[Objective] To establish an efficient, convenient and economical method for site-directed mutagenesis. [Method] The target mutation was introduced into primers designed by DNAMAN5.0 software. Through overlap extension...[Objective] To establish an efficient, convenient and economical method for site-directed mutagenesis. [Method] The target mutation was introduced into primers designed by DNAMAN5.0 software. Through overlap extension PCR for twice obtained the mutation gene which of the full length of the recombinant Human Tissue type plasminogen activator （Reteplase）. The mutation gene cloned it into pEASY- blunt simple cloning vector for sequencing. [Result] The sequencing results showed that three site mutations were fully consistent with the expected results （10~ site had been added a base-pair of A, C had been changed into G at 137~ site, G had been changed into A at 686~ site）.Three site mutations were introduced by using overlap extension PCR on one-step. The overall rate of obtaining the mutant sites was 100%. Site-directed mutagenesis will clone the recombinant Human Tissue type plas- minogen activator and laid the basis for the functional study. [Conclusion] Site-directed mutagenesis was successfully implemented based on the overlap extension PCR which is an efficient, convenient and economical DNA-directed mutagenesis method.展开更多
This study aimed to investigate the synergism of the TAT PTD （ protein transduction domain in HIV-1 transactivator of transcription protein） to antibacte- rial peptide tachyplesin from Tachp/eus tr/dentatus. Treated...This study aimed to investigate the synergism of the TAT PTD （ protein transduction domain in HIV-1 transactivator of transcription protein） to antibacte- rial peptide tachyplesin from Tachp/eus tr/dentatus. Treated with Pichia pastoris preferred codon optimization, using the eDNA sequence of tuchyplesin mature pep- tide （54 aa） harboring TAT FFD sequence （11 aa） as reference template, six single-stranded oligonueleotides were designed, the sequences of restriction sites EcoR I and Xba I were introduced to the 5＇ end of primers P1 and P6, respectively. TAT PTD ＋ Tachyplesin fusion gene with a full length of 219 bp was artificially synthesized by overlap extension PCR, which laid the preliminary foundation for subsequent functional and synergism studies.展开更多
Using transaction-level tick-by-tick data of same-and next-day settlement of the Russian Ruble versus the US Dollar exchange rate(RUB/USD)traded on the Moscow Exchange Market during the period 2005–2013,we analyze th...Using transaction-level tick-by-tick data of same-and next-day settlement of the Russian Ruble versus the US Dollar exchange rate(RUB/USD)traded on the Moscow Exchange Market during the period 2005–2013,we analyze the impact of trading hours extensions on volatility.During the sample period,the Moscow Exchange extended trading hours three times for the same-day settlement and two times for the next-day settlement of the RUB/USD rate.To analyze the effect of the implementations,various measures of historical and realized volatility are calculated for 5-and 15-min intraday intervals spanning a period of three months both prior to and following trading hours extensions.Besides historical volatility measures,we also examine volume and spread.We apply an autoregressive moving average-autoregressive conditional heteroscedasticity(ARMA-GARCH)model utilizing realized volatility and a trade classification rule to estimate the probability of informed trading.The extensions of trading hours cause a significant increase in both volatility and volume for further analyzing the reasons behind volatility changes.Volatility changes mostly occur after the opening of the market.The length of the extension has a significant positive effect on realized volatility.The results indicate that informed trading increased substantially after the opening for the rate of same-day settlement,whereas this is not observed for next-day settlement.Although trading hours extensions raise opportunities for more transactions and liquidity in foreign exchange markets,they may also lead to higher volatility in the market.Furthermore,this distortion is more significant at opening and midday.A potential explanation for the increased volatility mostly at the opening is that the trading hours extension attracts informed traders rather than liquidity providers.展开更多
[Objective] The aim of this study was to produce Streptomyces avermitilis strain with site-directed mutagenesis in aveD gene,and so as to provide theoretical basis for genetic breeding of S.avermitilis.[Method] PCR-dr...[Objective] The aim of this study was to produce Streptomyces avermitilis strain with site-directed mutagenesis in aveD gene,and so as to provide theoretical basis for genetic breeding of S.avermitilis.[Method] PCR-driven overlap extension was conducted for the site-directed mutagenesis in aveD gene;the mutated aveD gene then was used to construct vector pDC3（pKC1139∷aveD） via molecular manipulations like in vitro enzyme digestion and ligation;the vector pDC3（pKC1139∷aveD） was then introduced to aveD deletion mutant 489 of avermectin-producing strain S.avermitilis 76-9.[Result] Mutant strain 536 of site-directed mutagenesis of S.avermitilis 76-9 was obtained by homologous recombination.The sequencing results show that the sixty-ninth base C in aveD-coding region of mutant 536 was substituted by T,and the corresponding amino acid Thr was mutated to be Ile.[Conclusion] This study laid basis for the development of strains specifically producing avermectin B.展开更多
基金Supported by National Natural Science Foundation of China(31160032)~~
文摘[Objective] To establish an efficient, convenient and economical method for site-directed mutagenesis. [Method] The target mutation was introduced into primers designed by DNAMAN5.0 software. Through overlap extension PCR for twice obtained the mutation gene which of the full length of the recombinant Human Tissue type plasminogen activator （Reteplase）. The mutation gene cloned it into pEASY- blunt simple cloning vector for sequencing. [Result] The sequencing results showed that three site mutations were fully consistent with the expected results （10~ site had been added a base-pair of A, C had been changed into G at 137~ site, G had been changed into A at 686~ site）.Three site mutations were introduced by using overlap extension PCR on one-step. The overall rate of obtaining the mutant sites was 100%. Site-directed mutagenesis will clone the recombinant Human Tissue type plas- minogen activator and laid the basis for the functional study. [Conclusion] Site-directed mutagenesis was successfully implemented based on the overlap extension PCR which is an efficient, convenient and economical DNA-directed mutagenesis method.
基金Supported by Natural Science Foundation of Guangdong Province(06025389)Post-doctoral Project of Fujian Province
文摘This study aimed to investigate the synergism of the TAT PTD （ protein transduction domain in HIV-1 transactivator of transcription protein） to antibacte- rial peptide tachyplesin from Tachp/eus tr/dentatus. Treated with Pichia pastoris preferred codon optimization, using the eDNA sequence of tuchyplesin mature pep- tide （54 aa） harboring TAT FFD sequence （11 aa） as reference template, six single-stranded oligonueleotides were designed, the sequences of restriction sites EcoR I and Xba I were introduced to the 5＇ end of primers P1 and P6, respectively. TAT PTD ＋ Tachyplesin fusion gene with a full length of 219 bp was artificially synthesized by overlap extension PCR, which laid the preliminary foundation for subsequent functional and synergism studies.
文摘Using transaction-level tick-by-tick data of same-and next-day settlement of the Russian Ruble versus the US Dollar exchange rate(RUB/USD)traded on the Moscow Exchange Market during the period 2005–2013,we analyze the impact of trading hours extensions on volatility.During the sample period,the Moscow Exchange extended trading hours three times for the same-day settlement and two times for the next-day settlement of the RUB/USD rate.To analyze the effect of the implementations,various measures of historical and realized volatility are calculated for 5-and 15-min intraday intervals spanning a period of three months both prior to and following trading hours extensions.Besides historical volatility measures,we also examine volume and spread.We apply an autoregressive moving average-autoregressive conditional heteroscedasticity(ARMA-GARCH)model utilizing realized volatility and a trade classification rule to estimate the probability of informed trading.The extensions of trading hours cause a significant increase in both volatility and volume for further analyzing the reasons behind volatility changes.Volatility changes mostly occur after the opening of the market.The length of the extension has a significant positive effect on realized volatility.The results indicate that informed trading increased substantially after the opening for the rate of same-day settlement,whereas this is not observed for next-day settlement.Although trading hours extensions raise opportunities for more transactions and liquidity in foreign exchange markets,they may also lead to higher volatility in the market.Furthermore,this distortion is more significant at opening and midday.A potential explanation for the increased volatility mostly at the opening is that the trading hours extension attracts informed traders rather than liquidity providers.
基金Supported by Special Fund for Doctors in Universities in Hebei Province(B2003201)~~
文摘[Objective] The aim of this study was to produce Streptomyces avermitilis strain with site-directed mutagenesis in aveD gene,and so as to provide theoretical basis for genetic breeding of S.avermitilis.[Method] PCR-driven overlap extension was conducted for the site-directed mutagenesis in aveD gene;the mutated aveD gene then was used to construct vector pDC3（pKC1139∷aveD） via molecular manipulations like in vitro enzyme digestion and ligation;the vector pDC3（pKC1139∷aveD） was then introduced to aveD deletion mutant 489 of avermectin-producing strain S.avermitilis 76-9.[Result] Mutant strain 536 of site-directed mutagenesis of S.avermitilis 76-9 was obtained by homologous recombination.The sequencing results show that the sixty-ninth base C in aveD-coding region of mutant 536 was substituted by T,and the corresponding amino acid Thr was mutated to be Ile.[Conclusion] This study laid basis for the development of strains specifically producing avermectin B.
