• International Journal of Oral Biology
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• 제35권1호
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• pp.13-19
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• 2010

The DNA probes Pn17 and Pn34 were evaluated for their ability to specifically detect clinical strains of P. intermedia and P. nigrescens from a Korean population by dot blot hybridization. These probes were sequenced by extension termination and their specificity was determined by Southern blot analysis. The results revealed that the Pn17 sequence (2,517 bp) partially encodes an RNA polymerase beta subunit (rpoB) and that Pn34 (1,918 bp) partially encodes both rpoB (1-169 nts) and the RNA polymerase beta subunit (rpoB'; 695-1918 nts). These probes hybridized with both HindIII- and PstI-digested genomic DNAs from the strains of P. intermedia and P. nigrescens used in this study. Interestingly, each of the hybrid bands generated from the HindIII-digested genomic DNAs of the two bacterial species could be used to distinguish between them via restriction fragment length polymorphism. These results thus indicate that Pn17 and Pn34 can simultaneously detect P. intermedia and P. nigrescens.

• Genomics & Informatics
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• 제1권1호
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• pp.50-54
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• 2003

Genome of an extreme thermophile, Thermus caldophilus GK24 has been analyzed to construct the genomic map. The genomic DNAs encapsulated in agarose gel were digested with SspI, EcoRI, SpeI, and HpaI restriction endonucleases, and then the resulting genomic DNA fragments were analyzed by pulsed-field gel electrophoresis. Its restriction map has been constructed by analyzing sizes of the restriction fragments obtained from both complete and partial digestions. The circular form of its genome was composed of about 1.98 Mbp and a megaplasmid. The genomic loci for the genes of xylose isomerase, thioredoxin, tRNA-16S rRNA, 23S rRNA, L5 ribosomal protein, ADP-glucose pyrophosphorylase, DNA-ligase, and Tca DNA polymerase were determined by both Southern hybridization and PCR.

• Journal of Periodontal and Implant Science
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• 제24권3호
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• pp.541-560
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• 1994

The present study was performed to evaluate the relationship between the serotype or the genotype of Actnobacillus actinomycetemcomitans (A. a.) and the severity of periodontal disease. Total 64 A. a. clinical isolates were sampled from 46 sites of 20 subjects classified into the group I (1 periodontally healthy subject, 2 gingivitis patients, 5 ealry adult periodontitis patients), group II (3 moderatelly adult periodontitis patients) and group III (1 advanced adult periodontitis patient, 8 RPP patients). Southern bolt hybridization (fingerprinting) patterns of the five reference strains, A. a. strain ATCC 29523 (serotype a), ATCC 29522 (Serotype b), ATCC 43719 (serotype c), IDH 781 (serotype d) and IDH 1705 (serotype e), were used as the five basic genotypic patterns (A, B, C, D, E). NT type was designated as one which did dnot represent any of those five basic types. The serotypes were determined by ELISA technique with the serum samples from pre-immunized rabbit. Based on subject-based analysis, it was noted that genotypes A and C, NT, and B, D, E were significantly related to the disease groups I, II, and III, respectively. It was also noted that both the serotypes a and c were significantly related to the disease group I and II, while serotypes were significantly related bm), and serotypes b and nd were frequently found in sites with severe attachment loss (LA>6mm). The results indicated that the significant relationship can be delineated beteen the genotypes and the serotypes of Acinobacillus actinomycetemcomitans and the periodontal disease severity. The results also indicated that genotyping can provide more detailed information on its relationship with the disease severity based on both the patient-based and the site-based analyses.

• BMB Reports
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• 제40권6호
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• pp.845-852
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• 2007

The highly evolved flowers of orchids have colorful sepals and fused columns that offer an opportunity to discover new genes involved in floral development in monocotyledon species. In this investigation, we cloned and characterized the homologous PISTALLATA-like (PI-like) gene PhPI15 ($\underline{Ph}alaenopsis$ $\underline{PI}$ STILLATA # $\underline{15}$), from the Phalaenopsis hybrid cultivar. The protein sequence encoded by PhPI15 contains a typical PI-motif. Its sequence also formed a subclade with other monocot PI-type genes in phylogenetic analysis. Southern analysis showed that PhPI15 was present in the Phalaenopsis orchid genome as a single copy. Furthermore, it was expressed in all the whorls of the Phalaenopsis flower, while no expression was detected in vegetative organs. The flowers of transgenic tobacco plants ectopically expressing PhPI15 showed male-sterile phenotypes. Thus, as a Class-B MADS-box gene, PhPI15 specifies floral organ identity in orchids.

• 대한바이러스학회지
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• 제28권2호
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• pp.129-138
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• 1998

Bovine growth hormone (bGH) gene was expressed in an insect Spodoptera frugiperda cell line using a Baculovirus, Hyphantria cunea nuclear polyhedrosis virus (HcNPV). The bGH gene in pbGH plasmid was sequenced and amplified by PCR technique with two primers containing NcoI sites. The bGH gene consisted of 654 bp (217 amino acid residues), the 5'-untranslated region of the cloned bGH cDNA contains 56 bp, and the 3'-untranslated region contains 145 bp and two pallindromic regions. The amplified bGH gene DNA fragment (654 bp) was inserted into the NcoI site of the pHcEVII vector, which was named pHcbGH. The pHcbGH transfer vector DNA and the wild type HcNPV DNA were cotransfected into S. frugiperda cells to construct a recombinant virus. Eight recombinant viruses were selected and named HcbGH. One clone, HcbGH-4-1 showed largest plaque size, therefore the recombinant virus was further studied. The multiplication pattern of the recombinant HcbGH-4-1 was similar to that of the wild type HcNPV. The bGH gene DNA in the HcbGH-4-1 recombinant was confirmed by Southern blot hybridization. The amount of the bGH (217 amino acid residues, 21 kDa) produced in S. frugiperda cells infected with the HcbGH-4-1 recombinant was approximately 5.5 ng per ml ($10^6$ cells) by radioimmunoassay.

• Plant Biotechnology Reports
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• 제5권1호
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• pp.71-77
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• 2011

Drought and salinity are the most important abiotic stresses that affect the normal growth and development of plants. Glycine betaine is one of the most important osmolytes present in higher plants that enable them to cope with environmental stresses through osmotic adjustment. In this study, a betaine aldehyde dehydrogenase (BADH) gene from spinach under the control of the stress-induced promoter rd29A from Arabidopsis thaliana was introduced into potato cultivar Gannongshu 2 by the Agrobacterium tumefaciens system. Putative transgenic plants were confirmed by Southern blot analysis. Northern hybridization analysis demonstrated that expression of BADH gene was induced by drought and NaCl stress in the transgenic potato plants. The BADH activity in the transgenic potato plants was between 10.8 and 11.7 U. There was a negative relationship (y = -2.2083x + 43.329, r = 0.9495) between BADH activity and the relative electrical conductivity of the transgenic potato plant leaves. Plant height increased by 0.4-0.9 cm and fresh weight per plant increased by 17-29% for the transgenic potato plants under NaCl and polyethylene glycol stresses compared with the control potato plants. These results indicated that the ability of transgenic plants to tolerate drought and salt was increased when their BADH activity was increased.

• 미생물학회지
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• 제35권3호
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• pp.173-179
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• 1999

Rhizoctonia solani 분류를 위해 균사의 anastomosis group과 grouprks의 유전적 차이의 분석을 위해 rDNA 의 PCR-RFLP 와 RFLP를 실시하였다. rDNA 의 PCR-RFLP 결과 R. solani 는 크게 5 group 으로 나뉘어졌다. 균주 번호 1번과 3번은 AG-5 그룹과 0.976의 높은 상동성을 보였고, 12번, 13번은 AG-2-2와 0.976의 상동성을 보였다. 균주번호 7,8,11,13,15 는 AG-1에 속하였다. 제한효소 HaeIII로 절단하였을 때 균주번호 4,5,7,8은 700 bp에서 하나의 밴드가 나타나 AG-1에 속하였고, 균주번호 9는 AG-2-1과 517 bp에서 하나의 밴드가 나타났다. 도한 제한효소 Msp I을 사용하여 southern blotting을 한 결과 더 다양한 절단양상을 보이며 AG 간의 차이가 나타났다. AG-2-1과 균주번호 9는 200 bp에서 하나의 밴드가 나타났다. 균주 3,4,5,10,11,13은 AG-1과 1kb에서 하나의 밴드로 나타났다.

• Fisheries and Aquatic Sciences
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• 제18권2호
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• pp.183-193
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• 2015

Several transgenic marine medaka Oryzias dancena strains harboring a green fluorescent protein (GFP) reporter construct regulated by an endogenous ${\beta}$-actin promoter were established and their expression characteristics in relation to transgene copy numbers were examined in 21 transgene genotypes. Most of the transgenic strains displayed transgene insertion patterns typical of microinjection-mediated introduction of foreign DNA into fish embryos, characterized by the random integration of multiple transgene copies (ranging from 1 - 282 copies per cell), often accompanied by the formation of concatemer(s), as assessed by genomic Southern blot hybridization analysis and qPCR. Transgenic strains showed ubiquitous and continued temporal and spatial expression patterns of the transgenic GFP during most of their life cycle, from the embryonic stage to adulthood, enabling assessment of the expression pattern of the endogenous ${\beta}$-actin gene. However, a comparative evaluation of transgene copy numbers and expression levels showed that copy number-dependent expression, the stability of the ubiquitous distribution and expression efficiency per transgene copy varied among the transgenic strains. Fluorescence expression levels were positively correlated with absolute transgene copy numbers, whereas the expression efficiency per transgene copy was inversely related to the number of transgene integrant copies. Data from this study will guide the selection of potentially desirable transgenic strains with ubiquitous expression of a fluorescent transgene, not only in this marine medaka species but also in other related model fish species.

• Journal of Microbiology and Biotechnology
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• 제28권7호
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• pp.1133-1140
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• 2018

Pseudomonas fluorescens KLR101 was found to be capable of producing polyhydroxyalkanoate (PHA) using various sugars and fatty acids with carbon numbers ranging from 2 to 6. The PHA granules consisted mainly of a poly(3-hydroxybutyrate) homopolymer and/or poly(3-hydroxybutyrate-co-3-hydroxyvalerate) copolymer. Genomic DNA of P. fluorescens was fractionated and cloned into a lambda library, in which a 5.8-kb fragment that hybridized to a heterologous phaC probe from Ralstonia eutropha was identified. In vivo expression in Klebsiella aerogenes KC2671 (pUMS), restriction mapping, Southern hybridization experiments, and sequencing data revealed that PHA biosynthesis by P. fluorescens relied upon a polypeptide encoded by a 1,683-bp non-operonal ORF, which was preceded by a possible -24/-12 promoter and highly similar to DNA sequences of a gene encoding PHA synthase in the genus Pseudomonas. In vivo expression of the putative PHA synthase gene ($phaC_{Pf}$) in a recombinant Escherichia coli strain was investigated by using glucose and decanoate as substrates. E. coli (${phaC_{Pf}}^+$, pUMS) grown in medium containing glucose accumulated PHA granules consisting mainly of 3-hydroxybutyrate, whereas only a trace amount of 3-hydroxydecanoate was detected from an E. coli fadR mutant (${phaC_{Pf}}^+$) grown in medium containing decanoate. In vitro enzymatic assessment experiments showed that 3-hydroxybutyryl-CoA was efficiently used as a substrate of purified $PhaC_{Pf}$, suggesting that the putative PHA synthase of P. fluorescens utilizes mainly short-chain-length PHA precursors as a substrate.

• 한국미생물·생명공학회지
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• 제22권5호
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• pp.467-476
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• 1994

Lactobacillus casei HY 2782, prophage cured strain was characterized to be stable as much as L casei YIT 9018, parent strain. By southern hybridization, it was confirmed that the temperate phage was incorporated in chromosomal DNA of L. casei YIT 9018 as a prophage. It was also proved that the prophage was cured from chromosomal DNA of L casei HY 2782. The growth rate, lactic acid producing ability, carbohydrates fermentation, and enzymatic activity of L. casei HY 2782 were found to be similar to those of L. casei YIT 9018. When L casei HY 2782 was used as a host, the multiplicity of infection (M.O.I.) of the temperate phage for L. casei HY 2782 was 1.0~5.0. Restriction enzyme analysis of pLC90 plasmid from L. casei HY 2782 was shown that the size was an approximately 68.22 kb. The plasmid profiles, genomic DNA patterns, and cellular fatty acids composition of L. casei HY 2782 were similar to those of L casei YIT 9018. And the major fatty acids composition of these strains were C$_{14;0}$,C$_{16;1}$, C$_{16;0}$, C$_{18;1}$ and C$_{19;cyclo-}$ 10 sets of arbitrary primer in the PCR were screened to find differentiation against two strains of L. casei. Among them, b$_{5}-1/17-1 primer was produced an approximately 1.3 kb DNA band of only L casei YIT 9018. And b$_{5}-2/17-2 primer was produced an approximately 1.0 kb DNA band of only L casei HY 2782.