• Microbiology and Biotechnology Letters
• /
• v.48 no.2
• /
• pp.158-166
• /
• 2020

A gene coding for the xylanase was cloned from Paenibacillus woosongensis, followed by determination of its complete nucleotide sequence. This xylanase gene, designated as xyn10A, consists of 1,446 nucleotides encoding a polypeptide of 481 amino acid residues. Based on the deduced amino acid sequence, Xyn10A was identified to be a modular enzyme composed of a catalytic domain highly homologous to the glycosyl hydrolase family 10 xylanase and a putative carbohydrate-binding module (CBM) in the C-terminus. By using DEAE-sepharose and phenyl-sepharose column chromatography, Xyn10A was purified from the cellfree extract of recombinant Escherichia coli carrying a P. woosongensis xyn10A gene. The N-terminal amino acid sequence of the purified Xyn10A was identified to exactly match the sequence immediately following the signal peptide predicted by the Signal5.0 server. The purified Xyn10A was a truncated protein of 33 kDa, suggesting the deletion of CBM in the C-terminus by intracellular hydrolysis. The purified enzyme had an optimum pH and temperature of 6.0 and 55-60℃, respectively, with the kinetic parameters V_max and K_m of 298.8 U/mg and 2.47 mg/ml, respectively, for oat spelt xylan. The enzyme was more active on arabinoxylan than on oat spelt xylan and birchood xylan with low activity for p-nitrophenyl-β-xylopyranoside. Xylanase activity was significantly inhibited by 5 mM Cu²⁺, Mn²⁺, and SDS, and was noticeably enhanced by K⁺, Ni²⁺, and Ca²⁺. The enzyme could hydrolyze xylooligosaccharides larger than xylobiose. The predominant products resulting from xylooligosaccharide hydrolysis were xylobiose and xylose.

• Korean Journal of Microbiology
• /
• v.42 no.2
• /
• pp.142-148
• /
• 2006

In order to develop chitosanase for the production of chitosan oligosaccharides, a chitosanase-producing bacterium was isolated from the traditional fermented soybean, Meju, and identified as Bacillus amyloliquefaciene MJ-1. The cloned chitosanase gene, 825 bp in size, encoded a single peptide of 274 amino acids with a estimated molecular mass of 30.9 kDa. The deduced amino acid sequence showed significant homology with microbial chitosanases. The recombinant chitosanase was expressed in Escherichia coli upon induction with isopropyl-D-thiogalactopyranoside, and purified using $Ni^{2+}-NTA$ agarose column chromatography. The maximal activity of the recombinant chitosanase is at pH 5.0 and $60^{\circ}C$. The recombinant chitosanase is stable between pH 5.0 and pH 7.0 at $37^{\circ}C$ for 30 min, and more than 75% of the activity still remain at $80^{\circ}C$ for 30 min incubation.

• Korean Journal of Poultry Science
• /
• v.20 no.4
• /
• pp.177-188
• /
• 1993

This study was performed to find out the reasonable sexing methods In the chicken, obtain the basic information for the mechanisms related to chicken sexual differentiation and identify the genes which known to involved in chicken sex differentiation. The chromosome analysis of chicken embryonic fibroblast was a simple method to determine sex of chicken by means of Z and W chromosome identification. The bands of female chicken genomic DNA digested with Xho Ⅰ and Eco RI restriction endonuclease showed to be useful in direct sex determination and these repetitive sequences of Xho Ⅰ and Eco RI families were proposed to be very homologous in their sequences by colony hybridization analysis. Seven of 150 random primers were selected to amplify the W chromosome-specific band by using arbitrary primed PCR and three of them were useful to identify the sex of chicken. To identify the sex differentiation genes in the chicken, PCR for the amplification of ZFY and SRY sequences was performed. ZFY and SRY sequences were amplified successfully in the chicken genome, implying that chicken genome might have the sex-related conserved sequences similar to mammalian ones. The PCR products of ZFY amplification were the same in both sexes, suggesting that these sequences may be located on autosome or Z chromosome. The profile of PCR amplification for SRY sequences showed variation between sexes, but this result was not enough to specify whether the SRY gene in chicken is on the autosome or sex chromosome.

• Journal of Marine Life Science
• /
• v.1 no.2
• /
• pp.79-87
• /
• 2016

Heavy metals such as cadmium (Cd) are highly toxic to aquatic organisms and human, even at trace concentration. Herein we investigated the effect of Cd on the gene expression of ATP-binding cassette (ABC) transporters and glutathione S-transferase (GST) in marine ciliate Euplotes crassus. Seven ABC transporters and one GST genes were partially cloned and sequences, and thereafter, transcriptional modulation of these genes after exposure to Cd for 8 h was investigated using quantitative real time RT- PCR (qRT-PCR). As results, sequence analysis and phylogenetic study revealed that E. crassus ABCs are likely typical ABC transports, in particular, B/C family, and GST gene may be similar to GST theta isoform. A significant increase in the expression of ABCs, except for ABCB21 was observed in a concentration dependent manner after exposure to Cd (0.1 and 0.5 mg/l) for 8 h. The GST mRNA level was the highest at 0.5 mg/l Cd and then reduced until control level. These findings suggest that ABCs and GST may be involved in a protective mechanism against Cd-mediated toxicity in E. crassus.

• Journal of the Korea Academia-Industrial cooperation Society
• /
• v.14 no.7
• /
• pp.3582-3588
• /
• 2013

Expressed sequence tag (EST) library was constructed from A. tamarense. Base sequences of EST clones were analyzed and saxitoxin biosynthesis-related genes were cloned. Sequences of 827 clones were analyzed and 564 EST were functionally clustered using Blast searches against GenBank. Main genes in the EST had functions on cellular organization, cell metabolism, energy, cell cycle and DNA processing, cellular transport and transport, cell rescue, defense, death and aging, and transcription. Moreover, expression of S-adenosylmethionine synthetase and H2A histone family genes were increased in the toxic A. tamarense. These results show that two genes could be a good biomarkers for the detection of saxitoxin biosynthesis in the A. tamarense.

• Korean Journal of Microbiology
• /
• v.28 no.1
• /
• pp.1-5
• /
• 1990

The coding sequence of hepatitis B viral core antigen (HBcAg) (subtype adr) contains two in-phase initiation codons, one for precore and the other for core antigen gene. To study the expression of core antigen and the role of precore region, the coding sequence of HBcAg with or without precore (pre-C) region were subcloned into yeast expression vector containing phosphoglycerate kinase (PGK) promoter. To study the role of upstream region in the expression of the core antigen, a series of 5' deletion mutants were also subcloned into the vector. After transformation into various host strains, the expression of HBcAg were analysed by radio-immunoassat. Under optimal condition of core antigen gene expression in yeast, the highest amount of antigen was detected in the cell line SHY4 containing pGKHBc plasmid composed of the yeast PGK gene promoter, terminator and C-gene. Regardless of the presence of precore region, core antigen was not detected in the medium but in cell extract. These results suggest that precore region cannot affect the secretion of core antigen in Saccharomyces cerevisiae.

• Journal of Animal Science and Technology
• /
• v.50 no.1
• /
• pp.9-18
• /
• 2008

Species-specific polymorphisms in chicken, pheasant, turkey, and quail were identified by cloning and sequencing of the immunoglobulin constant domain (IgLC). A set of species-specific primers were then designed on the basis of polymorphisms in the IgLC between species, as well as two additional sets of primers for the cytochrome b and tapasin genes, for the purpose of species identification. Together, the primers successfully distinguished specific species from chicken by species-specific PCR. This simple but unambiguous method may be used to screen avian inter-species germline chimeras, which are valuable models for the conservation of endangered species.

• Korean Journal of Microbiology
• /
• v.30 no.6
• /
• pp.466-471
• /
• 1992

To determine the site at which -1 ribosomal frameshifting occurs within the gag-pro overlap of HTL V-I. DNA fragment corresponding to a portion of the gene overlap was cloned into a SP6 vector. The resultant plasmid harbors the hybrid gene consisting of a synthetic gene encoding 5 amino acids derived from chick prelysozyme including the initiator methionine plus 141 nucleotides of gag-pro overlapping region followed by Staphylococcus aurcus protein A gene fragment. In vitro transcription by SP6 RNA polymerase with this DNA template made an abundant amount of single species mRNA. Cell-free translation programmed with the RNA transcribed in vitro yielded a polypeptide of 21 kDal in size. which could be purified into homogeneity by IgG-Sepharose affinity chromatography. In vitro system described in this study must be useful for rapid purification and sequencing of the Gag-Pro transframe protein. allowing to determine the exact frameshift site on mRNA and to identify the tRNA involved in frameshifting event for the expression of pro gene.

• Korean Journal of Microbiology
• /
• v.30 no.1
• /
• pp.30-36
• /
• 1992

nif (nitrogen fixation)-H.D, K genes of Frankia sp. SNU 014201. a symbiotic strain isolated from root nodule of Alnus hirsura, were found to be located in the genome on 13.5 kb of EcoRI, 18.0 kb of BamHI, 10.5 kb of BglII and 4.5 kb of KpnI fragments. Using EMBL-3 BamHI arms of bacteriophage lambda. the genomic library was constructed. from which fourteen recombinant phage nif-clones were selected. Among them, Ahnif-I2 had insert DNA of 18 kb, in which 7.9 kb of BamHl fragment contained nif-H, D, K and 3.6 kb of HindlIl/KpnI had nif-H and partial -D. Therefore, the 7.9 kb and 3.6 kb fragments were subcloned and partial restriction maps were constructed. As the results, nif-F1, D.K genes were found to be located continuously on the 6.5 kb of HindII/BamHI and 5.2 kb of SalIIBamHI fragment in the genome of Frankia sp. SNU 014201.

• Korean Journal of Microbiology
• /
• v.29 no.6
• /
• pp.392-396
• /
• 1991

Mu d1(Ap lac) bacteriophage can be used to search for genes which are members of a common regulatory network without having to know the functions of the genes in advance. Aim was for obtaining the loci in the SOS network as well as temperature inducible loci. For this purpose, recA441 allele was used. This allele encodes a thermosensitive recA gene product; thus, the recA441 allele can be activated upon temperature upshift without by external DNA damage. Approximately 10, 000 colonies were screened, and then searched for the colonies which expressed .betha.-galactosidase higher level at 42.deg.C than at 30.deg.C. The strains identified fell into two dlasses; (i) ones in which the increased expression was $recA^{+}$ $lexA^{+}$ -dependent, that is, din(damage-inducible) genes which were due to the activation of recA441 allele and (ii) ones in which the increased expression was $recA^{+}$ $lexA^{+}$ -independent and only temperature-inducible, tin genes. Rough mapping position was obtained for these genes.