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Analysis of glcoamylase gene from Pholiota nameko
Yukiko Kinjo,Yan Li,Ruirong Yi,Jia ning Wan,Takeshi Yamaguchi,Norihiro Shimomura,Tadanori Aimi 한국버섯학회 2010 한국버섯학회지 Vol.8 No.4
In the past studies of Lyophlium shimeji, it was reported that the quantity of sufficient starch used as a carbon source was able to supply the factor that allows successful fruit-body formation without raising osmotic pressure in the medium. Glucoamylase are exo Glucosyl hydrolase, which catalyze the release glucose from the nonreducing ends of amylose, amylopectin, and other polysaccharides. Glucoamylase genes are found in many prokaryotic and eukaryotic microbes that use starch as a carbon source. It was believed to be important in the utilization of starch by the basidiomycetous fungus. Glucoamylase activity in the medium increased markedly during fruit-body formation. So study of the characteristic of glucoamylase in Pholiota nameko will provide the basis for P .nameko fruit body formation. In this research, in order to confirm the presence of glucoamylase gene in P. nameko genome, the genomic DNA was prepared from P. nameko NGW19-6 strain and was used as template to amplify the glucoamylases gene (PnGlu1). To prepare genomic DNA from the P. nameko NGW19-6 strain, the mycelium was grown on 10 ml of PD liquid medium (potato 200 g/l ,Glucose 20 g/l) prepared with tap water in a 100 ml Erlenmeyer flask and at 25°C for 7 days. Genomic DNA fragment encoding the glucoamylase protein (PnGlu1) were amplified by PCR with degenerate primer F15-GP2-AF/F15-GP2-BR. The primer pair was designed based on the amino acid sequences GLGEPKF and FDLWEEI, respectively, which are conserved in the glucoamylase protein of Laccaria bicolor. This produce fragments of approximately 400 bp. Next, to amplify the whole genomic clone of PnGlu1, oligonucleotide primer PnGP2F/ PnGP2R were designed based on the nucleotide sequence of DNA fragments amplified by cassette PCR method. The produced fragment has significant homology with glucoamylase of L. bicolor. To investigate the relationship between different composition of medium and glucoamylase expression, we checked the expression level of glucoamylase gene by realtime RT-PCR and measurement of glucoamylase enzyme activity.
Distribution of cytomegalovirus genotypes among ulcerative colitis patients in Okinawa, Japan
Saifun Nahar,Akira Hokama,Atsushi Iraha,Tetsuya Ohira,Tetsu Kinjo,Tetsuo Hirata,Takeshi Kinjo,Gretchen L. Parrott,Jiro Fujita 대한장연구학회 2018 Intestinal Research Vol.16 No.1
Background/Aims: To determine the prevalence of glycoprotein B (gB), glycoprotein N (gN), and glycoprotein H (gH) genotypes of human cytomegalovirus (HCMV) superimposed on ulcerative colitis (UC) patients in Japan. Methods: Four archivedstool samples and 7-archived extracted DNA from stool samples of 11 UC patients with positive multiplex polymerase chainreaction (PCR) results for HCMV were used UL55 gene encoding gB, UL73 gene encoding gN, and UL75 gene encoding gH were identified by PCR. Genotypes of gB and glycoprotein N were determined by sequencing. Results: Among 11 samples, 8samples were amplified through PCR. gB, gN, and gH genotypes were successfully detected in 3 of 8 (37.5%), 4 of 8 (50%), and8 of 8 (100%), respectively. The distribution of gB and gN genotypes analyzed through phylogenetic analysis were as follows:gB1 (2/3, 66.7%), gB3 (1/3, 33.3%), gN3a (2/4, 50%), and gN3b (2/4, 50%). Other gB genotypes (gB2 and gB4) and gN genotypes(gN1, gN2, and gN4) were not detected in this study. Out of successfully amplified 8 samples of gH genotype, gH1 and gH2 weredistributed in 12.5% and 75% samples, respectively. Only 1 sample revealed mixed infection of gH genotype. The distribution ofgH1 and gH2 differed significantly (1:6, P <0.05) in UC patients. The distribution of single gH genotype also revealed significantdifference in UC patients who were treated with immunosuppressive drug (P <0.05). Conclusions: In this study, gB1, gN3, andgH2 gene were determined as the most frequently observed genotypes in UC patients, which suggest that there might be an association between these genotypes of HCMV and UC.