• Korean Journal of Microbiology
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• v.40 no.2
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• pp.121-126
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• 2004

Comamonas sp. strain DJ-12 is a 4-chlobiphenyl(4CB)-degrading bacterium that was reidentified from Pseudomonas sp. DJ-12. The genomic DNA was isolated from the strain DJ-12 and amplified by PCR with primers for cloning pcbABCD genes responsible for degradation of 4CB. The amino acid sequences deduced from the nucleotide sequences of pcbA1, pcbA2, pcbA3, pcbA4, pcbB, pcbC2, and pcbD2 genes showed 91, 87, 99, 87, 97, 90 and 87％ homologies with those of Pseudomonas sp. KKS102, respectively. The pcbC1D1 genes that are involved in the degradation of (4-chloro)1,2-dihydroxybiphenyl produced from 4CB by pcbAB gene products were previously reported in the recombinant plasmid pCU1 from Pseudomonas sp. DJ-12. However, the pcbC2D2 genes in the plasmid pCT4 and pCT5 cloned from Comamonas sp. DJ-12 in this study showed 51 and 62％ homologies with those of pcbC1D1 in their nucleotide sequences. The pcbC1D1 and pcbC2D2 genes were found by Southern hybridization to be located at different loci on the chromosome of DJ-12 strain. These results indicate that Comamonas sp. strain DJ-12 has two different sets of pcbCD genes responsible for deg-radation of (4-chloro)1,2-dihydroxybiphenyl.

• Food Science of Animal Resources
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• v.25 no.2
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• pp.218-225
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• 2005

This study was performed to determine sequence variation and RFLP of the mt DNA D-loop region using Southern blot hybridization analysis and to develop mt DNA marker affecting milk production traits in Hanwoo cows. The PCR was used to amplify an 1142 bp fragment within the D-loop region of mt DNA using specific primers. Mt DNA were digested with seven restriction enzymes and hybridized using DIG-labeled D-loop probe. The mt DNA RFLP polymorphisms were observed in the four enzymes, BamHI, RsaI, XbaI and HpaII. Nucleotide substitutions were detected at positions 441 (G/C), 469 (T/C), 503 (C/T), 569 (G/A), 614 (C/A) and 644 (C/T) of the mt DNA D-loop region between two selected lines. Significant relationship between the XbaI RFLP type and breeding value was found(p<0.05). Cows with A type had higher estimated breeding values than those with B type (P<0.05) between high and low milk production lines. Therefore, the RFLP marker of mt DNA could be used as a selection assisted tool for individuals with high milk producing ability in Hanwoo.

• Journal of The Korean Society of Grassland and Forage Science
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• v.25 no.4
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• pp.281-286
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• 2005

To develop transgenic birdsfoot trefoil (Lotus corniculatus L.) plants tolerant to environmental stress, Arabidopsis NDPK gene (AtNDPK) was introduced into birdsfoot trefoil plants using Agrobacterium-mediated transformation and expressed powerfully under the control of the SWPA2 promoter. The expression vector, pCAMBIA2300 was used for introduction of AtNDPK gene into birdsfoot trefoil plants. The transformed calli were selected on kanamycin containing medium and then regenerated. The transformed birdsfoot trefoil plants were cultivated fur 4 months on BOi2Y medium. Genomic DNA PCR and Southern blot analysis confirmed the incorporation of AtNDPK into the birdsfoot trefoil genome.

• Journal of The Korean Society of Grassland and Forage Science
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• v.17 no.4
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• pp.379-386
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• 1997

Recombinant plasmid, pBKH4, containing NPT II and P35S-BcHSP17.6 was constructed by ligation of Bum H I -digested pBKSl-l and BcHSP 17.6 (thermotolerance gene) 6om pBLH4. The tobacco leaf disc was cocultivated with transformed Agmbacterium tumefaciens bearing pBKH4 for 24 hours and transformed shoots were selected on MS-n/B medium containing $100\;{\mu\textrm{g}}/ml$ of kanamycin. Heat-killing temperature of Nicotima tabacum was $50^{\circ}$ for >15min, and transformed tobacco plants with BcHSP17.6 cDNA exhibited thermotolerance at the heat-killing temperature. The transgenic plants were analyzed by Southern blot hybridization with the probe of ${\alpha}^{_32}P$ labelled BcHSP17.6 cDNA. Transcription and expression level of BcHSP17.6 cDNA were also continued by Northern blot analysis and Ouchterlony double immunodiffusion assay. In this study, we suggest that the BcHSP17.6 cDNA introduced to tobacco plant is related to thenuoto-lerance and 17.6-kD LMW HSP acts as a protector from heat damage in plants.

• KSBB Journal
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• v.25 no.2
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• pp.167-172
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• 2010

Streptomyces scabiei producing phytotoxin called thaxtomin, which cause scab disease on economically important crops such as potato. For molecular genetics study of S. scabiei an effective transformation method was established based on conjugal transfer from Escherichia coli ET12567 (pUZ8002) using a phiC31-derived integration vector, pSET152, containing oriT and attP fragments. The high frequency was obtained on MS medium containing 50 mM $MgCl_2$. In addition, the sequence and location of the chromosomal integration attB site of S. scabiei was identified for the first time in the strains producing thaxtomin by the southern blot analysis of exconjugants and the sequencing of plasmid containing DNA flanking the insertion sites from exconjugant chromosome. Similar to the case of Streptomyces species, a single phiC31 attB site of S. scabiei is present within an ORF encoding a pirin-homolog.

• Journal of Photoscience
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• v.9 no.2
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• pp.269-271
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• 2002

To investigate the visual and extra-ocular photoreception, we cloned the opsin genes in ayu (Plecoglossus allivelis). Amplified fragments encoding exon-4 (-5) of opsin cDNAs were cloned from the retina and brains of ayu, and sequenced. One clone was identified as rod (AYU-Rh), two as green cone (AYU-GI, -G2), one as red cone (A YU-R), two as ultraviolet cone (AYU-UVl, UV2), one as VA (AYU-VA), and one as extra-ocular rod (AYU-ExoRh) opsins. 335 amino acids sequence deduced from the full-length cDNA of AYU-Rh showed high identity with that of other fish. Southern blotting analysis indicated that ayu possess two 'rhodopsin' genes, one is visual rhodopsin and the other is non-visual extra-ocular rhodopsin. In situ hybridization showed that the mRNA of AYU-Rh was localized only in rod cells in the retina. On the other hands, AYU-ExoRh was expressed only in the pineal. We cloned two isoforms (AYU-VAM and -VAL) of VA opsin from ayu. The deduced amino acid sequences of these variants were identical to each other within the first 342 residues, but they showed divergence in the C-terminal sequence. AYU- VAL corresponded to the long isoform found in other fish, and AYU-VAM was identified as a new type of VA opsin variant. Pal-VAM is a new probably functional non-visual photoreceptive molecule in fish.

• Journal of Plant Biotechnology
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• v.7 no.4
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• pp.211-218
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• 2005

Genetic transformation was carried out by using biolistic gun method. The hypocotyl derived embryogenic calli (explants) of cotton (Gossypium hirsutum L.) cv. Cocker-312 were transformed with a recombinant pGreen II plasmid, in which both, bar (selection marker) and GUS (${\beta}$-glucuronidase) reporter genes were incorporated. Explants were arranged on osmoticum-containing medium (0.5M mannitol) 4 hours prior to and 16 hours after bombardment that was resulted into an increase about >80% for GUS stable expression. 3 days after bombardment, GUS assay was performed, which exhibited, $18.36{\pm}1.00$ calli showed blue spots. The transformed embryogenic calli were cultured on selection medium (@ 6 mg/L basta) for 3 months. The putative transgenic plants were developed via selective somatic embryogenesis (@1.50 mg/L basta); maximum $27.58{\pm}1.25$ somatic embryos were obtained while $17.47{\pm}1.00$ embryos developed into plantlets (@ 0.75mg/L basta). In five independent experiments, up to 7.24% transformation efficiency was recorded. The presence of the transgenes was analyzed by using PCR and southern hybridization analysis. The transgenic plants were developed with in 6-7 months, but mostly transformants were abnormal in morphology.

• Parasites, Hosts and Diseases
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• v.40 no.3
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• pp.131-138
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• 2002

The cyclophilins (Cyps) are family members of proteins that exhibit peptidylprolyl cis-trans isomerase (PPIase, EC 5.2.1.8) activity and bind the immunosuppressive agent cyclosprin A (CsA) in varying degrees. During the process of random sequencing of a cDNA library made from Giardia intestinalis WB strain, the cyclophilin gene (gicypl) was isolated. An open reading frame of gicyp1 gene was 576 nucleotides, which corresponded to a translation product of 176 amino acids (Gicypl). The identity with other Cyps was about 58-71%. The 13 residues that constituted the CsA binding site of human cyclophilin were also detected in the amino acid sequence of Gicypl, including tryptophan residue essential for the drug binding. The single copy of the gicypl gene was detected in the G. intestinalis chromosome by southern hybridization analysis. Recombinant Gicyp 1 protein clearly accelerated the rate of cis ${\rightarrow}$ trans isomerization of the peptide substrate and the catalysis was completely inhibited by the addition of $0.5{\;}{\mu}M$ CsA.

• Journal of Life Science
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• v.9 no.1
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• pp.54-57
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• 1999

We have got several clones from Serratia marcescens which stimulated the cells to use maltose as a carbon source in E. coli TP2139 (${\Delta}$lac, ${\Delta}$crp). One of the cloned genes, pCKB13, was further analyzed. In order to find whether the increased expression of the gene under the direction of maltose metabolism, we constructed several recombinant subclones. We have confirmed that the clone, pCKB13 codes Coenzyme A transferase gene by partial nucleotide sequencing in the terminal region. The enzyme activity of Coenzyme A transferase increased after introduction of the multicopy of the cloned gene in E. coli. The recombinant proteins expressed by multicopy and induction with IPTG, two polypeptide of 26-and 28-kDa, were confirmed by SDS-PAGE. Southern hybridization analysis confirmed that the cloned DNA fragment was originated from S. marcescens chromosomal DNA.

• Journal of Microbiology and Biotechnology
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• v.1 no.1
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• pp.17-21
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• 1991

A gene coding for ${\beta}-xylosidase$ in alkalophilic Bacillus sp. YC-335 isolated from soil was cloned into Escherichia coli HB101 using plasmid pBR322. The recombinant plasmid pYK40 was isolated, and the cloned HindIII fragment was 15 kilobases (kb). To reduce the size of the inserted DNA fragment of pYK40, the 15 kb HindIII fragment was subjected to a series of subclonings. A 6 kb subfragment was found to code for ${\beta}-xylosidase$ activity, and the recombinant plasmid was named pYK44. Southern hybridization analysis revealed that the cloned gene hybridized with 3.5 kb, 1.5 kb, and 1.0 kb of HindIII cleaved chromosomal DNA from Bacillus sp. YC-335. ${\beta}-xylosidase$ activity produced by recombinant E. coli was found to be 11 times higher than that produced by Bacillus sp. YC-335. Xylan was required to induce the production of ${\beta}-xylosidase$ in Bacillus sp. YC-335.