• Korean Journal of Microbiology
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• v.39 no.2
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• pp.114-117
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• 2003

In molecular biology, it is necessary to develop an easy and rapid method to identify a specific DNA sequence. Though Southern and Northern blot techniques have been used widely for the analysis of gene structure and function, those methods are inconvenient in the points that we need to control incubation temperature, time, and other parameters to get the final result. In this study, we report a new method for the rapid analysis of specific DNA sequence with the modification of an immunochromatographic method. The lateral flow DNA analysis strip is composed of a sample pad, a nitrocellulose membrane for the separation and propagation of analytes, and an absorption pad for the generation of capillary action. Capture DNA was immobilized on the membrane by UV cross-linking and target DNA was labeled with Cy-5 for signaling. The samples containing target DNA were applied onto the sample pad, incubated for 15 min for separation, and scanned with a GSI fluorescence scanner. Though the hybridization reaction occurs in a short time without any washing steps, there appears to be little cross hybridization between the different sequences. The result showed a possibility that the new method can be used for the rapid identification of specific DNA sequence among the samples.

• Journal of Periodontal and Implant Science
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• v.32 no.2
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• pp.281-290
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• 2002

The purpose of this study is to develop species-specific DNA probe for detection and identification of Prevotella intermedia (P. intermedia) G8-9K-3. This study procedure includes (1) whole-genomic DNA extraction of P. intermedia G8-9K-3 (2) construction of the genomic DNA library, (3) screening of strain-specific DNA probe by reverse dot hybridization, (4) confirmation of strain-specific DNA probe by Southern blot hybridization, (5) determination of nucleotide sequences of strain-specific DNA probe. Twenty-eight recombinant plasmids containing Hind III-digested DNA fragments of P. intermedia G8-9K-3 were obtained. Reverse Dot Hybridization and Southern blot analysis data showed that one of them, Pig3, could be P. intermedia G8-9K-3-specific DNA probe. This datum indicates that this Pig3 DNA probe could be useful in detection and identification of the P. intermedia G8-9K-3 strain.

• Applied Biological Chemistry
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• v.40 no.4
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• pp.271-276
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• 1997

We have prepared several chimeric constructs containing the bacteriophage T7 RNA polymerase gene under control of the wound-inducible potato proteinase inhibitor II (pin2) promoter and have transformed Nicotiana tabacum plants with these constructs. Southern blot analyses indicate that either one or two copies of the gene constructs are present in the transgenic plants. Northern blot analyses indicate that mRNA encoding T7 RNA polymerase is expressed in a wound-inducible manner. We purified T7 RNA polymerase and prepared antiserum. This antiserum was used for Western blot analyses to demonstrate that a protein which is cross reactive with T7 RNA polymerase is produced. The molecular mass of this protein is 80 kDa, a size which is consistant with the nicked form of the polymerase as is often seen when expressed in E. coli. RNA polymerase assays were used to indicate that the nicked form of T7 RNA polymerase is active and capable of incorporating labeled nucleotides into transcripts in vitro. Analysis of transgenic plants did indeed show that wound-inducible activation of the T7 RNA polymerase permits the establishment of a genetic system to overexpress genes in plants using T7 RNA polymerase(Received March 20, 1997; accepted May 2, 1997)

• Journal of Life Science
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• v.13 no.4
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• pp.522-528
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• 2003

The RAD4 gene of Saccharomyces cerevisiae is essential for the incision step of UV-induced excision repair. A yeast RAD4 gene has been previously isolated by functional complementation. In order to identify the RAD4 homologous gene from fungus Coprinus cinereus, we have constructed cosmid libraries from electrophoretically separated chromosomes of the C. cinereus. The 13 C. cinereus chromosomes were resolved by pulse-field gel electrophoresis, hybridized with S. cerevisiae RAD4 DNA, and then isolated homologous C. cinereus chromosome. The insert DNA of the RAD4 homolog was contained 3.2 kb. Here, we report the characterization of fungus C. cinereus homolog of yeast RAD4 gene. Southern blot analysis confirmed that C. cinereus contains the RAD4 homolog gene and this gene exists as a single copy in C. cinereus genome. When total RNA isolated from C. cinereus cells was hybridized with the 1.2 kb PvuII DNA fragment of the S. cerevisiae RAD4 gene, a 2.5 kb of transcript was detected. In order to investigation whether the increase of transcripts by DNA damaging agent, transcripts levels were examined after treating the cells. The level of transcript did not increase by untraviolet light (UV). This result indicated that the RAD4 homologous gene is not UV inducible gene. Gene deletion experiments indicate that the RAD4 homologous gene is essential for cell viability.

• Journal of Plant Biology
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• v.38 no.2
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• pp.137-141
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• 1995

We isolated a cDNA clone, BLSC1, encoding 5' exon of ATP synthase CF0 subunit I from broccoli. BLSC1 is 285 nucleotides long which consists of a 5' noncoding region of 34 nucleotides, a 5' exon of 145 nucleotides and an intron of 106 nucleotides. The 5' exon codes for 48 amino acids which reveals mostly hydrophobic. The amino acid sequence deduced from BLSC1 shares 83%, 83% and 91% identities with the genes coding for atpF from wheat, rice and spinach, respectively. Genomic Southern blot analysis for BLSC1 showed a typically strong signal for a gene located in the chloroplast genome. Northern blot analysis identified three major classes of transcripts showing strong positive signals in the leaves, but only trace amounts of the transcripts were identified in the other organs like stems, flowr buds and roots.

• Journal of Ginseng Research
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• v.19 no.1
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• pp.51-55
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• 1995

The DNA fragment containing ginseng ribulose-1,5-bisphosphate carboxytase/oxygenase large subunit(rbcL) gene was cloned from the ginseng chloroplast EcoRl library by colony lift hybridization with tobacco rbcL gene probe. From the screened clone, the DNA fragment containing ginseng rbcL gene was digested with several restriction enzyme and analyzed by Southern blot hybridization for the construction of restriction map. The ginseng rbcL gene fragment was subcloned in pBluescript II SK + vector and sequence analysis was performed. The nucleotide sequence of ginseng rbcL gene was compared with those of petunia, tobacco, alfalfa, rice and barley, which showed a homology of 93.1%, 95.2%, 90.5%, 85.5% and 84.3%, respectively.

• Journal of Life Science
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• v.12 no.4
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• pp.496-503
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• 2002

To develop a yeast strain of Pichia pastoris producing an antibacterial peptide, we have attempted the expression and secretion of an insect defensin. The nucleotide sequences corresponding to mature defensin were chemically synthesized by 6 oligomers, assembled in vitro and the synthesized gene was identified by nucleotide sequencing. The prepro sequence of yeast mating factor $\alpha$1 and the defensin gene were recombined into a Pichia expression vector, pPIC9K. The resulting plasmid, pPIDE, was transformed into P. pastoris GSl15 and transformants selected on histidine-deficient minimal plates were tested for antibacterial activity against Micrococcus luteus. Four strains with different antibacterial activity were selected for further analysis. Southern hybridization and RT-PCR verified the defensin gene was maintained and transcribed in a host. Four strains were cultivated in YPD broth for 96 hours to compare cell growth and antibacterial activity, They showed no difference in cell growth, however, each strain showed different antibacterial activity pattern with culture time. The maximal activity was about 550 AU/ $m\ell$.

• Korean Journal of Microbiology
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• v.30 no.4
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• pp.286-290
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• 1992

The present study was performed to isolate the fluorescent pseudomonads from Kwang-Ju soil and to construct a mutant strain defective in siderophore biosynthesis. The siderophore-secreting pseudomonads were screened on Blue agar (Chrome Azuol S agar) plates and one strain of them was designated to Pseudominas fluorescens (P. fluorescens) PY002. To construct a mutant defective in siderophore biosynthesis, P. fluorescens PY002 was randomly mutagenized with a transposon Tn5. The location of Tn5 integrated into chromosomal of the mutants strain was determined by Southern blot analysis. The mutagenized strain showed non-fluorescent on a King's B agar plate and were defective in iron (III) acquisition ability.

• Journal of Korean society of Dental Hygiene
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• v.6 no.4
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• pp.311-324
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• 2006

The purpose of this investigation was to evaluate of the specificity of Fusobacterium nucleatum subspecies-specific DNA probes using dot blot hybridization. To confirm whether the clinical isolates were F. nucleatum or not, 16S rDNA of them were cloned and sequenced. The sequencing data were used in homology search with database of GenBank. When the homology was above 98% compared with the nucleotide sequence of a certain bacteria, it was judged as the same species with the bacteria. 23 strains of F. nucleatum were isolates from subgingival plaque of periodontitis patient. The clinical isolates of F. nucleatum were classified into 10 groups using phylogenetic analysis of 16S rDNA sequence. F. nucleatum subspecies nucleatum-specific DNA probe Fu4(1.3 kb) reacted with genomic DNAs from 8 type strains of F. nucleatum and it reacted strongly with those from 8 clinical isolates. The Fp4(0.8 kb) reacted with F. nucleatum subsp. polymorphum ATCC 10953 and one clinical isolates. Fv35(1.9 kb) and Fs17(8.2 kb) probes reacted with genomic DNAs from F. nucleatum subsp. vincentii ATCC 49256 and F. nucleatum subsp. fusiform ATCC 51190, respectively. Our results showed that it is not enough to evaluate the specificity of F. nucleatum subspecies-specific DNA probes with only dot blot hybridization. Therefore, Southern blot analysis will be necessary to confirm the specificity of F. nucleatum subspecies-specific DNA probes.

• Journal of Microbiology and Biotechnology
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• v.22 no.4
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• pp.563-566
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• 2012

To investigate the possibility of horizontal gene transfer between agricultural microorganisms and soil microorganisms in the environment, Bacillus subtilis KB producing iturin and the PGPR recombinant strain Pseudomonas fluorescens MX1 were used as model microorganisms. The soil samples of cucumber or tomato plants cultivated in pots and the greenhouse for a six month period were investigated by PCR, real-time PCR, Southern hybridization, and terminal restriction fragment length polymorphism (T-RFLP) fingerprinting. Our data from Southern blotting and T-RFLP patterns suggest that the model bacteria do not give significant impacts on the other bacteria in the pots and greenhouse during cultivation.