• Journal of Microbiology and Biotechnology
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• v.20 no.4
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• pp.821-827
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• 2010

Gene delivery that provides targeted delivery of therapeutic genes to the cells of a lesion enhances therapeutic efficacy and reduces toxic side effects. This process is especially important in cancer therapy when it is advantageous to avoid unwanted damage to healthy normal cells. Incorporating cancer-specific ligands that recognize receptors overexpressed on cancer cells can increase selective binding and uptake and, as a result, increase targeted transgene expression. In this study, we investigated whether a peptide capable of homing to hepatocellular carcinoma (HCC) could facilitate targeted gene delivery by cationic liposomes. This homing peptide (HBP) exhibited selective binding to a human hepatocarcinoma cell line, HepG2, at a concentration ranging from 5 to 5,000 nM. When conjugated to a cationic liposome, HBP substantially increased cellular internalization of plasmid DNA to increase the transgene expression in HepG2 cells. In addition, there was no significant enhancement in gene transfer detected for other human cell lines tested, including THLE-3, AD293, and MCF-7 cells. Therefore, we demonstrate that HBP provides targeted gene delivery to HCC by cationic liposomes.

• Proceedings of the Korea Society of Poultry Science Conference
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• 1999.11a
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• pp.34-50
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• 1999

A cDNA encoding chicken interferon-gamma (chIFN-${\gamma}$) was amplified from P34, a CD4$^{+}$ T-cell hybridoma by reverse transcription-polymerase chain reaction (RT-PCR) and cloned into pUC18. THe sequences of cloned PCR products were determined to confirm the correct cloning. Using this cDNA as probe, chicken genomic library from White Leghorn spleen was screened. Phage clones harboring chicken interferon-gamma (chIFN-${\gamma}$) were isolated and their genomic structure elucidated. The chIFN-${\gamma}$ contains 4 exons and 3 introns spanning over 14 kb, and follows the GT/AG rule for correct splicing at the exon/intron boundaries. The four exons encode 41, 26, 57 and 40 amino acids, respectively, suggesting that the overall structure of IFN-${\gamma}$ is evolutionairly conserved in mammalian and avian species. The 5’-untranslated region and signal sequences are located in exon 1. Several AT-rich sequences located in the fourth exon may indicate a role in mRNA turnover. The 5’-flanking region contains sequences homologous to the potential binding sites for the mammalian transcription factors, activator protein-1(AP-1) activator protein-2(AP-2) cAMP-response element binding protein(CREB), activating transcription factor(ATF), GATA-binding fator(GATA), upstream stimulating factor(USF), This suggests that the mechanisms underlying transcriptional regulation of chicken and mammalian IFN-${\gamma}$ genes may be similar.r.

• The Korean Journal of Pharmacology
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• v.24 no.1
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• pp.1-10
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• 1988

To obtain information about the structure of the human phenylethanolamin N-methyltransferase (PNMT) and to further define the extent of the evolutionary relationships among PNMT molecules of several spesies, a full length cDNA clone for bovine adrenal PNMT was used to screen a charon 4A genomic library. One phage was isolated and identified, which included the entire PNMT gene. The length of inserted genomic DNA was 13.1-Kilobase (Kb) containing two internal EcoRI sites. Construction of a restriction map and subsequent Southern and dot blot analysis with 5'-and3'-specific cDNA probes allowed the identification of exon-containing fragments. This is the first report of the cloning of gene for human epinephrine synthesizing enzyme.

• Biomedical Science Letters
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• v.6 no.3
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• pp.165-170
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• 2000

Vacuolar (H$^{+}$)-ATPase (V-ATPase) is an intracellular protein which consists of multiple subunits. It carries out acidification by pumping protons in the cell. This enzyme has also been found in the synaptic vesicles and may play an important role in the neurotransmission. We cloned cDNA fragments encoding the 16 kDa subunit of V-ATPase from the rat brain by RT-PCR and PCR using total RNA or recombinant phage DNA as templates. They contained the full coding sequences (468 bp) and one nucleotide at 3' region turned out to be different (A to C) when compared to the liver counterpart. However, this polymorphic difference did not cause any significant change in the primary structure of the protein because both GCA and GCC code for alanine. Our study would contribute to the understanding of the function of 16 M)a V-ATPase in the brain and of the mechanisms of neurotransmission.

• BMB Reports
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• v.32 no.5
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• pp.461-467
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• 1999

Recombinant Mycobacterium smegmatis expressing ovalbumin was used to immunize C57BL/6(H-$2^b$) mice, and the humoral immunity against recombinant ovalbumin was analyzed. Antibodies were purified by denatured ovalbumin-conjugated affinity chromatography. The epitopes of the antibodies were screened with a random peptide library displayed on the tip of fUSE5 filamentous phage pIII minor coat proteins. Two peptides, IRLADR and SPGAEV, were selected predominantly by the recognition of purified antibodies using biopanning methods. The composition of the peptide sequence with the primary structure of OVA revealed that the peptide sequence analogizes to INEAGR, part of the $^{323}ISQAVHAAHAEINEAGR^{339}$ sequence previously reported as the antigenic determinant for murine Band also Th cell epitopes (I-$A^d$ binding). Also, the structures of these mimotopes obtained from restrained molecular dynamic computations resulted in the formation of a $\beta$-turn proven to be a secondary structure of the parent peptide within the ovalbumin molecule, enabling us to confirm the structural similarity. This study demonstrates that immunization with recombinant M. smegmatis can generate neutralizing antibodies identical with those induced by the administration of natural antigenic proteins and supports the potential use of mycobacteria as vaccine delivery vehicles.

• Journal of Microbiology and Biotechnology
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• v.15 no.6
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• pp.1189-1196
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• 2005

The cyclohexanol dehydrogenase (ChnA), produced by Rhodococcus sp. TK6, which is capable of growth on cyclohexanol as the sole carbon source, has been previously purified and characterized. However, the current study cloned the complete gene (chnA) for ChnA and its flanking regions using a combination of a polymerase chain reaction (PCR) based on the N-terminal amino acid sequence of the purified ChnA and plaque hybridization from a phage library of Rhodococcus sp. TK6. A sequence analysis of the 5,965-bp DNA fragment revealed five potential open reading frames (ORFs) designated as partial pte (phosphotriesterase), acs (acyl-CoA synthetase), scd (short chain dehydrogenase), stp (sugar transporter), and chnA (cyclohexanol dehydrogenase), respectively. The deduced amino acid sequence of the chnA gene exhibited a similarity of up to $53\%$ with members of the short-chain dehydrogenase/reductase (SDR) family. The chnA gene was expressed using the pET21 a(+) system in Escherichia coli. The activity of the expressed ChnA was then confirmed (13.6 U/mg of protein) and its properties investigated.

• The Korean Journal of Physiology and Pharmacology
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• v.1 no.2
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• pp.195-200
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• 1997

Water transport in highly-permeable membranes is facilitated by some specialized pathways, which are called aquaporins (AQP). AQP1 (AQP-CHIP) is the first recognized aquaporin identified from red cells and renal proximal tubules. Up until now 4 other aquaporin homologs have been reported. Each aquaporin has its unique tissue distribution and regulatory mechanims. To elucidate molecular mechanisms for their transcription regulation and tissue-specific expression isolation of aquaporin genes is required. To clone promoters of the AQP family mouse genomic library was screened by the 1st exon-specific probe of AQP4, and 5 different plaques were positively hybridized. Phage DNAs were purified and characterized by restriction mapping and sequencing. One of them is the mouse AQP-CD gene. The gene was consisted of 4 exons and the exon-intron boundaries of mouse AQP-CD gene were identified at identical positions in other related genes. The 5'-flanking region of AQP-CD gene contains one classic TATA box, a GATA consensus sequence, an E-box and a cyclic AMP-responsive element. The cloning of the mouse AQP-CD gene, of which product is expressed in the collecting duct and is responsible for antidiuresis by vasopressin, will contribute to understand the molecular mechanisms of tissue-specific expression and regulation of AQP-CD gene under various conditions.

• BMB Reports
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• v.36 no.6
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• pp.586-592
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• 2003

Four putative promoters of the temperate mycobacteriophage L1 were cloned by detecting the $\beta$-galactosidase reporter expression in E. coli transformants that carried L1 specific operon-fusion library. All of the four L1 promoters were also found to express differentially in the homologous environment of mycobacteria. Of the four promoters, two were suggested to be the putative early promoters of L1 since they express within 0 to 10 min of the initiation of the lytic growth of L1. One of the putative early promoters showed a relatively better and almost identical activity in both E. coli and M. smegmatis. By a sequence analysis, we suggest that the L1 insert that contained the stronger early promoter possibly carries two convergent E. coli $\sigma^{70}$-like L1 promoters, which are separated from each other by about 300 nucleotides. One of them is the early promoter of L1 as it showed a 100% similarity with the early $P_{left}$ promoter of the homoimmune phage L5. The second promoter, designated P4, was suggested for its appreciable level of reporter activity in the absence of the -10 element of the $P_{left}$ equivalent of L1. By analyzing most of the best characterized mycobacteriophages-specific promoters, including the L1 promoter P4, we suggest that both the -10 and -35 hexamers of the mycobacteriophage promoters are highly conserved and almost similar to the consensus -10 and -35 hexamers of the E. coli $\sigma^{70}$ promoters.

• Parasites, Hosts and Diseases
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• v.49 no.1
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• pp.79-83
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• 2011

Trichomoniasis is a sexually transmitted disease due to infection with Trichomonas vaginalis, and it can cause serious consequences for women's health. To study the virulence factors of this pathogen, T. vaginalis surface proteins were investigated using polyclonal antibodies specific to the membrane fractions of T. vaginalis. The T. vaginalis expression library was constructed by cloning the cDNA derived from mRNA of T. vaginalis into a phage ${\lambda}$ Uni-ZAP XR vector, and then used for immunoscreening with the anti-membrane proteins of T. vaginalis antibodies. The immunoreactive proteins identified included adhesion protein AP65-1, ${\alpha$-actinin, kinesin-associated protein, teneurin, and 2 independent hypothetical proteins. Immunofluorescence assays showed that AP65-1, one of the identified immunogenic clones, is prevalent in the whole body of T. vaginalis. This study led us to identify T. vaginalis proteins which may stimulate immune responses by human cells.

• Journal of Life Science
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• v.22 no.4
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• pp.437-446
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• 2012

A metagenomic library of cow rumen in the pCC1FOS phage vector was screened in $E.$ $coli$ EPI300 for cellulase activity on carboxymethyl cellulose agar plates. One clone was partially digested with $Sau$3AI, ligated into the $Bam$HI site of the pBluescript II SK+ vector, and transformed into $E.$ $coli$ $DH5{\alpha}$. We obtained a 1.5 kb insert DNA, designated $cel$5C, which hydrolyzes carboxymethyl cellulose. The cel5C gene has an open reading frame (ORF) of 1,125 bp encoding 374 amino acids. It belongs to the glycosyl hydrolase family 5 with the conserved domain LIMEGFNEIN. The molecular mass of the Cel5C protein induced from $E.$ $coli$ $DH5{\alpha}$, as analyzed by CMC SDS-PAGE, appeared to be approximately 42 kDa. The enzyme showed optimum cellulase activity at pH 4.0, and $50^{\circ}C$. We examined whether the $cel$5C gene comes from the 49 identified cow rumen bacteria using PCR. No PCR bands were identified, suggesting that the $cel$5C gene came from the unidentified cow rumen bacteria.