• Journal of Animal Science and Technology
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• v.46 no.3
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• pp.315-324
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• 2004

Cytochrome P450 aromatase is the enzyme responsible for biosynthesis of female sex hormone(estrogen) and 19-nortestosterone(nandrolone), a unique steroid hormone endogenously synthesized in the pig. By use of RT-PCR coupled with DHPLC technique (WAVE analysis), expression pattern of isoforms of porcine cytochrome P450 aromatase gene was investigated. Relatively higher expression of aromatase mRNA was observed in testis than in ovary and this result accounted for the previous findings of higher blood estrogen level in male compared with female in this species. The result from the DHPLC demonstrated that PCR amplified DNA fragments of ovary and testis tissues. using unique PCR primers for all three types of aromatase genes, were different from those of type II and ill genes. Further nucleotide sequence analyses of the plasmid clones containing the PCR products revealed that nucleotide sequences of all clones were identical to type I aromatase gene(ovary type). Thus, the result from the present study indicates that the ovary and testis express the same type of aromatase gene. Therefore, the efficacy of DHPLC techniques used for this study helped us to analyze tissue-specific expression of isoform of genes containing the nucleotide sequences with high homology.

• The Korean Journal of Pesticide Science
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• v.10 no.1
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• pp.56-65
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• 2006

An antagonistic bacterial isolate, that inhibits the growth of plant pathogens, was selected and identified from 5,000 isolates screened from the rhizosphere of various crop plants. An isolate Bacillus sp. N32, tested against Colletotrichum gloeosporioides causing anthracnose disease in hot pepper, produced both a heat resistant antifungal protein and a heat sensitive antifungal protein. The heat resistant protein was partially purified by Ammonium sulfate fractionation and gel filtration chromatography. The bioautography showed that the proteins possessed high antifungal activity. The biosynthetic gene cluster responsible for the heat resistant antifungal protein was cloned from cosmid library using DNA probe obtained from PCR product with the primers targeting the conserved nucleotide sequence of the synthetic genes reported earlier, Most of the clones obtained showed higher homology to fengycin antibiotic synthetic gene family reported earlier. On the other hand, the heat sensitive protein was isolated from SDS-PAGE and electroblotting to determine the N-terminal amino acid sequences. The heat sensitive antifungal protein gene was cloned from the ${\lambda}-ZAP$ libraries using a DNA probe based on the N-terminal amino acid sequences of the heat sensitive protein. We are contemplating to clone and sequence the whole gene cluster encoding the heat sensitive protein for further analysis.

• Journal of Aquaculture
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• v.8 no.3
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• pp.209-220
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• 1995

The nuclear matrix was isolated from Misgumus mizolepis liver nuclei by low salt extraction and restriction enzyme treatment. The structure was digested with proteinase K. After centrifugation, matrix attachment regions (MARs) were obtained by RNase treatment and phenol-chloroform extraction. The result leads to the appearance of smeared bands in the range of about 0.3-15 kb. pURY19 vector was constructed by inserting 2.13 kb Eco47 III fragment of the yeast uracil 3 gene into the unique Ssp I site of pUC19 plasmid vector as a selection marker. This vector is unable to be maintained in Sacrharomyces cerevisiae by itself since it cannot replicate as an extrachromosomal element. Using this system, we attempted cloning the ARS (autonomously replicating sequence) from M. mizelepis to develop an efficient expression vector for the transgenic fish. pURY19N_{l-62}$ were constructed by inserting MARs in pURY19 plasmid vector and transformation of E. coli $DH5\alpha$. Replication origins (ARS) of M. mizolepis were isolated, which enabled the vector to replicate autonomously in S. cerevisiae. The cloned DNA fragments were sequenced by Sanger's dideoxy-chain termination method. All clones were AT-rich. $pURY19N_6$, one of the clones, expecially contained ARS consensus sequence, Topoisomerase II consensus, near A-box and T-box.

• Journal of Life Science
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• v.14 no.1
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• pp.21-25
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• 2004

To identify genes involved in the decolorization of both crystal violet and malachite green, we isolated random mutants generated by transposon insertion in triphenylmethane-decolorizing bacterium, Citrobacter sp. The resulting mutant bank yielded 14 mutants with complete defect in color removal capability of both crystal violet and malachite green. Southern hybridization with a Tn5 fragment as a probe showed a single hybridized band in 5 mutants and these mutants appeared to have insertions at different sites of the chromosome. Tn5-inserted genes were isolated and the DNA sequence flanking Tn5 was determined. From comparison with a sequence database, putative protein products encoded by cmg genes were identified as follows. cmg 2 is MaIC protein in maltose transport system; cmg 6 is transcriptional regulator (LysR-type): cmg 12 is a putative oxidoreductase. The sequences deduced from two cmg genes, cmg 8 and cmg 11, showed no significant similarity to any protein with a known function. Therefore, these results indicate that these two cmg genes encode unidentified proteins responsible for decolorization of both crystal violet and malachite green.

• Weed & Turfgrass Science
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• v.4 no.1
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• pp.18-25
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• 2015

A universal DNA barcoding for agricultural noxious weeds is a powerful technique for species identification without morphological knowledge, by using short sections of DNA from a specific region of the genome. Two standard barcode markers, chloroplast rbcL and matK, and a supplementary nuclear ribosomal Internal Transcribed Spacer (ITS) region were used to examine the effectiveness of the markers for Pooideae barcoding using 163 individuals of 29 taxa across 16 genera of Korean Pooideae. The rbcL and ITS revealed a good level of amplification and sequencing success while matK did not. Barcode gaps were 78.6% for rbcL, 96.2% for matK, and 91.7% for ITS, respectively. Resolving powers were 89.3% for rbcL, 92.3% for matK, and 79.1% for ITS. The matK obtained the best both barcode gap and resolving power. However, it should be considered not to employ matK for Pooideae barcode because of low rate of PCR amplification and sequencing success. As a single DNA marker, rbcL and ITS were reasonable for Pooideae barcode. Barcode gap and resolving power were increased when ITS was incorporated into the rbcL. The barcode sequences were deposited to the National Center for Biotechnology Information (NCBI) database for public use.

• Korean Journal of Microbiology
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• v.37 no.1
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• pp.72-79
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• 2001

The effect of phenol on the change of bacterial community in the effluent water from a wastewater treatment plant was analyzed by PCR and terminal restriction fragment length polymorphism (T-RFLP). The fragments of 16S rDNA were amplified by PCR with bacterial primers, where one of the primers was biotinylated at the 5'-end. After digestion with restriction enzymes, HaeIII and AluI, the biotinylated terminal restriction tragments (T-RFs) of the digested products were selectively isolated by using streptavidin paramagnetic particles. The single-stranded DNA of T-RFs was separated by electrophoresis on a polyacrylamide gel and detected by silver staining technique. When 10 standard strains were analyzed by our method, each strain had a unique T-RF which corresponded to the calculated size from the known sequences of RDP database. The T-RFLP fingerprint generated from the effluent water was very complex, and the predominant T-RFs corresponded to members of the genus Acinetobacter, Bacillus and Pseudomonas. In addition, the perturbation of bacterial community was observed when phenol was added to the sample at the final concentration of 250 $l^{-1}$. The number of T-RFs increased and the major bacterial population could be assigned to the genus Acinetobacter, Comamonas, Cytophaga and Pseudomonas. A intense band assigned to the putative genera of Acinetobacter and Cytophaga was eluted, amplified, and sequenced. The nucleotide sequence of the T-RF showed close relationship with the sequence of Acinetobacter junii.

• Journal of Life Science
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• v.22 no.11
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• pp.1470-1476
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• 2012

A genetic variation of Lepista nuda and two genus Lepista species (L. irina and L. sordida) were analyzed by random amplified polymorphic DNA (RAPD) and internal transcribed spacer (ITS) sequence analysis. In the resulting RAPD analysis, 22 out of 40 random primers amplified polymorphic RAPD fragment patterns, the amplified bands were 355, and DNA fragment sizes were 200-400bp. Intraspecific genetic dissimilarity of the 10 L. nuda strains were calculated to range from 0% to 21.60%, L. sordida from 16.93% to 24.82%, L. irina were 20.62% to 25.54%, and intraspecific genetic dissimilarity of L. sordida and L. irina was 23.49%. The 673 base pairs were sequenced during the analysis of the ITS I and II region; six L. nuda strains intraspecific genetic dissimilarities ranged from 1.58% to 11.47%, L. nuda and L. sordida from 3.83% to 12.88%, L. nuda and L. irina from 7.11% to 15.61%, and intra-specific genetic variation between L. sordida and L. irina was 4.79%. The findings showed that RAPD and ITS sequencing could be used for developing molecular genetic markers and screening of unidentified genus Lepista species.

• KSBB Journal
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• v.10 no.5
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• pp.499-509
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• 1995

PU.1, a tissue-specific transcription activator, binds to a purine-rich sequence(5'-GAGGAA-3') called PU box. The PU.1 cDNA consists of an open reading frame of 816 nucleotides coding for 272 amino acids. The amino terminal end is highly acidic, while the carboxyl terminal end is highly basic. Transcriptional activation domain is located at the amino terminal end, while DNA binding domain is located at the carboxyl terminal end. Activation of PU.1 transcription factor is supposed to be accomplished by the phosphorylation of serine residue(s). There exist 22 serines in the PU.1. Five(the 41, 45, 132$.$133, and 148th) of the serines(plausible phosphorylation site by casein kinase II), are the primary targets of interest in elucidating the molecular mechanism(s) of the action of the PU.1 gene. In this study, PU.1 cDNA coding for the five serine residues(41th AGC, 45th AGC, 132$.$133th AGC$.$TCA, and 148th TCT), was mutated to alanine codon(41th GCC, 45th GCC, 132$.$133th GCC$.$GCA, and 1481h GCT), respectively, by Splicing-Overlapping-Extension(SOE) using Polymerase Chain Reaction(PCR). And each mutated cDNA fragments was ligated into pBluescript KS＋ digested with HindIII and Xba I, to generate mutant clones named pKKS41A, pRKS45A, pMKS132$.$133A, and pMKS148A. The clones will be informative to study the "Structure and Function" of the immu-nologically important gene, PU.1.

• KOREAN JOURNAL OF CROP SCIENCE
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• v.55 no.2
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• pp.151-158
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• 2010

Camelina sativa L., known as popular names "gold-of-pleasure" or "false flax" is an alternative oilseed crop that can be grown under different climatic and soil conditions. Up to date, however, the genomic information of Camelina has not been studied in detail. Therefore, a cDNA library was constructed and characterized from young leaves. The constructed cDNA library incorporated of 1334 cDNA clones and the size of the insertion fragments average was 736 base pair. We generated a total of 1269 high-quality expressed sequence tags (ESTs) sequences. The result of cluster analysis of EST sequences showed that the number of unigene was 851. According to subsequent analysis, the 476 (55.9%) unigenes were highly homologous to known function genes and the other 375 (44.1%) unigenes were unknown. Remaining 63 (7.4%) unigenes had no homology with any other peptide in NCBI database, indicating that these seemed to be novel genes expressed in leaves of Camelina. The database-matched ESTs were further classified into 17 categories according to their functional annotation. The most abundant of categories were "protein with binding function or cofactor requirement (27%)", "metabolism (11%)", "subcellular localization (11%)", "cellular transport, transport facilities and transport routes (7%)", "energy (6%)", "regulation of metabolism and protein function (6%)". Our result in this study provides an overview of mRNA expression profile and a basal genetic information of Camelina as an oilseed crop.

• Applied Biological Chemistry
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• v.40 no.5
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• pp.380-387
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• 1997

Phytopathogenic Erwinia carotovora subsp. carotovora (Ecc) LY34 causes plant tissue maceration by secretion of pectinolytic enzymes such as pectate Iyase (PL) existed as multiple isoenzyme form. Genomic DNA from Ecc LY34 was digested with Sau3Al and ligated into the BamHI site of pBluescript ll $SK^+$. Among them, a clone hydrolyzing polypectate was selected and its DNA was digested with BamHI. Through the subsequent subcloning the resulting 3.1 kb fragment, corresponding to a peICI, was subcloned into pLYPA 100. The structural organization of a peICI gene encoding a 374 amino acid residues consists of an open reading frame (ORF) of 1,122 bp commencing with a ATG start codon and followed by a TAA stop codon. PeICI contained a typical prokaryotic signal peptide of 22-amino acid. Since the deduced amino acid sequences of PeICl protein was very similar to those of PelIII of Erwinia carotovora subsp. carotovora, and to those of Pel3 of Erwinia carotovora subsp. atroseptica, and to those of PeIC of Erwinia carotovora subsp. carotovora, it belong to the same family PLbc group. The 374-amino acld PeICI had a calculated Mr of 40,507 and pI of 7.60.