• Microbiology and Biotechnology Letters
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• v.14 no.2
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• pp.155-160
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• 1986

$\alpha$-Amylase gene of B. amyloliquefaciens was cloned to E. coli-yeast shuttle vector YEp-13 and expressed in E. coli. Chromosomal DNA of B. amyloliquefaciens was partially digested with Sau3Al and YEp13 plasmid was cleaved with BamH1. The hybrid plasmid, pHA28, was constructed by shotgun method and transformed to E. coli C600 and HB101. The amount of $\alpha$-amylase produced by transformants of E. coli was about 20% to 30% of that produced by B. amyloli-quefaciens. About 65% of $\alpha$-amylase produced by transformant was secreted into periplasm and the others were located in cytoplasm. $\alpha$-Amylase production was maximal when transformants were cultivated for 15hr to 20hr. As the result of agarose gel electrophoresis, pHA28 plasmid was found to be various in its size. This result suggested that pHA28 plasmid was segregated.

• International Journal of Industrial Entomology and Biomaterials
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• v.18 no.2
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• pp.113-120
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• 2009

Embryonic lethal abnormal visual (elav) is a lethal gene in Drosophila inducing the abnormal development and function of nervous system. We cloned a Bm-elav gene by bioinformatics and biological experiment, based on sequence of ELAV protein and dbEST of Bombyx mori. The full-length of Bm-elav cDNA is 1498 bp, contains a 906 bp open read frame (ORF) encoding a precursor of 301 amino acid residues with a calculated molecular weight of 34 kDa and pI of 8.99. Bm-ELAV protein precursor contains three RNA recognition motifs (RRM) in $24{\sim}91$, $110{\sim}177$ and $222{\sim}295$ bit amino acid residues respectively, and belongs to RNA-binding protein family. Bm-ELAV shared varying positives, ranging from 56% to 60% (Identities from 41% to 45%), with RRM from other species of Xenopus tropicalis, Apis mellifera, Tribolium castaneum, Branchiostoma belcheri and Drosophila. Gene localization indicated that Bm-elav is a single-copy gene, gene mapping within 12-chromosome from 7916.68 knt to 7918.16 knt region of nscaf2993. Spatiotemporal expressions pattern analysis revealed that Bm-elav expressed higher in most tested tissues and developmental stages in whole generation, such as silk gland, fat body, midgut, hemopoietic organ and ovary, but almost no expression in terminated diapause eggs. This suggested that the expression of Bm-elav in early developmental embryonic stages might induce abnormal development like in Drosophila. Cloning of the Bm-elav gene enables us to test its potential role in controlling pests by transferring the gene into field lepidopteran insects in the future.

• Journal of Microbiology and Biotechnology
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• v.17 no.1
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• pp.74-80
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• 2007

An enantioselective lipase gene (esf) for the kinetic resolution of optically active (S)-flurbiprofen was cloned from the new strain Serratia marcescens ES-2. The esf gene was composed of a 1,845-bp open reading frame encoding 614 amino acid residues with a calculated molecular mass of 64,978 Da. The lipase expressed in E. coli was purified by a three-step procedure, and it showed preferential substrate specificity toward the medium-chain-length fatty acids. The esf gene encoding the enantioselective lipase was reintroduced into the parent strain S. marcescens ES-2 for secretory overexpression. The transformant S. marcescens BESF secreted up to 217kU/ml of the enantioselective lipase, about 54-fold more than the parent strain, after supplementing 3.0% Triton X-207. The kinetic resolution of (S)-flurbiprofen was carried out even at an extremely high (R,S)-flurbiprofen ethyl ester [(R,S)-FEE] concentration of 500 mM, 130 kU of the S. marcescens ES-2 lipase per mmol of (R,S)-FEE, and 1,000 mM of succinyl ${\beta}-cyclodextrin$ as the dispenser at $37^{\circ}C$ for 12h, achieving the high enantiomeric excess and conversion yield of 98% and 48%, respectively.

• Journal of Plant Biotechnology
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• v.37 no.2
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• pp.115-124
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• 2010

As the completion of genome sequencing, large collection of expression data and the great efforts in annotating plant genomes, the next challenge is to systematically assign functions to all predicted genes in the genome. Functional genome analysis of plants has entered the high-throughput stage. The generations and collections of mutants at the genome-wide level form technological platform of functional genomics. However, to identify the exact function of unknown genes it is necessary to understand each gene's role in the complex orchestration of all gene activities in the plant cell. Gene function analysis therefore necessitates the analysis of temporal and spatial gene expression patterns. The most conclusive information about changes in gene expression levels can be gained from analysis of the varying qualitative and quantitative changes of messenger RNAs, proteins and metabolites. New technologies have been developed to allow fast and highly parallel measurements of these constituents of the cell that make up gene activity. We have reviewed currently employed technologies to identify unknown functions of predicted genes including map-based cloning, insertional mutagenesis, reverse genetics, chemical mutagenesis, microarray analysis, FOX-hunting system, gene silencing mutagenesis, proteomics and chemical genomics. Recent improvements in technologies for functional genomics enable whole-genome functional analysis, and thus open new avenues for studies of the regulations and functions of unknown genes in plants.

• Journal of Microbiology and Biotechnology
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• v.19 no.12
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• pp.1542-1546
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• 2009

Zeaxanthin glucosyltransferase (CrtX) mediates the formation of zeaxanthin to zeaxanthin diglucoside. Here, we report cloning of the crtX gene responsible for zeaxanthin diglucoside biosynthesis from Paracoccus haeundaensis and the production of the corresponding carotenoids in transformed cells carrying this gene. An expression plasmid containing the crtX gene (pSTCRT-X) was constructed, and Escherichia coli cells containing this plasmid produced the recombinant protein of approximately 46 kDa. Biosynthesis of zeaxanthin diglucoside was obtained when the plasmid pSTCRT-X was co-transformed into E. coli containing the pET-44a(+)-CrtEBIYZ carrying crtE, crtB, crtI, crtY, and crtZ genes required for zeaxanthin $\beta$-D-diglucoside biosynthesis.

• BMB Reports
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• v.39 no.6
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• pp.749-758
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• 2006

The cytosolic members of the HSP70 family of proteins play key roles in the molecular chaperone machinery of the cell. In the study we cloned and sequenced the full-length cDNA of Delia antiqua HSP70 gene, which is 2461 bp long and encodes 643 a.a. with a calculated molecular mass of 70,787 Da. We investigated gene copies of cytosolic HSP70 members of 4 insect species with complete genome available, and found that they are quite variable with species. In order to characterize this protein we carried out an alignment and a phylogenetic analysis with 41 complete protein sequences from insects. The analysis divided the cytosolic members of the family into two classes, HSP70 and HSC70, distinguishable on the basis of 15 residues. HSP70 class members were slightly shorter in length and smaller in molecular mass relative to the HSC70 class members, and the conservative and functional regions in these sequences were documented. Mainly, we investigated the expression of Delia antiqua HSP70 gene, in response to diapauses and thermal stresses. Both summer and winter diapauses elevated HSP70 transcript levels. Cold-stress led to increased HSP70 expression levels in summer- and winter-diapausing pupae, but heat-stress elevated the levels only in the winter-diapausing pupae. In all cases, the expression levels, after being elevated, gradually decreased with time. HSP70 expression was low in non-diapausing pupae but was up-regulated following cold- and heat-stresses. Heat-stress gradually increased the mRNA level with time whereas cold-stress gradually decreased levels after an initial increase.

• Journal of Microbiology
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• v.44 no.6
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• pp.617-621
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• 2006

Coprinellus congregatus secreted a laccase isozyme when the culture was transferred to an acidic liquid medium (pH 4.1). The laccase cDNA gene (clac2) was used as a probe for cloning of the genomic laccase gene (lac2) including the promoter (Plac2). The open reading frame (ORF) of lac2 had 526 deduced amino acids and four conserved copper binding domains as other fungal laccases. Recombinant plasmid (pRSlac2p-cDNA) of lac2 cDNA with its own promoter was transformed in Saccharomyces cerevisiae. Expression of the transformed lac2 gene was induced by oxidative stress ($H_2O_2$) in yeast and the survival rate of the transformed yeast strain was greatly increased when compared with that of the control strain transformed with pRS316 yeast vector.

• Journal of Microbiology and Biotechnology
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• v.10 no.1
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• pp.86-90
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• 2000

We have earlier reported on the cloning and identification of bft-k from an enterotoxigenic strain of Bacteroides fragilis 419, which was isolated from the blood of a Korean patient who suffered from systemic infections [4,5]. The bft-k gene encodes a 397-amino-acids metalloprotease enterotoxin, and the protein has been identified as a new isoform of B. fragilis enterotoxins (BFTs), which are cytopathic to intestinal epithelial cells to induce fluid secretion and tissue damage in ligated intestinal loops [4, 6, 18, 20]. This report describes the cloning and sequencing of the enterotoxin pahogenicity islet of B. fragilis 419 which contains the bft-k gene. the cloned enterotoxin pathogenicity islet was found to have 6,045 bp in length and to contain 120bp direct repeats near its end. In the pathogenicity islet, in addition to the BFR-K, two putative open reading frames (ORFs) were identified; (1) the t-3 gene encoding a 396-amino-acids protein of a putative metalloprotease; (2) the third gene encoding an ORF of a 59-amino-acids protein, whose function has not yet beenn characterized. The expression of the t-3 gene in B. fragilis 419 was verified by western blot analysis.

• The Korean Journal of Mycology
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• v.17 no.1
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• pp.35-38
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• 1989

Gene libraries of DNA from Flammulina velutipes were constructed using Escherichia coli plasmid pBR 322. Leu 2 gene coding ${\beta}-isopropylmalate$ dehydrogenase from F. velutipes was cloned by complementation of leucine requiring mutant of E. coli. The size of inserted DNA fragment of this clone is about 1 Kbp. The fragment has Bam H1 and Ava 1 restriction sites.

• Microbiology and Biotechnology Letters
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• v.17 no.2
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• pp.160-164
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• 1989

A 5.7Kb EcoRI fragment containing alkaline amylase gene of Bacillus sp. AL-8 obtained in the previons experiment (10) was transformed in B. subtilis via plasmid pUB110. The enzymatic proper-ties of the amylase produced by the transformants were Identical to those of the donor strain. Thus, the alkaline amylase activity from the transformant was maximum at pH 10 and 5$0^{\circ}C$. And the enzyme was very stable over the ranges of alkaline pH. In order to determine the location of the alkaline amylase gene within the 5.7Kb DNA fragment, the fragment was subcloned in E. coli. It was found that the alkaline amylase gene was located k EcoRI fragment of 3.7Kb.