• Toxicological Research
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• v.26 no.1
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• pp.47-52
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• 2010

The Malassezia fungi are responsible for various human skin disorders including dandruff and seborrheic dermatitis. Of the Malassezia fungi, Malassezia globosa (M. globosa) is one of the most common in human scalp. The completed genome sequence of M. globosa contains four putative cytochrome P450 genes. To determine the roles of Malassezia P450 enzymes in the biosynthesis of ergosterol, we isolated MGL3996 gene from M. globosa chromosomal DNA by PCR. The MGL3996 gene encodes an enzyme of 616 amino acids, which shows strong similarity with known CYP52s of other species. MGL3996 gene was cloned and expressed in Pichia pastoris (P. pastoris) heterologous yeast expression system. Using the yeast microsomes expressing MGL3996 protein, a typical P450 CO-difference spectrum was shown with absorption maximum at 448 nm. SDS-PAGE analysis revealed a protein band of apparent molecular weight 69 kDa and Western blot with anti-histidine tag antibody showed that MGL3996 was successfully expressed in P. pastoris. Cloning and expression of a new P450 gene is an important step to study the P450 monooxygenase system of M. globosa and to understand the role of P450 enzymes in pathophysiology of dandruff.

• BMB Reports
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• v.39 no.6
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• pp.686-695
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• 2006

Interleukin-2 enhancer binding factor 2 (ILF2) was reported to regulate transcription of interleukin-2 (IL-2), a central cytokine in the regulation of T-cell responses. This property of ILF2 was well characterized in human and mammals, but little is known in bony fish. In this paper, an ILF2 homologue was cloned and well characterized from Tetraodon nigrovirid is for the further investigation of the function of ILF2 in bony fish. The full-length Tetraodon ILF2 cDNA was 1380 bp in size and contained an open reading frame (ORF) of 1164 bp that translates into a 387 amino-acid peptide with a molecular weight of 42.9 kDa, a 5' untranslated region (UTR) of 57 bp, and a 3' UTR of 159 bp containing a poly A tail. The deduced peptide of Tetraodon ILF2 shared an overall identity of 58%~93% with other known ILF2 sequences, and contained two N-glycosylation sites, two N-myristoylation sites, one RGD cell attachment sequence, six protein kinase C phosphorylation sites, one amino-terminal RGG-rich single-stranded RNA-binding domain, and a DZF zinc-finger nucleic acid binding domain, most of which were highly conserved through species compared. Constitutive expression of Tetraodon ILF2 was observed in all tissues examined, including gill, gut, head kidney, spleen, liver, brain and heart. The highest expression was detected in heart, followed by liver, head kidney and brain. Stimulation with LPS did not significantly alter the expression of Tetraodon ILF2. Gene organization analysis showed that the Tetraodon ILF2 gene have fifteen exons, one more than other known ILF2 genes in human and mouse. Genes up- and down-stream from the Tetraodon ILF2 were Rpa12, Peroxin-11b, Smad4, Snapap and Txnip homologue, which were different from that in human and mouse.

• Journal of Microbiology and Biotechnology
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• v.10 no.5
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• pp.690-693
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• 2000

Efficient intron deletion with the correct splicing of the two exons of the human proinsulin gene was accomplished by a novel stepwise method using genomic DNA [5]. The two exons were separately amplified in two steps, using the second step primers that incorporated additional bases complementary to the other exon. The fragments were combined in a third PCR reaction. Cloning and sequencing of the PCR product demonstrated the correct splicing of the two exons. Expression studies, using the pET9a vector, revealed a protein band with the correct size with respect to human proinsulin as confirmed by SDS-PAGe and Western blot. Proinsulin concentration was estimated to be around 200 mg per liter culture, expressed as inclusion bodies. Protein secretion to the culture medium and periplasmic space was achieved by cloning in the pEZZ18 vector.

• Journal of Microbiology and Biotechnology
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• v.11 no.1
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• pp.106-111
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• 2001

A ${\beta}-galactosidase$ gene of Bifidobacterium adolescentis Int57 (INT57) was cloned using the shotgun method. The sequence of the ${\beta}-galactosidase$ gene existing in the sequenced 3,260-bp fragment showed higher than 40% homology with other bacterial ${\beta}-galactosidase$ genes. The expression in Escherichia coli suggested that the ${\beta}-galactosidase$ might have a monomeric, dimeric, or tetrameric protein structure. This is probably the first peer-reviewed sequence analysis of the ${\beta}-galactosidase$ gene of the genus Bifidobacterium.

• Korean Journal of Microbiology
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• v.25 no.1
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• pp.40-45
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• 1987

The gene for the Bdi I modification enzyme, which is one of Bdi I restriction-modification system, fromBrevibacterium divaricatum FERM 5948 was cloned and expressed in E. coli. For cloning of the Bdi I methylase gene, we have initially used three cloning site(EcoRI, BamHI and Sal I) of plasmid vector pBR 322 and adopted the retransformation method after Bdi I restriction endonuclease cleavage. Selection of transformants carrying the gene was based on the resistance of the modified plasmid encoding the enzyme to cleavage by Bdi I restriction enzyme, and the recombinant plasmid pBDIM 116 containing 5.6kb EcoRI insery was proved to carry the gene. Crude cell extracts prepared from strains carrying the plasmid pBDIM 116 contained an S-adenosylmethionine-dependent methyltransferase activity specific for the Bdi I recognition site, ATCGAT. The restriction map was constructed with 11 restriction enzyme, and the Bdi I restriction-modification system was also discussed.

• BMB Reports
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• v.29 no.3
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• pp.183-191
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• 1996

DNA fragments, homologous to the dTDP-D-glucose 4,6-dehydratase gene, obtained from the genomic DNA of Streptomyces antibioticus $T\ddot{u}99$, a producer of the unusual macrolide antibiotic chlorothricin, were cloned and sequenced. This dehydratase gene was designated as oxil. The coding region of the oxil gene is composed of 987 bp, and analysis of the DNA sequence data reveals sequences for the gene products of 329 amino acids (molecular weight of 36,037). The deduced amino acids are 59% identical to the StrE, dTDP-D-glucose 4,6-dehydratase from the streptomycin pathway. The oxil's function was examined by expressing it in E. coli using the T7 RNA polymerase/promoter system (pRSET) to produce an active fusion protein including a his tag. This enzyme shows specificity of substrate, specific only to dTDP-D-glucose.

• Proceedings of the KSAR Conference
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• 2002.06a
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• pp.64-64
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• 2002

Peroxiredoxins (Prxs) possess protective activity against oxygen radicals generated by thiol-catalyzed oxidative systems. We already reported the genomic structure and its expression of mouse Prx Ⅰ, Ⅱ, and 1-Cys Prx. However, the Prx Ⅲ has not been determined. That was initially defined transiently expressed gene, mouse MER5, of murine erythroleukaemia cell differentiation. In addition, this protein was recently redefined a member of the thiol-specific antioxidant gene family. (omitted)

• BMB Reports
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• v.31 no.3
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• pp.303-306
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• 1998

Serine proteinase cDNA fragment from protozoan parasite Acanthamoeba culbertsoni was amplified by the reverse transcription-polymerase chain reaction (RTPCR) using degenerate oligonucleotide primers derived from conserved serine proteinase sequences. The amplified DNA fragment was subcloned and sequenced. The sequence analysis and alignment showed significant sequence similarity to other eukaryotic serine proteinases and conservation of the His, Asp, and Ser residues that form the catalytic triad. The cDNA fragment was cloned into the pGEMEX-1 expression vector and expressed in Escherichia coli. A resulting fusion protein of 56 kDa had proteolytic activity. The fusion protein reacted with sera of mice immunized with purified serine proteinase of A. culbertsoni in Western blot. Immune recognition of the fusion protein by mouse antisera suggested that the fusion protein may be valuable as a diagnostic reagent.

• Korean Journal of Microbiology
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• v.36 no.2
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• pp.112-118
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• 2000

A new gene encoding an acidophilic TEX>$\alpha$-amylase of Bacillus cil-culans KCTC3004 was cloned into Eschericlzia coli using pUC19 as a vector. The gene localized in the 5.8 kb PstI DNA fragment was expressed independently of its orientation in the cloning vector showing enzyme activity about 40 times greater than that produced by the original B, circulans The optimum pH and temperature of the cloned enzyme were pH 3.6 and 45^{\circ}C.$ respectively. The enzyme hydrolyzed starch to produce maltotriose and maltooligosaccharides. The SDS-PAGE and zymopram of the enzyme produced in E coli(p.4L850) indicated a molecular weight of 55,000.

• Fisheries and Aquatic Sciences
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• v.13 no.4
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• pp.263-271
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• 2010

G protein-coupled receptors (GPCR) constitute the largest superfamily of cell membrane receptors, mediating diverse signal-transduction pathways. The melanocortin 4 receptor (MC4R) has been of interest for its physiological role and size, one of the smallest among the GPCRs, which makes it a good model system for the structural study of GPCRs. To study the molecular structure and tissue-specific expression of MC4R in olive flounder (Paralichthys olivaceus), the full-length MC4R gene was obtained using PCR amplification of genomic DNA as well as cDNA synthesis. Sequence analysis of the gene indicates that 978 bp of the MC4R gene encodes 325 amino acids without introns. Sequence alignment with the MC4Rs from other fish shows the highest degree of identity (96%) between Paralichthys olivaceous and Verasper moseri, followed by Takifugu rubripes and Tetraodon nigroviridis (89%). RNA was isolated from various tissues to examine the tissue distribution of MC4R by using RT-PCR. The results showed major expression of MC4R in the liver, brain, and eye, which is consistent with the expression pattern in other fish belonging to the order Pleuronectiformes.