• Journal of Life Science
• /
• v.17 no.2 s.82
• /
• pp.241-247
• /
• 2007

The cycloinulooligosaccharide fructanotransferase (CFTase) gene (cft) from Paenibacillus macerans was subcloned into the surface display vector, pCTcon (GAL1 promoter). The constructed plasmid, pCTECFTN (9.0 kb) was introduced to S. cerevisiae EBY100 cell and then east transformants were selected on the synthetic defined medium lacking uracil and on the inulin containing medium. The surface display of CFTase was confirmed by immunofluorescence microscopy and its enzymatic ability to form cycloinulooligosaccharides(cyclofructans, CFs) from inulin. The total activity of the CFTase was reached about 5.52 unit/1 by cultivation of yeast transformant on YPDG medium. The optimized conditions determined were as follows; pH, 8.0; temperature, $50^{\circ}C$ ; substrate concentration, 5%; inulin source, Jerusalem artichoke. By the reaction with inulin, CFs consisting of cycloinulohexaose (CF6), cycloinuloheptaose (CF7), and cycloinulooctaose (CF8) were produced and CF6 was the major product.

• The Korean Journal of Mycology
• /
• v.24 no.2 s.77
• /
• pp.111-126
• /
• 1996

Transfer of the isolated nuclei from Lentinus edodes into protoplasts of Pleurotus florida was induced with polyethlene glycol (PEG) and $CaCl_2$. The intergeneric transfer products were classified into nuclear hybrid, heterokaryon or synkaryon, and reconstituted cell. These progenies except nuclear hybrids formed mature fruiting bodies on sawdust rice bran medium. Formation of fruit bodies was influenced by several factors such as light, temperature, nutrition and physic state of the culture media. Most of fruiting body characters were similar to those of P. florida in synkaryon and L. edodes in reconstituted cell, respectively. All these basidiocarps had clamp connections though initial heterokaryon colonies were lacking. Isozyme patterns of intergeneric progenies were quite different from those of parents. DNA polymorphisms of transfer products were also compared by random amplified polymorphic DNAs (RAPD) analysis based on polymerase chain reaction. The RAPD patterns were different from those of donor and recipient. DNA fingerprints ranged in size from 0.25 to 4.0 Kb. On the basis of RAPD, the transfer products were classified into five groups. Two synkaryon were analysed with distribution of progenies and segregation of genetic markers by random spore analyses. The genetic markers were segregated into wild type and riboflavine requiring auxotrophs.

• Korean Journal of Microbiology
• /
• v.25 no.2
• /
• pp.94-102
• /
• 1987

Rat liver LDH A-cDNA has been isolated from a .lambda.gt11-rat lover cDNA library and partially characterized. The size of the isolated rat liver LDH A-cDNA if shown to be 1.6Kb and restriction enzyme sites for the rat liver LDH A-cDNA are also mapped. 682-nucleotide sequence coding for 3'-end of rat liver LDH A-cDNA has been analyzed and compared to the nucleotide sequence of the same region of rat $C_6$-glioma cell LDH A-cDNA which has been cloned from the hormonally stimulated cell cultures. The result shows that 177 nucleotide sequences coding for the C-terminal 59-amino acids are identical but 505 nucleotide sequences of 3'-nontranslated region of the two LSH A-cDNA exhibit characteristic differences in thier nucleotide sequences. Computer analysis for the folding structures for 3'-end 400 nucleotide sequences of the two LDH A-cDNA shows a possibility implying that the two LDH A-mRNAs isolated from different tissues of rats may have different half life and therefore their translational efficiency may be different. It has been previously demonstrated that isoproterenol stimulated rat $C_6$ -glioma cell cultures produce LDH A-mRNA showing 2 to 3-fold longer half life in comparison to that of noninduced LHD A-mRNA. The result therefore support for the idea that hormonally stimulated rat $C_6$-glioma cells may produce LDH A-mRNA containing different nucleotide sequences at the 3'-end nontranslated region by which the hormonally induced LDH A-mRNA could have more stable secondary mRAN structure in comparison to that of noninduced LDH A-mRNA.

• Proceedings of the Korean Biophysical Society Conference
• /
• 2003.06a
• /
• pp.34-34
• /
• 2003

Protein unfolding is a key step in several cellular processes, including protein translocation across some membranes and protein degradation by ATP-dependent proteases. C1pAP protease and the proteasome can actively unfold proteins in a process that hydrolyzes ATP, These proteases catalyze unfolding by processively unraveling their substrates from the attachment point of the degradation signal. As a consequence, the ability of a protein to be degraded depends on its structure as well as its stability. An ${\alpha}$-helix is easier to unravel than a ${\beta}$-strand. In multidomain proteins, independently stable domains are unfolded sequentially. The steric constraints imposed on substrate proteins during their degradation by the proteasome were investigated by constructing a model protein in which specific parts of the polypeptide chain were covalently connected through disulfide bridges. The cross-linked model proteins were fully degraded by the proteasome, but two or more cross-links retarded the degradation slightly. Our results suggest that the pore of the proteasome allows the concurrent passage of at least three stretches of a polypeptide chain, and also explain the limited degradation by the proteasome that occurs in the processing of the transcription factor NF-KB, and also implicate difficulty in degradation of amyloidal aggregates by the proteasome

• Asian-Australasian Journal of Animal Sciences
• /
• v.16 no.7
• /
• pp.1055-1059
• /
• 2003

Keratins, the constituents of epithelial intermediate filaments, are precisely regulated in a tissue and development specific manner. There are two types of keratin in bovine. The type I is acidic keratin and the type II is neutral/basic keratin. 1.5 kb of 5' flanking sequence of Korean cattle Keratin IV gene, type II keratin (59 kDa), was cloned and sequenced. A symmetrical motif AApuCCAAA are located in a defined region upstream of the TATA box. Proximal SP1, AP1, E-box and CACC elements as the major determinants of transcription are identified. When it was compared to the bovine sequence from -600 bp to ATG upstream, the homology was 97% in nucleotide sequence. Several A and T sequences, located in the promoter region, are deleted in the Korean cattle. An expression vector consisted of Korean cattle Keratin IV gene promoter/SV40 large T antigen was transfected to HaCaT cell (Epithelial keratinocyte). The transformed HaCaT cells showed active proliferation when treated with PDGF (Platelet-derived growth factor) in 0.3% soft agar compared to control cells. These results indicate that Korean cattle Keratin IVgene promoter can be used as a promoter for transfection into epithelial cell.

• Parasites, Hosts and Diseases
• /
• v.39 no.2
• /
• pp.133-141
• /
• 2001

A cDNA of 1.1 kb comprising the gene encoding the peroxiredoxin of Toxo-plasma gondii(TgPrx) has been cloned. The open reading frame of 591 Up was translated into a protein of 196 amino acids with a molecular mass of 25 kDa. Conserved 2 cysteine domains of Phe-Val-Cys-Pro and Glu-Val-Cys-Pro indicated TgPrx belonged to 2-Cys Prx families. TgPrx showed the highest homology with that of Arabidopsis thaliana by 53.9% followed by Entamoeba histolytica with 39.5% by the amino acid sequence alignment. Polyclonal antibody against recombinant TgPrx detected 25 kDa band in T. gondii without binding to host cell proteins TgPrx was located in the cytoplasm of T. gondii extracellularly or intracellularly by immunofluorescence assay. The expression of TgPrx was increased as early as 30 min after the treatment with artemisinin in the intracellular stage, while no changes in those of host Prx I and TgSOD. This result implies that TgPrx may function as an antioxidant protecting the cell from the attack of reactive oxygen intermediates. It is also suggested that TgPrx is a possible target of chemotherapy.

• Biomedical Science Letters
• /
• v.8 no.3
• /
• pp.107-113
• /
• 2002

Enterohemorrhagic Escherichia coli (EHEC) strains of serotype O157:H7 have been shown to colonize the intestinal epithelial cell by the attaching and effacing (AE) mechanism. The AE lesion is mediated by an intimin, of which production and expression are controlled by a 3-Kb eaeA gene located EHEC chromosomal DNA. If the eaeA gene is mutated, EHEC O157:H7 strains lose capacity of adhesion to intestinal epithelial cells. In this study, a 891 bp of the 3'-end region of a gamma intimin was amplified by polymerase chain reaction (PCR). The PCR product was inserted into pSTBlue-1 cloning vector and transformed into DE3 (BL21) competent cell. After plasmid mini-preparation and restriction enzyme digestion of eaeA/891-pSTBlue-1 vector, target eaeA gene was re-inserted into pET-28a expression vector and was transformed. Then the expression of recombinant eaeA/891 (891 bp) gene was induced by isopropyl-$\beta$-D-thiogalactopyranoside (IPTG). The expression of the 40-KDa recombinant protein was identified in SDS-PAGE and confirmed by immunoblotting using the His.Tag$^{\circledR}$ and T$_{7}$.Tag$^{\circledR}$ monoclonal antibody. This recombinant protein expressed by eaeA gene could be applied in further studies on the mechanisms of E. coli O157:H7 infection and the development of recombinant vaccine.

• Biomolecules & Therapeutics
• /
• v.14 no.3
• /
• pp.148-151
• /
• 2006

We have previously reported that 2.4 kb of L-plastin promoter (LP) could regulate the expression of adenoviral vector (AV) exogenous genes in a tumor cell specific manner. In the present study, we tested if the replication competent AdLPE1A vector results in a direct cytotoxic effect in hepatocelluar carcinoma (HCC) cells. In vitro cytotoxicity tests were carried out with replication-competent (AdLPE1A) and -incompetent (AdLPCD) LP-driven vectors. AdLPE1A is an AV in which LP was inserted 5' to the E1A and E1B genes. The AdLPCD vector contains LP and the E. coli cytosine deaminase (CD) gene in transcription unit. Exposure of cells to AdLPE1A generated a significant cytotoxic effect as compared to the control. Almost 90% of the cell had manifested the characteristic cytopatic effect on day 9 after infection of cells with 10 MOI of AdLPE1A. On the other hand, almost 35% of the cells were left when the cells had been treated with 100 MOI of AdLPCD together with 5-FC on day 9 when compared with the cells which had never been exposed neither 5-FC nor AdLPCD. These results showed that the replication competent AdLPE1A vector could kill the HepG2 cells directly by the oncolytic effect of the virus. The replication competent AV vector carrying viral E1A generated greater cytotoxic effect than the replication incompetent AV, which contains the CD prodrug activation transcription unit without E1A, in HepG2 cells.

• Animal cells and systems
• /
• v.7 no.2
• /
• pp.159-164
• /
• 2003

The RAD3 gene of Saccharomyces cerevisiae is required for excision repair and is essential for cell viability. The RAD3 encoded protein possesses a single stranded DNA-dependent ATPase and DNA and DNA-RNA helicase activities. To examine the extent of conservation of structure and function of a S. pombe RAD3 during eukaryotic evolution, the RAD3 homolog gene was isolated by screening of genomic DNA library. The isolated gene was designated as HRD3 (homolog of RAD3 gene). Southern blot analysis confirmed that S. pombe chromosome contains the same DNA as HRD3 gene and this gene exists as a single copy in S. pombe. The transcript of 2.8 kb was detected by Northern blot analysis, The level of transcripts increased by ultraviolet (UV) irradiation, indicating that HRD3 is one of the UV-inducible genes in S. pombe. Furthermore, the predicted partial sequence of HRD3 protein has 60％ identity to S. cerevisiae RAD3 gene. This homology was particularly striking in the regions identified as being conserved in a group of DNA helicases. Gene deletion experiments indicate that the HRD3 gene is essential for viability and DNA repair function. These observations suggest evolutionary conservation of other protein components with which HRD3 might interact in mediating its DNA repair and viability functions.

• Journal of Microbiology and Biotechnology
• /
• v.10 no.5
• /
• pp.700-706
• /
• 2000

Iron- and zinc-containing superoxide dismutase (FeZnSOD) and nickel-containing superoxide dismutase (NiSOD) are cytoplamic enzymes in Streptomyces griseus. The sodF gene coding for FeZnSOD was cloned from genomic Southern hybridization analysis with a 0.5-kb DNA probe, which was PCR-amplified with facing primers corresponding to the N-terminal amino acid of the purified FeZnSOD of S. griseus and a C-terminal region which is conserved among bacterial FeSODs and MnSODs. The sodF open reading frame (ORF) was comprised of 213 amino acid (22,430 Da), and the deduced sequence of the protein was highly homologous (86% identity) to that of FeZnSOD of Streptomyces coelicolor. The FeZnSOD expression of exponentially growing S. griseus cell was approximately doubled as the cell growth reached the early stationary phase. The growth-associated expression of FeZnSOD was mainly controlled at the transcriptional level, and the regulation was exerted through the 110 bp regulatory DNA upstream from the ATG initiation codon of the sodF gene.