Nucleic Acids Research
Volume 4 Number 10 October 1977
The synthesis of high yields of full-length reverse transcripts of globin mRNA
4
E.Y. Friedman and M. Rosbash
*
Rosenstiel Basic Medical Sciences Research Center and Department of Biology, Brandeis University,
Waltham, MA 02154, USA
Received 17 June 1977
ABSTRACT
Conditions have been determined under which reverse transcriptase catalyzes the synthesis of high yields of full length complementary deoxyribonucleic acid (cDNA). These conditions depend not only on the concentration of
deoxynucleoside triphosphates (1) but also on the concentration of reverse
transcriptase. An analysis of the kinetics of cDNA synthesis and the size of
cDNA synthesized as a function of time under different conditions indicates
that the mechanism of action of reverse transcriptase is partially distributive. This accounts for the necessity of a high enzyme concentration to obtain high yields of full length cDNA. Additional experiments indicate that
the yield of cDNA is limited by the fact that the template mRNA is rapidly
inactivated. This is most likely due to the fact that the product cDNA is
hydrogen bonded to the template mRNA during synthesis.
INTRODUCTION
Reverse transcriptase catalyzes the synthesis of complementary DNA (cDNA)
from a poly(A)-containing messenger ribonucleic acid (mRNA) template (2,3,4).
The ability to synthesize cDNA of high specific activity provides an important
and useful tool for hybridization and nucleotide sequencing (5,6,7,8,24,26). In
addition, bacterial plasmids have been constructed containing a cDNA insert
(9,11,12,27).Several groups have shown that the size of cDNA is dependent on
the deoxynucleoside triphosphate (dNTP) concentration (1,5,6,13,14). "Full
size" cDNA is synthesized when all four dNTP are present at a "high" concentration (>50 PM).
For the purposes of molecular hybridization, the yield
(picomoles cDNA mRNA
of cDNA as well as its length (14) is an imporpicomoles
tant consideration. The interpretation of the kinetics of mRNA-cDNA hybridization in systems which contain heterogeneous mRNA populations is to a certain
synthesized)
extent dependent upon the assumption that there is no preferential synthesis
of some mRNA species (15). This possibility is difficult to exclude but, in
C) Information Retrieval Limited 1 Falconberg Court London Wl V 5FG England
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principle, should be excluded for each biological system under investigation.
One way of dealing with this problem is to adjust the reaction conditions
used for the synthesis of cDNA such that the yield of cDNA is high. Knowing
the mechanism of action of reverse transcriptase under these conditions, it
becomes possible to argue that each mRNA molecule is copied into cDNA, obviating the possibility of preferential transcription.
We have used duck globin mRNA as a template for cDNA synthesis. The reaction conditions are designed to obtain a maximal yield of cDNA. Surprisingly, we find that a maximal yield of full length cDNA product is dependent on
the levels of reverse transcriptase enzyme as well as dNTP concentration. By
examining the size of the cDNA product synthesized as a function of time
under different conditions, interesting aspects of the mechanism of action of
reverse transcriptase are revealed.
MATERIALS AND METHODS
Purification of Duck Globin mRNA: Duck globin mRNA was isolated as in
MacNaughton et al. (16). Briefly, polyribosomes were digested with proteinase K, and poly(A)-containing RNA was isolated by chromatography on oligo(dT)cellulose. 10S mRNA was purified by two cycles of centrifugation on 15-30%
(w/w) sucrose gradients containing 0.5% sodium dodecyl sulphate (SDS) in
(NTE) (100 mM NaCl, 10 mM Tris-HCl pH 7.5, 1 mM EDTA) in
a Beckman SW40 rotor at 39,000 rpm for 15 hr at 25 0C. An aliquot was analyzed
by electrophoresis on a 3.5% polyacrylamide-7M urea gel and stained with ethidium bromide. The only band present had the expected mobility of approximately
650 nucleotides. In all experiments employing globin mRNA as a template for
avian myeloblastosis virus (AMV) reverse transcriptase, it was assumed that
A260 of 1.0 was equivalent to 40 ug of globin mRNA.
Conditions for Enzymatic Synthesis of Globin cDNA: The standard reaction contained the following components: 50 mM Tris-HCl, pH 8.3, 60 mM NaCl,
6 mM MgCl2, 20 mM dithiothreitol, 100 ug/ml actinomycin D (Sigma), 5 ug/ml
oligo(dT)12 18 (Collaborative Research). Unlabeled deoxynucleotides (dATP,
dGTP, dTTP) were present at 1 mM while [ H] or [ P]dCTP was at 200 PM (unless
otherwise specified in the figure legends). Duck globin mRNA and reverse
transcriptase were added as indicated in the figure legends in each experiment. The reaction (25 ul final volume) was begun by the addition of reverse
transcriptase with gentle mixing and subsequent incubation at 37 C. Each reaction was covered with several drops of paraffin oil to prevent evaporation.
Incorporation as a function of time was assayed by diluting 1-2 ul aliquots
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into 200 ul of 0.2 M sodium pyrophosphate with 150 ug/ml single stranded calf
thymus DNA carrier. Ten percent (20 ul) was spotted on a GF/C filter (Whatman) and the remainder precipitated with 10% trichloroacetic acid, collected
on a GF/C filter, washed, dried and counted in a scintillation counter.
Picomoles of cDNA synthesized was calculated with the assumption that globin
cDNA contains 25% cytosine. For the analysis of cDNA size, fractions were
removed at different time points, diluted into a final volume of 100 ul of
0.5% SDS, 10 mM EDTA and incubated for 3 min at 370C. An aliquot was spotted
and precipitated with 10% TCA as described above. The remainder of the
sample was placed on an SP-50 (Pharmacia) column (0.7cmxlOcm) equilibrated
with 300 mM NaCl, 10 mM Na acetate pH 5.5. The excluded fraction was collected and NaOH added to a final concentration of 0.3 M. The sample was heated
to 100 C for 3 min, cooled in ice, and neutralized. The cDNA samples were
then brought to a final concentration of 10 mM MgCl2, 10 mM Tris-HCl pH 7.
50 ug/ml tRNA was added along with 3 vol of ethanol. The cDNA was centrifuged, resuspended in 200 ul of .3 M NaAc pH 6 and, after addition of 0.6 ml
ethanol, reprecipitated (A. Maxam, personal communication).
(a) Alkaline Sucrose Gradient Centrifugation: Ethanol precipitated
[3H]cDNA samples were resuspended in 100-200 ul of 100 mM NaOH, .5% SDS, 1 mM
EDTA. To each sample was added 2 [ P]lambda Hae III fragments (470, 150
base pairs). Each sample was layered onto a 5-20% (w/w) sucrose gradient in
100 mM NaOH, 0.5% SDS, 1 mM EDTA. Gradients were centrifuged in a Beckman SW40
rotor for 20 hr at 40,000 rpm at 200C. Fractions were collected and counted
in Aquasol (New England Nuclear).
(b) Polyacrylamide Gel Electrophoresis: 3.5% polyacrylamide-7M urea
acrylamide gels were prepared as described by Maniatis et al. (16). Ethanol
precipitated [3 P]cDNA samples were resuspended in 20 pl of 7 M urea, boiled
for 2 min and cooled in ice. After addition of glycerol to a final concentration of 10% and the marker dyes, xylene cyanol FF (X) and bromophenol blue (B),
the samples were layered in gel slots. Electrophoresis was performed at room
temperature for 10 hr at a constant voltage of 100 V. One slot contained 4
[ PI lambda Hae III restriction fragments (470, 350, 150, 90 base pairs).
In addition, globin mRNA was present in this same slot. The gel was stained
with ethidium bromide after electrophoresis, photographed and subjected to
autoradiography (Kodak XR-5 X-ray film).
Sl Nuclease Treatment: Ethanol precipitated cDNA was resuspended in 200
ul water. 100 ul was heated to 100 0C for 5 min and quickly cooled. The other
100 ul was kept at 40C. 150 ul S1 mixture was added to each sample with the
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final 250 ul volume containing 0.1 M NaCl, 0.15 M Na acetate pH 4.6, 1 mM Zn
S049 5% glycerol, 25 ug/ml double stranded DNA, 25 ug/ml single stranded DNA.
Each sample was divided into two 100 ul aliquots. 5 ul S1 nuclease was added
to one tube and incubated for 15 min at 370C. The second tube was incubated
in the absence of nuclease. The reactions were terminated with the addition
of 200 ul tRNA (1 mg/ml) and 600 ul 3% CETAB. Samples were collected on filters and washed with distilled water.
AMV Reverse Transcriptase: Reverse transcriptase was a generous gift of
Dr. J. Beard. The enzyme, purified by the procedure of Kacian and Spiegelman
(17), was stored in 50% glycerol, 0.2 M potassium phosphate, pH 7.2, 2 mM
dithiothreitol, 0.2% Triton X-100 at -200C. The enzyme preparation used in
these studies had an activity of 3,538 units/ml. (one unit of enzyme activity
will incorporate 1 nanomole dTMP into acid insoluble product in 10 min at 37 C.)
RESULTS
The cDNA yield and kinetics of cDNA synthesis are dependent on many factors including the concentration of oligo(dT) primer, globin poly(A)-contain-
ing mRNA, and reverse transcriptase. At a saturating enzyme concentration,
the yield of cDNA increased with an increasing concentration of oligo(dT)
primer (Figure 1). The concentration of oligo(dT) was saturating at slightly
less than the estimated concentration of the poly(A) segment of globin poly(A)
mRNA. For example, 0.01 ug oligo(dT) (0.4 ug/ml) was sufficient to saturate
0.133 ug globin mRNA (5.33 ug/ml) which contains 7-14% (0.37-0.74 ug/ml)
poly(A) (16; Friedman and Rosbash, unpublished observations). Increasing
concentrations of oligo(dT) did not increase the yield of cDNA. In subsequent
experiments, a concentration of 5 ug/ml oligo(dT) primer was used, approximately equivalent to a 10-fold mass excess of the poly(A) in 5 ug/ml globin
mRNA. The kinetics of cDNA synthesis shown in Figure 1A are representative
of experiments in which the enzyme was present at a saturating concentration.
The high yield of cDNA under these conditions should be noted.
In a second series of experiments, the synthesis of cDNA was assayed as a
function of increasing concentration of globin mRNA. The amount of cDNA product increased with increasing concentration of mRNA (Table 1). However, the
yield of cDNA decreased. Similar experiments were conducted by varying the
concentration of reverse transcriptase (Figure 2). At saturating concentrations of reverse transcriptase, cDNA synthesis terminated by 15 min. Higher
concentrations of reverse transcriptase did not lead to an increased yield
of cDNA or to a greater initial rate of synthesis (data not shown). However,
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4
0
z
.2
E
E
4
z
0
Time (Min)
B
0.6
0.4
q
z
E
4
z
a
0.2
0.2
0.4
(Primer]
8
itg/mi
Primer. Each reaction
FIGURE 1: Effect of Varying Amount of Oligo(dT)
mixture (25 ul volume) contained all the components gescribed in Materials
and Methods except oligo(dT) primer. Each reaction contained 0.133 ug globin
mRNA (5.3 ug/ml) and 7 units of reverse transcriptase (280 units/ml). Varying
Each
were added to each tube prior to incubation.
amounts of oligo(dT) 2
tube was incubated at 3'§C and samples removed as indicated.
A.
0.001 ug (.04 ug/ml)
0.002 ug (.08 ug/ml)
0.005 ug (.2 ug/ml)
(0-0)
0.025 ug (1 ug/ml)
(x - x)
U-0) 0.2 ug (8 ug/ml)
The yield of cDNA obtained at 60 min is plotted
concentration.
(o
(S
B.
-
o)
0)
as a
function of primer
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TABLE I:
Synthesis of Globin cDNA with Increasing RNA Concentrations.a
picomoles mRNA
picomoles cDNA
cDNA/mRNA
40
200
1000
2000
4000
33.4
141
482
638
738
.83
.71
.48
.32
.18
aThe
reaction conditions are described in Materials and Methods. [3 H]dCTP
was present at 200 uM at a specific activity of 300 cpm/pmole. 7 units of
reverse transcriptase (280 units/ml) were added to each reaction volume of
25 ul. After enzyme addition, the mixture was incubated at 370C for 60 min.
The concentration of oligo(dT) was saturating for all concentrations of mRNA.
with lower concentrations of enzyme, cDNA synthesis was linear for at least
1 hr.
The previous experiments emphasize the need for high concentrations of
enzyme to obtain high yields of cDNA under these conditions. In principle,
it might be possible to achieve even higher yields of cDNA. Consequently,
experiments were pursued to examine the reasons for which the reaction rapidly reaches terminal values. It is possible that there exists nuclease or
protease contamination in our mRNA or reverse transcriptase preparations. It
is also possible that incubation at 370C, under the conditions employed in
this study, was deleterious to the mDRNA and/or the enzyme activity. Therefore, reverse transcriptase or globin mRNA were preincubated alone or together in the cDNA reaction mixture for 15 min at 37 0C. Synthesis of cDNA was
initiated by the addition of globin mRNA, reverse transcriptase or oligo(dT)
(Figure 3). There appeared to be some effect upon preincubation of the mRNA
either in the presence or in the absence of enzyme. In all cases, however,
the kinetic pattern was similar to that observed in the control reaction in
the absence of any preincubation. This argues that the rapid approach to
plateau values is not entirely due to destruction of the template mRNA during
the incubation.
The rapid termination of the reaction must, however, be due to the fact
that one of the reaction components was rendered inactive with regard to cDNA
synthesis after approximately 15 min. To test such a hypothesis, additional
mRNA or enzyme was added to a reaction at 15 min (Figure 4). Enzyme addition
led to no increase in cDNA synthesis, suggesting that enzyme inactivation was
not the cause of reaction termination. Additional mRNA, however, stimulated
the incorporation of dCTP resulting in a second plateau approximately equal in
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0.6
10.4
0
z
4
E
E
49
z
0
10.2
Time (Min)
FIGURE 2: Kinetics of cDNA Synthesis with Varying Levels of Reverse Transcriptase. The reaction conditons were as described in Materials and Methods
with the addition of bovine serum albumin (BSA) at 60 ug/ml. [ H]dCTP was
present at 200 uM at a specific activity of 1000 cpm/pmole. The mRNA concentration was 5.3 ug/ml (400 picomoles/25pl). Different amounts of enzyme were
added to each reaction.
0.02 ul - 2.8 units/ml
(e - *)
(A - A) 0.05 ul - 7.1 units/ml
(D-DJ) 0.2 ul - 28.3 units/ml
0.5 ul - 70.8 units/ml
(V - V)
1 ul - 141.5 units/ml
(o - o)
3 ul - 424.5 units/ml
(x - x)
height to the first.
The data shown in Figure 4 suggest that the mRNA template was inactivated
under the reaction conditions employed. In addition, cDNA was largely resistant to single-strand specific Si nuclease (Table 2). Control experiments
indicated that in the presence of 100 ug/ml of actinomycin D the purified cDNA
was entirely single-stranded (data not shown). The most straightforward explanation for the inactivation of the template and for the inability to
achieve cDNA yields greater than 0.6-0.8 is that each mRNA molecule acts as a
template for the synthesis of at most one cDNA molecule which remains hydrogenbonded to its template and renders it unable to serve as a template for further synthesis.
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20
0.8
15
0.6
4
z
2
xon
I0
0.4 s
4
E
z
cL
0
0
5
0.2
15
30
45
Time (Min)
FIGURE 3: Effect of Preincubation on cDNA Synthesis. The enzyme and/or mRNA
was preincubated for 15 min at 370C in the reaction mixture. cDNA synthesis
was initiated by the addition of mRNA, enzyme, or oligo(dT), respectively. The
final volume was 25 ul with 200 uM [3H]dCTP (1600 cpm/pmole), 0.133 ug mRNA
(5.3 ug/ml) and 10.5 units reverse transcriptase (420 units/ml).
(o - o), enzyme and mRNA both present at t=0.
(A - A), enzyme preincubated 15 min; mRNA added at t=0.
(A - A), mRNA preincubated 15 min; enzyme added at t=0.
(o - *), enzyme and mRNA preincubated 15 min in absence of oligo(dT).
0.125 ug oligo(dT) added at t=0.
15-
10
jf
E
5-
20
40
Time (Min)
60
80
FIGURE 4: Effect of mRNA or Enzyme Addition to Reaction. The initial reaction
mixture (37.5 ul) contained 0.2 ug mRNA (5.3 ug/ml), 200 uM [3H]dCTP (300 cpm/
pmole) and 10.5 units of reverse transcriptase (280 units/ml). After 10 min
at 370C (indicated by arrow)(a) 10 ul were removed and added to 3.5 units of
enzyme, ( ---C]). (b) 10 ul were removed and added to 0.066 ug mRNA, (V. .V).
(c) the remainder of original reaction mixture was incubated further at 37 C,
(o- o). Samples were removed at the indicated times and assayed as described
in Materials and Methods.
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TABLE II:
Resistance of cDNA to Si Single-Strand Specific Nuclease.a
0
time
100°C - 5 min
3'
-
12,921
1,279
11,426
655
+
20'
~~~+Si
cpm
+
-Si
cpm
% duplex
S
+S1
17,370
18,337
74%
16,400
70%
p
cpm
7%
3.7%
17,560
The cDNA reaction mixture (50 ul) contained 0.267 ug mRNA (5.3 ug/ml), 420
units/ml reverse transcriptase, and 200 u-M [3H]dCTP (1150 cpm/pmole dCTP).
Aliquots were removed at 3 min and 20 min. Water was added to each sample
to a final volume of 200 ul. After phenol extraction and ether extraction
the cDNA was ethanol precipitated and treated with Si nuclease as described
in Materials and Methods. Percent duplex is cpm of sample treated with Si
divided by cpm of sample not treated with Si.
3
2
0
E
.
10
30
Time (Min)
60
FIGURE 5: Kinetics of cDNA Synthesis. Each reaction mixture (25 ul) contained
0.133 ug globin mRNA (5.3 ug/ml). The four reactions had the following enzyme
and dCTP concentrations.
dCTP
Enzyme
(a) 420 units/ml, 10 uM [( P]dCTP (10,000 cpm/pmole); (o-o)
(b) 420 units/ml, 200 uM3132P]dCTP (41,000 cpm/pmole); (. ).
(c) 7 units/ml, 10 uM [ P]dCTP (68,000 cpm/pmole); (U-U).
(d) 7 units/ml, 200 uM [32P]dCTP (34,000 cpm/pmole); (0-0).
7 ul fractions were removed at 3, 8 and 30 min from (a) and (b) and at 10, 30
and 60 min in (c) and (d) and added to a final volume of 100 ul containing 0.5%
SDS, 10 mM EDTA. Two ul was assayed for trichloroacetic acid (TCA) insoluble
radioactivity as described in Materials and Methods. The remainder was passed
through an SP50 column. The fractions containing cDNA were pooled, brought to
0.3 M NaOH, heated to 1000C for 3 min, neutralized, and precipitated in ethanol
with tRNA carrier at 50 ug/ml as in Materials and Methods.
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i2I H1
10 9
7 8 6
I
5 4
3
2
0 M
I
-
Hb mRNA
470
wl.
XCGFF
350
-150
-.90
-BPB
32
FIGURE 6: Autoradiograph of Acrylamide Gel of Samples from Figure 5. [ P1
cDNA prepared in Figure 5 was resuspended in 7 M urea, glycerol and marker
dyes as described in Materials and Methods. The marker dyes, globin mRNA and
marker DNAs are indicated on the ordinate. Approximately 30,000 cpm was
layered in each gel slot except for lane 10 which contained approximately
15,000 cpm.
[dCTP]
Enzyme
M, marker DNAs and Hb mRNA
10
420
30
8
min.
units/ml
JM
3
3,
min;
min; 2,
1,
200 pM
420 units/ml
4, 3 min; 5, 8 min; 6, 30 min.
7 units/ml
10 JM
7, 10 min; 8, 30 min; 9, 60 min.
7 units/ml
200 VM
10, 10 min; 11, 30 min; 12, 60 min.
It was of some interest to consider the size of cDNA synthesized under
these conditions of enzyme saturation and high yields of cDNA. If the mechanism of action of the enzyme under these conditions were entirely processive,
the enzyme concentration would have no effect on the product size. However,
if the enzyme acted in a distributive or partially distributive fashion, the
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Indeed, evienzyme concentration might affect the resultant product size.
dence for both processive and distributive mechanisms have been presented for
homopolymer templates (18,19). Therefore, the cDNA size was examined under
conditions of both saturating and limiting enzyme concentrations. For a comparison with previous reports (1,5,6), these conditions were also compared
with conditions in which one deoxytriphosphate was present at a low concentration (10 PM).
At saturating enzyme concentrations, the synthesis of cDNA followed the
previously described kinetic pattern and terminated within 30 min at both high
(200 PM) and low (10 PM) concentrations of dCTP. At limiting enzyme concentrations, cDNA was synthesized with linear kinetics as shown earlier in Figure 2
A
B
Tx
90
150
350
470
Hb
C
x
90
350
470
Hb
0
90
IGO
360 470 Hb
90
150
350
470 Hb
I
FIGURE 7: Densitometer Tracing of Autoradiograph of Figure 6.
graph in Figure 6 was scanned with a Joyce-Loebl densitomerer.
lane 3, (---).
);
lane 2, (
);
(A) lane 1, (
lane 6, (---).
);
(B) lane 4, (. . .); lane 5, (
lane 9, (---).
);
(C) lane 7, (. . .); lane 8, (
lane 11, (
);
); lane 12,
(---).
(D) lane 10, (
II
.
The autoradio-
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E
0
8
16
24
0
Froction Number
8
16
24
FIGURE 8: Akaline Sucrose Gradients. i[ 3HI labeled cDNA, synthesized and prepared as in Figure 5, was resuspended in a volume of 250 ul of 0.1 M NaOH, 0.5%
SDS, 1 mM EDTA containing two [32P]-lambda DNA restriction markers (150 nucleotides and 470 nucleotides). The cDNA was centrifuged as described in Materials
and Methods. Individual sucrose gradients from each reaction were aligned with
the [32P]-restriction markers.
(A) 420 units/ml enzyme 10 uM [3H]dCTP: 3 min, (o.. o); 8 min, (O-0); 30
min, (A---A).
3
(B) 420 units/ml enzyme, 200 uM [ H]dCTP: 3 min, (o. o); 8 min, (C-j);
30 min, (A---A).
3
(C) 7 units/ml enzyme, 10 uM [ H]dCTP: 10 min, (o. o); 30 min, (E -g);
60 min, (A---A).
3
(D) 7 units/ml enzyme, 200 uM [ H]dCTP: 10 min, (o **o); 30 min, (Q-E);
60 min, (A---A).
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(Figure 5). The cDNA synthesized under these four conditions was analyzed by
denaturing polyacrylamide gel electrophoresis (Figure 6, 7) and alkaline sucrose gradient centrifugation (Figure 8). At a saturating enzyme concentration,
full length globin cDNA was synthesized at both high (200 PM) and low (10 PM)
concentrations of dCTP (Figures 6, 7). However, at limiting enzyme concentrations, a small fraction of full length cDNA was observed only in the presence
of a high concentration of dCTP. Similar conclusions were obtained with alkaline sucrose gradient centrifugation (Figure 8).
Under optimal conditions, a large fraction of the cDNA was "full-size"
(Fig. 7B). Surprisingly, a high concentration of reverse transcriptase resulted in the synthesis of a large fraction of full-length cDNA at low (10 PM) as
well as high (200 vM) concentrations of dCTP (Fig. 7A). These results indicate that the desired goal of a high yield of full-length cDNA can be achieved
by maintaining a high enzyme concentration even when one of the four triphosphates is present at sub-optimal concentrations.
DISCUSSION
The major goal of this study was to determine reaction conditions for the
synthesis of cDNA which would result in high yields of full length cDNA. We
have confirmed the observations of Efstratiadis et al. (1), that at a low
concentration of enzyme the synthesis of full length globin cDNA is dependent
on the concentration of deoxynucleoside triphosphates. At an enzyme concentration which catalyzes the linear synthesis of DNA, full-length cDNA is not
synthesized when the dCTP concentration is 10 vM and is synthesized at 200 vM
dCTP (Fig. 7C & D). At saturating concentrations of reverse transcriptase,
however, full-length cDNA is synthesized even at 10 vlM dCTP (Fig. 7A). Unlike the study previously cited (1), we did not observe discrete bands of
partial cDNA transcripts (Fig. 6 & 7). This is perhaps due to slight differences in reaction conditions. These include the use of dithiothreitol and
actinomycin D in the reactions reported here. Alternatively, the inability
to resolve discrete partial transcripts may be due to the use of 7 molar urea
as compared to 98% formamide as the denaturing agent during polyacrylamide
gel electrophoresis.
In the presence of a low concentration of reverse transcriptase, the kinetics of synthesis are linear (Fig. 2, 5) for approximately one hour. At
limiting enzyme concentrations, the level of synthesis does not attain values
achieved with a high concentration of enzyme even after a 5 hour incubation
(unpublished results). During these relatively long incubations, the enzyme
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may be inactivated. Alternatively, a small amount of contaminating nucleases
in the reaction may be responsible for premature termination of the reaction
under these conditions of low enzyme concentration and long incubation times.
Indeed, preincubation of the mRNA alone or the mRNA and enzyme for 15 min prior
to initiation of synthesis decreases the yield of cDNA (Fig. 3).
The data suggest that the yield of cDNA approaches 1.0 as a maximum value
(Fig. 2 and Table 1). This limit suggests that each mRNA molecule is used
only once as a template for the synthesis of a single cDNA molecule. Consistent with this hypothesis is the experiment presented in Figure 4 which clearly indicates that under conditions of enzyme saturation, the absence of template mRNA is responsible for reaction termination. Also consistent with this
hypothesis is the data presented in Table 2 in which the newly-synthesized
cDNA is shown to be present in duplex form. The cDNA is in an RNA-DNA duplex
since treatment with alkalai liberates single stranded cDNA with no capacity
to form duplex structures when allowed to self-anneal (data not shown). It
is possible that the double stranded nature of the cDNA is due to the low
sequence complexity of the globin mRNA species which might permit annealing
of the cDNA with the template mRNA. Such a possibility is excluded by almost
identical data (data not shown) obtained when a similar assay is performed
on cDNA complementary to yeast poly(A)-containing RNA, a situation in which
there exists a relatively large number of mRNA species and, therefore, a relatively high sequence complexity (20). These data indicate that it is likely
that each cDNA molecule remains hydrogen-bonded to its template mRNA after synthesis. The actual state of the template mRNA in these experiments has not
been directly assayed and is only inferred from the apparent double-stranded
character of most of the cDNA. It is unclear why this template RNA has not
been degraded by the RNAse H associated with reverse transcriptase (21).
The data presented in this report not only suggest conditions for the
synthesis of high yields of full length cDNA, but also contribute to an understanding of the mechanism of action of purified reverse transcriptase with
template poly(A)-containing RNA. Template dependent polynucleotide polymerases can act in either a processive or distributive fashion, a distinction
which may be dependent upon reaction conditions (22,23). For reverse transcriptase, evidence for both mechanisms has been presented in the literature
(18,19). Any mechanism of action of this enzyme with this template must account for the following data presented-in this report. 1) At a low enzyme
concentration and at a low (10 vM) concentration of dCTP, conditions under
which the reaction kinetics are linear (Fig. 5), there is no change in the
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size of the product as a function of time (Fig. 7C & 8C). Therefore, the reaction cannot be entirely distributive and must have some processive character.
(2) Under these conditions the final size of the cDNA product is heterogeneous
and considerably shorter than full-length, suggesting that the enzyme must ter-
minate synthesis before it reaches the end of the RNA molecule. Therefore, the
synthesis reaction is processive to some points along the polynucleotide chain
and the 'terminates, presumably because of specific nucleotide sequences or
secondary structures which occur at these points (1,24). Consistent with this
interpretation is the fact that, at the same low enzyme concentration, increasing the concentration of dCTP to 200 plM increases the average size of the
cDNA product and causes the appearance of a small fraction of full length cDNA
(Fig. 7C,D; Fig. 8C,D). This is presumably due to the fact that at higher
concentrations of dCTP, this premature termination occurs somewhat less frequently and/or reverse transcriptase can successfully reinitiate synthesis
more frequently on these partial cDNA transcripts. The cDNA size increase as
a function of time at 200 PM dCTP favors the second possibility (Fig. 7D, 8D).
The fact that the rate of DNA synthesis is relatively unaffected by increasing
the concentration of dCTP from 10 iM to 200 pM (Fig. 5) argues that the rate
of chain initiation on poly(rA):oligo(dT) template primer, as well as the
general rate of polymerization, is relatively unaffected by the increase in
concentration of dCTP over this range. (3) At high concentrations of reverse
transcriptase, there occurs a very definite increase in the size of cDNA as a
function of incubation time. A large fraction of the cDNA product is full
length, both at 10 pM dCTP or at 200 iM dCTP. It follows that at this high
enzyme concentration, transcription through these termination regions is a
much more likely event.
All of these considerations are consistent with a model in which reverse
transcriptase synthesizes DNA in a partially processive and partially distributive manner. Polymerization is mostly processive except at specific regions
determined by the sequence or secondary structure of the template RNA. Consistent with the results presented in this report is that the Km for continued
polymerization past these stop points is high and considerably higher than the
Km for chain initiation at poly(A):oligo-(dT)(25). Increasing the concentration of dCTP lowers the Km for polymerization past the stop points. A significant increase in enzyme concentration, however, is much more efficient in
overcoming the apparent high Km at these blocked regions.
In addition to mechanistic considerations, the results suggest conditions
under which one can obtain high yields of full length cDNA. Furthermore, the
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Nucleic Acids Research
high concentration of enzyme obviates the need for a high concentration of all
four triphosphates and therefore, permits the use of one radioactive triphosphate at relatively low concentration and, therefore, considerable incorporation of input radioactivity.
ACKNOWLEDGEMENTS
colleagues Lynna Hereford, Stanley Perlman, Fiona Gibson and
John Woolford for helpful discussion and comments. We are also grateful to Will
McClure, David Baltimore, Ray White and John Morrow for helpful discussions. We thank
We thank
our
J.W. Beard and the National Cancer Institute for purified AMV
scriptase and
are
reverse
tran-
indebted to S. Kaplan for gifts of Hae III lambda restricThis research wassupported by grants from the
tion fragments of defined size.
National Institutes of Health and the National Science Foundation
to M.R.
*
Present address
Department of Biochemistry,
Johns Hopkins School of Hygiene and Public Health
615 North Wolfe Street
Baltimore, Maryland 21205
To whom correspondence should be addressed
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