Characterization of CEP131, a novel centriolar satellite protein

Thesis(masters) --서울대학교 대학원 :생명과학부,2009.2.Maste

Direct electron transfer between frhAGB-encoding hydrogenase and TrxR in a non-methanogenic hyperthermophilic archaeon

Previously, a gene cluster homologous to the frhAGB-encoding F420-reducing hydrogenase (Frh) was identified in a non-methanogen Thermococcus onnurineus NA1. The heterotrimeric enzyme complex exhibited hydrogenase activity with methyl viologen, FMN or FAD as electron acceptor, but not with deazaflavin coenzyme F420, implicating that it has a role distinct from that of homologues from methanogens. Herein, we describe that Frh from T. onnurineus NA1 can reduce thioredoxin reductase (TrxR). In the course of understanding the role of TrxR in T. onnurineus NA1, we employed protein-protein interaction tools and some protein bands, of which one band was identified as a protein coded by frhG afterwards, were pulled down with TrxR in the immunoprecipitation experiment using an antibody of TrxR. The relationship between the hydrogenase and TrxR was further investigated, and TrxR could be reduced directly by Frh, coupled with H2 oxidation in the absence of any other electron carriers. Subsequently, the reduced TrxR by Frh relayed cascade of electron transfer to a sulfur response regulator, SurR. The mechanism behind the phenomena will be discussed.en, FMN or FAD as electron acceptor, but not with deazaflavin coenzyme F420, implicating that it has a role distinct from that of homologues from methanogens. Herein, we describe that Frh from T. onnurineus NA1 can reduce thioredoxin reductase (TrxR). In the course of understanding the role of TrxR in T. onnurineus NA1, we employed protein-protein interaction tools and some protein bands, of which one band was identified as a protein coded by frhG afterwards, were pulled down with TrxR in the immunoprecipitation experiment using an antibody of TrxR. The relationship between the hydrogenase and TrxR was further investigated, and TrxR could be reduced directly by Frh, coupled with H2 oxidation in the absence of any other electron carriers. Subsequently, the reduced TrxR by Frh relayed cascade of electron transfer to a sulfur response regulator, SurR. The mechanism behind the phenomena will be discussed.2

Direct electron transfer between frhAGB-encoding hydrogenase and TrxR in a non-methanogenic hyperthermophilic archaeon

Previously, a gene cluster homologous to the frhAGB encoding F420-reducing hydrogenase (Frh) was identified in a non-methanogen Thermococcus onnurineus NA.The heterotrimeric enzyme complex exhibited hydrogenase activity with methyl viologen, FMN or FAD as electron acceptor, but not with deazaflavin coenzyme F420, implicating that it has a role distinct from that of homologues from methanogens. Herein, we describe that Frh from T. onnurineus NA1 can reduce thioredoxin reductase (TrxR). In the course of understanding the role of TrxR in T. onnurineus NA1, we employed protein-protein interaction tools and some protein bands, of which one band was identified as a protein coded by frhG afterwards, were pulled down with TrxR in the immunoprecipitation experiment using an antibody against TrxR. The relationship between the hydrogenase and TrxR was further investigated, and TrxR could be reduced directly by Frh, coupled with H2 oxidation in the absence of any other electron carriers. Subsequently, the reduced TrxR by Frh relayed cascade of electron transfer to a sulfur response regulator, SurR. The mechanism behind the phenomena will be discussed., FMN or FAD as electron acceptor, but not with deazaflavin coenzyme F420, implicating that it has a role distinct from that of homologues from methanogens. Herein, we describe that Frh from T. onnurineus NA1 can reduce thioredoxin reductase (TrxR). In the course of understanding the role of TrxR in T. onnurineus NA1, we employed protein-protein interaction tools and some protein bands, of which one band was identified as a protein coded by frhG afterwards, were pulled down with TrxR in the immunoprecipitation experiment using an antibody against TrxR. The relationship between the hydrogenase and TrxR was further investigated, and TrxR could be reduced directly by Frh, coupled with H2 oxidation in the absence of any other electron carriers. Subsequently, the reduced TrxR by Frh relayed cascade of electron transfer to a sulfur response regulator, SurR. The mechanism behind the phenomena will be discussed.1

Performance analysis of modified digital costas loop

학위논문(석사) - 한국과학기술원 : 전기 및 전자공학과, 1980.2, [ v, 97 p. ]A new type of digital phase-locked loop(DPLL) that employs a phase error detector with linear characteristic is proposed and analyzed for the first-and second-order loops in the absence and presence of noise. By inserting the function $\tan^{-1}(\cdot)$ in the loop, the DPLL phase error detector characteristic becomes linear. Consequently, the system equation describing the behavior of the loop is also linear. In the absence of noise, the first-and second-order loops have been analyzed by plotting the phase planes. Also, the false lock and oscillation phenomena occurring under some initial conditions have been considered. In addition, the locking range for the DPLL to achieve exact locking independently of initial conditions is obtained in a closed form for the firstand second- order systems. These results are verified by computer simulation. In the presence of noise, the loop is analyzed by solving Chapmann-Kolmogorov(C-K) equation. The steady state probability density function(pdf) of the phase error has been obtained by solving the C-K equation numerically. The mean and variance of the phase error in the steady state have been obtained analytically, and are compared with the results obtained by computer simulation. Finally, the conditions that cycle slipping occurs have been derived for the first-and second-order loops mathematically. The probability of cycle slipping has been obtained by computer simulation for the first- and second-order loops.한국과학기술원 : 전기 및 전자공학과

Enhanced hydrogen production by a laboratory-evolved archaeon in formate

The hyperthermophilic archaeon Thermococcus onnurineus NA1 has been reported to grow on formate coupled with H2 production and ATP generation (HCOO- + H2O→HCO3- + H2, ΔG80oC = -2.6 kJ/mol). In this study, we applied adaptive evolution toimprove H2 productivity of the strain. The strain was grown in a medium containing formate and then transferred to a fresh medium every 15 hours. Through serial transfers, physiological changes were monitored and gradual increases in cell density, H2 production and formate consumption were observed. After 156 transfers, the evolved strain showed 1.7-, 1.9- and 1.9-fold higher values in cell density, H2 production and formate consumption in comparison with those of the parental strain. In order to obtain an integrative understanding of the genetic and phenotypic changes during evolution, genome sequencing was performed and 11 mutation sites, including 2 insertions, 2 deletions and 7 substitutions either at the intergenic (2 sites) or coding regions (9 sites), were observed. When we introduced each reverse mutation in the evolved strain, two revertants for TON_1561 and TON_1573 encoding F420-reducing hydrogenase β subunit and formate transporter,respectively exhibited significant decreases in the cell density and hydrogen production. On the other hand, the mutations in TON_1561 or TON_1573 were introduced to the wild-type strain and only TON_1573 A52T mutant displayed increases ofmprove H2 productivity of the strain. The strain was grown in a medium containing formate and then transferred to a fresh medium every 15 hours. Through serial transfers, physiological changes were monitored and gradual increases in cell density, H2 production and formate consumption were observed. After 156 transfers, the evolved strain showed 1.7-, 1.9- and 1.9-fold higher values in cell density, H2 production and formate consumption in comparison with those of the parental strain. In order to obtain an integrative understanding of the genetic and phenotypic changes during evolution, genome sequencing was performed and 11 mutation sites, including 2 insertions, 2 deletions and 7 substitutions either at the intergenic (2 sites) or coding regions (9 sites), were observed. When we introduced each reverse mutation in the evolved strain, two revertants for TON_1561 and TON_1573 encoding F420-reducing hydrogenase β subunit and formate transporter,respectively exhibited significant decreases in the cell density and hydrogen production. On the other hand, the mutations in TON_1561 or TON_1573 were introduced to the wild-type strain and only TON_1573 A52T mutant displayed increases of2

Direct Electron Transfer between the frhAGB-Encoding Hydrogenase and Thioredoxin Reductase in a Non-Methanogenic Archaeon Thermococcus onnurnieus NA1

Thioredoxin reductase (TrxR), a dimeric flavin-containing enzyme, plays several important roles in maintaining redox status inside cell by reducing thioredoxin (Trx). Previously, we reported that TrxR in a hyperthermophilic archaeon Thermococcus onnurineus NA1, could be reduced by NAD(P)H and provide reducing power to protein disulfide oxidoreductase (Pdo) and other Trx homologous proteins. In the course of understanding the role of TrxR in T. onnurineus NA1, it was found that a protein co-immunoprecipitated with TrxR using an anti-TrxR antibody. The protein was identified as frhG subunit of the frhAGB-encoding hydrogenase by mass spectrometric analysis. The interaction between TrxR and frhAGB-encoding hydrogenase was confirmed by in vitro pull-down assays. Based on the interaction data, the possibility of electron transfer from frhAGB-encoding hydrogenase to TrxR was investigated. The frhAGB-encoding hydrogenase was capable of reducing TrxR only in the presence of H2. These results indicate that the frhAGB-encoding hydrogenase in the non-methanogenic T. onnurineus NA1 has a distinguished way of electron transfer from those in methanogens employing coenzyme F420. Our findings provide insight into the functionality of the frhAGB-encoding hydrogenase in non-methanogens and the underlying mechanism for the electron transfer in the absence of F420 cofactor.1

Direct Electron Transfer between the frhAGB-Encoding Hydrogenase and Thioredoxin Reductase in a Non-Methanogenic Archaeon Thermococcus onnurnieus NA1

The thioredoxin reductase (TrxR) of the hyperthermophilic archaeon T. onnurineus NA1 is known to be an NADP-dependent enzyme that uses NAD(P)H as an electron donor. In this study, we demonstrate that electrons generated via hydrogenase-mediated H2 oxidation can be utilized by TrxR directly without the use of other electron carriers. The frhAGB-encoding hydrogenase, a homolog of the F420-reducing hydrogenase of methanogens, that lacks F420-reducing activity, was shown to interact with TrxR in coimmunoprecipitation experiments and in vitro pull-down assays. Furthermore, the transfer of electrons from H2 between the frhAGB-encoding hydrogenase and TrxR was demonstrated by reduction of a redox partner of TrxR, Pdo, identified in the redox cascades of T. onnurineus NA1. Interaction and electron transfer were observed between TrxR and the heterodimeric (FrhAG) hydrogenase complex as well as the heterotrimeric complex (FrhAGB). This study revealed the functionality of the frhAGB-encoding hydrogenase in a non-methanogen and provided insight into the mechanism underlying electron transfer by the hydrogenase to a target in the absence of the F420 cofactor.1

Adaptive evolution of a hyperthermophilic archaeon Thermococcus onnurineus NA1 during growth on formate

The hyperthermophilic archaeon Thermococcus onnurineus NA1 has been reported to grow on formate coupled with H&not 2 production and ATP generation (HCOO- + H2O → HCO3- + H2, ΔG80oC = -2.6 kJ/mol). In an attempt to develop the strain for the enhanced H2 production on formate, adaptive evolution was adopted. T. onnurineus NA1 was grown to stationary phase to trigger spontaneous mutation and then transferred to a fresh medium containing formate. Through serial transfers of batch cultures, physiological changes were monitored and gradual increases in cell density, H2 production rate and formate consumption rate were observed. After 156 transfers, the evolved strain showed 1.4-, 1.3- and 1.3-fold higher values in maximum cell density, H2 production rate and formate consumption rate, respectively, in comparison with those of parental strain. In order to obtain an integrative understanding of the genetic and phenotypic changes during evolution on formate, genome sequencing was performed using the PacBio Single Molecule Real-Time (SMRT) sequencing technology. As a result, we discovered eleven mutation sites, including insertion (2 sites), deletion (2 sites) and substitution (7 sites) either at the intergenic (2 sites) or coding regions (9 sites) in a DNA sequence. Further mutational studies would be required to assess the contribution of gene mutations to the phenotypic changes during evolution. The potential of the enginehe enhanced H2 production on formate, adaptive evolution was adopted. T. onnurineus NA1 was grown to stationary phase to trigger spontaneous mutation and then transferred to a fresh medium containing formate. Through serial transfers of batch cultures, physiological changes were monitored and gradual increases in cell density, H2 production rate and formate consumption rate were observed. After 156 transfers, the evolved strain showed 1.4-, 1.3- and 1.3-fold higher values in maximum cell density, H2 production rate and formate consumption rate, respectively, in comparison with those of parental strain. In order to obtain an integrative understanding of the genetic and phenotypic changes during evolution on formate, genome sequencing was performed using the PacBio Single Molecule Real-Time (SMRT) sequencing technology. As a result, we discovered eleven mutation sites, including insertion (2 sites), deletion (2 sites) and substitution (7 sites) either at the intergenic (2 sites) or coding regions (9 sites) in a DNA sequence. Further mutational studies would be required to assess the contribution of gene mutations to the phenotypic changes during evolution. The potential of the engine2