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Kuniyo Inouye - One of the best experts on this subject based on the ideXlab platform.
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Improving the thermal stability of Avian Myeloblastosis Virus reverse transcriptase α-subunit by site-directed mutagenesis
Biotechnology Letters, 2012Co-Authors: Atsushi Konishi, Kiyoshi Yasukawa, Kuniyo InouyeAbstract:Avian Myeloblastosis Virus reverse transcriptase (AMV RT) is a heterodimer consisting of a 63 kDa α-subunit and a 95 kDa β subunit. Moloney murine leukaemia Virus reverse transcriptase (MMLV RT) is a 75 kDa monomer. These two RTs are the most extensively used for conversion of RNA to DNA. We previously developed several mutations that increase the thermostability of MMLV RT and generated a highly stable MMLV RT variant E286R/E302K/L435R/D524A by combining three of them (Glu286→Arg, Glu302→Lys, and Leu435→Arg) and the mutation to abolish RNase H activity (Asp524→Ala) [Yasukawa et al. (2010) J Biotechnol 150:299–306]. To generate a highly stable AMV RT variant, we have introduced the triple mutation of Val238→Arg, Leu388→Arg, and Asp450→Ala into AMV RT α-subunit and the resulted variant V238R/L388R/D450A, was expressed in insect cells and purified. The temperature decreasing the initial activity by 50 %, measured over 10 min, of the variant with or without template primer (T/P), poly(rA)-p(dT)_15, was 50 °C; for the wild-type AMV RT α-subunit (WT) this was 44 °C. The highest temperature at which the variant exhibited cDNA synthesis activity was 64 °C; the WT was 60 °C. A highly stable AMV RT α-subunit is therefore generated by the same mutation strategy as applied to MMLV RT and that positive charges are introduced into RT at positions that have been implicated to interact with T/P by site-directed mutagenesis.
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comparison of the thermal stabilities of the αβ heterodimer and the α subunit of Avian Myeloblastosis Virus reverse transcriptase
Bioscience Biotechnology and Biochemistry, 2011Co-Authors: Atsushi Konishi, Kiyoshi Yasukawa, Daisuke Nemoto, Kuniyo InouyeAbstract:Avian Myeloblastosis Virus reverse transcriptase (AMV RT) is a heterodimer consisting of a 63-kDa α subunit and a 95-kDa β subunit. In this study, we explored the role of the interaction between the α and β subunits on AMV RT stability. The recombinant AMV RT α subunit was expressed in insect cells and purified. It exhibited lower thermal stability than the native AMV RT αβ heterodimer. Unlike the αβ heterodimer, the α subunit was not stabilized by template-primer. These results suggest that interaction between the α and β subunits is important for AMV RT stability.
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effects of organic solvents on the reverse transcription reaction catalyzed by reverse transcriptases from Avian Myeloblastosis Virus and moloney murine leukemia Virus
Bioscience Biotechnology and Biochemistry, 2010Co-Authors: Kiyoshi Yasukawa, Atsushi Konishi, Kuniyo InouyeAbstract:The use of certain organic chemicals has been found to improve yields and specificity in PCR. In this study, we examined the effects of dimethyl sulfoxide (DMSO), formamide, and glycerol on the reverse transcription reaction catalyzed by reverse transcriptases (RTs) from Avian Myeloblastosis Virus (AMV) and Moloney murine leukemia Virus (MMLV). At 42 °C, DMSO at 24% v/v and formamide at 12-14% inhibited the cDNA synthesis reaction, but DMSO at 12% and formamide at 6-8% improved the efficiency of the cDNA synthesis reaction at low temperatures (25-34 °C). Glycerol at 10% improved the efficiency of the cDNA synthesis reaction at high temperatures (49-61 °C). The effects of DMSO and formamide appeared to be accompanied by decreases in the melting temperatures of the primers, and the effect of glycerol was due to increases in the thermal stabilities of AMV RT and MMLV RT.
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characterization of moloney murine leukaemia Virus Avian Myeloblastosis Virus chimeric reverse transcriptases
Journal of Biochemistry, 2009Co-Authors: Kiyoshi Yasukawa, Masaki Mizuno, Kuniyo InouyeAbstract:Reverse transcriptases (RTs) from Moloney murine leukaemia Virus (MMLV) and Avian Myeloblastosis Virus (AMV) contain all the fingers, palm, thumb, connection and RNase H domains. The fingers, palm and thumb domains are thought to be involved in the reverse transcription activity, and the RNase H domain is in the RNase H activity. In this study, we characterized four chimeric RTs which comprise one of the fingers, palm, thumb and RNase H domains originated from AMV RT and the other three and connection domains originated from MMLV RT. Unexpectedly, all chimeric RTs exhibited the same characteristics: their specific reverse transcription activities decreased to less than 0.1% of that of MMLV RT, while their specific RNase H activities were approximately 20% of that of MMLV RT. The decreases in the two activities of the chimeric RTs were ascribed to the decreases in k(cat). Based on that the reverse transcription activity of MMLV RT was impaired by substituting its RNase H domain with that from AMV RT, we propose that in MMLV RT, there might be an interaction between the fingers/palm/thumb domain and the RNase H domain.
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comparison of the thermal stabilities of reverse transcriptases from Avian Myeloblastosis Virus and moloney murine leukaemia Virus
Journal of Biochemistry, 2008Co-Authors: Kiyoshi Yasukawa, Daisuke Nemoto, Kuniyo InouyeAbstract:Reverse transcriptases (RTs) from Avian Myeloblastosis Virus (AMV) and Moloney murine leukaemia Virus (MMLV) have been most extensively used as a tool for conversion of RNA to DNA. In this study, we compared the thermal stabilities of AMV RT and MMLV RT by observing their irreversible thermal inactivation. The temperatures reducing initial activity by 50% in 10-min incubation, T(50), of AMV RT were 47 degrees C without the template-primer (T/P), poly(rA)-p(dT)(12-18), and 52 degrees C with the T/P (28 microM). T(50) of MMLV RT were 44 degrees C without the T/P and 47 degrees C with the T/P (28 microM). Unexpectedly, AMV RT was considerably activated when incubated with the T/P at 45 and 48 degrees C. Such activation was not observed in MMLV RT. These results suggest that AMV RT and MMLV RT are different in the following: (i) The intrinsic thermal stability of AMV RT is higher than that of MMLV RT; (ii) AMV RT is activated by thermal treatment with the T/P at 45-48 degrees C; and (iii) AMV RT is stabilized by the T/P more potently than MMLV RT. Thermodynamic analysis indicates that thermal inactivation of AMV RT and MMLV RT is due to the large entropy change of activation for thermal inactivation.
Kiyoshi Yasukawa - One of the best experts on this subject based on the ideXlab platform.
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Improving the thermal stability of Avian Myeloblastosis Virus reverse transcriptase α-subunit by site-directed mutagenesis
Biotechnology Letters, 2012Co-Authors: Atsushi Konishi, Kiyoshi Yasukawa, Kuniyo InouyeAbstract:Avian Myeloblastosis Virus reverse transcriptase (AMV RT) is a heterodimer consisting of a 63 kDa α-subunit and a 95 kDa β subunit. Moloney murine leukaemia Virus reverse transcriptase (MMLV RT) is a 75 kDa monomer. These two RTs are the most extensively used for conversion of RNA to DNA. We previously developed several mutations that increase the thermostability of MMLV RT and generated a highly stable MMLV RT variant E286R/E302K/L435R/D524A by combining three of them (Glu286→Arg, Glu302→Lys, and Leu435→Arg) and the mutation to abolish RNase H activity (Asp524→Ala) [Yasukawa et al. (2010) J Biotechnol 150:299–306]. To generate a highly stable AMV RT variant, we have introduced the triple mutation of Val238→Arg, Leu388→Arg, and Asp450→Ala into AMV RT α-subunit and the resulted variant V238R/L388R/D450A, was expressed in insect cells and purified. The temperature decreasing the initial activity by 50 %, measured over 10 min, of the variant with or without template primer (T/P), poly(rA)-p(dT)_15, was 50 °C; for the wild-type AMV RT α-subunit (WT) this was 44 °C. The highest temperature at which the variant exhibited cDNA synthesis activity was 64 °C; the WT was 60 °C. A highly stable AMV RT α-subunit is therefore generated by the same mutation strategy as applied to MMLV RT and that positive charges are introduced into RT at positions that have been implicated to interact with T/P by site-directed mutagenesis.
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comparison of the thermal stabilities of the αβ heterodimer and the α subunit of Avian Myeloblastosis Virus reverse transcriptase
Bioscience Biotechnology and Biochemistry, 2011Co-Authors: Atsushi Konishi, Kiyoshi Yasukawa, Daisuke Nemoto, Kuniyo InouyeAbstract:Avian Myeloblastosis Virus reverse transcriptase (AMV RT) is a heterodimer consisting of a 63-kDa α subunit and a 95-kDa β subunit. In this study, we explored the role of the interaction between the α and β subunits on AMV RT stability. The recombinant AMV RT α subunit was expressed in insect cells and purified. It exhibited lower thermal stability than the native AMV RT αβ heterodimer. Unlike the αβ heterodimer, the α subunit was not stabilized by template-primer. These results suggest that interaction between the α and β subunits is important for AMV RT stability.
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effects of organic solvents on the reverse transcription reaction catalyzed by reverse transcriptases from Avian Myeloblastosis Virus and moloney murine leukemia Virus
Bioscience Biotechnology and Biochemistry, 2010Co-Authors: Kiyoshi Yasukawa, Atsushi Konishi, Kuniyo InouyeAbstract:The use of certain organic chemicals has been found to improve yields and specificity in PCR. In this study, we examined the effects of dimethyl sulfoxide (DMSO), formamide, and glycerol on the reverse transcription reaction catalyzed by reverse transcriptases (RTs) from Avian Myeloblastosis Virus (AMV) and Moloney murine leukemia Virus (MMLV). At 42 °C, DMSO at 24% v/v and formamide at 12-14% inhibited the cDNA synthesis reaction, but DMSO at 12% and formamide at 6-8% improved the efficiency of the cDNA synthesis reaction at low temperatures (25-34 °C). Glycerol at 10% improved the efficiency of the cDNA synthesis reaction at high temperatures (49-61 °C). The effects of DMSO and formamide appeared to be accompanied by decreases in the melting temperatures of the primers, and the effect of glycerol was due to increases in the thermal stabilities of AMV RT and MMLV RT.
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characterization of moloney murine leukaemia Virus Avian Myeloblastosis Virus chimeric reverse transcriptases
Journal of Biochemistry, 2009Co-Authors: Kiyoshi Yasukawa, Masaki Mizuno, Kuniyo InouyeAbstract:Reverse transcriptases (RTs) from Moloney murine leukaemia Virus (MMLV) and Avian Myeloblastosis Virus (AMV) contain all the fingers, palm, thumb, connection and RNase H domains. The fingers, palm and thumb domains are thought to be involved in the reverse transcription activity, and the RNase H domain is in the RNase H activity. In this study, we characterized four chimeric RTs which comprise one of the fingers, palm, thumb and RNase H domains originated from AMV RT and the other three and connection domains originated from MMLV RT. Unexpectedly, all chimeric RTs exhibited the same characteristics: their specific reverse transcription activities decreased to less than 0.1% of that of MMLV RT, while their specific RNase H activities were approximately 20% of that of MMLV RT. The decreases in the two activities of the chimeric RTs were ascribed to the decreases in k(cat). Based on that the reverse transcription activity of MMLV RT was impaired by substituting its RNase H domain with that from AMV RT, we propose that in MMLV RT, there might be an interaction between the fingers/palm/thumb domain and the RNase H domain.
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comparison of the thermal stabilities of reverse transcriptases from Avian Myeloblastosis Virus and moloney murine leukaemia Virus
Journal of Biochemistry, 2008Co-Authors: Kiyoshi Yasukawa, Daisuke Nemoto, Kuniyo InouyeAbstract:Reverse transcriptases (RTs) from Avian Myeloblastosis Virus (AMV) and Moloney murine leukaemia Virus (MMLV) have been most extensively used as a tool for conversion of RNA to DNA. In this study, we compared the thermal stabilities of AMV RT and MMLV RT by observing their irreversible thermal inactivation. The temperatures reducing initial activity by 50% in 10-min incubation, T(50), of AMV RT were 47 degrees C without the template-primer (T/P), poly(rA)-p(dT)(12-18), and 52 degrees C with the T/P (28 microM). T(50) of MMLV RT were 44 degrees C without the T/P and 47 degrees C with the T/P (28 microM). Unexpectedly, AMV RT was considerably activated when incubated with the T/P at 45 and 48 degrees C. Such activation was not observed in MMLV RT. These results suggest that AMV RT and MMLV RT are different in the following: (i) The intrinsic thermal stability of AMV RT is higher than that of MMLV RT; (ii) AMV RT is activated by thermal treatment with the T/P at 45-48 degrees C; and (iii) AMV RT is stabilized by the T/P more potently than MMLV RT. Thermodynamic analysis indicates that thermal inactivation of AMV RT and MMLV RT is due to the large entropy change of activation for thermal inactivation.
Duane P. Grandgenett - One of the best experts on this subject based on the ideXlab platform.
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efficient concerted integration of retroVirus like dna in vitro by Avian Myeloblastosis Virus integrase
Nucleic Acids Research, 1994Co-Authors: Ajaykumar C. Vora, R B Inman, Michael L Fitzgerald, Mark Mccord, Duane P. GrandgenettAbstract:Abstract We report the efficient concerted integration of a linear Virus-like DNA donor into a 2.8 kbp circular DNA target by integrase (IN) purified from Avian Myeloblastosis Virus. The donor was 528 bp, contained recessed 3' OH ends, was 5' end labeled, and had a unique restriction site not found in the target. Analysis of concerted (full-site) and half-site integration events was accomplished by restriction enzyme analysis and agarose gel electrophoresis. The donor also contained the SupF gene that was used for genetic selection of individual full-site recombinants to determine the host duplication size. Two different pathways, involving either one donor or two donor molecules, were used to produce full-site recombinants. About 90% of the full-site recombinants were the result of using two donor molecules per target. These results imply that juxtapositioning an end from each of two donors by IN was more efficient than the juxtapositioning of two ends of a single donor for the full-site reaction. The formation of preintegration complexes containing integrase and donor on ice prior to the addition of target enhanced the full-site reaction. After a 30 min reaction at 37 degrees C, approximately 20-25% of all donor/target recombinants were the result of concerted integration events. The efficient production of full-site recombinants required Mg2+; Mn2+ was only efficient for the production of half-site recombinants. We suggest that these preintegration complexes can be used to investigate the relationships between the 3' OH trimming and strand transfer reactions.
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comparison of dna binding and integration half site selection by Avian Myeloblastosis Virus integrase
Journal of Virology, 1993Co-Authors: Duane P. Grandgenett, Ajaykumar C. Vora, R B Inman, Michael L FitzgeraldAbstract:Insertion of the linear retroVirus DNA genome into the host DNA by the Virus-encoded integrase (IN) is essential for efficient replication. We devised an efficient Virus-like DNA plasmid integration assay which mimics the standard oligonucleotide assay for integration. It permitted us to study, by electron microscopy and sequence analysis, insertion of a single long terminal repeat terminus (LTR half-site) of one plasmid into another linearized plasmid. The reaction was catalyzed by purified Avian Myeloblastosis Virus IN in the presence of Mg2+. The recombinant molecules were easily visualized and quantitated by agarose gel electrophoresis. Agarose gel-purified recombinants could be genetically selected by transformation of ligated recombinants into Escherichia coli HB101 cells. Electron microscopy also permitted the identification and localization of IN-DNA complexes on the Virus-like substrate in the absence of the joining reaction. Intramolecular and intermolecular DNA looping by IN was visualized. Although IN preferentially bound to AT-rich regions in the absence of the joining reaction, there was a bias towards GC-rich regions for the joining reaction. Alignment of 70 target site sequences 5' of the LTR half-site insertions with 68 target sites previously identified for the concerted insertion of both LTR termini (LTR full-site reaction) indicated similar GC inflection patterns with both insertional events. Comparison of the data suggested that IN recognized only half of the target sequences necessary for integration with the LTR half-site reaction.
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Avian Myeloblastosis Virus reverse transcriptase effect of glycerol on its hydrodynamic properties
Journal of Biological Chemistry, 1991Co-Authors: Thyhou Lin, Duane P. Grandgenett, Thomas P Quinn, Mary Walsh, James C LeeAbstract:Although reverse transcriptase has been the subject of intensive investigation, minimal information is available regarding the physical properties of the enzyme. The basic hydrodynamic properties of Avian Myeloblastosis Virus reverse transcriptase in solution were measured by both sedimentation velocity and equilibrium measurements in two buffer systems. In a 0.3 M potassium phosphate buffer system, pH 7.8, the enzyme sedimented as a homogenous particle with a sedimentation coefficient of (7.1 +/- 0.3) S with a weight-average molecular weight, Mw, of (1.52 +/- 0.05) x 10(5). Since the enzyme consists of an alpha and beta subunit of equal molar ratio with Mw of 6.3 x 10(4) and 9.4 x 10(4), respectively, it was concluded that the enzyme exists as an alpha beta heterodimer in this buffer system. In a Tris buffer system, pH 7.9, containing 0.46 M NaCl and 4% glycerol, the native enzyme also sedimented as a homogeneous particle with an apparent sedimentation coefficient of (10.1 +/- 0.5) S, without considering the effect of glycerol on solvent-protein interaction. Based on the results of Gekko and Timasheff (Gekko, K., and Timasheff, S. N. (1981) Biochemistry 20, 4667-4676) and the polarity of the enzyme, it was estimated that there is significant solvent-protein interaction even at 4% glycerol leading to a value of -0.06 g/g in the preferential solvent interaction parameter. When the solvent effect was taken into consideration, the value for s020,w increased from 10.1 to 11.9 S, implying that the native enzyme dimerizes in the presence of 4% glycerol. The combined results of gel filtration and sedimentation velocity showed that the dimerization of the enzyme to form (alpha beta)2 is favored at 20 degrees C with the alpha beta form predominating at 4 degrees C. The secondary structure of the reverse transcriptase was measured by circular dichroism. Results showed that the enzyme consists of (16 +/- 2)% alpha-helix, (24 +/- 2)% beta-sheet, (24 +/- 2)% beta-turn, and (36 +/- 4)% undefined structures.
Michael L Fitzgerald - One of the best experts on this subject based on the ideXlab platform.
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efficient concerted integration of retroVirus like dna in vitro by Avian Myeloblastosis Virus integrase
Nucleic Acids Research, 1994Co-Authors: Ajaykumar C. Vora, R B Inman, Michael L Fitzgerald, Mark Mccord, Duane P. GrandgenettAbstract:Abstract We report the efficient concerted integration of a linear Virus-like DNA donor into a 2.8 kbp circular DNA target by integrase (IN) purified from Avian Myeloblastosis Virus. The donor was 528 bp, contained recessed 3' OH ends, was 5' end labeled, and had a unique restriction site not found in the target. Analysis of concerted (full-site) and half-site integration events was accomplished by restriction enzyme analysis and agarose gel electrophoresis. The donor also contained the SupF gene that was used for genetic selection of individual full-site recombinants to determine the host duplication size. Two different pathways, involving either one donor or two donor molecules, were used to produce full-site recombinants. About 90% of the full-site recombinants were the result of using two donor molecules per target. These results imply that juxtapositioning an end from each of two donors by IN was more efficient than the juxtapositioning of two ends of a single donor for the full-site reaction. The formation of preintegration complexes containing integrase and donor on ice prior to the addition of target enhanced the full-site reaction. After a 30 min reaction at 37 degrees C, approximately 20-25% of all donor/target recombinants were the result of concerted integration events. The efficient production of full-site recombinants required Mg2+; Mn2+ was only efficient for the production of half-site recombinants. We suggest that these preintegration complexes can be used to investigate the relationships between the 3' OH trimming and strand transfer reactions.
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comparison of dna binding and integration half site selection by Avian Myeloblastosis Virus integrase
Journal of Virology, 1993Co-Authors: Duane P. Grandgenett, Ajaykumar C. Vora, R B Inman, Michael L FitzgeraldAbstract:Insertion of the linear retroVirus DNA genome into the host DNA by the Virus-encoded integrase (IN) is essential for efficient replication. We devised an efficient Virus-like DNA plasmid integration assay which mimics the standard oligonucleotide assay for integration. It permitted us to study, by electron microscopy and sequence analysis, insertion of a single long terminal repeat terminus (LTR half-site) of one plasmid into another linearized plasmid. The reaction was catalyzed by purified Avian Myeloblastosis Virus IN in the presence of Mg2+. The recombinant molecules were easily visualized and quantitated by agarose gel electrophoresis. Agarose gel-purified recombinants could be genetically selected by transformation of ligated recombinants into Escherichia coli HB101 cells. Electron microscopy also permitted the identification and localization of IN-DNA complexes on the Virus-like substrate in the absence of the joining reaction. Intramolecular and intermolecular DNA looping by IN was visualized. Although IN preferentially bound to AT-rich regions in the absence of the joining reaction, there was a bias towards GC-rich regions for the joining reaction. Alignment of 70 target site sequences 5' of the LTR half-site insertions with 68 target sites previously identified for the concerted insertion of both LTR termini (LTR full-site reaction) indicated similar GC inflection patterns with both insertional events. Comparison of the data suggested that IN recognized only half of the target sequences necessary for integration with the LTR half-site reaction.
Joseph S. Lipsick - One of the best experts on this subject based on the ideXlab platform.
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transformation of myelomonocytic cells by the Avian Myeloblastosis Virus is determined by the v myb oncogene not by the unique long terminal repeats of the Virus
Journal of Virology, 1994Co-Authors: U Engelke, Joseph S. LipsickAbstract:The Avian Myeloblastosis Virus (AMV) induces acute monoblastic leukemia in chickens and transforms only myelomonocytic cells in vitro. The long terminal repeat (LTR) regulatory region of AMV is unique among the known classes of Avian retroVirus LTRs. We demonstrate that the substitution of the AMV LTRs by Rous sarcoma Virus LTRs did not alter the cell type specificity or the transforming ability of the Virus.
