• The Korean Journal of Mycology
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• v.23 no.2 s.73
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• pp.129-138
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• 1995

DNA fragment being able to restore in vitro activity of ${\beta}-1,3-glucan$ synthase was cloned by transformation of the Saccharomyces cerevisiae LP353 mutant strain with genomic library constructed in the YCp50. For the selection of transformants which showed no detectable phenotype linked to recovery of the defect in ${\beta}-1,3-glucan$ synthase activity, the colony autoradiography was succesfully applied. The restriction map of the cloned DNA fragment, which is 8.5-kb in length, was constructed. Both the YEplac195 and the YCp50 carrying the 8.5-kb fragment increased ${\beta}-1,3-glucan$ synthase activity of LP353 by two fold. Neither the YEplac195 nor the YCp50 carrying the 8.5-kb DNA fragment, however, complemented the temperature-dependent osmotic sensitivity which is another distinctive phenotype of LP353. Subcloning experiments indicated that a functional region was located in 4.8-kb BglII-KpnI fragment. The 4.8-kb fragment was also able to increase the level of ${\beta}-1,3-glucan$ content in cell wall as well as the resistance of cells to cell wall lytic enzyme, ${\beta}-1,3-glucanase$. The growth rate of the LP353 with 4.8-kb fragment was almost same as that of wild type strain in liquid medium with 1.2 M sorbitol at nonpermissive temperature. Taken these results together, the 4.8-kb fragment seemed to contain the BGS2 gene for ${\beta}-1,3-glucan$ synthase activity in yeast S. cerevisiae.

• Journal of Plant Biotechnology
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• v.37 no.2
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• pp.174-185
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• 2010

Full-length cDNAs are essential for the correct annotation of genomic sequences and for the functional analysis of genes and their products. To elucidate the functions of a large population of Chinese cabbage (Brassica rapa) genes and to search efficiently for agriculturally useful genes, we have been taking advantage of the full-length cDNA Over-eXpresser (FOX) gene hunting system. With oligo dT column it purify the each mRNA from the flower organs, leaf and stem tissue. And about 120,000 cDNAs from the library were transformed into $\lambda$-pFLCIII-F vector. Of which 115,000 cDNAs from the library were transformed into T-DNA binary vector, pBigs for transformation study. We used normalized full-length cDNA and introduced each cDNA into Arabidopsis by in planta transformation. Full-length Chinese cabbage cDNAs were expressed independently under the CaMV 35S promoter in Arabidopsis. Selfed seeds were harvested from transgenic Arabidopsis. We had selected 2,500 transgenic plants by hygromycin antibiotic tolerant test, and obtained a number of transgenic mutants. Each transgenic Arabidopsis was investigated in morphological changes, fertility and leaf colour. As a result, 285 possible morphological mutants were identified. Introduced cDNA was isolated by PCR amplification of the genomic DNA from the transgenic mutants. Sequencing result and BLAST analysis showed that most of the introduced cDNA were complete cDNAs and functional genes. Also, we examined the effect of Bromelain on enhancing resistance to soft rot in transgenic Chinese cabbage 'Osome'. The bromelain gene identified from FOX hunting system was transformed into Chinese cabbage using Agrobacterium methods. Transformants were screened by PCR, then RT-PCR and real time PCR were performed to analyze gene expression of cysteine protease in the T1 and T2 generations. The anti-bacterial activity of bromelain was tested in Chinese cabbages infected with soft rot bacteria. The results showed that the over-expressed bromelain gene from pineapple conferred enhanced resistance to soft rot in Chinese cabbage.

• Journal of Microbiology and Biotechnology
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• v.15 no.4
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• pp.838-843
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• 2005

Brain-derived neurotrophic factor (BDNF) is a small secretory protein and a member of the nerve growth factor (NGF) gene family. We cloned the flounder BDNF gene from a flounder brain cDNA library. The nucleotide sequence of the cloned gene showed an open reading frame (ORF) consisting of 810 bp, corresponding to 269 amino acid residues. The tissue distribution of flounder BDNF was determined by reverse transcription-polymerase chain reaction (RT-PCR) in brain, embryo, and muscle tissues. To express fBDNF using a eukaryotic expression system, we constructed the vector mpCTV-BDNF containing the fBDNF gene and transformed this vector into Chlorella ellipsoidea. Stable integration of introduced DNA was confirmed by PCR analysis of genomic DNA, and mRNA expression in C. ellipsoidae was confirmed by RT-PCR analysis.

• Microbiology and Biotechnology Letters
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• v.23 no.2
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• pp.178-186
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• 1995

Streptomyces albus wild type ATCC 21838 produced salinomycin, polyether antibiotic. To clone genes related salinomycin production, a genomic library was screened using actI as a DNA hybridization probe. pWHM 210 was isolated, which contained an approximately 24 kb of insert DNA. A 3.8 kb region in the 24 kb insert DNA was hybridized to actI and the nucleotide sequence of this region was determinied. Two open reading frames found in the same direction were homologous to genes for $\beta$-keto acyl synthase/acyl transferase and chain length determining factor in type II PKS (polyketide synthase). The genes were components of minimal type II PKS genes, highly conserved and showed the strong simiarity to other type II PKS genes known today.

• Journal of the korean academy of Pediatric Dentistry
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• v.33 no.2
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• pp.304-310
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• 2006

Prevotella intermedia is one of the most frequently implicated pathogens in human periodontal disease and has a requirement for hemin for growth. This study has identified a hemin-binding P. intermedia protein by expression of a P. intermedia genomic library in Escherichia coli, a bacterium which does not require or transport exogenous hemin. The genomic library of P. intermedia was constructed into plasmid pUC18, transformed into Escherichia coli strain $DH5{\alpha}$, and screened for recombinant clones using heminbinding activity by plating onto hemin-containing agar. Approximately 5,000 recombinant E. coli colonies were screened onto LB-amp-hemin agar, single clone(pHem1) was exhibited a clearly pigmented phonotype. The 2.5kb insert DNA of pHem1 was determined by restriction enzyme mapping. Southern blot analysis of BamHI, BglII, EcoRI, HindIII and PstI-digested P. intermedia DNA indicated that single copy of the gene was present in the genome. Northern blot analysis revealed that the size of transcript was approximately 1.8 kb. The cloned gene contained a single ORF, consisting of approximately 850-residue amino acids. A BLAST search of the Institute for Genomic Research genes with similar nucleotide sequence revealed no significant similarity It needs further investigation to clarify the mechanisms of heme uptake in P. intermedia.

• BMB Reports
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• v.29 no.1
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• pp.52-57
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• 1996

A specific DNA fragment from Korean radish (Raphanus sativus L.) was amplified by performing PCR with oligonucleotide primers which correspond to the highly conserved regions of plant peroxidases. The size of the PCR product was ca. 400 bp, as expected from the known plant peroxidase genes. Comparison of the nucleotide and deduced amino acid sequences of the PCR product to those of other plant peroxidase-encoding genes revealed that the amplified fragment corresponded to the highly conserved region I and III of plant peroxidases. By screening a genomic library of Korean radish using the amplified fragment as a probe, two positive clones, named prxK1 and prxK2, were isolated. Restriction mapping studies indicated that the 5.2 kb Sail fragment of the prxK1 clone and the 4.0 kb EcoRI fragment of the prxK2 clone encode separate isoperoxidase genes. Analyses of the promoter region of the prxK1 clone shows that putative CAAT box, CMT box, and TGA1b binding sequence (5' TGACGT) are present 718 bp upstream from the start codon.

• Korean Journal of Microbiology
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• v.32 no.4
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• pp.264-270
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• 1994

A gene coding for a novel putative $\sigma$ factor of RNA polymerase has been identified from Streptomyces coelicolor A3(2) using Escherichia coli rpoS gene fragment as a probe. The 486 bp rpoS gene fragment was amplified from E. coli genomic DNA by PCR with two synthetic oligonucleotides, the sequences of which were deduced from the amino acid sequences in the regions 2.3 and 4.2 conserved among various bacterial factors. When E. coli genomic DNA fragments were hybridized with cloned rpoS probe, only one band corresponding to rpoS gene (3.2 kb PvuII fragment or 2.3 kb KpnI fragment) was detected. In S. coelicolor, however, two bands were detected both in PvuII digested DNA and SalI digested DNA. 3.5 kb PvuII fragment which binds the rpoS gene probe was cloned (pMS1) from the sublibrary, and the nucleotide sequences of 1.0 kb BamH'/HincII subclone (pBH2) was partially determined. The nucleotide sequences revealed extensive similarity to other $\sigma$ factor genes of S. coelicolor (hrdA, hrdB, hrdC, hrdD), S. aureofaciens (hrdA, hrdB, hrdC, hrdD), Synechococcus species, Pseudomonas aeruginosa, Stigmatella aurantiaca, and Anabaena species. The nucleotide sequences in regions 1.2 and 4 were compared with the corresponding regions of 5 known ${\sigma}$ factor genes of S. coelicolor by multiple alignment. It turned out that the cloned gene is most closely related to hrdA showing 88% amino acid similarity in region 1.2 and 75% in region 4.

• The Korean Journal of Mycology
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• v.42 no.1
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• pp.50-56
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• 2014

Transposable elements (TEs) are mobile DNA elements that often cause mutations in genes and alterations in the chromosome structure. In order to identify and characterize transposable elements (TEs) in Pleurotus eryngii, a TE-enriched library was constructed using two sets of TE-specific degenerated primers, which target conserved sequences of RT and RVE domains in fungal LTR retrotransposons. A total of 256 clones were randomly chosen from the library and their insert sequences were determined. Comparative investigation of the insert sequences with those in repeat element database, Repbase, revealed that 71 of them were found to be TE-related fragments with significant similarity to LTR retrotransposons from other species. Among the TE sequences, the 70 TEs were Gypsy-type LTR retrotransposons, including 20 of MarY1 from Tricholoma matsutake, 26 of Gypsy-8_SLL from Serpula lacrymans, and 16 of RMER17D_MM from mouse, whereas a single sequence, Copia-48-PTR, was found as only Copia-type LTR retrotransposon. Southern blot analysis of the HindIII-digested P. eryngii genomic DNA showed that the retrotransposon sequences similar to MarY1 and Gypsy-8_SLL were contained as high as 14 and 18 copies per genome, respectively, whereas other retrotransposons were remained low. Moreover, both of the two Gypsy retrotransposons were expressed in full length mRNA as shown by Northern blot analysis, suggesting that they were functionally active retrotransposons.

• Microbiology and Biotechnology Letters
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• v.36 no.3
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• pp.189-194
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• 2008

The phospholipase B (PLB) families are enzymes sharing phospholipase (PL), lysophospholipase (LPL) and lysophospholipase-transacylase (LPTA) activities. In this study, we report the putative gene encoding phospholipase B (plbA) containing lipase motifs was cloned for the first time from the filamentous fungus, Aspergillus nidulans. plbA was isolated from A. nidulans genomic DNA library using a PCR-amplified probe, which is designed on the basis of sequence information derived from the conserved lipase regions of various PLBs. The deduced product of plbA is of 626 amino acids. From the assigned sequence, PlbA showed 72% identity with Penicillium notatum PLB but have low similarity with phospholipase A of other organisms.

• Microbiology and Biotechnology Letters
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• v.16 no.5
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• pp.363-368
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• 1988

Molecular cloning of gene for $\alpha$-acylamino-$\beta$-lactam acylhydrolase (ALAH) III from Acetobacter turbidans has been attempted by immunochemical detection method, in which polyclonal antibody from mouse Balb/c against this enzyme was employed as a probe. As a cloning vector, λ gtll was chosen for this purpose. Two positive clones has been selected from genomic libraries of A. turbidans, which had somewhat different binding affinities on anti-ALAH III umm and anti-$\beta$-galactosidase. By restriction analysis, both clones has been turned out to lose one of EeoRI sites. From these results, it concluded that deletion of DNA between lacZ gene and inserted DNA has occurred during replication of these clones in host cells.