• Journal of Ginseng Research
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• 제19권1호
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• pp.56-61
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• 1995

Molecular cloning and restriction mapping on ATPase $\alpha$-subunit gene (atpA) were carried out to obtain genomic information concerned with the gene structure and organization in Korean ginseng mitochondria. Two different clones containing the homologous sequence of atpA gene were selected from SalI and PstI libraries of mitochondrial DNA (mtDNA) of Korean ginseng. The sizes of mtDNA fragments inserted in SalI and PstI clones were 3.4 kb and 13 kb, respectively. Southern blot analysis with [$^{32}P$] labelled Oenothera atPA gene probe showed that atpA gene sequence was located in 2.0 kb XkaI fragment in PstI clone and in 1.7 kb XbaI fragment in SalI clone. A partial sequening ascertained that the SalI clone included about 1.2 kb fragment from SalI restriction site to C-terminal sequence of this gene but about 0.3 kb N-terminal sequence of open reading frame was abscent. The PstI fragment was enough large to cover the full sequence of atpA gene. The same restriction pattern of the overlapped region suggests that both clones include the same fragment of atiA locus. Data of Southern blot analysis and partial nucleotide sequencing suggested that mtDNA of Korean ginseng has a single copy of atpA gene. Key words ATPase a-subunit, mitochondrial DNA, Panax ginseng.

• 한국미생물·생명공학회지
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• 제22권5호
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• pp.485-490
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• 1994

A restriction endonuclease, PsyI, has been isolated from Pseudomonas syringae pv. pha- seolicola, and its catalytic properties have been studied. This enzyme was purified through strepto- mycin sulfate and ammonium sulfate fractionation, phosphocellulose Pll, DEAE-cellulose, hydroxy- apatite and Sephadex G-100 column chromatography. It's molecular weight was about 50,000 dalton as determined by 7.5% polyacrylamide gel electrophoresis containing 0.1% SDS. In catalytic proper- ties, PsyI shows stable at wide ranges of pH between 7.0 and 10.0, of temperature between 30$\circ$C and 37$\circ$C, and its thermal stability is between 25$\circ$C, and 45$\circ$C, at the presence Of 10 mM MgCl$_{2}$-PsyI essentially require Na salt for enzyme reaction, is rather inhibited in the high Na salt concent- ration. The presence of 2-mercaptoethanol is absolutely required for the enzyme activity. This endonuclease, PsyI was determined to be an isoschizomer of SalI from the results of the restriction mapping and DNA sequencing.

• Journal of Plant Biology
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• 제32권2호
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• pp.79-87
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• 1989

Southern hybridization of genomic DNAs with radioactively labeled cDNA of tomato proteinase inhibitor II revealed that proteinase inhibitor II proteins in potato plants are encoded by a family of about 10 related sequences. Screening of potato EcoRI genomic library with the cDNA resulted in isolation of 13 recombinant phage clones which carry 3 different genomic regions. Of these clones, clones 8, 18, and 39 were subjected to restriction mapping and subcloning. Further characterization of the subclones of clones 8, 18 and 39 indicated that two inhibitor II genes are present on a 8.0 kb EcoRI fragment of clone 8, one on 3.3 and 0.8 kb EcoRI fragments of clone 18 and two genes on a 13.5 kb EcoRI fragment of clone 39.

• Journal of Microbiology and Biotechnology
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• 제6권5호
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• pp.309-314
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• 1996

Alcaligenes sp. SH-69 can synthesize poly(3-hydroxybutyrate-co-3-hydroxyvalerate) from a single carbon source such as glucose. To clone the phaC gene from Alcaligenes sp. SH-69, a polymerase chain reaction was performed using the oligomers synthesized based on the conserved regions of the phaC genes from other bacteria. A PCR product (550 bp) was partially sequenced and the deduced amino acid sequence was found to be homologous to that of the phaC gene from Alcaligenes eutrophus. Using the PCR fragment Southern blotting of Alcaligenes sp. SH-69 genomic DNA digested with several restriction enzymes was carried out. To prepare a partial genomic library, about 5-Kb genomic DNA fragments digested with EcoRI, which showed a positive signal in the Southern blotting, were eluted from an agarose gel, ligated with pUC19 cleaved with EcoRI, and transformed into Escherichia coli. The partial library was screened using the PCR fragment as a probe and a plasmid, named pPHA11, showing a strong hybridization signal was selected. Restriction mapping of the insert DNA in pPHA11 was performed. Cotransformation into E. coli of the plasmid pPHA11 and the plasmid pPHA21 which has phaA and phaB from A. eutrophus resulted in turbid E. coli colonies which are indicative of PHA accumulation. This result tells us that the Alcaligenes sp. SH-69 phaC gene in the pPHA11 is functionally active in E. coli and can synthesize PHA in the presence of the A. eutrophus phaA and phaB genes.

• 한국초지조사료학회지
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• 제17권3호
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• pp.285-292
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• 1997

This study was conducted to obtain the transgenic Brnssica napus plants with tobacco Apase gene using the binary vector system of Agrobacteriurn fumefociens. The results obtained were summarized as follows: A repressible acid phosphatase gene of Saccharon~yces cerevisiae, pho105 was used for screening of tobacco Apase cDNA. In order to identify Apase gene in tobacco genome, Southern blot analysis was pcrformed and the Apase gcnc may be present as a single copy, or at most two or three copies, in tobacco genome. To isolate the tobacco Apase gene, tobacco cDNA library was constructed using purifed mRNA from -Pi treated tobacco root and the plaque forming unit of the library was 2.8 x $10^5$ pfu/m${\ell}$, therefore the library might cover all expressed mRNAs. Using pho5 as a probe. tobacco Apase cDNA was cloned, and restriction mapping and Southern blot analysis of cDNA insert were revealed that the 3.6 kb cDNA contained tobacco acid phosphatase cDNA. Plasmid pGA695 -tcAPl was constructed by subcloning tobacco Apase cDNA into the Hind site of pGA695 with 35s promoter which can be expressed constitutively in plants. The Brassica napus cotyledonary petioles were cocultivated with the ,4 grobacteriunz and transferred to the selection medium. The transformed and regenerated plants were transplanted to soil medium. Southern blot analysis was done on the transformed plants, and it was confirmed that a foregin gene was stably integrated into the genonies of B. nnpus plants.

• 미생물학회지
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• 제28권1호
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• pp.27-34
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• 1990

지속성 및 유발성 발한의 두 macrolide-lincosamide-streptogramin B 저항성 인자가 한 Staphylococcus aureus DHI 균주의 염색체 DNA 및 plasmid pDE1(7.4kb)로부터 각각 분리되었다. pDE1상의 유발성 Em 저항성 인자의 염기서열은 이미 보고 된 바 있는 pE194상의 ermC와 동일하였으며 지속성 Em 저항성 인자의 경우는 그 제한효소 인식부위의 mapping 결과로 보아 ermCdb전자에서 유발성 기구에 관여하는 leader peptide 부위가 결여된 인자인 것으로 밝혀졌다.

• 한국미생물·생명공학회지
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• 제23권2호
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• pp.145-149
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• 1995

For the purpose of developing a new biodegradable detergent, we have isolated a gene encoding wide-range temperature applicable alkaline protease from Xanthomonas sp. YL-37 (Lee et al., 1994, Kor. J. Appl. Microbiol. Biotechnol.). An alkaline protease gene was isolated from the gene bank that was prepared from the chromosomal DNA of Xanthomonas sp. YL-37. From the results of agarose gel electrophoresis and a restriction enzyme mapping, a 2.7 kb DNA fragment containing the alkaline protease gene was inserted in the plasmid pUC9. Extracellular activity of a clone having alkaline protease gene was detected on SDS-polyacrylamide gel with activity staining assay. The molecular weight of alkaline protease was determined to be about 64 kDa from 11% SDS-PAGE analysis. Alkaline protease activity, produced from E. coli which harboring the plasmid, showed no difference at reaction temperature 20, 30 and 40$\circ$C, respectively. This result showed that alkaline protease produced from E. coli harboring the plasmid was apparently the same as that of Xanthomonas sp. YL-37.

• 한국식물학회:학술대회논문집
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• 한국식물학회 1995년도 제9회 식물생명공학 심포지움 식물육종과 분자생물학의 만남 The 9th Plant Biotechnology Symposium -Breeding and Molecular Biology-
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• pp.57-86
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• 1995

Molecular mapping of plant genomes has progressed rapidly since Bostein et al.(1980) introduced the idea of constructing linkage maps of human genome based on restriction fragment length polymorphism (RFLP) markers. In recent years, the development of protein and DNA markers has stimulated interest for the new approaches to plant improvement. While classical maps based on morphological mutant markers have provided important insights into the plant genetics and cytology, the molecular maps based on molecular markers have a number of inherent advatages over classical genetic maps for the applications in genetic studies and/or breeding schemes. Isozymes and DNA markers are numerous, discrete, non-deleterious, codominant, and almost entirely free of environmental and epistatic interactions. For these reasons, they are widely used in constructing detailed linkage maps in a number of plant species. Plant breeders improve crops by selecting plants with desirable phenotypes. However a plant's phenotyes is often under genetic control, positioning at different "quantitative trait loci" (QTLs) together with environmental effects. Molecular maps provide a possible way to determine the effect of the individual gene that combines to produce a quantitative trait because the segregation of a large number of markers can be followed in a single genetic cross. Using market-assisted selection, plants that contain several favorable genes for the trait and do not contain unfavourable segments can be obtained during early breeding processes. Providing molecular maps are available, valuable data relevant to the taxonomic relationships and chromosome evolution can be accumulated by comparative mapping and also the structural relationships between linkage map and physical map can be identified by cDNA sequencing. After constructing high density maps, it will be possible to clone genes, whose products are unknown, such as semidwarf and disease resistance genes. However, much attention has to be paid to level-up the basic knowledge of genetics, physiology, biochemistry, plant pathology, entomology, microbiology, and so on. It must also be kept in mind that scientists in various fields will have to make another take off by intensive cooperation together for early integration and utilization of these newly emerging high-techs in practical breeding. breeding.

• Journal of Microbiology and Biotechnology
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• 제11권5호
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• pp.819-824
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• 2001

The principal DNA modification systems of the amino-acid-producing bacteria Corynebacterium glutamicum AS019, Brevibacterium flavum BF4, and B. lactofermentum BL1 was investigated using two approaches; digestion of plasmid DNA isolated from these species TseI and Fnu4HI, and sequence analysis of the putative methyltransferase target sites following the derivatization of DNA using metabisulfite treatment. The C. glutamicum and B. flavum strains showed similar digestion patterns to the two enzymes, indicating that the target for cytidine methyltransferase recognizes 5'-GCSGC-3'(where S is either G or C). Mapping the methylated cytidine sites by bisulfite derivatization, followed by PCR amplification and sequencing, was only possible when the protocol included an additional step eliminating any underivatized DNA after PCR amplification, thereby indicating that the derivatization was not $100\%$ efficient. This may have been due to the high G0C content of this genus. It was confirmed that C. glutamicum AS019 and B. flavum BF4 methylated the cytidine in the $Gm^5CCGC$ sequences, yet there were no similar patterns of methylation in B. lactofermentum, which was consistent with the distinctive degradation pattern seen for the above enzymes. These findings demonstrate the successful application of a modified bisulfite derivatization method with the Corynebacterium species for determining methylation patterns, and showed that different species in the geneus contain distinctive restriction and modification systems.

• 농업과학연구
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• 제44권4호
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• pp.495-503
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• 2017

Genotyping-by-sequencing (GBS) is an outstanding technology for genotyping and single nucleotide polymorphism (SNP) discovery compared to next generation sequencing (NGS) because it can save time when analyzing large-scale samples and carries a low cost per sample. Recently, studies using GBS have been conducted on major crops and, to a greater extent, on fruit crops. However, many researchers have some problems due to low GBS efficiency resulting from low quality GBS libraries. To overcome this limitation, we developed an efficient GBS library construction method that regulates important conditions such as restriction enzymes (RE) digestion and a PCR procedure for grapevine. For RE digestion, DNA samples are digested with ApeKI (3.6U) at $75^{\circ}C$ for 5 hours and adapters are ligated to the ends of gDNA products. To produce suitable PCR fragments for sequencing, we modified the PCR amplification conditions; temperature cycling consisted of $72^{\circ}C$ (5 min), $98^{\circ}C$ (30 s), followed by 16 cycles of $98^{\circ}C$ (30 s), $65^{\circ}C$ (30 s), $72^{\circ}C$ (20 s) with a final extension step. As a result, we had obtained optimal library construct sizes (200 to 400 bp) for GBS analysis. Furthermore, it not only increased the mapping efficiency by approximately 10.17% compared to the previous method, but also produced mapped reads which were distributed equally on the19 chromosomes in the grape genome. Therefore, we suggest that this system can be used for various fruit crops and is expected to increase the efficiency of various genomic analysis performed.