. Sequence comparison between plant and other known Rho family genes suggests that they are closely related (67 to 82% amino acid identity). Sequence analysis and southern blot analysis of rab and rho in mung-bean suggest than thses genes are encoded by multigene family in mung-bean.

• Journal of Microbiology and Biotechnology
• /
• v.9 no.1
• /
• pp.91-97
• /
• 1999

In this work, a 1.9-kb region of enterococcal plasmid p703/5 was isolated and the nucleotide sequence analysis of the region was performed. One major open reading frame (ORF) was identified encoding a polypeptide of 28 kDa. Database comparisons suggested that the protein showed some homology with other bacterial RepA proteins. Upstream of the ORF, a potential dnaA box, AT-rich region and 22-bp tandemly repeated sequences (DNA iterons), a feature typical for many replication ori sites, were recognized. Deletion analysis using Exonuclease III and several restriction enzymes indicated that the three elements and the gene product from the ORF were essential for replication and that the minimum unit of DNA required for replication resided on the 1.2-kb AvaII subfragment. Thus, this gene product was referred to as RepA.

• Journal of Aquaculture
• /
• v.17 no.1
• /
• pp.62-68
• /
• 2004

A cDNA encoding the masu salmon, Oncorhynchus masou, estrogen receptor $\alpha$ (msER$\alpha$) was cloned from the pituitary gland by polymerase chain reaction (PCR). This cDNA contains an open reading frame encoding 513 amino acid residues, and the calculated molecular weight of this protein is about 56,430 Dalton. The amino acid sequences of the DNA binding and ligand binding domains of msER$\alpha$ showed high homology to those of other fish species (84-100%). Reverse transcription PCR analysis showed that the mRNA level of msER$\alpha$ in the pituitary was slightly higher in estradiol-17$\beta$(E2) injected masu salmon than that of control fish. To test the biological activity of msER$\alpha$, the cDNA was ligated to a mammalian expression vector and transfected into a gonadotrope-derived cell line, L$\beta$T2, with a reporter plasmid including estrogen responsive element. Expression of the reporter protein, luciferase, was E2 and msER$\alpha$-dependent. The masu salmon ER$\alpha$ is structurally conserved among teleost species and functions as a transcriptional activator in the pituitary cells.

• Biotechnology and Bioprocess Engineering:BBE
• /
• v.5 no.1
• /
• pp.23-26
• /
• 2000

In order to develop the specific genetic marker for Korean native cattle (Hanwoo), randomly amplified polymorphic DNA (RAPD) analysis of 6 different cattle breeds was attempted by using 38 decamer primers. In comparison of RAPD patterns, two distinctive DNA bands specific for Hanwoo were detected. One was 296 bp of DNA fragment found to be specific only for female Hanwoo when primer GTCCACACGG was employed. In individual analysis of this RAPD marker was observed only in female individuals with the possibility of $85.3\%$. The other was 521 bp of RAPD marker amplified using TCGGCGATAG and AGCCAGCGAA primers, which showed $83.0\%$ of genetic frequency in 85 male and 68 female individuals tested. Nucleotide sequencing of these genetic markers revealed that 296 bp marker has a short micro satellite-like sequence, ACCACCACAC, and a tandem repeat sequence of microsatellite GAAAAATG in the determined sequence. Two distinctive tandem repeats of microsatellite sequences, MC and GAAGA, were also appeared in 521 bp DNA marker. In BLAST search, any gene having high homology with these markers was not found.

• The Plant Pathology Journal
• /
• v.17 no.3
• /
• pp.164-168
• /
• 2001

Rice black-streaked dwarf virus (RBSDV) and Maize rough dwarf virus (MRDV) are closely related viruses. Since the two viruses produce identical symptoms on maize, barley, and wheat, diagnosis of infected plants is difficult. Previously, we reported that partial cDNA clones of RBSDV S5 and S6 from the Japanese isolate (RBSDV-H) have lower sequence homology to MRDV than do cDNA clones from other genomic segments. In order to test whether cDNA clones of RBSDV-H S5 and S6 can be used for molecular diagnosis, RBSDV field isolates from Korea and from Hokkaido, Japan were tested in dot blot hybridizations probed with RBSDV-H S5 and S6 cDNA colnes. Hybridization with these probes was more intense against the RBSDV genome than against the MRDV genome. Therefore, RBSDV-H S5 and S6 cDNA clones can be used to differentiate between the two viruses. Furthermore, RBSDV-H S5 and S6 clones reacted more strongly against the viruses from stunted maize plants from Korean fields than to MRDV, indicating that RBSDV may be the causal disease agent in maize plants in Korea.

• Journal of Life Science
• /
• v.8 no.6
• /
• pp.673-680
• /
• 1998

Adenosine deaminase gene from Nocardioides sp. J-326TK was cloned by polymerase chain reaction using primers (PI, PII and PIII) constructed from the highly conserved amino acid sequences among Escherichia coli, mouse and human. A PCR product of about 800bp, as expected from the sequence of E. coli adenosine deaminase gene, was obtained from Nocardioides sp. J-326TK chromosomal DNA double-digested with EcoRI and Pst I. DNA sequencing of the PCR product after cloning into pT7Blue T-vector shows 99.5% and 98.9% homologies in nucleotide and amino acid sequences, respectively, with the E. coli adenosine deaminase whereas 59.5% and 46.8% homologies with the human adenosine deaminase, indicating the evolutionarily relationship of these organisms.

• Animal cells and systems
• /
• v.1 no.1
• /
• pp.135-142
• /
• 1997

Random sequencing of expressed sequence tags in roots of Chinese cabbage led to isolation of a partial cDNA clone, BR77, which encoded a putative protein kinase. Using the BR77 cDNA as a probe, we isolated a full-length cDNA encoding the Brassica campestris protein kinase 1 (Bcpk1). The Bcpt1 cDNA contained one open reading frame encoding a polypeptide of 439 amino acids. The putative polypeptide consisted of a short N-terminal region and a protein kinase catalytic domain. The catalytic domain of Bcpkl showed a high homology to cAMP- and calcium- phospholipid-dependent subfamilies of serine/threonine protein kineses. Eleven major catalytic domains in protein kineses were well conserved in Bcpk1. However, Bcpk1 contained a unique nonhomologous intervening sequence between subdomains VII and VIII, which was not found in protein kineses of animals and lower eukaryotes. Genomic DNA gel blot analysis showed that Bcpt1 genes might be present as three copies in the Chinese cabbage genome. These imply that Bcpk1 belongs to a plant-specific serine/threonine protein kinase subfamily.

• The Korean Journal of Zoology
• /
• v.35 no.2
• /
• pp.219-225
• /
• 1992

The genetic variation in mitochondrial DNA (mtDNA) was analysed within and between two species of tree frogs. Hyla japonica and H. suweonensis from South Korea. Purified mtDNAs were digested with each of 11 restriction enLvmes which cleave at six base recognition sequences. The genome size of H. iaponica revealed ho types (20.0 $\pm$ 0.3 and 19.6 $\pm$ 0.3 kb) and this difference is explained by either addition or deletion of about 0.4 kb fragment. On the other hand, the genome sire of H. suueonensis was about 19.0 $\pm$ 0.4 kb only. For the analysis, level of fragment homology (F) and nucleotide sequence divergence (p) were estimated from comparisons of digestion profiles. Among four populations of H. iaponica, substantial mean sequence divergence was 0.017 (range 0.001-0.026); between identical types, 0.001 IslilaRl type) and 0.004 (Large type) respectively; between different ones, 0.024 (range 0.023-0.026). The level of sequence divergence between he species was 0.142 (range 0.131-0.146). This result suggested that he species ㅂwere distinctly differentiated species. The divergence time between ko species was estimated 7.1 million years.

• Journal of fish pathology
• /
• v.7 no.2
• /
• pp.95-103
• /
• 1994

We have prepared cDNA libray of loach. M. mizolepis in order to isolate cDNA clone of growth hormone gene. Total RNA was isolated from pituitary of loach, and then mRNA was further purified from total RNA by oligo (dT)-coupled magnetic beads. The purified mRNA was used as substrates to prepare cDNA. The resulting cDNA was ligated into the EcoRV/Smal site of pBlueKS+. The ligation mixture have transformed E. coli JM109 strain with electroporator to obtain high yield of transformation efficiency. All the transformants was screened with DIG-labeled Tilapia growth hormone gene by high density colony hybridization. After isolating 10 putative colonies showing the positive signals, secondary colony hybridization and southern hybridization could confirm it as true clones. The nucleotide sequence of one candidate, pCGHI, was compared with 312 bp DNA fragment used as DNA probe and show 52% relative homology to Tilapia growth hormone gene.

• Applied Biological Chemistry
• /
• v.38 no.5
• /
• pp.382-386
• /
• 1995

The potato proteinase inhibitor II (PI-II) protein contains chymotrypsin and trypsin inhibitory site. Among several PI-II genes isolated from genomic library, amino acid sequence deduced from PI-IIT gene has 84% identity with that of the polypeptide chymotrypsin inhibitor (PCI). Therefore a gene fragment having homology with the PCI was cloned into a vector using polymerase chain reaction(PCR) from the potato proteinase inhibitor IIT gene. Two different primers were utilized for cloning; primer A contains NdeI restriction site and 30 nucleotides, which has AUG N-terminal methionine codon, primer B contains BclI restriction site and 28 nucleotides, which has TAG translation stop codon. After PCR, about 160 bp-long DNA fragment was cloned into pRT146, derivative of pUC118, and sequenced. The sequenced NdeI/BclI fragment was moved to pET3a, containing bacteriophage T7 promoter and terminator. The expressed proteins in E. coli BL2l(DE3) were determined on a polyacrylamide gel containing sodium dodecyl sulfate. The expected size of protein deduced from the sequenced gene fragment is about 6,500 dalton whose size was similar to the IPTG-induced protein (6,000 dalton) on a gel. However the expression level was much lower than expected.