• Korean Journal of Environmental Biology
• /
• v.27 no.4
• /
• pp.406-413
• /
• 2009

The enzyme invertase contributes to sugar unloading, pathogen defense, differentiation and development in plants. We cloned the complete cDNA of a soluble acid invertase from pea seedlings (Pisum sativum) via RT-PCR and the rapid amplification of the cDNA end (RACE) technique. The full-length cDNA of the soluble pea invertase comprised 2237 bp and contained a complete open reading frame encoding 647 amino acids. The deduced amino acid sequence showed high homology to soluble acid invertases from various plants. Northern blot analysis demonstrated the soluble acid invertase gene of P. sativum was strongly expressed in sink organs such as shoot tips and root tips, and induced by abscisic acid, gibberellic acid and jasmonic acid in shoots. Especially, gibberellic acid enhanced the gene expression of the soluble acid invertase in a time-dependent manner. This study presents that the gene expression patterns of a soluble acid invertase from pea are strongly consistent with the suggestion that individual invertase gene product has different functions in the growing plant.

• Journal of agriculture & life science
• /
• v.45 no.6
• /
• pp.201-212
• /
• 2011

The alkaline protease gene was cloned from a halo-tolerant alkalophilic Bacillus clausii I-52 isolated from the heavily polluted tidal mud flat of West Sea in Inchon Korea, which produced a strong extracellular alkaline protease (BCAP). Based on the full genome sequence of Bacillus subtilis, PCR primers were designed to allow for the amplification and cloning of the intact pro-BCAP gene including promoter region. The full-length gene consists of 1,143 bp and encodes 381 amino acids, which includes 29 residues of a putative signal peptide and an additional 77-amino-acid propeptide at its N-terminus. The mature BCAP deduced from the nucleotide sequence consists of 275 amino acids with a N-terminal amino acid of Ala, and a relative molecular weight and pI value was 27698.7 Da and 6.3, respectively. The amino acid sequence shares the highest similarity (99%) to the nattokinase precursor from B. subtilis and subtilisin E precursor from B. subtilis BSn5. The substrate specificity indicated that the recombinant BCAP could hydrolyze efficiently the synthetic substrate, N-Suc-Ala-Ala-Pro-Phe-pNA,and did not hydrolyze the substrates with basic amino acids at the P1 site. The recombinant BCAP was strongly inhibited by typical serine protease inhibitor, PMSF, indicating that BCAP is a member of the serine proteases.

• The Korean Journal of Physiology and Pharmacology
• /
• v.1 no.1
• /
• pp.19-25
• /
• 1997

The C-terminus ends of the second putative transmembrane domains of both $M_1$ and $M_2$ muscarinic receptors contain a triplet of amino acid residues consisting of leucine (L), tyrosine (Y) and threonine (T). This triplet is repeated as LYT-TYL in $M_1$ receptors at the interface between the second transmembrane domain and the first extracellular loop. Interestingly, however, it is repeated in a transposedfashion (LYT-LYT) in the sequence of $M_2$ receptors. In our previous work, we investigated the possible significance of this unique sequence diversity for determining the distinct differential receptor function at the two receptor subtypes. However, we found mutation of the LYTTYL sequence of $M_1$ receptors to the corresponding $M_2$ receptor LYTLYT sequence demonstrated markedly enhanced the stimulation of phosphoinositide (PI) hydrolysis by carbachol without a change in its coupling to increased cyclic AMP formation. In this work, thus, the enhanced stimulation of PI hydrolysis in the LYTLYT $M_1$ receptor mutant was further investigated. The stimulation of PI hydrolysis by carbachol was enhanced in the mutant $M_1$ receptor, and this change was not due to alterations in the rate of receptor desensitization or sequestration. The observed larger response to carbachol at mutant $M_1$ receptors was also not due to an artifact resulting from selection of CHO cells which express higher levels of G-proteins or phospholipase C. Our data suggest that although the LYTTYL sequence in $M_1$ muscarinic receptors is not involved in determining receptor pharmacology, mutation of the sequence enhanced the coupling of $M_1$ receptors to the stimulation of phospholipase C.

• Asian-Australasian Journal of Animal Sciences
• /
• v.20 no.6
• /
• pp.850-855
• /
• 2007

Leptin is the adipocyte-specific product of the obese gene and plays a major role in food intake and energy metabolism. Leptin research was mainly focused on mammalian species, but understanding of leptin and its function in poultry is very poor. In this study, the duck leptin gene was amplified using the reverse transcription-polymerase chain reaction (RT-PCR) from duck liver RNA. The cDNA fragment was inserted into the pET-28a expression vector, and the resulting plasmid was expressed in Escherichia coli BL21 (DE3). Experimental mice were given an intraperitoneal injection of 10 mg/kg leptin dissolved in phosphate buffered saline (PBS), while the control mice were injected with PBS. The effect of leptin on food intake, body weight and fatty deposition in mice was detected. Sequence analysis revealed that duck leptin had a length of 438 nucleotides which encoded a peptide with 146 amino acid residues. The sequence shares highly homology to other animals. The coding sequence of duck leptin was 84 and 86% identical to human and pig leptin nucleotides sequence. Highest identity was with the rat coding sequence (95%). The identity of the amino acid sequence was 84, 82 and 96% respectively compared to that of the human, pig and rat. Results of SDS-PAGE analysis indicated that a fusion protein was specifically expressed in E. coli BL21 (DE3). The purified product was found to be biologically active during tests. Continuous administration of recombinant duck leptin inhibited food intake. Despite the decrease of food intake, leptin significantly induced body weight and fatty deposition. These changes were accompanied by a significant down-secretion of plasma glucose, cholesterol, triglyceride and insulin levels in mice. The observations provide evidence for an inhibitory effect of leptin in the regulation of food intake and for a potential role of duck leptin in the regulation of lipogenesis.

• Microbiology and Biotechnology Letters
• /
• v.35 no.4
• /
• pp.352-356
• /
• 2007

A Lactobacillus sp. has been isolated from infant feces and characterized according to its physiological properties and identified as Lactobacillus acidophilus KLA1012. A gene coding surface layer protein (SLP) has been cloned and the sequence has been determined. The nucleotide sequence of slpA was 1,338 bp in size and was identical to that of L. acidophilus ATCC 4356 (100%). Amino acid sequence of SLP-A was deduced from the nucleotide sequence and it had signal sequence at N-terminal, consisting of positively charged amino acid mainly lysine. slpA was cloned and heterologously expressed in E. coli M15 and the 45.2 kDa surface-layer protein band was examined by SDS-PAGE and confirmed by Western blotting using polyclonal antibody against L. acidophilus KLA 1012 SLP-A protein.

• The Plant Pathology Journal
• /
• v.16 no.1
• /
• pp.9-18
• /
• 2000

Using differential display techniques, a new acidic pathogenesis-related (PR) protein-1 cDNA (GMPRla) gene was isolated from a cDNA library of soybean (Glycinemax L.Merr, cultivar Jangyup) hypocotyls infected by Phytophthora sojae f. sp. glycines. The 741 bp of fulllength GMPRla clone contains an open reading frame of 525 nucleotides encoding 174 amino acid residues (pI 4.23) with a putative signal peptide of 27 amino acids in the N-terminus. Predicted molecular weight of the protein is 18,767 Da. The deduced amino acid sequence of GMPRla has a high level of identity with PR-1 proteins from Brassica napus, Nicotiana tabacum, and Sambucus nigra. The GMPRla mRNA was more strongly expressed in the incompatible than the compatible interaction. The transcript accumulation was induced in the soybbean hypocotyls by treatment with ethephon or DL-$\beta$-amino-n-butyric acid, but not by wounding. In situ hybridization data showed that GMPRIa mRNAs were usually localized in the vascular bundle of hypocotyl tissues, especially phloem tissue. Differences between compatible and incompatible interactions in the timing of GMPRla mRNA accumulation were remarkable, but the spatial distribution of GMPRla mRNA was similar in both interactions. However, more GMPRla mRNA was accumulated in soybean hypocotyls at 6 and 24 h after inoculation.

• Korean Journal of Microbiology
• /
• v.32 no.3
• /
• pp.215-221
• /
• 1994

The complete nucleotide sequence of the cloned pga gene encoding the penicillin G acylase of Bacillus megaterium ATCC 14945 and its 5'- and 3'-flanking regions was determined. The sequence revealed only one large open reading frame (2,406 hp) of the penicillin G acylase (pga) gene. Upstream from ATG of the pga gene, there was a putative ribosome binding site, Shine-Dalgarno sequence. The promoter-like structure, - 10 and - 35 sequences, was also found. Following the stop codon, TAG, a structure reminiscent of the E. coli rho-independent transcription terminator was present. The amino acid sequence was deduced from the nucleotide sequence. The molecular mass of the polypeptide was 91,983 Da. There was a potential signal sequence in its amino-terminal region. A comparison of its deduced amino acid sequence with other characterized penicillin G acylases and the result of SDS-polyacrylamide gel electrophoresis of the purified enzyme showed that a precursor polypeptide of 92 kDa was processed into two dissimilar ${\alpha}$ and ${\beta}$-subunits of 25 and 61 kDa.

• Journal of Photoscience
• /
• v.9 no.2
• /
• pp.17-20
• /
• 2002

We have cloned a cDNA for an opsin (Boceropsin) from the silkworm larval brain which was suggested to contain the photoperiodic receptor. Its deduced amino acid sequence was composed of 381 amino acids and included amino acid residues highly conserved in insect visual pigments. This opsin belonged to the long wavelength photoreceptor group of insect opsins, and are presumed to be photoperiodic receptor. RT-PCR analysis revealed that Boceropsin mRNA is expressed in the larval brain, but not in the subesophageal (Sg) and thoracic ganglion. Immunohistochemical analyses demonstrated that Boceropsin protein is present bilaterally in some defined cells localized in the brain of the Bombyx larva. Boceropsin was considered not to be involved in the circadian photoreception, because carotenoids are not indispensable for the photoreception and formation of circadian rhythms in the silkworm.

• BMB Reports
• /
• v.30 no.6
• /
• pp.410-414
• /
• 1997

We purified a protein carboxyl O-methyltransferase (protein methylase II) from porcine spleen to homogeneity. The molecular weight of the porcine spleen protein methylase II (ps-PM II) was estimated to be 27,500 daltons on SDS-PAGE. Amino acid sequence of N-terminal 28 residues for ps-PM II was identified. Amino-terminal three amino acid residues of ps-PM II were deleted when compared to those of other protein carboxyl methytransferase. S-Adenosyl-L-homocysteine competitively inhibits ps-PM II with a K, value of $1.63{\times}10^{-7}M$. Myelin basic protein exhibited the highest methyl-accepting capacity among the proteins tested.

• Journal of Microbiology and Biotechnology
• /
• v.18 no.6
• /
• pp.1050-1058
• /
• 2008

A gene encoding a dextransucrase (dsrBCB4) that synthesizes only ${\alpha}$-1,6-linked dextran was cloned from Leuconostoc mesenteroides B-1299CB4. The coding region consisted of an open reading frame (ORF) of 4,395 bp that coded a 1,465-amino-acids protein with a molecular mass of 163,581 Da. The expressed recombinant DSRBCB4 (rDSRBCB4) synthesized oligosaccharides in the presence of maltose or isomaltose as an acceptor, plus the products included ${\alpha}$-1,6-linked glucosyl residues in addition to the maltosyl or isomaltosyl residue. Alignments of the amino acid sequence of DSRBCB4 with glucansucrases from Streptococcus and Leuconostoc identified conserved amino acid residues in the catalytic core that are critical for enzyme activity. The mutants D530N, E568Q, and D641N displayed a 98- to 10,000-fold reduction of total enzyme activity.