• Asian-Australasian Journal of Animal Sciences
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• v.25 no.6
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• pp.758-763
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• 2012

As a member of a subclass of immunophilins, it is controversial that FKBP38 acts an upstream regulator of mTOR signaling pathway, which control the process of cell-growth, proliferation and differentiation. In order to explore the relationship between FKBP38 and mTOR in the Cashmere goat (Capra hircus) cells, a full-length cDNA was cloned (GenBank accession number JF714970) and expression pattern was analyzed. The cloned FKBP38 gene is 1,248 bp in length, containing an open reading frame (ORF) from nucleotide 13 to 1,248 which encodes 411 amino acids, and 12 nucleotides in front of the initiation codon. The full cDNA sequence shares 98% identity with cattle, 94% with horse and 90% with human. The putative amino acid sequence shows the higher homology which is 98%, 97% and 94%, correspondingly. The bioinformatics analysis showed that FKBP38 contained a FKBP_C domain, two TPR domains and a TM domain. Psite analysis suggested that the ORF encoding protein contained a leucine-zipper pattern and a Prenyl group binding site (CAAX box). Tissue-specific expression analysis was performed by semi-quantitative RT-PCR and showed that the FKBP38 expression was detected in all the tested tissues and the highest level of mRNA accumulation was detected in testis, suggesting that FKBP38 plays an important role in goat cells.

• Journal of The Korean Society of Inherited Metabolic disease
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• v.1 no.1
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• pp.23-27
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• 2001

Fanconi-Bickel Syndrome (FBS) is a rare autosomal recessive disorder of carbohydrate metabolism recently demonstrated to be caused by mutations in the GLUT 2 gene for the glucose transporter protein 2 expressed in liver, pancreas, intestine, and kidney. This disease is characterized by hepatorenal glycogen accumulation, both fasting hypoglycemia as well as postprandial hyperglycemia and hyperglactosemia, and generalized proximal renal tubular dysfunctions. We report the first Korean patient with FBS diagnosed based on clinical manifestations and identification of a novel mutation in the GLUT 2 gene. She was initially diagnosed having a neonatal diabetes mellitus due to hyperglycemia and glycosuria at 3 days after birth. In addition, newborn screening for galactosemia revealed hypergalactosemia. Thereafter, she has been managed with lactose free milk, insulin therapy. However, she failed to grow and her liver has been progressively enlarging. Her liver functions were progressively deteriorated with increased prothrombin time. Liver biopsy done at age 9 months indicated micronodular cirrhosis with marked fatty changes. She succubmed to hepatic failiure with pneumonia at 10 months of age. Laboratory tests indicated she had generalized proximal renal tubular dysfuctions; renal tubular acidosis, hypophosphatemic rickets, and generalized aminoaciduria. Given aforementioned findings, the diagnosis of FBS was appreciated at age of 2 months. The DNA sequencing analysis of the GLUT 2 gene using her genomic DNA showed a novel mutation at 5th codon; Lysine5 Stop (K5X).

• Journal of Microbiology and Biotechnology
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• v.11 no.1
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• pp.85-91
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• 2001

To construct an efficient Bacillus subtilis expression vector, strong promoters were isolated from the chromosomal DNA libraries of Clostridium acetobutylicum ATCC 4259, Thermoactinomyces sp. E79, and Bacillus thermoglucosidasius KCTC 3400. The $P_{C27}$ promoter cloned from the clostridial chromosmal DNA showed a 5-fold higher promoter strength than the $P_{SP02}$ promoter in the expression of the cat gene, and its sequence was estimated as an upstream region of the predicted hypothetical gene (tet-R family bacterial transcription regulator gene) in C. acetobutylicum. As a promoter element, $P_{C27}$ exhibited putative nucleotide sequences that can bind with bacterial RNAP and the 3'end of the 16S rRNA just upstream of the start codon. In addition, the promoter activity of $P_{C27}$ was distinctively repressed in the presence of glucose. Using $P_{C27}$ as the promoter element, a glucose controllable B. subtilis expression vector was constructed and the lipase gene from Staphylococcus haemolyticus KCTC 8957P was expressed in B. subtilis. When compared with the lipase expression by the T7 promoter induced by IPTG in E. coli, the $P_{C27}$ promoter showed about a 1.5-fold higher expression level in B. subtilis than that without induction.

• Journal of Life Science
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• v.11 no.4
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• pp.385-391
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• 2001

Filamentous hemagglutinin (FHA) is considered as an essential immunogenic component for incorporation into acellular vaccines against Bordetella pertussis, the causative agent of whooping cough. Classically, antipertussis vaccination has employed an intramuscular route. An alternative approach to stimulate mucosal and systemic immune responses is oral immunization with recombinant live vaccine carrier strains of Salmonella typhimurium. An attenuated live Salmonella vaccine sgrain($\Delta$cya $\Delta$crp) expressing recombinant FHA(rFHA) was developed. Stable expressionof rFHA was achieved by the use of balanced-lethal vector-host system. which employs an asd deletion in the host chromosome to impose in obligate requirement for diaminopimelic acid. The chromosomal $\Delta$asd mutation was complemented by a plasmid vector possessing the asd$^{+}$ gene. A 3 kb DNA fragment encoding immuno dominant regionof FHA was subcloned in-frame downstream to the ATG translation initiation codon in the multicopy Asd$^{+}$ pYA3341 vector to create pYA3457. Salmonella vaccine harboring pYA3457 expressed approximately 105kDa rFHA protein. The 100% maintenance of [YA3457 in vaccine strain was confirmed by stability examinations. Additionally, a recombinant plasmid pYA3458 was constructed to overpress His(8X)-tagged rFHA in Essherichia coli. His-tagged rFHA was purified from the E. coli strain harboring pYA3458 using Ni$^{2+}$-NTA affinity purification system.>$^{2+}$-NTA affinity purification system.

• The Plant Pathology Journal
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• v.22 no.4
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• pp.369-374
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• 2006

To characterize benzimidazole-resistant and -sensitive Monilinia fructicola populations, the fungal isolates were obtained from peach plants showing brown rot and bloosom blight. Benzimidazole-sensitive isolates did not grow on potato dextrose agar(PDA) amended with $\geq1.0{\mu}g$ active ingredient(a.i.)/ml of the fungicides. However, benzimidazole-resistant isolates grew on PDA regardless of the tested concentrations of fungicides. Benzimidazole-resistant isolates did not grow on diethofencarb-PDA, but sensitive isolates grew on the same PDA. In the nucleotide sequences of $\beta$-tubulin gene, only codon 198(GAG: glutamic acid), a target site for benzimidazole, was replaced with GCG(alanine) in all of the resistant isolates, and this substitution seems to play an important role in the development of resistance. Other interesting codons such as 165(GCT), 200(TTC), and 241(GCT) were not changed among the isolates. Benzimidazole-resistant and -sensitive isolates were clustered clearly in random amplified polymerphic DNA analyses and the results revealed that low levels of genetic diversity between benzimidazole-sensitive and -resistant isolates of M. fructicola in the investigated regions.

• Journal of Microbiology and Biotechnology
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• v.8 no.1
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• pp.28-33
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• 1998

A gene coding for ${\beta}$-xylosidase from thermophilic xylanolytic Bacillus sp. KK-1 was cloned into Escherichia coli using plasmid pBR322. Recombinant plasmid DNAs were isloated from E. coli clones which were capable of hydrolyzing 4-methylumbelliferyl-${\beta}$-D xylopyranoside. Restriction analysis showed the DNAs to share a common insert DNA. Xylo-oligosaccharides, including xylotriose, xylotetraose, xylopentaose, and xylobiose were hydrolyzed to form xylose as an end product by cell-free extracts of the E. coli clones, confirming that the cloned gene from strain KK-1 is ${\beta}$-xylosidase gene. The ${\beta}$-xylosidase gene of strain KK-1 designated as xylB was completely sequenced. The xylB gene consisted of an open reading frame of 1,602 nucleotides encoding a polypeptide of 533 amino acid residues, and a TGA stop codon. The 3' flanking region contained one stem-loop structure which may be involved in transcriptional termination. The deduced amino acid sequence of the KK-1 ${\beta}$-xylosidase was highly homologous to the ${\beta}$-xylosidases of Bacillus subtilis and Bacillus pumilus, but it showed no similarity to a thermostable ${\beta}$-xylosidase from Bacillus stearothermophilus.

• Journal of Microbiology and Biotechnology
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• v.3 no.3
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• pp.139-145
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• 1993

The nucleotide sequence of the xylanase gene (xynS) from alkali-tolerant Bacillus sp. YA.14 was determined and analyzed. A 639 base pairs open reading frame for xynS gene was observed and encoded for a protein of 213 amino acids with a molecular weight of 23, 339. S1 nuclease mapping showed that the transcription initiation site of the xynS gene did not exist in the cloned DNA. Ribosome binding site sequence with the free energy of -18.8 Kcal/mol was observed 8 base pairs upstream from the initiation codon, ATG. The proposed signal sequence consisted of 28 amino acids, of which 3 were basic amino acid residues and 21 were hydrophobic amino acid residues. When the amino acid sequences of xylanases were compared, Bacillus sp. YA-14 xylanase showed 48% homology with Bacillus sp. YC-335 xylanase and 96% homology with xylanases from B. subtilis and B. circulans.

• Journal of Plant Biotechnology
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• v.40 no.1
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• pp.27-36
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• 2013

We isolated and functionally characterized a Dendrobium Actin1 (DmACT1) promoter that drives strong gene expression in the orchid genus Dendrobium. A genomic fragment containing the region 3227 bp upstream of the coding region of DmACT1 was obtained by inverse PCR. Detailed comparison of the full-length cDNA and genomic sequences revealed that DmACT1 has a 1374 bp first intron in the 5' UTR. However, the 5' flanking sequences upstream of the coding region showed no obvious sequence similarities compared to those of known promoters, including plant actin promoters. Serial deletion constructs of the 5' flanking region from the translation initiation codon were fused to the coding sequence of a GUS/luciferase fusion reporter to identify the regulatory elements necessary for promoter activity. Transient assays in the flowers of Dendrobium revealed that the 5' UTR-intron greatly enhanced promoter activity. Moreover, the DmACT1 promoter with its 5' UTR-intron yielded approximately 10-fold higher reporter activity than the rice Act1 promoter-intron. Our data suggest that the DmACT1 promoter with its 5' UTR-intron is a useful tool for strong expression of transgenes in Dendrobium orchids.

• BMB Reports
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• v.37 no.5
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• pp.574-581
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• 2004

The full-length cDNA of the lumbrokinase fraction 6 (F6) protease gene of Lumbricus rubellus was amplified using an mRNA template, sequenced and expressed in E. coli cells. The F6 protease gene consisted of pro- and mature sequences by gene sequence analysis, and the protease was translated and modified into active mature polypeptide by N-terminal amino acid sequence analysis of the F6 protease. The pro-region of F6 protease consisted of the 44 residues from methionine-1 to lysine-44, and the mature polypeptide sequence (239 amino acid residues and one stop codon; 720 bp) started from isoleucine-45 and continued to the terminal residue. F6 protease gene clones having pro-mature sequence and mature sequence produced inclusion bodies in E. coli cells. When inclusion bodies were orally administrated rats, generated thrombus weight in the rat' venous was reduced by approximately 60% versus controls. When the inclusion bodies were solubilized in pepsin and/or trypsin solutions, the solubilized enzymes showed hemolytic activity in vitro. It was concluded the F6 protease has hemolytic activity, and that it is composed of pro- and mature regions.

• Journal of Life Science
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• v.10 no.4
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• pp.422-430
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• 2000

In order to search for the effects of Shine-Dalgarno (SD) sequence and nucleotide sequence of spacer region (SD-ATG) on bGH expression, oligonucleotides containing random SD sequences and a spacer region were chemically synthesized. The distance between SD region and initiation codon (ATG) was fixed to 9 nucleotides in length. The expression vectors have been constructed using pT7-1 vector containing a T7 promoter. Positive clones were screened with colony hybridization and named pT7A or pT7B plasmid series. The selected clones were confirmed by DNA sequencing and finally, 19 clones having various SD combinations were obtained. When bovine growth hormone was induced by IPTG in E. coli BL21(DE3), all cells harboring these plasmids produced a detectable level of bGH in western blot analysis. However, various SD sequences did not affect on bGH expression, indicating that the sequences of SD and the spacer region did not sufficiently destabilize mRNA secondary structure of bGH gene. Therefore, these results indicate that the disruption of mRNA secondary structure might be a major factor for regulating bGH expression in the translational initiation process.