• Korean journal of applied entomology
• /
• v.32 no.3
• /
• pp.300-306
• /
• 1993

The CryIIA gene encoding the insecticidal crystal protein of Bacillus thuringiens!s subsp. kurstalri HD-l has been cloned in Escherichia col!, and its nucleotide sequences were determined completely. 5kb Hindlli fragment harboring CryIIA gene was screened in the large ca. 225kb plasmid DNA by southern blot. HindlIT digested 5kb fragment was ligated into pUC19 and transformed in E. coli. The 4kb BamHI-HindlIT fragment containing the CryIIA gene was subcloned and named pSKIIA. DNA sequence analysis demonstrates that pSKIIA is the gene of an operon which is comprised of Lhree open reading frames (designated orn, orf2 and or£3). The CrylIA gene is composed of 3,952bp-long BamHI-Hindill DNA restriction fragment. The orf3 code for a polypeptide of 633 amino acid residues. The protoxin protein has a predicted molecular weight of 70,780. The E. coli derived protoxin gene product is biologICally active against three species of Lepidopteran (Plu.lelia maculipennis, He/iolhis assulta, Spodoptera litura) and a species of Dip Leran( Culex pipines) larvae in bioassay.

• Applied Biological Chemistry
• /
• v.38 no.2
• /
• pp.118-122
• /
• 1995

Escherichia Coli ornithine transcarbamylase is the enzyme which catalyzes the L-citrulline biosynthesis from L-ornithine and carbamyl phosphate. To facilitate the purification of enzyme which will be used for many biochemical studies such as structure and function relationships and catalytic mechanisms, the cloning and expression of E. coli argI gene for ornithine transcarbamylase was conducted. argI was amplified from genomic DNA of E. coli strain of $DH5{\alpha}$, by polymerization chain reaction (PCR) method. The amplified argI gene was ligated to the prokaryotic expression vector pKK223-3 and used for transformation of E. coli TB2 which was deficient of ornithine transcarbamylase. The over-produced enzyme by the tnansformant was purified by ammonium sulfate fractionation, heat denaturation and affinity chromatography. The result of SDS denaturation gel electrophoresis for the purified enzyme showed a single band of about 38 kDa of ornithine transcarbamylase. Kinetic data for the expressed enzyme gave almost the s?????? values as those of the wild type enzyme. The $k_{cat}$, of the enzyme was $1.0{\times}10^5min^{-1}$, and $K_ms$ for ornithine and carbamyl phosphate were 0.35 mM and 0.06 mM, respectively.

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

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

• Journal of Mushroom
• /
• v.2 no.3
• /
• pp.145-148
• /
• 2004

This study was carried out to screen the fruiting body formation-specific genes from the medicinal mushroom Cordyceps militaris. A cDNA synthesized using total RNA from 4 stages of mushroom development, mycelium, primordium, immature fruiting body and mature fruiting body. Differential expression gene screening was performed by DD-PCR(Differential Display Arbitrary Primer PCR) with cDNA, we sequenced partial 6 genes using pGEM cloning vector. The DNA Sequence of the six DD-PCR products derived from differentially expressed genes was compared to that in the GenBank database by using the NCBI BLAST search to identify similarities to known sequences. Sequence analysis showed that six of DD-PCR products have unknown sequence.

• Korean Journal of Microbiology
• /
• v.41 no.4
• /
• pp.306-311
• /
• 2005

In this study, the amino-terminal half truncated lump and the whole lump genes from Photobacterium phosphoreum coding for the lumazine protein were cloned by polymerase chain reaction and expressed in Escherichia coli. To identifiy of the binding site of the ligand or substrate, the amino acid identities from the sequences of the lumazine protein, yellow fluorescent protein, and riboflavin synthase from different organisms were also compared and analyzed.

• Applied Biological Chemistry
• /
• v.39 no.6
• /
• pp.443-448
• /
• 1996

In order to investigate the function of xylA promoter(Pxyl) as regulatory region Pxyl-lacZ fusion gene was constructed by the insertion of xylA promoter to the multiple cloning site of upstream of lacZ gene in a multicopy numbered plasmid pMC1403 containing promoterless lac operon, which was designated pMCX191, and Pxyl-lacZ fragment from pMCX191 was inserted to low copy numbered plasmid pLG339, designated pLGX191. The expressions of ${\beta}-galactosidase$ in these recombinant plasmids containing Pxyl-lacZ fusion gene were induced strongly by the addition of xylose, repressed by the addition of 0.2% glucose in the presence of xylose. The catabolite repressions were derepressed by the addition of 1 mM cAMP as same as native xylA gene. The fragment of xylA promoter was partially deleted from the upstream of xylA promoter by exonuclease III to investigate the regulation site of xylA promoter and the degrees of deletion derivatives of xylA promoter were analyzed by the DNA base sequencing. By the investigations of the induction by xylose, repression by glucose and derepression by cAMP on xylose isomerase production, the regulation site of xylA promoter may be located in segment between -165 and -59 bp upstream from the initiation site of xylA translation.

• Journal of Korean Society of Forest Science
• /
• v.87 no.3
• /
• pp.334-340
• /
• 1998

This research was conducted to identify plant regulatory regions by gene tagging method. A promoterless GUS coding sequence was introduced to Populus nigra ${\times}$ P. maximowiczii via Agrobacterium strains(LBA4404/EHA101), and putative transgenic poplars were selected by culturing on medium containing G418($60mg/{\ell}$) and by GUS assay. Among them one positive plant was to amplify the native sequences flanking to the introduced GUS gene in plant genome by inverse PCR method and from this 730 by DNA product was obtained. After subcloning and sequencing, it has 88% homology to the Eucalyptus gunnii CAD(cinnamyl alcohol dehydrogenase) gene. The GUS gene fused with the putative promoter reinserted into poplar leaves by particle bombardment method to test the funtional promoter activity. Upon staining with X-gluc, many blue spots appeared on the leaf segments bombarded by the chimeric gene 2-3 days, thus the isolated DNA fragment contain some possible coding region as well as a putative regulatory sequences of poplar CAD gene.

• Journal of Life Science
• /
• v.19 no.6
• /
• pp.809-817
• /
• 2009

The phenotypes associated with a gene function are often the best clue to its role in the plant. Transgenic plants ectopically expressing or suppressing a gene can provide useful information related to the gene function. In this study, we constructed three vectors - pFGL571, pFGL846 and pFGL847 - for the Agrobacterium-mediated ectopic expression of plant genes using pPZP211 and modified CaMV 35S, UBQ3 or UBQ10 promoters. The three vectors have several merits such as small size, high copy in bacteria, enough restriction enzyme sites in multi cloning sites and nucleotide sequence information. Analysis of transgenic plants containing GUS or sGFP reporter genes under the control of modified CaMV 35S, UBQ3 or UBQI0 promoter revealed that all of the three promoters showed high activities during most developmental stages after germination and in floral organs. Furthermore, we generated a RNAi module vector, pFGL727, to suppress plant gene expressions and confirmed that pFGL727 is useful for the suppression of a gene expression using rice transgenic plants. Taken together, our new vectors would be very useful for the ectopic expression or the suppression of plant genes.

• The Microorganisms and Industry
• /
• v.15 no.2
• /
• pp.24-28
• /
• 1989

폐염균의 특징은 폐염, 류마티스성 심장병, 수막염 등의 질병을 일으키는 병원균이라는 것과 아주 높은 효율로 형질전환을 할 수 있다는 사실이다 ($10^{8}$ 개의 균에 염색체 DNA 1.mu.g을 써서 실험하였을 때 $10^{6}$개의 transformant를 얻을 수 있음). 폐염균이 자연상태에서 이렇게 높은 효율의 형질전환을 할 수 있는 이유는 정상생리의 일부로서 형질전환에 관계된 유전자가 형질전환을 할때 발현되기 때문이다. 그러나 폐염균 형질전환에 대한 연구는 아직도 초기단계라고 할 수 있으며 그 이유는 현재 전세계적으로 극소수의 연구자들만이 연구를 수행하고 있기 때문이다. 즉, 미국 Brookhaven National Laboratory의 Lacks와 프랑스 CNRS의 Claverys는 mismatch repair에 대한 연구를, Illinois 대학의 Morrison은 competence에 대해, 미국 Rockefeller 대학의 Hotchkiss와 Tomasz는 DNA binding을 연구하고 있을 뿐이다. 본 논고에서는 폐염균 형질전환에 대해 지금까지의 연구 상태와 문제점, recP의 클로닝에 대해 소개하고자 한다.