• BMB Reports
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• v.37 no.3
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• pp.356-361
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• 2004

To develop a simplified method that can rapidly prepare DNA microarray probes in a massive scale, a lambda phage genomic DNA-fragments library was constructed for the microarray-probes collection. Four methods of DNA band recovery from the first PCR products were tested and compared. The DNA microarray probes were collected by a novel method of nested PCR that was mediated by gel isolation of the first PCR products. This method was named GIN-PCR. The probes that were prepared by this GIN-PCR technique were used as subjects to fabricate a DNA microarray. The results showed that a wooden toothpick was superior to the other 3 methods, since this technique can steadily transfer the DNA bands as the template of the second PCR after the first PCR. A group of probes were successfully collected and DNA microarrays were constructed using these probes. Hybridization results demonstrated that this technique of DNA recovery and probe preparation was rapid, efficient, and effective. We developed a cost-effective and less labor-intensive method for DNA microarray probe preparation by nested PCR that is mediated by wooden toothpick transfer of the DNA bands in the gel after electrophoresis.

• Proceedings of the Korean Society of Plant Biotechnology Conference
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• 2002.04a
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• pp.101-109
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• 2002

A lot of SSRs (simple sequence repeats) in peach and pear from enriched genomic libraries and in peach from a cDNA library were developed. These SSRs were applied to other related species, giving phenograms of 52 Prunus and 60 pear accessions. Apple SSRs could also be successfully used in Pyrus spp. Thirteen morphological traits were characterized on the basis of the linkage map obtained from an $F_2$ population of peach. This map was compiled with those morphological markers and 83 DNA markers, including SSR markers used as anchor loci, to compare different peach maps. Molecular markers tightly linked to new root-knot nematode resistance genes were also found. A linkage map including disease-related genes, pear scab resistance and black spot susceptibility, in the Japanese pear Kinchaku were constructed using 118 RAPD markers. Another linkage map, of the European pear Bartlett, was also constructed with 226 markers, including 49 SSRs from pear, apple, peach and chewy. Maps of other Japanese pear cultivars, i.e., Kousui and Housui, were also constructed. These maps were the first results of pear species.

• Journal of Microbiology
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• v.33 no.2
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• pp.142-145
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• 1995

The pumb gene encoding a basic amino acid transport protein in Neurospora crassa could be cloned by using a mutant strain defective in pmb gene as a host strain, using a negative selection on the media containing amino acid analogue canavanine. To select positive transformants of the genes for cloning, an auxotrophic marker (his-2) was added to a pmb mutant strain by mating ; a triple mutant (pmn : pmb : his-2) was constructued by crossing a strain defective in basic amino acid transport system (# 1683-bat um 535 "A") to a double mutant strain defective in neutral amino acid transport and histidine production (mitrol : his-2 "a"). Crossing was performed on synthetic crossing (SC) media containing histidine. The pmn : pmb and pmn :pmb : his-2 strains were selected among the progeny colonies from crosses on plates containing 5- .mu.g/ml para-fluoro-phenylalanine (PFPA), 200 .mu.g/ml canavanine, and 500 .mu.g/ml histidine. The selected colonies were cultured on minimal media with or without histidine for discarding pmn : pmb strain, because the pmn : pmb : his -2 strain grows only on histidine containing media. The pmn :pmb : his-2 strain selected can be used as a host strain for the cloning of the pmb and the pmn genes from a Neurospora genomic library by means of positive selections.

• BMB Reports
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• v.29 no.2
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• pp.146-150
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• 1996

A 2.1 kb cDNA clone for rat transketolase was isolated from rat liver ${\lambda}gt11$ cDNA library and its sequence was determined. The predicted rat transketolase (655 amino acids with $M_r$ 71,186) is highly similar (92%) to that of the human enzyme except that it contains an extra 32 amino acids at its N-terminus. Although it is less similar (<27%) to transketolases from non-mammalian species, the functional motifs such as the catalytic sites and thiamine binding domain are well conserved in the rat enzyme. Southern blot analysis of genomic DNA verified that transketolase appears to be derived from a single gene. Immunoblot and Northern blot analyses suggested that hepatic transketolase was activated pretranslationally by a 2.1-fold while little change was observed in brain enzyme, indicating a tissue-specific pretranslational activation during postnatal development.

• Parasites, Hosts and Diseases
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• v.46 no.2
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• pp.59-63
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• 2008

In order to obtain greater insight into the relevant genomic expression patterns of Trichinella spiralis, 992 expressed sequence tags (ESTs) were collected from a cDNA library of T. spiralis muscle stage larvae and assembled into 60 clusters and 385 singletons. Of them, 445 (44.7%) ESTs were annotated to their homologous genes, and small fractions were matched to known genes of nematodes. The annotated ESTs were classified into 25 eukaryotic orthologous groups (KOG). Cytochrome C oxidase (34 clones) was found to be most frequent species.

• Korean Journal Plant Pathology
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• v.10 no.3
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• pp.151-156
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• 1994

Pseudomonas fluorescens produces the antibiotic, 2,4-diacetylphloroglucinol (Phl), which promotes plant growth by inhibiting bacteria and fungi. Cosmids (genomic library) were mobilized into Phl-nonproducing mutants through the triparental matings with pRK2013 as the helper plasmid at the frequency of 8.37$\times$10-4. Complemented mutants that showed antibiotic activity were selected among about 2,000 transconjugants. The complemented mutants were confirmed by acquired drug resistances (kanamycin and tetracycline). The antibiotic substances of wild type and complemented mutants showed the most excellent anti-bacterial activity. Inhibitory effects of complemented P. fluorescens against phytopathogenic fungi were equal to the parental strain. Complemented mutant and wild type of P. fluorescens were causal microbes of fungal morphological abnormalities. Complemented mutants in potato dextrose agar supplemented with bromothymol blue also showed restoration of glucose utilization as wild type. Plasmids of complemented mutants were isolated from transconjugant sand transformed into competent cells of E. coli DH5$\alpha$. The plamid DNA was reisolated from transformed E. coli DH5$\alpha$.

• Journal of fish pathology
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• v.19 no.3
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• pp.227-233
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• 2006

Olive flounder immunoreceptor DAP10 homologue cDNA was cloned from a peripheral blood lymphocytes (PBLs) cDNA library. The length of the olive flounder DAP10 cDNA is 473bp and it contains an open reading frame of 234bp. The predicted polypeptide sequence is 78 amino acids, consisting of a 22-amino acid leader, an 11-amino acid extracellular domain, a 21-amino acid transmembrane segment, and a 24-amino acid cytoplasmic domain. The amino acid sequence of olive flounder DAP10 has 56%, 50%, 32%, 31%, and 31% sequence identity with zebrafish DAP10, catfish DAP10, cattle DAP10, rat DAP10 and Monkey DAP10, respectively. Olive flounder DAP10 has a conserved aspartic acid in the transmembrane domain and a phophatidylinositol-3 kinase-binding site (YxxM/V) in the cytoplasmic region. Genomic organization reveals that olive flounder DAP10 comprises five exons and four introns. A phylogenetic analysis based on the deduced amino acid sequence grouped the olive flounder DAP10 with other species DAP10. In RT-PCR analysis, DAP10 transcripts were detected predominantly in PBLs, kidney, spleen and intestine.

• Microbiology and Biotechnology Letters
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• v.29 no.4
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• pp.201-205
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• 2001

A bacteria strain producing extracellular $\beta$-mannanase was isolated from soil and was identified as Aeromonas sp. A genomic DNA library constructed from Aeromonas, sp that secrets a $\beta$-mannanase was screened for mannan hydrolytic acticity. Recombinant $\beta$-mannanase activity was detercted on the basis of the clear zones around Escherichia coli colonies grown on a LB medium supplemented locust bean gum, EcoRI restriction analysis of plasmid prepared from recombinant E. coli which showed a $\beta$-mannanase activity revealed 10 kb DNA insert, The optimum pH and temperature for the activity of reconmbinant $\beta$-mannanase were 6.0 and $50^{\circ}C$ respectively and were identical to those of the native enzyme.

• Journal of Microbiology and Biotechnology
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• v.14 no.3
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• pp.470-473
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• 2004

In the yeast Saccharomyces cerevisiae, iron can be toxic. Because of this phenomenon, its metabolism of iron is strictly regulated. We have constructed a model system in which cell growth is defected during periods of iron over-load. When $Aft1-1^{up}$ protein was overexpressed with Ga110 promoter, a galactose inducible promoter, cell growth was defected and levels of CLN2 transcript decreased. However transcript levels of AFT1 and FET3 genes increased over time in a consistent manner throughout the course of $AFT1-1^{up}$ overexpression. We have screened to find genes to suppress cell growth defect by iron overload with YEp-derived high copy yeast genomic DNA library and found that high copy of Rmelp suppressed cell growth defects. Rme1p has been known as an activator protein of CLN2 gene expression. Taking these results together, we suggest that the yeast cell cycle is arrested at the $G_1$, phase by iron overload via Cln2p.

• Journal of Microbiology and Biotechnology
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• v.17 no.10
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• pp.1675-1681
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• 2007

The pseudodisaccharide salbostatin, which consists of valienamine linked to 2-amino-1,5-anhydro-2-deoxyglucitol, is a strong trehalase inhibitor. From our Streptomyces albus ATCC 21838 genomic library, we identified thirty-two ORFs in a 37-kb gene cluster. Twenty-one genes are supposed to be a complete set of modules responsible for the salbostatin biosynthesis. Through sequence analysis of the gene cluster, some of the upstream gene products (SalB, SalC, SalD, SalE, and SalF) revealed functional resemblance with trehalose biosynthetic enzymes. On the basis of this rationale, we isolated the five genes (salB, salC, salD, salE, and salF) from the S. albus ATCC 21838 and cloned them into the expression vector pWHM3. We demonstrated the noticeable expression and accumulation of trehalose, using only the five upstream biosynthetic gene cluster of salbostatin, in the transformed Streptomyces lividans TK24. Finally, 490 mg/l trehalose was produced by fermentation of the transformant with sucrosedepleted R2YE media.