AIM To explore a rapid and easy sequencing method for hepatitis C virus (HCV) genome, and establish a new sequencing method in China. METHODS Polymerase Chain Reaction (PCR) was combined with DNA sequencing techn...AIM To explore a rapid and easy sequencing method for hepatitis C virus (HCV) genome, and establish a new sequencing method in China. METHODS Polymerase Chain Reaction (PCR) was combined with DNA sequencing technique. PCR products were purified by agarose gel electrophoresis, polyacrylamide gel electrophoresis (PAGE), Polyethylene glycol (PEG) respectively. Then in the presence of a 5′ labeling PCR primer, purified PCR products were directly sequenced. By this method, HCV NS5b cDNA from two HCV infected individuals (HC 42 and HC 49) were sequenced.展开更多
AIM： To establish the role of human T Cell Factor-4 （hTCF-4） gene exons 3-9 mutation status in association with sporadic rectal cancer with microsatellite instability （MSI）. METHODS： Microsatellite markers were ...AIM： To establish the role of human T Cell Factor-4 （hTCF-4） gene exons 3-9 mutation status in association with sporadic rectal cancer with microsatellite instability （MSI）. METHODS： Microsatellite markers were genotyped in 93 sporadic rectal cancer patients. Eleven cases were found to be high-frequency MSI （MSI-H）. Sequence analysis of the coding region of the exons 3-9 of hTCF-4 gene was carried out for the 11 MSI-H cases and 10 controls （5 microsatellite stability （MSS） cases and 5 cases with normal mucosa）. The sequencing and MSI identification were used. RESULTS： Several novel mutations and variants were revealed. In exon 4, one is a 4-position continuous alteration which caused amino acid change from Q131T and S132I （391insA, 392 G 〉 A, 393 A 〉 G and 395delC） and another nucleotide deletion （395delC） is present in MSI-H cases （5/10 and 4/10, respectively） but completely absent in the controls.CONCLUSION： Novel mutations in exon 4 of hTCF-4 gene were revealed in this study, which might be of importance in the pathogenesis of sporadic rectal cancer patients with MSI-H.展开更多
The subgenomic mRNA transcription and genomic replication of the porcine reproductive and respiratory syndrome virus (PRRSV) are directed by the viral replicase. The replicase is expressed in the form of two polyprote...The subgenomic mRNA transcription and genomic replication of the porcine reproductive and respiratory syndrome virus (PRRSV) are directed by the viral replicase. The replicase is expressed in the form of two polyproteins and is subsequently processed into smaller nonstructural proteins (nsps). nsp9, containing the viral replicase, has characteristic sequence motifs conserved among the RNA-dependent RNA polymerases (RdRp) of positive-strand (PS) RNA viruses. To test whether the conserved SDD motif can tolerate other conserved motifs of RNA viruses and the influence of every residue on RdRp catalytic activity, many amino acids substitutions were introduced into it. Only one nsp9 substitution, of serine by glycine (S3050G), could rescue mutant viruses. The rescued virus was genetically stable. Alteration of either aspartate residue was not tolerated, destroyed the polymerase activity, and abolished virus transcription, but did not eliminate virus replication. We also found that the SDD motif was essentially invariant for the signature sequence of PRRSV RdRp. It could not accommodate other conserved motifs found in other RNA viral polymerases, except the GDD motif, which is conserved in all the other PS RNA viruses. These findings indicated that nidoviruses are evolutionarily related to other PS RNA viruses. Our studies support the idea that the two aspartate residues of the SDD motif are critical and essential for PRRSV transcription and represent a sequence variant of the GDD motif in PS RNA viruses.展开更多
文摘AIM To explore a rapid and easy sequencing method for hepatitis C virus (HCV) genome, and establish a new sequencing method in China. METHODS Polymerase Chain Reaction (PCR) was combined with DNA sequencing technique. PCR products were purified by agarose gel electrophoresis, polyacrylamide gel electrophoresis (PAGE), Polyethylene glycol (PEG) respectively. Then in the presence of a 5′ labeling PCR primer, purified PCR products were directly sequenced. By this method, HCV NS5b cDNA from two HCV infected individuals (HC 42 and HC 49) were sequenced.
基金the National Natural Science Foundation of China, No. 39925032
文摘AIM： To establish the role of human T Cell Factor-4 （hTCF-4） gene exons 3-9 mutation status in association with sporadic rectal cancer with microsatellite instability （MSI）. METHODS： Microsatellite markers were genotyped in 93 sporadic rectal cancer patients. Eleven cases were found to be high-frequency MSI （MSI-H）. Sequence analysis of the coding region of the exons 3-9 of hTCF-4 gene was carried out for the 11 MSI-H cases and 10 controls （5 microsatellite stability （MSS） cases and 5 cases with normal mucosa）. The sequencing and MSI identification were used. RESULTS： Several novel mutations and variants were revealed. In exon 4, one is a 4-position continuous alteration which caused amino acid change from Q131T and S132I （391insA, 392 G 〉 A, 393 A 〉 G and 395delC） and another nucleotide deletion （395delC） is present in MSI-H cases （5/10 and 4/10, respectively） but completely absent in the controls.CONCLUSION： Novel mutations in exon 4 of hTCF-4 gene were revealed in this study, which might be of importance in the pathogenesis of sporadic rectal cancer patients with MSI-H.
基金supported by the National Natural Science Foundation of China (Grant Nos. 30972204 and 30901078)the EU Frame 7 Project (Grant No. 245141)the Shanghai Municipal Science and Technology Special Programs (Grant No. 08ZR1422800) to Yuan ShiShan
文摘The subgenomic mRNA transcription and genomic replication of the porcine reproductive and respiratory syndrome virus (PRRSV) are directed by the viral replicase. The replicase is expressed in the form of two polyproteins and is subsequently processed into smaller nonstructural proteins (nsps). nsp9, containing the viral replicase, has characteristic sequence motifs conserved among the RNA-dependent RNA polymerases (RdRp) of positive-strand (PS) RNA viruses. To test whether the conserved SDD motif can tolerate other conserved motifs of RNA viruses and the influence of every residue on RdRp catalytic activity, many amino acids substitutions were introduced into it. Only one nsp9 substitution, of serine by glycine (S3050G), could rescue mutant viruses. The rescued virus was genetically stable. Alteration of either aspartate residue was not tolerated, destroyed the polymerase activity, and abolished virus transcription, but did not eliminate virus replication. We also found that the SDD motif was essentially invariant for the signature sequence of PRRSV RdRp. It could not accommodate other conserved motifs found in other RNA viral polymerases, except the GDD motif, which is conserved in all the other PS RNA viruses. These findings indicated that nidoviruses are evolutionarily related to other PS RNA viruses. Our studies support the idea that the two aspartate residues of the SDD motif are critical and essential for PRRSV transcription and represent a sequence variant of the GDD motif in PS RNA viruses.
