MoLV reverse transcriptase

This chapter focuses on a few selected DNA polymerases that are most widely used in recombinant DNA technology. In the category of DNA-directed DNA polymerases (Section II), four DNA polymerases are described that originate from bacteria . coli and T. aquaticus) and bacteriophages (T4 and T7). In the category of RNA-directed DNA polymerases (Section III), two reverse transcriptases, one from AMV and the other from MoLV, are described. As the typical enzyme of template-independent DNA polymerases (Section IV), the terminal deoxynucleoti-dyltransferase (TDTase) from calf thymus is described. 

At least partly due to this, RTases exhibit relatively high frequencies of misincorpo-ration. Reverse transcriptases do not catalyze dNTP turnover (10). They utilize mispaired primer termini complexed to either RNA or DNA templates as functional sites for polyermization (61). Substitution of Mn for Mg increases the error frequency of RTases (62,63). The error rates for AMV and MoLV RTases are similar. On a poly(A) or poly(C) template, the two RTases exhibit an error rate of 1/3500-1/7400 at a dNTP concentration of 250 pM, the condition often employed for cDNA synthesis reactions in vitro (64). On DNA template-primers, the frequencies of misincorporation at 50 pM dNTPs are 10 -10 (65-67). On certain RNA templates, however, AMV RTase shows higher error rates than MoLV RTase (68). 

Reverse transcriptases are also valuable reagents in the direct dideoxynucleotide sequencing of RNA (110). In fact, RTases have been instrumental in obtaining nucleotide sequence information directly from viral RNA genomes, 16S rRNA (111), and mRNAs (112). The higher thermostability of AMV RTase compared with that of MoLV RTase makes the AMV RTase more useful for nucleotide sequencing at elevated temperatures. 

The reverse transcriptase (RTase) from the Moloney murine leukemia virus (MoLV) possesses both DNA polymerase and RNase (H and D) activities. It is monomeric (80 kDa) in solution but is presumed to function as a dimer. The ratio of the polymerase to RNase H activities and many of MoLV RTase functions are similar to those of AMV RTase. In contrast to AMV RTase, however, MoLV RTase does not possess DNA endonuclease activity. The gene for MoLV RTase has been cloned and expressed in . colt, and both wild-type RTase (RNase H" ) and modified RTase (RNase H ) are commercially available. MoLV RTase is thus a reliable reagent which shares with AMV RTase much of the role of cDNA synthesis in molecular cloning. Modified MoLV RTase has proven to be particularly useful in the synthesis of full-length cDNAs. 

Reverse transcriptases, e.g., from AMV and MoLV, are frequently used for dideoxy sequencing of DNA and are indispensable tools for dideoxy sequencing of RNA. RNA sequencing is typically performed via reverse transcriptase-catalyzed... 

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