74 Gerard and D'Alessio
been established for proper handling and storage of the enzyme. Con-
ditions for second-strand cDNA synthesis with first-strand product
synthesized by AMV RT have also been optimized (9,10).
In 1985, Moloney murine leukemia virus (M-MLV) RT was cloned,
overexpressed, and purified from E. coli (11-14). A series of reports
have been published delineating optimal conditions for use of this
alternative enzyme in first- and second-strand cDNA synthesis (15-
18). M-MLV RT has been used widely to synthesize cDNA and is
frequently used instead ofAMV RT for carrying out cDNA synthesis.
In 1989, a modified version of M-MLV RT lacking RNase H activ-
ity became available (7,19). RT without interfering RNase H activity
catalyzes more efficient synthesis of full-length cDNA than either
native AMV or cloned M-MLV RT possessing RNase H activity (7).
The enzyme lacking RNase H activity is designated M-MLV H- RT.
We describe here some of the properties of both forms of M-MLV RT
and protocols for using M-MLV H- RT in first- and second-strand
cDNA synthesis.
- The Enzyme
2.1. Sources
Commercial preparations of purified M-MLV RT are derived from
two sources (12-14). Both proteins differ from the native enzyme in
the amino acids at their amino and carboxy termini because of changes
in sequence information introduced into the gene during the cloning
procedure. The protein encoded by plasmid pB6B15.23 (14) has 25
additional amino acids fused at the amino terminus, is missing seven
amino acids present at the carboxy terminus of the native enzyme, and has
nine additional carboxy-terminal amino acids not found in the M-MLV
pol gene. The RT encoded by pRT601 (12,13) has one additional amino
acid (methionine) at its amino terminus, the 671 amino acids present
in the native enzyme, and 12 additional amino acids at its carboxy
terminus. The same number of molecules of each enzyme is needed to
catalyze cDNA synthesis, despite their structural and apparent cata-
lytic distinctions (see Table 1). Ten times as many units (see Section
2.2.) of pRT601 RT as pB6B 15.23 RT are required to copy efficiently
an mRNA population (11). Not surprisingly, the specific activity of
pRT601 RT (350,000 U/mg of protein) is 10 times greater than that of
