Comparison of the Thermal Stabilities of Reverse Transcriptases from Avian Myeloblastosis Virus and Moloney Murine Leukaemia Virus

Abstract

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.

Insight into the Mechanism of the Stabilization of Moloney Murine Leukaemia Virus Reverse Transcriptase by Eliminating RNase H Activity

“…Gerard et al 8) and we 9) have found that T/P-mediated stabilization of AMV RT was more potent than that of MMLV RT, suggesting that AMV RT has higher affinity toward the T/P than MMLV RT. Recently, Arezi et al discovered five novel mutations (Glu69 !…”

“…6,9) As shown in Fig. 1, the plot showed a curve with an optimal temperature of 46 C for each enzyme.…”

“…6,9) On SDS-PAGE under a reducing condition, WT and D524A yielded a single band with a molecular mass of 75 kDa (data not shown). Their reverse transcription activities were measured at various temperatures ranging from 37 to 58 C ( Fig.…”

Effects of Organic Solvents on the Reverse Transcription Reaction Catalyzed by Reverse Transcriptases from Avian Myeloblastosis Virus and Moloney Murine Leukemia Virus

“…Both Gerard et al 22) and our own laboratory group 13) have found that T/P-mediated stabilization of AMV RT was more potent than that of MMLV RT, suggesting that AMV RT has higher affinity toward the T/P than does MMLV RT. Arezi et al discovered five novel mutations (Glu69!…”

“…The MMLV RT gene was expressed under the T7 promoter in Escherichia coli, and recombinant MMLV RT was purified from the cells as described previously. 13,14) p(dT) 15 was from Sigma-Aldrich Japan (Ishikari, Japan). [methyl-…”