• Parasites, Hosts and Diseases
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• 제42권4호
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• pp.159-167
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• 2004

Sequences of the complete protein-coding portions of the mitochondrial (mt) genome were analysed for 6 species of cestodes (including hydatid tapeworms and the pork tapeworm) and 5 species of trematodes (blood flukes and liver- and lung-flukes). A near-complete sequence was also available for an additional trematode (the blood fluke Schistosoma malayensis). All of these parasites belong to a large flatworm taxon named the Neodermata. Considerable variation was found in the base composition of the protein-coding genes among these neodermatans. This variation was reflected in statistically-significant differences in numbers of each inferred amino acid between many pairs of species. Both convergence and divergence in nucleotide, and hence amino acid, composition was noted among groups within the Neodermata. Considerable variation in skew (unequal representation of complementary bases on the same strand) was found among the species studied. A pattern is thus emerging of diversity in the mt genome in neodermatans that may cast light on evolution of mt genomes generally.

• Journal of Microbiology
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• 제39권1호
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• pp.37-41
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• 2001

The fission yeast Schizosaccharomyces pombe contains two distinct superoxide dismutase (SOD) activities, one in the cytosol encoded by the $sod2^{+}$ gene and the other in mitochondria. The $sod2^{+}$ gene encoding putative mitochondrial manganese superoxide dismutase (MnSOD) was isolated from the S. pombe genomic library using a PCR fragment as the probe. The nucleotide sequence of the $sod2^{+}$ gene and its flanking region (4051 bp HindIII fragment) was determined. An intron of 123 nt in size was predicted and confirmed by sequencing the cDNA following reverse transcription PCR. The predicted Sod2p consists of 218 amino acid residues with a molecular mass of 24,346 Da. The deduced amino acid sequence showed a high degree of homology with other MnSODs, especially in the metal binding residues at the active site and their relative positions. The transcriptional start site was mapped by primer extension at 231 at upstream from the ATG codon. A putative TATA box(TATAAAA) was located 58 nt upstream from the transcriptional start site and putative polyadenylation sites were located at 1000, 1062, and 1074 nt downstream from the ATG start codon.

• Journal of Microbiology
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• 제37권3호
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• pp.154-158
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• 1999

The arginine residue at position 243 (Arg 243) of the yeast transcription factor, Gcn4p, is invariably conserved among bZIP transcription factors. Using site-directed oligonucleotide saturation mutagenesis involving two-step polymerase chain reaction (PCR) amplification, random mutations were successfully introduced at the codon of 243 in the basic domain of Gcn4p. This mutant library was transformed ito Gcn4p defective yeast strain and selected for the transcriptionally active colonies. All colonies which were transcriptionally active had arginines in the codon 243. In this study, the strand preference by Taq polymerase during mutagenesis was also tested. Oligonucleotides were specially designed to test whether or not the polymerase was preferred using the strand as a template. A population of randomly mutated products were cloned into an appropriate vector and characterized by DNA sequencing analysis. Saturation mutagenesis which was performed efficiently by this method revealed a strong bias in terms of strand preference of Taq polymerase by an approximate ratio of 3 to 1 in this study.

• 한국미생물·생명공학회지
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• 제22권2호
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• pp.134-142
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• 1994

The neucleotide sequences of the xylA gene encoding $\beta $-xylosidase of Bacillus stearothermophilus and is its flanking regions were datermined. Three open reading frame(ORFs) were found, one of which(ORF1) appeared to code for the $\beta $-xylosidase. The 1830 base pair ORF1 encoded 609 amino acids starting from a TTG initiation codon. The molecular weight deduced from the nucleotide sequence(68 KD) was in agreement with that estimated by SDS-polyacrylamide gel electrophoresis of the purified enzyme(66 KD). The Shine-Dalgarno sequence(5'-AGGAGG-3') was found 11 bp upstream of the initiation codon. Further 15 bp upstream, there observed a potential transcription initiation signals. The putative -10 sequence(CATAAT) and -35 sequence(TTGTTA) coresponded closely to the consensus sequences for Bacillus subtilis RNA polymerase with major sigma factor. The guanine-plus-cytosine content of the coding region of the xylA gene was 56mol% while that of the third position of the codons was 63 mol%. Based on the comparison with the amino acid sequences of several other carbohydrate degrading enzymes, two conserved regions, possibly participating in the catalytic mechamism of $\beta $-xylosidase xylA, were identified in 278-298 and 329-350 regions of the translated xylA gene. The nucleotide sequence of the xylA was found to exhibit no homology to any other genes so far reproted.

• Journal of Microbiology and Biotechnology
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• 제9권1호
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• pp.122-125
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• 1999

Gcn4p, a transcriptional activator protein of the yeast, Sacchromyces cerevisiae, binds to the specific sequence in the promoters of many amino acid biosynthetic genes for general control. The serine residue (Ser 242) of Gcn4p directly contacts the DNA. Here, for inspecting the DNA binding properties and the level of transcriptional activation of Gcn4p, we introduced a polymerase chain reaction (PCR) site-directed saturation mutation library into the Ser 242 site using 2 outside primers and 2 oligonucleotides with its codons fully degenerated. The sequencing analysis of 146 samples revealed the even nucleotide distribution within the experimental error showing 23, 26, 25, and 26％ frequency of U, C, A, and G bases, respectively. This method turned out to be a simple, fast, and economical method for constructing a library of all 20 amino acids at specific codon.

• Journal of Microbiology and Biotechnology
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• 제30권7호
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• pp.1082-1091
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• 2020

Microbial transglutaminases (MTGs) are widely used in the food industry. In this study, the MTG gene of Streptomyces sp. TYQ1024 was cloned and expressed in a food-grade bacterial strain, Bacillus subtilis SCK6. Extracellular activity of the MTG after codon and signal peptide (SP Ync M) optimization was 20 times that of the pre-optimized enzyme. After purification, the molecular weight of the MTG was 38 kDa and the specific activity was 63.75 U/mg. The optimal temperature and pH for the recombinant MTG activity were 50℃ and 8.0, respectively. MTG activity increased 1.42-fold in the presence of β-ME and 1.6-fold in the presence of DTT. Moreover, 18% sodium chloride still resulted in 83% enzyme activity, which showed good salt tolerance. Cross-linking gelatin with the MTG increased the strength of gelatin 1.67 times and increased the thermal denaturation temperature from 61.8 to 75.8℃. The MTG also significantly increased the strength and thermal stability of gelatin. These characteristics demonstrated the huge commercial potential of MTG, such as for applications in salted protein foods.

• Journal of Microbiology and Biotechnology
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• 제10권2호
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• pp.244-250
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• 2000

An ${\alpha}-glucosidase$ gene (aglA) from thermophilic Bacillus sp. DG0303 was cloned, sequenced, and expressed in Escherichia coli. The aglA was localized to the 2.1-kb PvuI-XmnI region within the 5.9-kb DNA insert of the gybrid plasmid pAG1. The gene consisted of an open reading frame of 1,686 bp with an unusual GTG initiation codon and TGA termination codon. The amino acid sequence deduced from the nucleotide sequence predicted a protein of 562 amino acid residues with a M, of 66,551 dalton. A comparative amino acid sequence analysis revealed that DG0303 ${\alpha}-glucosidase$ is related to bacillary oligo-1, 6-glucosidases. The Bacillus sp. DG0303 ${\alpha}-glucosidase$ showed a high sequence identity (36-59%) to the B. flavocaldarius, B. cereus, and B. thermoglucosidasius oligo-1, 6-glucosidases. The number of prolines in theses four ${\alpha}-glucosidases. was observed to increase with increasing thermostability of these enzymes. The cloned ${\alpha}-glucosidase was purified from E. coli $DH5{\alpha}$ bearing pAG1 and characterized. The recombinant enzyme was identical with the native enzyme in its optimum pH and in its molecular mass, estimated by sodium dodecy1 sulfate-polyacrylamide gel electrophoresis. The temperature optimum of the cloned ${\alpha}-glucosidase$ was lower than that of the native enzyme.

• Journal of Photoscience
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• 제12권3호
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• pp.129-133
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• 2005

The psbC gene, encoding the intrinsic chlorophyll-binding protein of CP43, one of the PS core complex polypeptides, was cloned from the Panax ginseng chloroplast, which is composed of 1,422 nucleotides and the overall nucleotide sequence shows more than 84% identity to those of eukaryotic photosynthetic organisms. The predicted topology of CP43, based on hydropathy analysis, includes six membrane-spanning ${\alpha}-helices$ resulting in three lumenal and four stromal loops. The putative translation start codon for the psbC gene is located at 48 nucleotides upstream from the stop codon of the psbD gene whose product is also a component of the PSII reaction center, implying that the promoter of the psbC gene is possibly located in the middle of the structural gene of the psbD gene. Northern blot analysis of the in vivo accumulation of the psbC transcript from the plants grown under the various growth light intensities (5%, 10%, 20%, and 100%) of daylight indicated that the steady-state level of the psbC transcript was not significantly affected by light intensity.

• 한국수산과학회지
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• 제28권6호
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• pp.699-707
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• 1995

Eurasian yellow perch (Perca fluviatilis) and American yellow perch (Perca flavescens) are known to be endemic species in Eurasia and North America, respectively. The presence of endemic species on each continent suggests their independent evolutionary history. However, because of the morphological similarity, distribution pattern, and only recent fossil record, their divergence time and speciation of the two Perca species has long been controversial. Here, from the comparison of the entire nucleotide sequences of cytochrome b gene, large genetic divergence between the two Perca species is observed although they are morphologically similar each other. Among 1,140 base pairs, interspecific nucleotide differences are found at 130 sites $(11.4\%)$. The differences varies with codon position, showing 22 sites in the first, 5 sites in the second, and 103 sites in the third codon position. Considering the types of nucleotide changes, transitional differences are much more than transversional differences and its ratio turned out to be 5.19. The estimated divergence time of the two Perca species indicates that they were separated each other approximately in the late Miocene period, which implies the long history of speciation. With comparison of the inferred amino acid sequences, strong structural and functional constraints which seem to be maintained by the highly conservative amino acid residues or protein regions, as found in other taxonomic groups of organisms, are also recognized in the cytochrome b of the fishes examined.

• Journal of Microbiology and Biotechnology
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• 제13권2호
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• pp.287-291
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• 2003

Botulinum neurotoxin type A (BONT/A) is an extremely potent toxin, which is produced by Clostridium botulinum. The light chain of this protein (BONT/A LC), which is known as a zinc endopeptidase, cleaves SNAP-25 involved in the exocytosis process. In this work, the expression of recombinant BoNT/A LC in E. coli is described. The BONT/A LC gene of C. botulinum contains a high frequency of the arginine AGA and isoleucine ATA codons that are rarely used in genes of E. coli, hampering the translation of recombinant protein. The argD and ilex tRNA genes were cloned into pACYC184 vector, resulting in pAAD131X plasmid. The translational stress of the toxin gene related to codon bias was reversed by fupplernentation of the AGA arginyl tRNA of T4 phage and AUA isoleucyl tRNA of E. coli. This system may be applicable for the expression of a variety of AT-rich heterologous genes in E. coli.