109
In addition to the DNA polymerase activity, reverse transcriptases also show
RNase H activity involved in the degradation of RNA in an RNA-DNA hybrid, such
as those obtained during reverse transcription using an RNA template. It has both
endonuclease and exonuclease activities. Reverse transcriptase has become an
essential component of molecular biology with the introduction of reverse transcription polymerase chain reaction (RT-PCR) used for the synthesis of singlestranded “complementary” cDNA of a RNA template.
4.7.1 AMV/MAV Reverse Transcriptase
It is the most widely used reverse transcriptase in molecular biology and is isolated
from Avian Myeoloblastosis Virus (AMV), an alpha retrovirus that causes acute
myeloblastic leukemia (AML) in chicken (Baluda et al. 1983). Being a deficient
virus, AMV requires a helper virus, Myeloblastosis Associated Virus (MAV) for the
synthesis of envelope and RT proteins. The actual source of RT in AMV life cycle
is MAV (Perbal 2008). The AMV/MAV RT is composed of two structurally similar
subunits, α of 65 kDa and β of 95 kDa, which assemble to form a holoenzyme, the
active reverse transcriptase. The α subunit contributes to the RT and RNase H activities, the latter of which is conferred by a 24 kDa fragment of the α subunit by which
RNA strands can be specifically degraded from RNA-DNA hybrids in both directions. AMV/MAV RT is extensively used in molecular cloning for the synthesis of
cDNA from mRNA using poly dT or random primers, for techniques such as quantitative PCR, rapid amplification of cDNA ends (RACE), and nucleotide
sequencing.
4.7.2 M-MuLV Reverse Transcriptase
This reverse transcriptase is isolated from Moloney Murine Leukemia Virus
(M-MuLV), is devoid of DNA endonuclease activity and 3′ → 5′ exonuclease activity. RNAse H activity of this enzyme is comparatively much lower than AMV/MAV
RT (Moelling 1974). It has four times less efficiency and lower stability than AMV/
MAV RT. The main advantage of M-MuLV RT is its ability to synthesize transcripts
of higher length than AMV/MAV RT (Houts et al. 1979). A recombinant M-MuLV
reverse transcriptase with lower RNase H activity and higher thermostability has
been produced under the name ProtoScript II Reverse Transcriptase (New England
Biolabs), which is active up to 48 °C, shows higher specificity and cDNA yield, and
can synthesize cDNA of up to 12 kb size.
4.7.3 Tth DNA Polymerase
This thermostable DNA polymerase of ~94 kDa size, isolated from Thermus thermophilus strain HB8, can efficiently carry out reverse transcription of RNA in a
4 Enzymes as Molecular Tools
In addition to the DNA polymerase activity, reverse transcriptases also show
RNase H activity involved in the degradation of RNA in an RNA-DNA hybrid, such
as those obtained during reverse transcription using an RNA template. It has both
endonuclease and exonuclease activities. Reverse transcriptase has become an
essential component of molecular biology with the introduction of reverse transcription polymerase chain reaction (RT-PCR) used for the synthesis of singlestranded “complementary” cDNA of a RNA template.
4.7.1 AMV/MAV Reverse Transcriptase
It is the most widely used reverse transcriptase in molecular biology and is isolated
from Avian Myeoloblastosis Virus (AMV), an alpha retrovirus that causes acute
myeloblastic leukemia (AML) in chicken (Baluda et al. 1983). Being a deficient
virus, AMV requires a helper virus, Myeloblastosis Associated Virus (MAV) for the
synthesis of envelope and RT proteins. The actual source of RT in AMV life cycle
is MAV (Perbal 2008). The AMV/MAV RT is composed of two structurally similar
subunits, α of 65 kDa and β of 95 kDa, which assemble to form a holoenzyme, the
active reverse transcriptase. The α subunit contributes to the RT and RNase H activities, the latter of which is conferred by a 24 kDa fragment of the α subunit by which
RNA strands can be specifically degraded from RNA-DNA hybrids in both directions. AMV/MAV RT is extensively used in molecular cloning for the synthesis of
cDNA from mRNA using poly dT or random primers, for techniques such as quantitative PCR, rapid amplification of cDNA ends (RACE), and nucleotide
sequencing.
4.7.2 M-MuLV Reverse Transcriptase
This reverse transcriptase is isolated from Moloney Murine Leukemia Virus
(M-MuLV), is devoid of DNA endonuclease activity and 3′ → 5′ exonuclease activity. RNAse H activity of this enzyme is comparatively much lower than AMV/MAV
RT (Moelling 1974). It has four times less efficiency and lower stability than AMV/
MAV RT. The main advantage of M-MuLV RT is its ability to synthesize transcripts
of higher length than AMV/MAV RT (Houts et al. 1979). A recombinant M-MuLV
reverse transcriptase with lower RNase H activity and higher thermostability has
been produced under the name ProtoScript II Reverse Transcriptase (New England
Biolabs), which is active up to 48 °C, shows higher specificity and cDNA yield, and
can synthesize cDNA of up to 12 kb size.
4.7.3 Tth DNA Polymerase
This thermostable DNA polymerase of ~94 kDa size, isolated from Thermus thermophilus strain HB8, can efficiently carry out reverse transcription of RNA in a
4 Enzymes as Molecular Tools