U.S. patent application number 15/089749 was filed with the patent office on 2016-11-24 for mutant reverse transcriptase and methods of use.
This patent application is currently assigned to Agilent Technologies, Inc.. The applicant listed for this patent is Agilent Technologies, Inc.. Invention is credited to Bahram Arezi, Holly H. Hogrefe, Weimei Xing.
Application Number | 20160340657 15/089749 |
Document ID | / |
Family ID | 39763306 |
Filed Date | 2016-11-24 |
United States Patent
Application |
20160340657 |
Kind Code |
A1 |
Hogrefe; Holly H. ; et
al. |
November 24, 2016 |
Mutant Reverse Transcriptase and Methods of Use
Abstract
The invention relates to the generation and characterization of
stable MMLV reverse transcriptase mutants. The invention also
discloses methods of using stable MMLV reverse transcriptase
mutants.
Inventors: |
Hogrefe; Holly H.; (San
Diego, CA) ; Arezi; Bahram; (Carlsbad, CA) ;
Xing; Weimei; (Palo Alto, CA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Agilent Technologies, Inc. |
Santa Clara |
CA |
US |
|
|
Assignee: |
Agilent Technologies, Inc.
Santa Clara
CA
|
Family ID: |
39763306 |
Appl. No.: |
15/089749 |
Filed: |
April 4, 2016 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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11502819 |
Aug 10, 2006 |
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15089749 |
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60707019 |
Aug 10, 2005 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
C12Y 207/07049 20130101;
C40B 40/08 20130101; C12Q 1/686 20130101; C12N 15/1096 20130101;
C40B 50/06 20130101; C12N 9/1276 20130101 |
International
Class: |
C12N 9/12 20060101
C12N009/12; C12Q 1/68 20060101 C12Q001/68; C12N 15/10 20060101
C12N015/10 |
Claims
1-41. (canceled)
42. An isolated mutant MMLV reverse transcriptase having reverse
transcriptase activity comprising the sequence of SEQ ID NO:19 with
the exception of mutations at at least three of the following amino
acid positions: E69, E302, W313, L435, N454, and M651.
43. The isolated mutant MMLV reverse transcriptase of claim 42,
wherein the reverse transcriptase comprises at least one of the
following mutations: a glutamic acid to lysine mutation at position
E69, a glutamic acid to lysine mutation at position E302, a
glutamic acid to arginine mutation at position E302, a tryptophan
to phenylalanine mutation at position W313, a leucine to glycine
mutation at position L435, a leucine to methionine mutation at
position L435, an asparagine to lysine mutation at position N454,
an asparagine to arginine mutation at position N454, and a
methionine to leucine mutation at position M651.
44. The isolated mutant MMLV reverse transcriptase of claim 42,
wherein the reverse transcriptase comprises a combination of
mutations selected from the following groups of combinations of
mutations: E302R/E69K/W313F/L435G/N454K; E302R/W313F/L435G/N454K;
E302R/W313F/L435G; E302R/E69K/N454K; E302R/W313F; and
E69K/E302R/W313F/L435G/N454K/D524N.
45. The isolated mutant MMLV reverse transcriptase of claim 42,
further comprising a C-terminal extension.
46. The isolated mutant MMLV reverse transcriptase of claim 45,
wherein said C-terminal extension is RDRNKNNDRRKAKENE (SEQ ID
NO:1).
47. The isolated mutant MMLV reverse transcriptase of claim 42,
wherein said reverse transcriptase lacks RNase H activity.
48. The isolated mutant MMLV reverse transcriptase of claim 42,
wherein the reverse transcriptase has at least one of the following
characteristics: increased stability, increased accuracy, increased
processivity, and increased specificity.
49. A composition comprising the isolated mutant MMLV reverse
transcriptase of claim 42.
50. The composition of claim 49, wherein the reverse transcriptase
comprises at least one of the following mutations: a glutamic acid
to lysine mutation at position E69, a glutamic acid to lysine
mutation at position E302, a glutamic acid to arginine mutation at
position E302, a tryptophan to phenylalanine mutation at position
W313, a leucine to glycine mutation at position L435, a leucine to
methionine mutation at position L435, an asparagine to lysine
mutation at position N454, an asparagine to arginine mutation at
position N454, and a methionine to leucine mutation at position
M651.
51. The composition of claim 49, wherein the reverse transcriptase
comprises a combination of mutations selected from the following
groups of combinations of mutations: E302R/E69K/W313F/L435G/N454K;
E302R/W313F/L435G/N454K; E302R/W313F/L435G; E302R/E69K/N454K;
E302R/W313F; and E69K/E302R/W313F/L435G/N454K/D524N.
52. The composition of claim 49, wherein the reverse transcriptase
further comprises a C-terminal extension.
53. The composition of claim 52, wherein said C-terminal extension
is RDRNKNNDRRKAKENE (SEQ ID NO:1).
54. The composition of claim of claim 49, wherein the reverse
transcriptase has at least one of the following characteristics:
increased stability, increased accuracy, increased processivity,
and increased specificity.
55. The composition of claim 49, wherein said reverse transcriptase
lacks RNase H activity.
56. The composition of claim 49, further comprising an epsilon
subunit from a eubacteria.
57. The composition of claim 56, wherein said epsilon subunit is
from Escherichia coli.
58. The composition of claim 56, wherein said epsilon subunit is
epsilon 186 from Escherichia coli.
59. The composition of claim 49, further comprising formamide,
betaine, or DMSO.
60. A kit comprising the isolated mutant MMLV reverse transcriptase
of claim 42 and packaging materials thereof.
61. The kit of claim 60, wherein the transcriptase comprises at
least one of the following mutations: a glutamic acid to lysine
mutation at position E69, a glutamic acid to lysine mutation at
position E302, a glutamic acid to arginine mutation at position
E302, a tryptophan to phenylalanine mutation at position W313, a
leucine to glycine mutation at position L435, a leucine to
methionine mutation at position L435, an asparagine to lysine
mutation at position N454, an asparagine to arginine mutation at
position N454, and a methionine to leucine mutation at position
M651.
62. The kit of claim 60, wherein the reverse transcriptase
comprises a combination of mutations selected from the following
groups of combinations of mutations: E302R/E69K/W313F/L435G/N454K;
E302R/W313F/L435G/N454K; E302R/W313F/L435G; E302R/E69K/N454K;
E302R/W313F; and E69K/E302R/W313F/L435G/N454K/D524N.
63. The kit of claim 60, wherein said reverse transcriptase lacks
RNase H activity.
64. The kit of claim 60, wherein said mutant MMLV reverse
transcriptase further comprises a C-terminal extension.
65. The kit of claim 64, wherein said C-terminal extension is
RDRNKNNDRRKAKENE (SEQ ID NO:1).
66. The kit of claim 60, wherein said reverse transcriptase has at
least one of the following characteristics: increased stability,
increased accuracy, increased processivity, and increased
specificity.
67. The kit of claim 60, further comprising an epsilon subunit from
a eubacteria.
68. The kit of claim 67, wherein said epsilon subunit is from
Escherichia coli.
69. The kit of claim 67, wherein said epsilon subunit is epsilon
186 from Escherichia coli.
70. The kit of claim 60, further comprising formamide, betaine, or
DMSO.
Description
[0001] This application is a Continuation of U.S. patent
application Ser. No. 11/502,819, filed Aug. 10, 2006, which claims
the benefit of U.S. Provisional Application No. 60/707,019, filed
on Aug. 10, 2005, the disclosures of which are incorporated herein
by reference in their entirety.
FIELD OF THE INVENTION
[0002] The invention relates to mutant reverse transcriptases with
increased stability.
BACKGROUND
[0003] Three prototypical forms of retroviral reverse transcriptase
have been studied thoroughly. Moloney Murine Leukemia Virus (M-MLV)
reverse transcriptase contains a single subunit of 78 kDa with
RNA-dependent DNA polymerase and RNase H activity. This enzyme has
been cloned and expressed in a fully active form in E. coli
(reviewed in Prasad, V. R., Reverse Transcriptase, Cold Spring
Harbor, N.Y.: Cold Spring Harbor Laboratory Press, p. 135 (1993)).
Human Immunodeficiency Virus (HIV) reverse transcriptase is a
heterodimer of p66 and p51 subunits in which the smaller subunit is
derived from the larger by proteolytic cleavage. The p66 subunit
has both an RNA-dependent DNA polymerase and an RNase H domain,
while the p51 subunit has only a DNA polymerase domain. Active HIV
p66/p51 reverse transcriptase has been cloned and expressed
successfully in a number of expression hosts, including E. coli
(reviewed in Le Grice, S. F. J., Reverse Transcriptase, Cold Spring
Harbor, N.Y.: Cold Spring Harbor Laboratory press, p. 163 (1993)).
Within the HIV p66/p51 heterodimer, the 51-kD subunit is
catalytically inactive, and the 66-kD subunit has both DNA
polymerase and RNase H activity (Le Grice, S. F. J., et al., EMBO
Journal 10:3905 (1991); Hostomsky, Z., et al., J. Virol. 66:3179
(1992)). Avian Sarcoma-Leukosis Virus (ASLV) reverse transcriptase,
which includes but is not limited to Rous Sarcoma Virus (RSV)
reverse transcriptase, Avian Myeloblastosis Virus (AMV) reverse
transcriptase, Avian Erythroblastosis Virus (AEV) Helper Virus MCAV
reverse transcriptase, Avian Myelocytomatosis Virus MC29 Helper
Virus MCAV reverse transcriptase, Avian Reticuloendotheliosis Virus
(REV-T) Helper Virus REV-A reverse transcriptase, Avian Sarcoma
Virus UR2 Helper Virus UR2AV reverse transcriptase, Avian Sarcoma
Virus Y73 Helper Virus YAV reverse transcriptase, Rous Associated
Virus (RAV) reverse transcriptase, and Myeloblastosis Associated
Virus (MAV) reverse transcriptase, is also a heterodimer of two
subunits, alpha (approximately 62 kDa) and beta (approximately 94
kDa), in which alpha is derived from beta by proteolytic cleavage
(reviewed in Prasad, V. R., Reverse Transcriptase, Cold Spring
Harbor, N.Y.: Cold Spring Harbor Laboratory Press (1993), p. 135).
ASLV reverse transcriptase can exist in two additional
catalytically active structural forms, beta beta and alpha (Hizi,
A. and Joklik, W. K., J. Biol. Chem. 252: 2281 (1977)).
Sedimentation analysis suggests alpha beta and beta beta are dimers
and that the alpha form exists in an equilibrium between monomeric
and dimeric forms (Grandgenett, D. P., et al., Proc. Nat. Acad.
Sci. USA 70:230 (1973); Hizi, A. and Joklik, W. K., J. Biol. Chem.
252:2281 (1977); and Soltis, D. A. and Skalka, A. M., Proc. Nat.
Acad. Sci. USA 85:3372 (1988)). The ASLV alpha beta. and beta beta
reverse transcriptases are the only known examples of retroviral
reverse transcriptase that include three different activities in
the same protein complex: DNA polymerase, RNase H, and DNA
endonuclease (integrase) activities (reviewed in Skalka, A. M.,
Reverse Transcriptase, Cold Spring Harbor, N.Y.: Cold Spring Harbor
Laboratory Press (1993), p. 193). The alpha form lacks the
integrase domain and activity.
[0004] The conversion of mRNA into cDNA by reverse
transcriptase-mediated reverse transcription is an essential step
in many gene expression studies. However, the use of unmodified
reverse transcriptase (RT) to catalyze reverse transcription is
inefficient for a number of reasons. First, reverse transcriptase
sometimes degrades an RNA template before the first strand reaction
is initiated or completed, primarily due to the intrinsic RNase H
activity present in reverse transcriptase. In addition, mis-priming
of the mRNA template molecule can lead to the introduction of
errors in the cDNA first strand. RTs have in fact been shown to
incorporate one base error per 3000-6000 nucleotides for HIV RT,
and 1/10,000 nucleotide for AMV RT during cDNA synthesis (Berger,
S. L., et al., Biochemistry 22:2365-2372 (1983); Krug, M. S., and
Berger, S. L., Meth. Enzymol. 152:316 (1987); Berger et al. Meth.
Enzymol. 275: 523 (1996)). Secondary structure of the mRNA molecule
itself may make some mRNAs refractory to first strand synthesis.
Another factor which influences the efficiency of reverse
transcription is the ability of RNA to form secondary structures.
Such secondary structures can form, for example, when regions of
RNA molecules have sufficient complementarity to hybridize and form
double stranded RNA. Generally, the formation of RNA secondary
structures can be reduced by raising the temperature of solutions
which contain the RNA molecules. Thus, in many instances, it is
desirable to reverse transcribe RNA at temperatures above
37.degree. C. However, art known reverse transcriptases generally
lose activity when incubated at temperatures much above 37.degree.
C. (e.g., 50.degree. C.).
[0005] A variety of methods of attempting to engineer a
thermostable reverse transcriptase are known in the art. These
methods include using thermostable DNA polymerases that contain
reverse transcriptase activity (Shandilya et al., Extremophiles,
2004 8:243), mutagenizing thermostable DNA polymerases to increase
their reverse transcriptase activity (U.S. 2002/0012970),
mutagenizing thermolabile reverse transcriptases (US 2004/0209276),
using Mn.sup.2+ instead of Mg.sup.2+ in the presence of Taq/Tth DNA
polymerases (Myers et al., Biochemistry 1991 30:7661), and using
additives such as trehalose with thermolabile reverse
transcriptases (Carninci et al., 1999 Proc Natl Acad Sci USA
95:520).
[0006] Scientists in the field have also tried different enzyme
compositions and methods for increasing the fidelity of
polymerization on DNA or RNA templates. For example, Shevelev et
al., Nature Rev. Mol. Cell Biol. 3:364 (2002) provides a review on
3'-5' exonucleases. Perrino et al., PNAS, 86:3085 (1989) reports
the use of epsilon subunit of E. coli DNA polymerase III to
increase the fidelity of calf thymus DNA polymerase a.
Bakhanashvili, Eur. J. Biochem. 268:2047 (2001) describes the
proofreading activity of p53 protein and Huang et al., Oncogene,
17:261 (1998) describes the ability of p53 to enhance DNA
replication fidelity. Bakhanashvili, Oncogene, 20:7635 (2001) also
reports that p53 enhances the fidelity of DNA synthesis by HIV type
I reverse transcriptase. Hawkins et al. describes the synthesis of
full length cDNA from long mRNA transcripts (2002, Biotechniques,
34:768).
[0007] U.S. Patent Application 2003/0198944A1 and U.S. Pat. No.
6,518,019 provide an enzyme mixture containing two or more reverse
transcriptases (e.g., each reverse transcriptase having a different
transcription pause site) and optionally one or more DNA
polymerases. U.S. Patent Application 2002/0119465A1 discloses a
composition that includes a mutant thermostable DNA polymerase and
a mutant reverse transcriptase (e.g., a mutant Taq DNA polymerase
and a mutant MMLV-RT). U.S. Pat. No. 6,485,917B1 and U.S. Patent
application 2003/0077762 and EP patent application EP1132470
provide a method for synthesizing cDNA in the presence of an enzyme
having a reverse transcriptional activity and an .alpha.-type DNA
polymerase having a 3'-5' exonuclease activity.
[0008] Removal of the RNase H activity of reverse transcriptase can
eliminate the problem of RNA degradation of the RNA template and
improve the efficiency of reverse transcription (Gerard, G. F., et
al., FOCUS 11(4):60 (1989); Gerard, G. F., et al., FOCUS 14(3):91
(1992)). However such reverse transcriptases ("RNase H-" forms) do
not address the additional problems of mis-priming and mRNA
secondary structure.
[0009] There is a need in the art for a reverse transcriptase that
exhibits increased stability.
SUMMARY OF THE INVENTION
[0010] The invention relates to the construction and
characterization of thermostable MMLV reverse transcriptase. The
invention also relates to methods of using the thermostable MMLV
reverse transcriptase described herein, as well as kits comprising
this enzyme.
[0011] The invention relates to a mutant MMLV reverse
transcriptase, wherein at least one of the following amino acid
positions comprises a mutation: E69, E302, W313, L435, N454 and
M651.
[0012] The invention also relates to a mutant MMLV reverse
transcriptase, comprising at least one of a glutamic acid to lysine
mutation at position E69, a glutamic acid to lysine mutation at
position E302, a glutamic acid to arginine mutation at position
E302, a tryptophan to phenylalanine mutation at position W313, a
leucine to glycine mutation at position L435, a leucine to
methionine mutation at position L435, an asparagine to lysine
mutation at position N454, an asparagine to arginine mutation at
position N454, and a methionine to leucine mutation at position
M651.
[0013] The invention also relates to a mutant MMLV reverse
transcriptase, selected from the group consisting of:
E302R/E69K/W313F/L435G/N454K; E302R/W313F/L435G/N454K;
E302R/W313F/L435G; E302R/E69K/N454K; E302R/W313F; and
E69K/E302R/W313F/L435G/N454K/D524N.
[0014] In one embodiment, the mutant MMLV reverse transcriptase
further comprises a C-terminal extension.
[0015] In another embodiment, the C-terminal extension is
RDRNKNNDRRKAKENE. (SEQ ID NO: 1)
[0016] In another embodiment, the mutant MMLV reverse transcriptase
lacks RNase H activity.
[0017] In another embodiment, the mutant MMLV reverse transcriptase
further comprises at least one of increased stability, increased
accuracy, increased processivity, and increased specificity.
[0018] The invention also relates to an isolated polynucleotide
comprising a nucleotide sequence encoding a mutant MMLV reverse
transcriptase, wherein at least one of the following amino acid
positions comprises a mutation: E69, E302, W313, L435, N454 and
M651.
[0019] The invention also relates to an isolated polynucleotide
comprising a nucleotide sequence encoding a mutant MMLV reverse
transcriptase, comprising at least one of a glutamic acid to lysine
mutation at position E69, a glutamic acid to lysine mutation at
position E302, a glutamic acid to arginine mutation at position
E302, a tryptophan to phenylalanine mutation at position W313, a
leucine to glycine mutation at position L435, a leucine to
methionine mutation at position L435, an asparagine to lysine
mutation at position N454, an asparagine to arginine mutation at
position N454, and a methionine to leucine mutation at position
M651.
[0020] The invention also relates to an isolated polynucleotide
comprising a nucleotide sequence encoding a mutant MMLV reverse
transcriptase, selected from the group consisting of:
E302R/E69K/W313F/L435G/N454K; E302R/W313F/L435G/N454K;
E302R/W313F/L435G; E302R/E69K/N454K; E302R/W313F; and
E69K/E302R/W313F/L435G/N454K/D524N.
[0021] In one embodiment, the isolated polynucleotide, further
encodes a C-terminal extension.
[0022] In one embodiment, the C-terminal extension is
RDRNKNNDRRKAKENE. (SEQ ID NO: 1)
[0023] The invention also relates to a composition comprising a
mutant MMLV reverse transcriptase, wherein at least one of the
following amino acid positions comprises a mutation: E69, E302,
W313, L435, N454 and M651.
[0024] The invention also relates to a composition comprising a
mutant MMLV reverse transcriptase, comprising at least one of a
glutamic acid to lysine mutation at position E69, a glutamic acid
to lysine mutation at position E302, a glutamic acid to arginine
mutation at position E302, a tryptophan to phenylalanine mutation
at position W313, a leucine to glycine mutation at position L435, a
leucine to methionine mutation at position L435, an asparagine to
lysine mutation at position N454, an asparagine to arginine
mutation at position N454, and a methionine to leucine mutation at
position M651.
[0025] The invention also relates to a composition comprising a
mutant MMLV reverse transcriptase, selected from the group
consisting of: E302R/E69K/W313F/L435G/N454K;
E302R/W313F/L435G/N454K; E302R/W313F/L435G; E302R/E69K/N454K;
E302R/W313F; and E69K/E302R/W313F/L435G/N454K/D524N.
[0026] In one embodiment, the mutant reverse transcriptase of the
composition further comprises a C-terminal extension.
[0027] In another embodiment, the C-terminal extension is
RDRNKNNDRRKAKENE. (SEQ ID NO: 1)
[0028] In another embodiment, the mutant reverse transcriptase
further comprises at least one of increased stability, increased
accuracy, increased processivity, and increased specificity.
[0029] In another embodiment, the reverse transcriptase lacks RNase
H activity.
[0030] In another embodiment, the composition further comprises an
epsilon subunit from an eubacteria.
[0031] In another embodiment, the epsilon subunit is from
Eschericia coli.
[0032] In another embodiment, the epsilon subunit is epsilon 186
from Eschericia coli.
[0033] In another embodiment, the composition further comprises
formamide, betaine or DMSO.
[0034] The invention also provides for a kit comprising a mutant
MMLV reverse transcriptase, wherein at least one of the following
amino acid positions comprises a mutation: E69, E302, W313, L435,
N454 and M651, and packaging materials thereof.
[0035] The invention also provides for a kit comprising a mutant
MMLV reverse transcriptase, comprising at least one of a glutamic
acid to lysine mutation at position E69, a glutamic acid to lysine
mutation at position E302, a glutamic acid to arginine mutation at
position E302, a tryptophan to phenylalanine mutation at position
W313, a leucine to glycine mutation at position L435, a leucine to
methionine mutation at position L435, an asparagine to lysine
mutation at position N454, an asparagine to arginine mutation at
position N454, and a methionine to leucine mutation at position
M651, and packaging materials thereof.
[0036] The invention also provides for a kit comprising a mutant
MMLV reverse transcriptase, selected from the group consisting of:
E302R/E69K/W313F/L435G/N454K; E302R/W313F/L435G/N454K;
E302R/W313F/L435G; E302R/E69K/N454K; E302R/W313F; and
E69K/E302R/W313F/L435G/N454K/D524N, and packaging materials
thereof.
[0037] In one embodiment, the mutant reverse transcriptase of the
kit lacks RNase H activity.
[0038] In another embodiment, the mutant MMLV-reverse transcriptase
of the kit, further comprises a C-terminal extension.
[0039] In another embodiment, the C-terminal extension is
RDRNKNNDRRKAKENE. (SEQ ED NO: 1)
[0040] In another embodiment, the mutant reverse transcriptase of
the kit further comprises at least one of increased stability,
increased accuracy, increased processivity, and increased
specificity.
[0041] In another embodiment, the kit further comprises an epsilon
subunit from an eubacteria.
[0042] In another embodiment, the epsilon subunit is from
Eschericia coli.
[0043] In another embodiment, the epsilon subunit is epsilon 186
from Eschericia coli. In another embodiment, the kit further
comprises formamide, betaine or DMSO.
[0044] The invention also provides for a method for cDNA synthesis
comprising providing a mutant reverse transcriptase of the
invention; and contacting the mutant reverse transcriptase with a
nucleic acid template to permit cDNA synthesis.
[0045] The invention also provides for a method for cloning
comprising providing a mutant reverse transcriptase of the
invention; contacting the mutant reverse transcriptase with a
nucleic acid template to generate a synthesized cDNA product and
inserting the synthesized cDNA product into a cloning vector.
[0046] The invention also provides for a method for RT-PCR
comprising: providing a mutant reverse transcriptase of the
invention; and contacting the mutant reverse transcriptase with a
nucleic acid template to replicate and amplify the nucleic acid
template.
[0047] In one embodiment, the RT-PCR comprises end-point
RT-PCR.
[0048] In another embodiment, the RT-PCR is performed in
real-time.
[0049] The invention also provides for a method for cDNA library
construction comprising providing a mutant reverse transcriptase of
the invention; contacting the mutant reverse transcriptase with a
nucleic acid template to generate a synthesized cDNA product and
inserting the synthesized cDNA product into a vector.
[0050] The invention also provides for a method for preparing a
microarray comprising providing a mutant reverse transcriptase of
the invention; contacting the mutant reverse transcriptase with a
nucleic acid template to generate a synthesized cDNA product and
attaching the cDNA product to a substrate.
DEFINITIONS
[0051] As used herein, "reverse transcriptase activity" and
"reverse transcription" refer to the ability of an enzyme to
synthesize a DNA strand (i.e. complementary DNA or cDNA) utilizing
an RNA strand as a template. Reverse transcriptase activity may be
measured by incubating an enzyme in the presence of an RNA template
and deoxynucleotides, in the presence of an appropriate buffer,
under appropriate conditions, for example as described in Example
3.
[0052] As used herein, the term "reverse transcriptase (RT)" is
used in its broadest sense to refer to any enzyme that exhibits
reverse transcription activity as measured by methods disclosed
herein or known in the art. A "reverse transcriptase" of the
present invention, therefore, includes reverse transcriptases from
retroviruses, other viruses, as well as a DNA polymerase exhibiting
reverse transcriptase activity, such as Tth DNA polymerase, Taq DNA
polymerase, Tne DNA polymerase, Tma DNA polymerase, etc. RT from
retroviruses include, but are not limited to, Moloney Murine
Leukemia Virus (M-MLV) RT, Human Immunodeficiency Virus (HIV) RT,
Avian Sarcoma-Leukosis Virus (ASLV) RT, Rous Sarcoma Virus (RSV)
RT, Avian Myeloblastosis Virus (AMV) RT, Avian Erythroblastosis
Virus (AEV) Helper Virus MCAV RT, Avian Myelocytomatosis Virus MC29
Helper Virus MCAV RT, Avian Reticuloendotheliosis Virus (REV-T)
Helper Virus REV-A RT, Avian Sarcoma Virus UR2 Helper Virus UR2AV
RT, Avian Sarcoma Virus Y73 Helper Virus YAV RT, Rous Associated
Virus (RAV) RT, and Myeloblastosis Associated Virus (MAV) RT, and
as described in U.S. Patent Application 2003/0198944 (hereby
incorporated by reference in its entirety). For review, see e.g.
Levin, 1997, Cell, 88:5-8; Brosius et al., 1995, Virus Genes
11:163-79. Known reverse transcriptases from viruses require a
primer to synthesize a DNA transcript from an RNA template. Reverse
transcriptase has been used primarily to transcribe RNA into cDNA,
which can then be cloned into a vector for further manipulation or
used in various amplification methods such as polymerase chain
reaction (PCR), nucleic acid sequence-based amplification (NASBA),
transcription mediated amplification (TMA), or self-sustained
sequence replication (3 SR).
[0053] As used herein, the terms "reverse transcription activity"
and "reverse transcriptase activity" are used interchangeably to
refer to the ability of an enzyme (e.g., a reverse transcriptase or
a DNA polymerase) to synthesize a DNA strand (i.e., cDNA) utilizing
an RNA strand as a template. Methods for measuring RT activity are
provided herein below and also are well known in the art. For
example, the Quan-T-RT assay system is commercially available from
Amersham (Arlington Heights, Ill.) and is described in Bosworth, et
al., Nature 1989, 341:167-168.
[0054] As used herein, the term "increased" reverse transcriptase
activity refers to the level of reverse transcriptase activity of a
mutant enzyme (e.g., a mutant reverse transcriptase) as compared to
its wild-type form. A mutant enzyme is said to have an "increased"
reverse transcriptase activity if the level of its reverse
transcriptase activity (as measured by methods described herein or
known in the art) is at least 10% or more than its wild-type form,
for example, at least 10%, 15%, 20%, 25%, 30%, 40%, 50%, 60%, 70%,
80%, 90%, 100% more or at least 2-fold, 3-fold, 4-fold, 5-fold, or
10-fold or more.
[0055] Reverse transcriptases of the invention include any reverse
transcriptase having one or a combination of the properties
described herein. Such properties include, but are not limited to,
enhanced stability, enhanced thermostability, reduced or eliminated
RNase H activity, reduced terminal deoxynucleotidyl transferase
activity, increased accuracy, increased processivity, increased
specificity and/or increased fidelity.
[0056] "Complementary" refers to the broad concept of sequence
complementarity between regions of two polynucleotide strands or
between two nucleotides through base-pairing. It is known that an
adenine nucleotide is capable of forming specific hydrogen bonds
("base pairing") with a nucleotide which is thymine or uracil.
Similarly, it is known that a cytosine nucleotide is capable of
base pairing with a guanine nucleotide.
[0057] As used herein, "mutation" refers to a change introduced
into a parental or wild type DNA sequence that changes the amino
acid sequence encoded by the DNA, including, but not limited to,
substitutions, insertions, deletions, point mutations, mutation of
multiple nucleotides or amino acids, transposition, inversion,
frame shift, nonsense mutations, truncations or other forms of
aberration that differentiate the polynucleotide or protein
sequence from that of a wild-type sequence of a gene or gene
product. The consequences of a mutation include, but are not
limited to, the creation of a new character, property, function, or
trait not found in the protein encoded by the parental DNA,
including, but not limited to, N terminal truncation, C terminal
truncation or chemical modification. A "mutation" also includes an
N- or C-terminal extension.
[0058] The present invention relates in particular to mutant or
modified reverse transcriptases wherein one or more (e.g., one,
two, three, four, five, ten, twelve, fifteen, twenty, etc.) amino
acid changes have been made which renders the enzyme more stable in
nucleic acid synthesis, as compared to the unmutated or unmodified
reverse transcriptases. As will be appreciated by those skilled in
the art, one or more of the amino acids identified may be deleted
and/or replaced with one or a number of amino acid residues. In a
preferred aspect, any one or more of the amino acids may be
substituted with any one or more amino acid residues such as Ala,
Arg, Asn, Asp, Cys, Gln, Glu, Gly, His, Ile, Leu, Lys, Met, Phe,
Pro, Ser, Thr, Trp, Tyr, and/or Val.
[0059] A reverse transcriptase of the present invention may have
one or more of the following properties: (a) increased stability or
increased half-life at elevated temperatures; (b) reduced,
substantially reduced, or no detectable RNase H activity, (c)
reduced or substantially reduced terminal deoxynucleotidyl
transferase activity, (d) increased accuracy, (e) increased
specificity, (f) increased processivity and/or (d) increased
fidelity. In some embodiments, a reverse transcriptase of the
invention may have a plurality of the properties listed above
(e.g., a reverse transcriptase may have enhanced thermostability,
reduced RNase H activity, and enhanced accuracy). Reverse
transcriptases of the invention may have one or more of the
following properties: (a) increased thermostability or increased
half-life at elevated temperatures; (b) reduced, substantially
reduced, or no detectable RNase H activity, (c) reduced or
substantially reduced terminal deoxynucleotidyl transferase
activity, and/or (d) increased fidelity.
[0060] The term "wild-type" refers to a gene or gene product which
has the characteristics of that gene or gene product when isolated
from a naturally occurring source. In contrast, the term "modified"
or "mutant" refers to a gene or gene product which displays altered
characteristics when compared to the wild-type gene or gene
product. For example, a mutant DNA polymerase in the present
invention is a DNA polymerase which exhibits a reduced uracil
detection activity.
[0061] As used herein, "increased" refers to greater than 10%
(e.g., 11%, 12%, 13%, 14%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%,
55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% or more), as
compared to a wild-type enzyme. "Increased" also refers to greater
than at least 2-fold or more, (for example, 3, 4, 5, 10, 20, 50,
100, 1000, 10,000-fold or more), as compared to a wild-type
enzyme.
[0062] As used herein "stable" refers to exhibiting increased
activity, as defined herein, under denaturing conditions, including
but not limited to higher temperatures (for example greater than
37.degree. C. (for example 38, 39, 40, 50, 55, 60, 65, 70, 75, 80,
85.degree. C. or more), or in the presence of denaturing agents,
including but not limited to DMSO or formamide or betaine, as
compared to the activity of a wild-type enzyme subjected to
identical denaturing conditions.
[0063] As used herein, "stable" includes "thermostable" as defined
herein.
[0064] As used herein, "thermostable refers to an enzyme which is
resistant to inactivation by heat. "Thermostable" also refers to an
enzyme which is stable and active at temperatures as great as
preferably between about 38-100.degree. C., for example 38, 39, 40,
45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100.degree. C. and more
preferably between about 40-80.degree. C. (for example 40, 41, 42,
43, 44, 45, 50, 55, 60, 65, 70, 75, 80.degree. C.) to heat as
compared, for example, to a non-thermostable form of an enzyme with
a similar activity. Thermostable is further defined
hereinbelow.
[0065] In one embodiment, the present invention provides a modified
or mutated reverse transcriptase having a reverse transcriptase
activity that has a half-life of greater than that of the
corresponding unmodified or un-mutated reverse transcriptase at an
elevated temperature, i.e., greater than 37.degree. C. In some
embodiments, the half-life of a reverse transcriptase of the
present invention may be 5 minutes or greater and preferably 10
minutes or greater at 50.degree. C. In some embodiments, the
reverse transcriptases of the invention may have a half-life (e.g.,
at 50.degree. C.) equal to or greater than about 25 minutes,
preferably equal to or greater than about 50 minutes, more
preferably equal to or greater than about 100 minutes, and most
preferably, equal to or greater than about 200 minutes.
[0066] In some embodiments, the reverse transcriptases of the
invention may have a half-life at 50.degree. C. that is from about
10 minutes to about 200 minutes, from about 10 minutes to about 150
minutes, from about 10 minutes to about 100 minutes, from about 10
minutes to about 75 minutes, from about 10 minutes to about 50
minutes, from about 10 minutes to about 40 minutes, from about 10
minutes to about 30 minutes, or from about 10 minutes to about 20
minutes.
[0067] Mutated or modified reverse transcriptases of the present
invention may have a reverse transcriptase activity (e.g.,
RNA-dependent DNA polymerase activity) that has a longer half-life
at 55.degree. C. than the reverse transcriptase activity of a
corresponding un-mutated or unmodified reverse transcriptase. At
55.degree. C., the half-life of reverse transcriptase activity of a
mutated or modified reverse transcriptase of the invention may be
greater than about 2 minutes, greater than about 3 minutes, greater
than about 4 minutes, greater than about 5 minutes, greater than
about 6 minutes, greater than about 7 minutes, greater than about 8
minutes, greater than about 10 minutes, greater than about 15
minutes, greater than about 20 minutes, or greater than about 30
minutes. At 55.degree. C., the half-life of reverse transcriptase
activity of a reverse transcriptase of the invention may be from
about 2 minutes to about 60 minutes, from about 2 minutes to about
45 minutes, from about 2 minutes to about 30 minutes, from about 2
minutes to about 20 minutes, from about 2 minutes to about 15
minutes, from about 2 minutes to about 10 minutes, from about 2
minutes to about 8 minutes, from about 2 minutes to about 7
minutes, from about 2 minutes to about 6 minutes, from about 2
minutes to about 5 minutes, from about 2 minutes to about 4
minutes, or from about 2 minutes to about 3 minutes.
[0068] Reverse transcriptases of the present invention may produce
more product (e.g., full length product) at elevated temperatures
than other reverse transcriptases. In one aspect, comparisons of
full length product synthesis is made at different temperatures
(e.g., one temperature being lower, such as between 37.degree. C.
and 50.degree. C., and one temperature being higher, such as
between 50.degree. C. and 78.degree. C.) while keeping all other
reaction conditions similar or the same. The amount of full length
product produced may be determined using techniques well known in
the art, for example, by conducting a reverse transcription
reaction at a first temperature (e.g., 37.degree. C., 38.degree.
C., 39.degree. C., 40.degree. C., etc.) and determining the amount
of full length transcript produced, conducting a second reverse
transcription reaction at a temperature higher than the first
temperature (e.g., 45.degree. C., 50.degree. C., 52.5.degree. C.,
55.degree. C., etc.) and determining the amount of full length
product produced, and comparing the amounts produced at the two
temperatures. A convenient form of comparison is to determine the
percentage of the amount of full length product at the first
temperature that is produced at the second (i.e., elevated)
temperature. The reaction conditions used for the two reactions
(e.g., salt concentration, buffer concentration, pH, divalent metal
ion concentration, nucleoside triphosphate concentration, template
concentration, reverse transcriptase concentration, primer
concentration, length of time the reaction is conducted, etc.) are
preferably the same for both reactions. Suitable reaction
conditions include, but are not limited to, a template
concentration of from about 1 nM to about 1 .mu.M, from about 100
nM to 1 .mu.M, from about 300 nM to about 750 nM, or from about 400
nM to about 600 nM, and a reverse transcriptase concentration of
from about 1 nM to about 1 .mu.M, from about 10 nM to 500 nM, from
about 50 nM to about 250 nM, or from about 75 nM to about 125 nM.
The ratio of the template concentration to the reverse
transcriptase concentration may be from about 100:1 to about 1:1,
from about 50:1 to about 1:1, from about 25:1 to about 1:1, from
about 10:1 to about 1:1, from about 5:1 to about 1:1, or from about
2.5:1 to 1:1. A reaction may be conducted from about 5 minutes to
about 5 hours, from about 10 minutes to about 2.5 hours, from about
30 minutes to about 2 hours, from about 45 minutes to about 1.5
hours, or from about 45 minutes to about 1 hour. A suitable
reaction time is about one hour. Other suitable reaction conditions
may be determined by those skilled in the art using routine
techniques and examples of such conditions are provided below.
[0069] When the amount of full length product produced by a reverse
transcriptase of the invention at an elevated temperature is
compared to the amount of full length product produced by the same
reverse transcriptase at a lower temperature, at an elevated
temperature, the reverse transcriptases of the invention may
produce not less than about 25%, 35%, 45%, 55%, 65%, 75%, 85%, 95%,
100% of the amount of full length product produced at the lower
temperature. In some cases, the reverse transcriptases of the
invention may produce an amount of full length product at a higher
temperature that is greater than the amount of full length product
produced by the reverse transcriptase at a lower temperature (e.g.,
1% to about 100% greater). In one aspect, reverse transcriptases of
the invention produce approximately the same amount (e.g., no more
than a 25% difference) of full length product at the lower
temperature compared to the amount of full length product made at
the higher temperature.
[0070] A reverse transcriptase of the present invention may be one
that synthesizes an amount of full length product, wherein the
amount of full length product synthesized at 50.degree. C. is no
less than 10% (e.g., from about 10% to about 95%, from about 10% to
about 80%, from about 10% to about 70%, from about 10% to about
60%, from about 10% to about 50%, from about 10% to about 40%, from
about 10% to about 30%, or from about 10% to about 20%) of the
amount of full length product it synthesizes at 40.degree. C. In
some embodiments, a reverse transcriptase of the invention is one
wherein the amount of full length product synthesized at 50.degree.
C. is no less than 50% (e.g., from about 50% to about 95%, from
about 50% to about 80%, from about 50% to about 70%, or from about
50% to about 60%) of the amount of full length product it
synthesizes at 40.degree. C. In some embodiments, a reverse
transcriptase of the invention is one wherein the amount of full
length product synthesized at 50.degree. C. is no less than 75%
(e.g., from about 75% to about 95%, from about 75%, to about 90%,
from about 75% to about 85%, or from about 75% to about 80%) of the
amount of full length product it synthesizes at 40.degree. C. In
other embodiments, a reverse transcriptase of the invention is one
wherein the amount of full length product synthesized at 50.degree.
C. is no less than 85% (e.g., from about 85% to about 95%, or from
about 85% to about 90%) of the amount of full length product it
synthesizes at 40.degree. C.
[0071] A reverse transcriptase of the invention may be one that
synthesizes an amount of full length product, wherein the amount of
full length product synthesized at 52.5.degree. C. is no less than
10% (e.g., from about 10% to about 30%, from about 10% to about
25%, from about 10% to about 20%, from about 10% to about 15%, from
about 20% to about 60%, from about 20% to about 40%, from about 20%
to about 30%, from about 30% to about 80%, from about 30% to about
60%, from about 30% to about 45%, from about 40% to about 90%, from
about 40% to about 80%, from about 40% to about 60%, from about 40%
to about 50% from about 50% to about 90%, or from about 50% to
about 70%), of the amount of full length product it synthesizes at
40.degree. C. In some embodiments, the amount of full length
product synthesized at 52.5.degree. C. is no less than 30% (e.g.,
from about 30% to about 70%, from about 30% to about 60%, from
about 30% to about 50%, or from about 30% to about 40%) of the
amount of full length product it synthesizes at 40.degree. C. In
some embodiments, the amount of full length product synthesized at
52.5.degree. C. is no less than 50% (e.g., from about 50% to about
70%, from about 50% to about 65%, from about 50% to about 60%, or
from about 50% to about 55%), of the amount of full length product
it synthesizes at 40.degree. C.
[0072] A reverse transcriptase of the invention may be one that
synthesizes an amount of full length product, wherein the amount of
full length product synthesized at 55.degree. C. is no less than 1%
(e.g., from about 1% to about 30%, from about 1% to about 25%, from
about 1% to about 20%, from about 1% to about 15%, from about 1% to
about 10%, or from about 1% to about 5%) of the amount of full
length product it synthesizes at 40.degree. C. In some embodiments,
the amount of full length product synthesized at 55.degree. C. is
no less than 5% (e.g., from about 5% to about 30%, from about 5% to
about to about 25%, from about 5% to about 20%, from about 5% to
about 15%, or from about 5% to about 10%) of the amount of full
length product it synthesizes at 40.degree. C. In some embodiments,
the amount of full length product synthesized at 55.degree. C. is
no less than 10% (e.g., from about 10% to about 30%, from about 10%
to about to about 25%, from about 10% to about 20%, from about 10%
to about 15%, from about 20% to about 60%, from about 20% to about
40%, from about 20% to about 30%, from about 30% to about 80%, from
about 30% to about 60%, from about 30% to about 45%, from about 40%
to about 90%, from about 40% to about 80%, from about 40% to about
60%, from about 40% to about 50% from about 50% to about 90%, or
from about 50% to about 70%) of the amount of full length product
it synthesizes at 40.degree. C.
[0073] In another aspect, the reverse transcriptases of the
invention are capable of producing more nucleic acid product (e.g.,
cDNA) and, preferably, more full length product, at one or a number
of elevated temperatures (typically between 40.degree. C. and
78.degree. C.) compared to the corresponding un-mutated or
unmodified reverse transcriptase (e.g., the control reverse
transcriptase). Such comparisons are typically made under similar
or the same reaction conditions and the amount of product
synthesized by the control reverse transcriptase is compared to the
amount of product synthesized by the reverse transcriptase of the
invention. Preferably, the reverse transcriptases of the invention
produce at least about 5%, at least 10%, at least 15%, at least
25%, at least 50%, at least 75%, at least 100%, or at least 200%
more product or full length product compared to the corresponding
control reverse transcriptase under the same reaction conditions
and temperature. The reverse transcriptases of the invention
preferably produce from about 10% to about 200%, from about 25% to
about 200%, from about 50% to about 200%, from about 75% to about
200%, or from about 100% to about 200% more product or full length
product compared to a control reverse transcriptase under the same
reaction conditions and incubation temperature. The reverse
transcriptases of the invention preferably produce at least 2
times, at least 3 times, at least 4 times, at least 5 times, at
least 6 times, at least 7 times, at least 8 times, at least 9
times, at least 10 times, at least 25 times, at least 50 times, at
least 75 times, at least 100 times, at least 150 times, at least
200 times, at least 300 times, at least 400 times, at least 500
times, at least 1000 times, at least 5,000 times, or at least
10,000 times more product or full length product compared to a
control reverse transcriptase (e.g., the corresponding un-mutated
or unmodified reverse transcriptase) under the same reaction
conditions and temperature. The reverse transcriptases of the
invention preferably produce from 2 to 10,000, 5 to 10,000, 10 to
5,000, 50 to 5,000, 50 to 500, 2 to 500, 5 to 500, 5 to 200, 5 to
100, or 5 to 75 times more product or full length product than a
control reverse transcriptase under the same reaction conditions
and temperature.
[0074] In one aspect, the reverse transcriptases of the invention
produce, at 50.degree. C., at least 25% more, preferably at least
50% more and more preferably at least 100% more nucleic acid
product or full length product than a control reverse transcriptase
(which is preferably the corresponding wild-type reverse
transcriptase). In another aspect, at 52.5.degree. C., the reverse
transcriptases of the invention produce at least 1.5 times, at
least 2 times, at least 2.5 times, at least 3 times, at least 4
times, at least 5 times, at least 6 times, at least 7 times, at
least 8 times, at least 9 times, at least 10 times the amount of
nucleic acid product or full length product compared to a control
reverse transcriptase. In another aspect, at 55.degree. C., the
reverse transcriptases of the invention produce at least 2 times,
at least 5 times, at least 10 times, at least 15 times, at least 20
times, at least 25 times, at least 50 times, at least 75 times, at
least 100 times the amount of nucleic acid product or full length
product compared to a control reverse transcriptase. Such
comparisons are preferably made under the same reaction conditions
and temperature.
[0075] Modified or mutated reverse transcriptases of the present
invention may have an increased thermostability at elevated
temperatures as compared to corresponding unmodified or un-mutated
reverse transcriptases. They may show increased thermostability in
the presence or absence an RNA template. In some instances, reverse
transcriptases of the invention may show an increased
thermostability in both the presence and absence of an RNA
template. Those skilled in the art will appreciate that reverse
transcriptase enzymes are typically more thermostable in the
presence of an RNA template. The increase in thermostability may be
measured by comparing suitable parameters of the modified or
mutated reverse transcriptase of the invention to those of a
corresponding unmodified or un-mutated reverse transcriptase.
Suitable parameters to compare include, but are not limited to, the
amount of product and/or full length product synthesized by the
modified or mutated reverse transcriptase at an elevated
temperature compared to the amount or product and/or full length
product synthesized by the corresponding un-modified or un-mutated
reverse transcriptase at the same temperature, and/or the half-life
of reverse transcriptase activity at an elevated temperature of a
modified or mutated reverse transcriptase at an elevated
temperature compared to that of a corresponding unmodified or
un-mutated reverse transcriptase.
[0076] A modified or mutated reverse transcriptase of the invention
may have an increase in thermostability at 50.degree. C. of at
least about 1.5 fold (e.g., from about 1.5 fold to about 100 fold,
from about 1.5 fold to about 50 fold, from about 1.5 fold to about
25 fold, from about 1.5 fold to about 10 fold) compared, for
example, to the corresponding un-mutated or unmodified reverse
transcriptase. A reverse transcriptase of the invention may have an
increase in thermostability at 50.degree. C. of at least about 10
fold (e.g., from about 10 fold to about 100 fold, from about 10
fold to about 50 fold, from about 10 fold to about 25 fold, or from
about 10 fold to about 15 fold) compared, for example, to the
corresponding un-mutated or unmodified reverse transcriptase. A
reverse transcriptase of the invention may have an increase in
thermostability at 50.degree. C. of at least about 25 fold (e.g.,
from about 25 fold to about 100 fold, from about 25 fold to about
75 fold, from about 25 fold to about 50 fold, or from about 25 fold
to about 35 fold) compared to a corresponding un-mutated or
unmodified reverse transcriptase.
[0077] The present invention also contemplates a modified or
mutated thermostable reverse transcriptase, wherein the reverse
transcriptase has an increase in thermostability of greater than
about 1.5 fold at 52.5.degree. C. (e.g., from about 1.5 fold to
about 100 fold, from about 1.5 fold to about 50 fold, from about
1.5 fold to about 25 fold, or from about 1.5 fold to about 10 fold)
compared, for example, to the corresponding un-mutated or
unmodified reverse transcriptase. A reverse transcriptase of the
invention may have an increase in thermostability at 52.5.degree.
C. of at least about 10 fold (e.g., from about 10 fold to about 100
fold, from about 10 fold to about 50 fold, from about 10 fold to
about 25 fold, or from about 10 fold to about 15 fold) compared,
for example, to the corresponding un-mutated or unmodified reverse
transcriptase. A reverse transcriptase of the invention may have an
increase in thermostability at 52.5.degree. C. of at least about 25
fold (e.g., from about 25 fold to about 100 fold, from about 25
fold to about 75 fold, from about 25 fold to about 50 fold, or from
about 25 fold to about 35 fold) compared, for example, to the
corresponding un-mutated or unmodified reverse transcriptase.
[0078] In other embodiments, the present invention provides a
reverse transcriptase, wherein the reverse transcriptase has an
increase in thermostability of greater than about 1.5 fold at
55.degree. C. (e.g., from about 1.5 fold to about 100 fold, from
about 1.5 fold to about 50 fold, from about 1.5 fold to about 25
fold, or from about 1.5 fold to about 10 fold) compared to a
corresponding un-mutated or unmodified reverse transcriptase. In
some embodiments, a reverse transcriptase of the invention may have
an increase in thermostability at 55.degree. C. of at least about
10 fold (e.g., from about 10 fold to about 100 fold, from about 10
fold to about 50 fold, from about 10 fold to about 25 fold, or from
about 10 fold to about 15 fold) compared to a corresponding
un-mutated or unmodified reverse transcriptase. In some
embodiments, a reverse transcriptase of the invention may have an
increase in thermostability at 55.degree. C. of at least about 25
fold (e.g., from about 25 fold to about 100 fold, from about 25
fold to about 75 fold, from about 25 fold to about 50 fold, or from
about 25 fold to about 35 fold) compared to a corresponding
un-mutated or unmodified reverse transcriptase.
[0079] Nucleic acid templates suitable for reverse transcription
according to this aspect of the invention include any nucleic acid
molecule or population of nucleic acid molecules (preferably RNA
and most preferably mRNA), particularly those derived from a cell
or tissue. In a specific aspect, a population of mRNA molecules (a
number of different mRNA molecules, typically obtained from a
particular cell or tissue type) is used to make a cDNA library, in
accordance with the invention. Examples of cellular sources of
nucleic acid templates include bacterial cells, fungal cells, plant
cells and animal cells.
[0080] As used herein, a "C-terminal extension" refers to a peptide
tail of random sequence. A C-terminal extension is preferably from
1 to 500 amino acids, more preferably from 1 to 100, amino acids,
and most preferably from 2 to 50 amino acids.
[0081] As used herein, "random" means relating to an amino acid
sequence, wherein each amino acid of the sequence has an equal
probability of occurring.
[0082] As used herein, "RNase H activity" refers to
endoribonuclease degradation of the RNA of a DNA-RNA hybrid to
produce 5' phosphate terminated oligonucleotides that are 2-9 bases
in length. RNase H activity does not include degradation of
single-stranded nucleic acids, duplex DNA or double-stranded
RNA.
[0083] As used herein, the phrase "substantially lacks RNase H
activity" means having less than 10%, 5%, 1%, 0.5%, or 0.1% of the
activity of a wild type enzyme. The phrase "lacking RNase H
activity" means having undetectable RNase H activity or having less
than about 1%, 0.5%, or 0.1% of the RNase H activity of a wild type
enzyme.
[0084] An enzyme with "reduced" RNase H activity is meant that the
enzyme has less than 50%, e.g., less than 40%, 30%, or less than
25%, 20%, more preferably less than 15%, less than 10%, or less
than 7.5%, and most preferably less than 5% or less than 2%, of the
RNase H activity of the corresponding wild type enzyme containing
RNase H activity. The RNase H activity of an enzyme may be
determined by a variety of assays, such as those described, for
example, in U.S. Pat. Nos. 5,405,776; 6,063,608; 5,244,797; and
5,668,005 in Kotewicz, M. L., et al., Nucl. Acids Res. 16:265
(1988) and Gerard, G. F., et al., FOCUS 14(5):91 (1992), the
disclosures of all of which are fully incorporated herein by
reference.
[0085] As used herein, "processivity" refers to the ability of a
nucleic acid modifying enzyme, for example a reverse transcriptase,
to remain attached to the template or substrate and perform
multiple modification reactions. "Modification reactions" include
but are not limited to synthesis. "Processivity" also refers to the
ability of a nucleic acid modifying enzyme, for example a reverse
transcriptase, to perform a sequence of steps without intervening
dissociation of the enzyme from the growing DNA chains.
"Processivity" can depend on the nature of the nucleic-acid
modifying enzyme, the sequence of a nucleic acid template, and
reaction conditions, for example, salt concentration, temperature
or the presence of specific proteins.
[0086] As used herein, "increased processivity" refers to an
increase of 5-10%, preferably 10-50%, more preferably 50-100% or
more, as compared to a wild type reverse transcriptase.
Processivity and increased processivity can be measured as
described in Malboeuf et al., 2001, Biotechniques 30: 1074.
[0087] As used herein, "accuracy" refers to "fidelity", defined
hereinbelow. Accuracy or fidelity can be measured as described in
U.S. Patent Application Nos. 60/559,810 and Ser. No. 11,100,183,
incorporated by reference it their entirety herein.
[0088] As used herein, "specificity" refers to a decrease in the
amount of mispriming by the reverse transcriptase at the cDNA
synthesis level when the reaction is performed at higher
temperature, as compared to the amount of mispriming by a wild-type
reverse transcriptase performing under identical conditions.
Specificity can be measured as described in Mizuno Y, et al.,
Nucleic Acids Research, 1999, 27: 1345-1349.
[0089] As used herein, "decrease" refers to at least 1-fold or
more, for example, 1, 2, 3, 4, 5, 10, 20, 30, 40, 50, 60, 70, 80,
90, 100, 1000-fold or more, less than a wild-type enzyme performing
under identical conditions. "Decrease" also refers to at least 5%
or more (for example 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80,
90, 99, 100%) less than a wild-type enzyme performing under
identical conditions.
[0090] The term "fidelity," as used herein, refers to the accuracy
of nucleotide synthesis by reverse transcriptase or
template-dependent DNA polymerase, e.g., RNA-dependent or
DNA-dependent DNA polymerase. The fidelity of a DNA polymerase,
including a reverse transcriptase, is measured by the error rate
(the frequency of incorporating an inaccurate nucleotide, i.e., a
nucleotide that is not incorporated in a template-dependent
manner). The accuracy or fidelity of DNA polymerization is
maintained by both the polymerase activity and the 3'-5'
exonuclease activity. The term "high fidelity" refers to an error
rate equal to or lower than 33.times.10.sup.-6 per base pair (see
Roberts J. D. et al., Science, 1988, 242: 1171-1173, the entirety
hereby incorporated by reference). The fidelity or error rate of a
DNA polymerase may be measured using assays known to the art (see
for example, Lundburg et al., 1991 Gene, 108:1-6).
[0091] A reverse transcriptase having an "increased (or enhanced or
higher) fidelity" is defined as a mutant or modified reverse
transcriptase (including a DNA polymerase exhibiting reverse
transcriptase activity) having any increase in fidelity compared to
its wild type or unmodified form, i.e., a reduction in the number
of misincorporated nucleotides during synthesis of any given
nucleic acid molecule of a given length. Preferably there is 1.5 to
1,000 fold (more preferably 2 to 100 fold, more preferably 3 to 10
fold) reduction in the number of misincorporated nucleotides during
synthesis of any given nucleic acid molecule of a given length. For
example, a mutated reverse transcriptase may misincorporate one
nucleotide in the synthesis of a nucleic acid molecule segment of
1000 bases compared to an unmutated reverse transcriptase
misincorporating 10 nucleotides in the same size segment. Such a
mutant reverse transcriptase would be said to have a 10-fold
increase in fidelity.
[0092] As used herein, the terms "nucleic acid", "polynucleotide"
and "oligonucleotide" refer to primers, probes, and oligomer
fragments to be detected, and shall be generic to
polydeoxyribonucleotides (containing 2-deoxy-D-ribose), to
polyribonucleotides (containing D-ribose), and to any other type of
polynucleotide which is an N-glycoside of a purine or pyrimidine
base, or modified purine or pyrimidine bases (including abasic
sites). There is no intended distinction in length between the term
"nucleic acid", "polynucleotide" and "oligonucleotide", and these
terms will be used interchangeably. These terms refer only to the
primary structure of the molecule. Thus, these terms include
double- and single-stranded DNA, as well as double- and
single-stranded RNA.
[0093] As used herein, "nucleotide" refers to a
base-sugar-phosphate combination. Nucleotides are monomeric units
of a nucleic acid sequence (DNA and RNA) and deoxyribonucleotides
are "incorporated" into DNA by DNA polymerases. The term nucleotide
includes, but is not limited to, deoxyribonucleoside triphosphates
such as dATP, dCTP, dITP, dUTP, dGTP, dTTP, or derivatives thereof.
Such derivatives include, for example, [aS]dATP, 7-deaza-dGTP,
7-deaza-dATP, amino-allyl dNTPs, fluorescent labeled dNTPs
including Cy3, Cy5 labeled dNTPs. The term nucleotide as used
herein also refers to dideoxyribonucleoside triphosphates (ddNTPs
and acyclic nucleotides) and their derivatives (e.g., as described
in Martinez et al., 1999, Nucl. Acids Res. 27: 1271-1274, hereby
incorporated by reference in its entirety).
[0094] As used herein, a "primer" refers to a sequence of
deoxyribonucleotides or ribonucleotides comprising at least 3
nucleotides. Generally, the primer comprises from about 3 to about
100 nucleotides, preferably from about 5 to about 50 nucleotides
and even more preferably from about 5 to about 25 nucleotides. A
primer having less than 50 nucleotides may also be referred to
herein as an "oligonucleotide primer". The primers of the present
invention may be synthetically produced by, for example, the
stepwise addition of nucleotides or may be fragments, parts,
portions or extension products of other nucleotide acid molecules.
The term "primer" is used in its most general sense to include any
length of nucleotides which, when used for amplification purposes,
can provide a free 3' hydroxyl group for the initiation of DNA
synthesis by a DNA polymerase, either using an RNA or a DNA
template. DNA synthesis results in the extension of the primer to
produce a primer extension product complementary to the nucleic
acid strand to which the primer has hybridized.
[0095] As used herein, the term "homology" refers to the optimal
alignment of sequences (either nucleotides or amino acids), which
may be conducted by computerized implementations of algorithms.
"Homology", with regard to polynucleotides, for example, may be
determined by analysis with BLASTN version 2.0 using the default
parameters. "Homology", with respect to polypeptides (i.e., amino
acids), may be determined using a program, such as BLASTP version
2.2.2 with the default parameters, which aligns the polypeptides or
fragments being compared and determines the extent of amino acid
identity or similarity between them. It will be appreciated that
amino acid "homology" includes conservative substitutions, i.e.
those that substitute a given amino acid in a polypeptide by
another amino acid of similar characteristics. Typically seen as
conservative substitutions are the following replacements:
replacements of an aliphatic amino acid such as Ala, Val, Leu and
Ile with another aliphatic amino acid; replacement of a Ser with a
Thr or vice versa; replacement of an acidic residue such as Asp or
Glu with another acidic residue; replacement of a residue bearing
an amide group, such as Asn or Gln, with another residue bearing an
amide group; exchange of a basic residue such as Lys or Arg with
another basic residue; and replacement of an aromatic residue such
as Phe or Tyr with another aromatic residue. A polypeptide sequence
(i.e., amino acid sequence) or a polynucleotide sequence comprising
at least 50% homology to another amino acid sequence or another
nucleotide sequence respectively has a homology of 50% or greater
than 50%, e.g., 60%, 70%, 80%, 90% or 100% (i.e., identical).
[0096] The term "E. coli DNA polymerase III holoenzyme" refers to
an E. coli polymerase III holoenzyme composed of ten subunits
assembled in two catalytic cores, two sliding clamps and a clamp
loader, e.g., as described in Kelman, Z. & O'Donnell, M.
(1995). Annu. Rev. Biochem. 64, 171200 (the entirety is hereby
incorporated by reference).
[0097] The term "epsilon (c) subunit," according to the present
invention, refers to a c subunit having 3'-5' exonuclease activity.
An epsilon subunit may be from any eubacteria, such as from E.
coli, or from other organisms. The epsilon (c) subunit of the E.
coli DNA polymerase III holoenzyme is the 3'-5' exonuclease of the
holoenzyme and interacts with the a (polymerase unit) and .theta.
(unknown function) subunits (see, e.g., Fijalkowska et al., 1996,
Proc. Natl. Acad. Sci. USA, 93: 2856-2861, the entirety is hereby
incorporated by reference). The epsilon (c) subunit of E. coli DNA
polymerase III holoenzyme (see FIG. 9) is encoded by dnaQ gene (see
FIG. 9). The epsilon subunit of the present invention also includes
a wild type polypeptide which is at least 50% homologous (e.g.,
60%, 70%, 80%, 90%, or identical) to the sequences presented in
FIG. 9 and contains 3'-5' exonuclease activity. The epsilon (c)
subunit, according to the present invention, further includes a
mutant epsilon (c) subunit which still contains 3'-5' exonuclease
activity. Such mutant epsilon may contain a deletion (e.g.,
truncation), substitution, point mutation, mutation of multiple
amino acids, or insertion to the wild type epsilon subunit. For
example, a truncated epsilon useful according to the invention may
be, for example, as disclosed in Hamdan S. et al., Biochemistry
2002, 41: 5266-5275, the entirety hereby incorporated by
reference.
[0098] As used herein, "epsilon 186" refers to the N-terminal
domain of the epsilon subunit (codons 2-186 of dnaQ), as disclosed
in Hamdan et al., supra.
[0099] The term ".theta..epsilon. subunit complex" or
".theta..epsilon. 186 subunit complex" refers to the combination of
the epsilon subunit or a mutant epsilon subunit of E. coli DNA
polymerase III holoenzyme (for example epsilon 186) in combination
with the .theta. subunit of E. coli DNA polymerase III
holoenzyme.
[0100] As used herein, the term "eubacteria" refers to unicelled
organisms which are prokaryotes (e.g., as described in Garrity, et
al., 2001, Taxonomic outline of the procaryotic genera. Bergey's
Manual.RTM. of Systematic Bacteriology, Second Edition. Release
1.0, April 2001, and in Werren, 1997, Annual Review of Entomology
42: 587-609). Eubacteria include the following genera: Escherichia,
Pseudomonas, Proteus, Micrococcus, Acinetobacter, Klebsiella,
Legionella, Neisseria, Bordetella, Vibrio, Staphylococcus,
Lactobaccilus, Streptococcus, Bacillus, Corynebacteria,
Mycobacteria, Clostridium, and others (see Kandler, O., Zbl. Bakt.
Hyg., I. Abt. Orig. C3, 149-160 (1982)), as well as major
sub-groups of eubacteria such as Aquifex (extremely thermophilic
chemolithotrophs), Thermotoga (extremely thermophilic
chemoorganotrophs), Chloroflexus (thermophilic photosynthetic
bacteria), Deinococcus (radiation resistant bacteria), Thermus
(thermophilic chemoheterotrophs), Spirochaetes (helical bacteria
with periplasmic flagella), Proteobacteria (Gram-negative and
purple photosynthetic bacteria), Cyanobacteria (blue-green
photosynthetic bacteria), Gram-positives (Gram-positive bacteria),
Bacteroides/Flavobacterium (strict anaerobes/strict aerobes with
gliding motility), Chlorobium (photoautotrophic sulphur-oxidisers),
Planctomyces (budding bacteria with no peptidoglycan), Chlamydia
(intracellular parasites). Eubacteria include all thermostable
bacteria.
[0101] As used herein, "synthesis" refers to any in vitro method
for making a new strand of polynucleotide or elongating existing
polynucleotide (i.e., DNA or RNA) in a template dependent manner.
Synthesis, according to the invention, may include amplification,
which increases the number of copies of a polynucleotide template
sequence with the use of a polymerase. Polynucleotide synthesis
results in the incorporation of nucleotides into a polynucleotide
(i.e., a primer), thereby forming a new polynucleotide molecule
complementary to the polynucleotide template. The formed
polynucleotide molecule and its template can be used as templates
to synthesize additional polynucleotide molecules.
[0102] As used herein, an "amplified product" refers to the single-
or double-strand polynucleotide population at the end of an
amplification reaction. The amplified product contains the original
polynucleotide template and polynucleotide synthesized by DNA
polymerase using the polynucleotide template during the
amplification reaction. An amplified product preferably is produced
by a reverse transcriptase and/or a DNA polymerase.
[0103] As used herein, "polynucleotide template" or "target
polynucleotide template" refers to a polynucleotide (RNA or DNA)
which serves as a template for a DNA polymerase to synthesize DNA
in a template-dependent manner. The "amplified region," as used
herein, is a region of a polynucleotide that is to be either
synthesized by reverse transcription or amplified by polymerase
chain reaction (PCR). For example, an amplified region of a
polynucleotide template may reside between two sequences to which
two PCR primers are complementary to.
[0104] As used herein, the term "template dependent manner" refers
to a process that involves the template dependent extension of a
primer molecule (e.g., DNA synthesis by DNA polymerase). "Template
dependent manner" refers to polynucleotide synthesis of RNA or DNA
wherein the sequence of the newly synthesized strand of
polynucleotide is dictated by the well-known rules of complementary
base pairing (see, for example, Watson, J. D. et al., In: Molecular
Biology of the Gene, 4th Ed., W. A. Benjamin, Inc., Menlo Park,
Calif. (1987)).
[0105] As used herein, the term "polymerase chain reaction" ("PCR")
refers to the method of K. B. Mullis, e.g., as described in U.S.
Pat. No. 4,683,195 4,683,202, and 4,965,188 (each hereby
incorporated in its entirety by reference) and any other improved
method known in the art. PCR is a method for increasing the
concentration of a segment of a target sequence in a mixture of
genomic DNA without cloning or purification. This process for
amplifying the target sequence typically consists of introducing a
large excess of two oligonucleotide primers to the DNA mixture
containing the desired target sequence, followed by a precise
sequence of thermal cycling in the presence of a DNA polymerase.
The two primers are complementary to their respective strands of
the double stranded target sequence. To effect amplification, the
mixture is denatured and the primers then annealed to their
complementary sequences within the target molecule. Following
annealing, the primers are extended with a polymerase so as to form
a new pair of complementary strands. The steps of denaturation,
primer annealing and polymerase extension can be repeated many
times (i.e., denaturation, annealing and extension constitute one
"cycle"; there can be numerous "cycles") to obtain a high
concentration of an amplified segment of the desired target
sequence. The length of the amplified segment of the desired target
sequence is determined by the relative positions of the primers
with respect to each other, and therefore, this length is a
controllable parameter. By virtue of the repeating aspect of the
process, the method is referred to as the "polymerase chain
reaction" (hereinafter "PCR"). Because the desired amplified
segments of the target sequence become the predominant sequences
(in terms of concentration) in the mixture, they are said to be
"PCR amplified".
[0106] As used herein, the term "RT-PCR" refers to the replication
and amplification of RNA sequences. In this method, reverse
transcription is coupled to PCR, e.g., as described in U.S. Pat.
No. 5,322,770, herein incorporated by reference in its entirety. In
RT-PCR, the RNA template is converted to cDNA due to the reverse
transcriptase activity of an enzyme, and then amplified using the
polymerizing activity of the same or a different enzyme. Stable,
thermostable or thermolabile reverse transcriptase and polymerase
can be used.
[0107] Amino acid residues identified herein are preferred in the
natural L-configuration. In keeping with standard polypeptide
nomenclature, J. Biol. Chem., 243:3557-3559, 1969, abbreviations
for amino acid residues are as shown in the following Table 1.
TABLE-US-00001 TABLE 1 1-Letter 3-Letter AMINO ACID Y Tyr
L-tyrosine G Gly glycine F Phe L-phenylalanine M Met L-methionine A
Ala L-alanine S Ser L-serine I Ile L-isoleucine L Leu L-leucine T
Thr L-threonine V Val L-valine P Pro L-proline K Lys L-lysine H His
L-histidine Q Gln L-glutamine E Glu L-glutamic acid W Trp
L-tryptophan R Arg L-arginine D Asp L-aspartic acid N Asn
L-asparagine C Cys L-cysteine
[0108] A "double tube RT-PCR" or "two step RT-PCR" refers to a
reaction wherein the RT step is performed in a first tube, and then
the cDNA is transferred to a second tube for amplification.
Therefore, the cDNA synthesis and PCR occur in two separate tubes.
A "single tube RT-PCR" or "one step RT-PCR" refers to a reaction
wherein both cDNA synthesis and PCR are performed in the same
tube.
[0109] As used herein, "end-point RT-PCR" refers to RT-PCR wherein
a template is added at the beginning of a PCR reaction and the
reaction is carried out in multiple cycles, usually 20 to 50
cycles. It is the end product of the amplification reaction which
is detected and/or quantitated.
[0110] As used herein, "real time RT-PCR" or "quantitative" or
"QRT-PCR" refers to an RT-PCR process wherein the progress of an
RT-PCR amplification is measured or detected as it is occurring. In
real-time RT-PCR techniques, signals (generally fluorescent) are
monitored as they are generated and are tracked after they rise
above background but before the reaction reaches a plateau.
[0111] As used herein, the term "real-time" refers to that which is
performed contemporaneously with the monitored, measured or
observed events and which yields as a result of the monitoring,
measurement or observation to one who performs it simultaneously,
or effectively so, with the occurrence of a monitored, measured or
observed event. Thus, a "real time" assay or measurement contains
not only the measured and quantitated result, such as generated
signal, but expresses this in real time, that is, in hours,
minutes, seconds, milliseconds, nanoseconds, picoseconds, etc.
[0112] As used herein, "microarray" refers to a plurality of
nucleic acid members stably associated with a substrate. The term
"array" is used interchangeably with the term "microarray,"
however, the term "microarray" is used to define an array which has
the additional property of being viewable microscopically.
[0113] As used herein, "viewable microscopically" refers to an
object which can be placed on the stage of a dissecting or compound
microscope and comprises at least a portion which can be viewed
using an ocular of the microscope.
[0114] As used herein, "stably associated" refers to an association
with a position on a substrate that does not change under nucleic
acid hybridization and washing conditions.
BRIEF DESCRIPTION OF THE DRAWINGS
[0115] FIG. 1 presents the results of an MMLV-RT thermostability
screen.
[0116] FIG. 2 presents the thermostability of His-tagged purified
MMLV-RT point mutants.
[0117] FIG. 3 presents the thermostability of C-terminally extended
mutants. HSRRRLKRHIFN=SEQ ID NO:64; SKRTNPINIHTNK=SEQ ID NO:65;
QEGKNRQGEGQT=SEQ ID NO:66; RDRNKNNDRRKAKENE=SEQ ID NO:1;
RDRNKNNDRRKAKRDRNKNNDRRKAK=SEQ ID NO:67;
RDRNKNNDRRKAKENEENEENEENEENE=SEQ ID NO:68.
[0118] FIG. 4 presents the results of an activity assay for an RT
comprising multiple mutations. RFGK=SEQ ID NO:37; RKFGK=SEQ ID
NO:36.
[0119] FIG. 5 presents cDNA ladder synthesis by His-tagged RTs.
RKFGK=SEQ ID NO:36.
[0120] FIG. 6 presents the thermostability of RTs of the invention.
RDRNKNNDRRKAKENE=SEQ ID NO:1.
[0121] FIGS. 7A, 7B, and 7C represent the half-life of mutant RTs
according to the invention.
[0122] FIGS. 8A, 8B, 8C, 8D, 8E, 8F, 8G, 8H, 8I, 8J, 8K, 8L, 8M,
8N, 80, 8P, 8Q, 8R, 8S, 8T, 8U, 8V, 8W, 8X, 8Y, 8Z, 8AA, 8BB, 8CC,
8DD, 8EE, 8FF, 8GG, and 8HH present the nucleic acid and amino acid
sequences of the mutant RTs according to the invention. Optional
codons are represented by XXX in FIGS. 8B, 8C, 8D, 8E, 8F, 8G, 8H,
8I, 8J, 8K, 8L, 8M, 8N, 80, 8P, and 8Q. Possible nucleic acids
sequences for XXX are presented above the XXX. Mutant amino acids
are circled in FIGS. 8S, 8T, 8U, 8V, 8W, 8X, 8Y, 8Z, 8AA, 8BB, 8CC,
8DD, 8EE, 8FF, 8GG, and 8HH.
[0123] FIG. 8A. WT-MMLV-RT nucleic acid sequence (SEQ ID NO: 2)
[0124] FIG. 8B. MMLV-RT E69K nucleic acid sequence (SEQ ID NO:
3)
[0125] FIG. 8C. MMLV-RT E302K nucleic acid sequence (SEQ ID NO:
4)
[0126] FIG. 8D. MMLV-RT E302R nucleic acid sequence (SEQ ID NO:
5)
[0127] FIG. 8E. MMLV-RT W313F nucleic acid sequence (SEQ ID NO:
6)
[0128] FIG. 8F. MMLV-RT L435G nucleic acid sequence (SEQ ID NO:
7)
[0129] FIG. 8G. MMLV-RT L435M nucleic acid sequence (SEQ ID NO:
8)
[0130] FIG. 8H. MMLV-RT N454K nucleic acid sequence (SEQ ID NO:
9)
[0131] FIG. 8I. MMLV-RT N454R nucleic acid sequence (SEQ ED NO:
10)
[0132] FIG. 8J. MMLV-RT D524N nucleic acid sequence (SEQ ID NO:
11)
[0133] FIG. 8K. MMLV-RT M651L nucleic acid sequence (SEQ ID NO:
12)
[0134] FIG. 8L. MMLV-RT E302R/E69K/W313F/L435G/N454K nucleic acid
sequence (SEQ ID NO: 13)
[0135] FIG. 8M. MMLV-RT E302R/W313F/L435G/N454K nucleic acid
sequence (SEQ ID NO: 14)
[0136] FIG. 8N. MMLV-RT E302R/W313F/L435G nucleic acid sequence
(SEQ ID NO: 15)
[0137] FIG. 8O. MMLV-RT E302R/E69K/N454K nucleic acid sequence (SEQ
ID NO: 16)
[0138] FIG. 8P. MMLV-RT E302R/W313F nucleic acid sequence (SEQ ID
NO: 17)
[0139] FIG. 8Q. MMLV-RT E302R/E69K/W313F/L435G/N454K/D524N nucleic
acid sequence (SEQ ID NO: 18)
[0140] FIG. 8R. WT-MMLV-RT protein sequence (SEQ ID NO: 19)
[0141] FIG. 8S. MMLV-RT E69K protein sequence (SEQ ID NO: 20)
[0142] FIG. 8T. MMLV-RT E302K protein sequence (SEQ ID NO: 21)
[0143] FIG. 8U. MMLV-RT E302R protein sequence (SEQ ID NO: 22)
[0144] FIG. 8V. MMLV-RT W313F protein sequence (SEQ ID NO: 23)
[0145] FIG. 8W. MMLV-RT L435G protein sequence (SEQ ID NO: 24)
[0146] FIG. 8X. MMLV-RT L435M protein sequence (SEQ ID NO: 25)
[0147] FIG. 8Y. MMLV-RT N454K protein sequence (SEQ ID NO: 26)
[0148] FIG. 8Z. MMLV-RT N454R protein sequence (SEQ ID NO: 27)
[0149] FIG. 8AA. MMLV-RT D524N protein sequence (SEQ ID NO: 28)
[0150] FIG. 8BB. MMLV-RT M651L protein sequence (SEQ ID NO: 29)
[0151] FIG. 8CC. MMLV-RT E302R/E69K/W313F/L435G/N454K protein
sequence (SEQ ID NO: 30)
[0152] FIG. 8DD. MMLV-RT E302R/W313F/L435G/N454K protein sequence
(SEQ ID NO: 31)
[0153] FIG. 8EE. MMLV-RT E302R/W313F/L435G protein sequence (SEQ ID
NO: 32)
[0154] FIG. 8FF. MMLV-RT E302R/E69K/N454K protein sequence (SEQ ID
NO: 33)
[0155] FIG. 8GG. MMLV-RT E302R/W313F protein sequence (SEQ ID NO:
34)
[0156] FIG. 8HH. MMLV-RT E302R/E69K/W313F/L435G/N454K/D524N protein
sequence (SEQ ID NO: 35)
[0157] FIG. 9A and FIG. 9B present the sequences of the epsilon
subunit (c) of E. coli DNA polymerase III holoenzyme (FIG. 9A) and
the dnaQ gene (FIG. 9B).
DETAILED DESCRIPTION
[0158] The invention relates to mutant reverse transcriptases
(RTs). In one embodiment the mutant RTs exhibit increased
stability, for example thermostability, as compared to a wild-type
enzyme. The mutant RTs of the invention are useful for cDNA
synthesis, cloning, production of cDNA libraries or microarrays and
RT-PCR.
I. REVERSE TRANSCRIPTASES
[0159] One common approach to the study of gene expression is the
production of complementary DNA (cDNA). Discovery of an
RNA-dependent DNA polymerase, a so-called reverse transcriptase
(RT), from a retrovirus has enabled a reverse transcription
reaction in which a cDNA is synthesized using an RNA as a template.
As a result of identifying RT, methods for analyzing mRNA molecules
have made rapid progress. The methods for analyzing mRNA molecules
using reverse transcriptase have now become indispensable
experimental methods for studying gene expression and function.
Subsequently, these methods, which have been applied to cloning and
PCR techniques, have also become indispensable techniques in a wide
variety of fields including biology, medicine and agriculture.
[0160] The invention relates to a reverse transcriptase (RT)
selected from the group consisting of: Moloney Murine Leukemia
Virus (M-MLV) RT, Human Immunodeficiency Virus (HIV) RT, Avian
Sarcoma-Leukosis Virus (ASLV) RT, Rous Sarcoma Virus (RSV) RT,
Avian Myeloblastosis Virus (AMV) RT, Avian Erythroblastosis Virus
(AEV) Helper Virus MCAV RT, Avian Myelocytomatosis Virus MC29
Helper Virus MCAV RT, Avian Reticuloendotheliosis Virus (REV-T)
Helper Virus REV-A RT, Avian Sarcoma Virus UR2 Helper Virus UR2AV
RT, Avian Sarcoma Virus Y73 Helper Virus YAV RT, Rous Associated
Virus (RAV) RT, and Myeloblastosis Associated Virus (MAV) RT.
[0161] Enzymes for use in the compositions, methods and kits of the
present invention include any enzyme having reverse transcriptase
activity. Such enzymes include, but are not limited to, retroviral
reverse transcriptase, retrotransposon reverse transcriptase,
hepatitis B reverse transcriptase, cauliflower mosaic virus reverse
transcriptase, E. coli DNA polymerase and klenow fragment, Tth DNA
polymerase, Taq DNA polymerase (Saiki, R. K., et al., Science
239:487-491 (1988); U.S. Pat. Nos. 4,889,818 and 4,965,188), Tne
DNA polymerase (WO 96/10640), Tma DNA polymerase (U.S. Pat. No.
5,374,553), C. Therm DNA polymerase from Carboxydothermus
hydrogenoformans (EP0921196A1, Roche, Pleasanton, Calif., Cat. No.
2016338), ThermoScript (Invitrogen, Carsbad, Calif. Cat. No.
11731-015) and mutants, fragments, variants or derivatives thereof.
As will be understood by one of ordinary skill in the art, modified
reverse transcriptases may be obtained by recombinant or genetic
engineering techniques that are routine and well-known in the art.
Mutant reverse transcriptases can, for example, be obtained by
mutating the gene or genes encoding the reverse transcriptase of
interest by site-directed or random mutagenesis. Such mutations may
include point mutations, deletion mutations and insertional
mutations. Preferably, one or more point mutations (e.g.,
substitution of one or more amino acids with one or more different
amino acids) are used to construct mutant reverse transcriptases of
the invention. Fragments of reverse transcriptases may be obtained
by deletion mutation by recombinant techniques that are routine and
well-known in the art, or by enzymatic digestion of the reverse
transcriptase(s) of interest using any of a number of well-known
proteolytic enzymes. Mutant DNA polymerases containing reverse
transcriptase activity, for example, as described in U.S. patent
application Ser. No. 10/435,766, incorporated by reference in its
entirety, are also useful according to the invention.
[0162] Polypeptides having reverse transcriptase activity that may
be advantageously used in the present methods include, but are not
limited to, Moloney Murine Leukemia Virus (M-MLV) reverse
transcriptase, Rous Sarcoma Virus (RSV) reverse transcriptase,
Avian Myeloblastosis Virus (AMV) reverse transcriptase,
Rous-Associated Virus (RAV) reverse transcriptase, Myeloblastosis
Associated Virus (MAV) reverse transcriptase, Human
Immunodeficiency Virus (HIV) reverse transcriptase, Avian
Sarcoma-Leukosis Virus (ASLV) reverse transcriptase, retroviral
reverse transcriptase, retrotransposon reverse transcriptase,
hepatitis B reverse transcriptase, cauliflower mosaic virus reverse
transcriptase, Thermus thermophilus (Tth) DNA polymerase, Thermus
aquaticus (Taq) DNA polymerase, Thermotoga neopolitana (Tne) DNA
polymerase, Thermotoga maritima (Tma) DNA polymerase, Thermococcus
litoralis (Tli or VENT.RTM.) DNA polymerase, Pyrococcus furiosus
(Pfu) DNA polymerase, DEEPVENT.TM.. Pyrococcus species GB-D DNA
polymerase, Pyrococcus woesi (Pwo) DNA polymerase, Bacillus
sterothermophilus (Bst) DNA polymerase, Bacillus caldophilus (Bca)
DNA polymerase, Sulfoloblus acidocaldarius (Sac) DNA polymerase,
Thermoplasma acidophilum (Tac) DNA polymerase, Thermus flavus
(Tfl/Tub) DNA polymerase, Thermus ruber (Tru) DNA polymerase,
Thermus brockianus (DYNAZYME.TM.) DNA polymerase, Methanobacterium
thermoautotrophicum (Mth) DNA polymerase, and mutants, variants and
derivatives thereof. The invention also encompasses bacterial DNA
polymerases comprising residual reverse transcriptase activity,
such as Taq DNA polymerase (for a description see, for example,
Shadilya et al., 2004 Extremophiles, 8:243).
[0163] Particularly preferred for use in the invention are the
variants of these enzymes that are reduced in RNase H activity
(i.e., RNase H-enzymes). Preferably, the enzyme has less than 20%,
more preferably less than 15%, 10% or 5%, and most preferably less
than 2%, of the RNase H activity of a wildtype or "RNase H.sup.+"
enzyme such as wildtype M-MLV reverse transcriptase. The RNase H
activity of any enzyme may be determined by a variety of assays,
such as those described, for example, in U.S. Pat. Nos. 5,244,797;
5,405,776; 5,668,005; and 6,063,608; in Kotewicz, M. L., et al.,
Nucl. Acids Res. 16:265 (1988) and in Gerard, G. F., et al., FOCUS
14(5):91 (1992), the disclosures of all of which are fully
incorporated herein by reference.
[0164] Particularly preferred RNase H-reverse transcriptase enzymes
for use in the invention include, but are not limited to, M-MLV
H-reverse transcriptase, RSV H-reverse transcriptase, AMV H-reverse
transcriptase, RAV H-reverse transcriptase, MAV H-reverse
transcriptase and HIV H-reverse transcriptase for example as
previously described (see U.S. Pat. Nos. 5,244,797; 5,405,776;
5,668,005 and 6,063,608; and WO 98/47912, the entirety of each is
incorporated by reference). The RNase H activity of any enzyme may
be determined by a variety of assays, such as those described, for
example, in U.S. Pat. Nos. 5,244,797; 5,405,776; 5,668,005 and
6,063,608; in Kotewicz, M. L., et al., Nucl. Acids Res. 16:265
(1988); and in Gerard, G. F., et al., FOCUS 14(5):91 (1992), the
disclosures of all of which are fully incorporated herein by
reference. It will be understood by one of ordinary skill, however,
that any enzyme capable of producing a DNA molecule from a
ribonucleic acid molecule (i.e., having reverse transcriptase
activity) that is substantially reduced in RNase H activity may be
equivalently used in the compositions, methods and kits of the
invention.
[0165] Polypeptides having reverse transcriptase activity for use
in the invention may be obtained commercially, for example, from
Invitrogen, Inc. (Carlsbad, Calif.), Pharmacia (Piscataway, N.J.),
Sigma (Saint Louis, Mo.) or Roche Molecular System (Pleasanton,
Calif.). Alternatively, polypeptides having reverse transcriptase
activity may be isolated from their natural viral or bacterial
sources according to standard procedures for isolating and
purifying natural proteins that are well-known to one of ordinary
skill in the art (see, e.g., Houts, G. E., et al., J. Virol. 29:517
(1979)). In addition, the polypeptides having reverse transcriptase
activity may be prepared by recombinant DNA techniques that are
familiar to one of ordinary skill in the art (see, e.g., Kotewicz,
M. L., et al., Nucl. Acids Res. 16:265 (1988); Soltis, D. A., and
Skalka, A. M., Proc. Natl. Acad. Sci. USA 85:3372-3376 (1988)). The
entire teaching of the above references is hereby incorporated by
reference.
[0166] Enzymes that are reduced in RNase H activity may be obtained
by methods known in the art, e.g., by mutating the RNase H domain
within the reverse transcriptase of interest, preferably by one or
more point mutations, one or more deletion mutations, and/or one or
more insertion mutations as described above, e.g., as described in
U.S. Pat. No. 6,063,608 hereby incorporated in its entirety by
reference.
[0167] Two or more enzymes with reverse transcriptase activity may
be used in a single composition, e.g., the same reaction mixture.
Enzymes used in this fashion may have distinct reverse
transcription pause sites with respect to the template nucleic
acid, as described in U.S. Patent Application 2003/0198944A1,
hereby incorporated in its entirety by reference.
[0168] The enzyme containing reverse transcriptase activity of the
present invention may also include a mutant or modified reverse
transcriptase where one or more amino acid changes have been made
which renders the enzyme more faithful (higher fidelity) in nucleic
acid synthesis, e.g., as described in U.S. Patent Application
2003/0003452A1, hereby incorporated in its entirety by
reference.
[0169] Epsilon Subunits
[0170] The invention provide for a reverse transcriptase of the
invention in combination with a complex comprising the .theta.
subunit of E. coli DNA polymerase III and the epsilon subunit of E.
coli DNA polymerase III (e.g., see Hamdan et al., 2002,
Biochemistry, 41:5266-5275). The .theta. subunit may also be used
with any other mutant form of the epsilon subunit, for example the
epsilon 186 truncated version of the epsilon subunit, to increase
stability of the enzyme and/or to improve the accuracy, specificity
and or processivity of the reverse transcriptases.
[0171] In one embodiment of the invention, a mutant reverse
transcriptase is provided in combination with the .theta. epsilon
subunit complex. Alternatively, a mutant reverse transcriptase is
provided in combination with a complex comprising .theta. and a
mutant form of the epsilon subunit, for example .epsilon.186
[0172] Denaturing Agents and Organic Solvents
[0173] The invention also provides for a reverse transcriptase in
combination with a denaturing agent or organic solvent including
but not limited to formamide and DMSO.
[0174] The invention also provides for a reverse transcriptase in
combination with a PCR enhancing factor, for example, betaine.
II. GENETIC MODIFICATIONS
Mutagenesis
[0175] The preferred method of preparing a mutant reverse
transcriptase is by genetic modification (e.g., by modifying the
DNA sequence of a wild-type reverse transcriptase). A number of
methods are known in the art that permit the random as well as
targeted mutation of DNA sequences (see for example, Ausubel et.
al. Short Protocols in Molecular Biology (1995) 3rd Ed. John Wiley
& Sons, Inc.). In addition, there are a number of commercially
available kits for site-directed mutagenesis, including both
conventional and PCR-based methods. Examples include the GeneMorph
Random mutagenesis kit (Stratagene Catalog No. 600550 or 200550),
EXSITE.TM. PCR-Based Site-directed Mutagenesis Kit available from
Stratagene (Catalog No. 200502) and the QUIKCHANGE.TM.
Site-directed mutagenesis Kit from Stratagene (Catalog No. 200518),
and the CHAMELEON.RTM. double-stranded Site-directed mutagenesis
kit, also from Stratagene (Catalog No. 200509).
[0176] In addition mutant reverse transcriptases may be generated
by insertional mutation or truncation (N-terminal, internal or
C-terminal) according to methodology known to one skilled in the
art.
[0177] Older methods of site-directed mutagenesis known in the art
rely on sub-cloning of the sequence to be mutated into a vector,
such as an M13 bacteriophage vector, that allows the isolation of
single-stranded DNA template. In these methods, one anneals a
mutagenic primer (i.e., a primer capable of annealing to the site
to be mutated but bearing one or more mismatched nucleotides at the
site to be mutated) to the single-stranded template and then
polymerizes the complement of the template starting from the 3' end
of the mutagenic primer. The resulting duplexes are then
transformed into host bacteria and plaques are screened for the
desired mutation.
[0178] More recently, site-directed mutagenesis has employed PCR
methodologies, which have the advantage of not requiring a
single-stranded template. In addition, methods have been developed
that do not require sub-cloning. Several issues must be considered
when PCR-based site-directed mutagenesis is performed. First, in
these methods it is desirable to reduce the number of PCR cycles to
prevent expansion of undesired mutations introduced by the
polymerase. Second, a selection must be employed in order to reduce
the number of non-mutated parental molecules persisting in the
reaction. Third, an extended-length PCR method is preferred in
order to allow the use of a single PCR primer set. And fourth,
because of the non-template-dependent terminal extension activity
of some thermostable polymerases it is often necessary to
incorporate an end-polishing step into the procedure prior to
blunt-end ligation of the PCR-generated mutant product.
[0179] Non-limiting examples for the isolation of mutant reverse
transcriptases useful according to the invention are described in
detail in Examples 1 and 2.
[0180] Methods of random mutagenesis, which will result in a panel
of mutants bearing one or more randomly situated mutations, exist
in the art. Such a panel of mutants may then be screened for those
exhibiting the desired properties, for example, increased
stability, relative to a wild-type reverse transcriptase. An
example of a method for random mutagenesis is the so-called
"error-prone PCR method". As the name implies, the method amplifies
a given sequence under conditions in which the DNA polymerase does
not support high fidelity incorporation. Although the conditions
encouraging error-prone incorporation for different DNA polymerases
vary, one skilled in the art may determine such conditions for a
given enzyme. A key variable for many DNA polymerases in the
fidelity of amplification is, for example, the type and
concentration of divalent metal ion in the buffer. The use of
manganese ion and/or variation of the magnesium or manganese ion
concentration may therefore be applied to influence the error rate
of the polymerase.
[0181] Genes for desired mutant reverse transcriptases generated by
mutagenesis may be sequenced to identify the sites and number of
mutations. For those mutants comprising more than one mutation, the
effect of a given mutation may be evaluated by introduction of the
identified mutation to the wild-type gene by site-directed
mutagenesis in isolation from the other mutations borne by the
particular mutant. Screening assays of the single mutant thus
produced will then allow the determination of the effect of that
mutation alone.
[0182] The amino acid and DNA coding sequence of wild-type
MMLV-reverse transcriptase are shown in FIG. 8. Non-limiting
detailed procedures for preparing a mutant MMLV-reverse
transcriptase useful according to the invention are provided in
Examples 1 and 2.
[0183] A person of average skill in the art having the benefit of
this disclosure will recognize that mutant reverse transcriptases
polymerases derived from other reverse transcriptases, including
but not limited to Moloney Murine Leukemia Virus (M-MLV); Human
Immunodeficiency Virus (HIV) reverse transcriptase and avian
Sarcoma-Leukosis Virus (ASLV) reverse transcriptase, which includes
but is not limited to Rous Sarcoma Virus (RSV) reverse
transcriptase, Avian Myeloblastosis Virus (AMV) reverse
transcriptase, Avian Erythroblastosis Virus (AEV) Helper Virus MCAV
reverse transcriptase, Avian Myelocytomatosis Virus MC29 Helper
Virus MCAV reverse transcriptase, Avian Reticuloendotheliosis Virus
(REV-T) Helper Virus REV-A reverse transcriptase, Avian Sarcoma
Virus UR2 Helper Virus UR2AV reverse transcriptase, Avian Sarcoma
Virus Y73 Helper Virus YAV reverse transcriptase, Rous Associated
Virus (RAV) reverse transcriptase, and Myeloblastosis Associated
Virus (MAV) reverse transcriptase may be suitably used in the
subject compositions.
[0184] The enzyme of the subject composition may comprise reverse
transcriptases that have not yet been isolated.
[0185] A method employing the addition of peptide tails with random
sequences to the C-terminus of Bacillus stearothermophilus Catalase
I, in an attempt to increase enzyme thermostability has been
described (Matsuura et al., 1999 Nature Biotechnology 17:58). The
invention contemplates mutant reverse transcriptases comprising a
C-terminal extension.
[0186] As used herein, a "C-terminal extension" refers to a peptide
tail of random sequence. A C-terminal extension is preferably from
1 to 500 amino acids, more preferably from 1 to 100 amino acids,
and most preferably from 2 to 50 amino acids.
III. METHODS OF EVALUATING MUTANTS FOR INCREASED
THERMOSTABILITY
[0187] Random or site-directed mutants generated as known in the
art or as described herein and expressed in bacteria may be
screened for RT activity and increased stability of RT activity by
several different assays. Preferably, an RT enzyme is screened in
an RT thermostability screen as described in Example 3,
hereinbelow.
IV. EXPRESSION OF WILD-TYPE OR MUTANT ENZYMES ACCORDING TO THE
INVENTION
[0188] Methods known in the art may be applied to express and
isolate the mutated forms of reverse transcriptase according to the
invention. The methods described here can be also applied for the
expression of wild-type enzymes useful in the invention. Many
bacterial
[0189] expression vectors contain sequence elements or combinations
of sequence elements allowing high level inducible expression of
the protein encoded by a foreign sequence. For example, bacteria
expressing an integrated inducible form of the T7 RNA polymerase
gene may be transformed with an expression vector bearing a mutated
DNA polymerase gene linked to the T7 promoter. Induction of the T7
RNA polymerase by addition of an appropriate inducer, for example,
isopropyl-.beta.-D-thiogalactopyranoside (IPTG) for a lac-inducible
promoter, induces the high level expression of the mutated gene
from the T7 promoter.
[0190] Appropriate host strains of bacteria may be selected from
those available in the art by one of skill in the art. As a
non-limiting example, E. coli strain BL-21 is commonly used for
expression of exogenous proteins since it is protease deficient
relative to other strains of E. coli. BL-21 strains bearing an
inducible T7 RNA polymerase gene include WJ56 and ER2566 (Gardner
& Jack, 1999, supra). For situations in which codon usage for
the particular reverse transcriptase gene differs from that
normally seen in E. coli genes, there are strains of BL-21 that are
modified to carry tRNA genes encoding tRNAs with rarer anticodons
(for example, argU, ileY, leuW, and proL tRNA genes), allowing high
efficiency expression of cloned protein genes, for example, cloned
archaeal enzyme genes (several BL21-CODON PLUS.TM. cell strains
carrying rare-codon tRNAs are available from Stratagene, for
example).
V. APPLICATIONS OF THE SUBJECT INVENTION
[0191] cDNA Synthesis
[0192] In accordance with the invention, cDNA molecules
(single-stranded or double-stranded) may be prepared from a variety
of nucleic acid template molecules. Preferred nucleic acid
molecules for use in the present invention include single-stranded
or double-stranded DNA and RNA molecules, as well as
double-stranded DNA:RNA hybrids. More preferred nucleic acid
molecules include messenger RNA (mRNA), transfer RNA (tRNA) and
ribosomal RNA (rRNA) molecules, although mRNA molecules are the
preferred template according to the invention.
[0193] The invention provides compositions and methods for cDNA
synthesis with increased specificity and accuracy. The present
invention provides compositions and methods for high fidelity cDNA
synthesis. The subject compositions and methods may also increase
the efficiency of the reverse transcription as well as the length
of the cDNA synthesized. As a result, the fidelity, efficiency, and
yield of subsequent manipulations of the synthesized cDNA (e.g.,
amplification, sequencing, cloning, etc.) are also increased. The
nucleic acid molecules that are used to prepare cDNA molecules
according to the methods of the present invention may be prepared
synthetically according to standard organic chemical synthesis
methods that will be familiar to one of ordinary skill. More
preferably, the nucleic acid molecules may be obtained from natural
sources, such as a variety of cells, tissues, organs or organisms.
Cells that may be used as sources of nucleic acid molecules may be
prokaryotic (bacterial cells, including but not limited to those of
species of the genera Escherichia, Bacillus, Serratia, Salmonella,
Staphylococcus, Streptococcus, Clostridium, Chlamydia, Neisseria,
Treponema, Mycoplasma, Borrelia, Legionella, Pseudomonas,
Mycobacterium, Helicobacter, Erwinia, Agrobacterium, Rhizobium,
Xanthomonas and Streptomyces) or eukaryotic (including fungi
(especially yeasts), plants, protozoans and other parasites, and
animals including insects (particularly Drosophila spp. cells),
nematodes (particularly Caenorhabditis elegans cells), and mammals
(particularly human cells)).
[0194] Mammalian somatic cells that may be used as sources of
nucleic acids include blood cells (reticulocytes and leukocytes),
endothelial cells, epithelial cells, neuronal cells (from the
central or peripheral nervous systems), muscle cells (including
myocytes and myoblasts from skeletal, smooth or cardiac muscle),
connective tissue cells (including fibroblasts, adipocytes,
chondrocytes, chondroblasts, osteocytes and osteoblasts) and other
stromal cells (e.g., macrophages, dendritic cells, Schwann cells).
Mammalian germ cells (spermatocytes and oocytes) may also be used
as sources of nucleic acids for use in the invention, as may the
progenitors, precursors and stem cells that give rise to the above
somatic and germ cells. Also suitable for use as nucleic acid
sources are mammalian tissues or organs such as those derived from
brain, kidney, liver, pancreas, blood, bone marrow, muscle,
nervous, skin, genitourinary, circulatory, lymphoid,
gastrointestinal and connective tissue sources, as well as those
derived from a mammalian (including human) embryo or fetus.
[0195] Any of the above prokaryotic or eukaryotic cells, tissues
and organs may be normal, diseased, transformed, established,
progenitors, precursors, fetal or embryonic. Diseased cells may,
for example, include those involved in infectious diseases (caused
by bacteria, fungi or yeast, viruses (including AIDS, HIV, HTLV,
herpes, hepatitis and the like) or parasites), in genetic or
biochemical pathologies (e.g., cystic fibrosis, hemophilia,
Alzheimer's disease, muscular dystrophy or multiple sclerosis) or
in cancerous processes. Transformed or established animal cell
lines may include, for example, COS cells, CHO cells, VERO cells,
BHK cells, HeLa cells, HepG2 cells, K562 cells, 293 cells, L929
cells, F9 cells, and the like. Other cells, cell lines, tissues,
organs and organisms suitable as sources of nucleic acids for use
in the present invention will be apparent to one of ordinary skill
in the art.
[0196] Once the starting cells, tissues, organs or other samples
are obtained, nucleic acid molecules (such as mRNA) may be isolated
therefrom by methods that are well-known in the art (See, e.g.,
Maniatis, T., et al., Cell 15:687-701 (1978); Okayama, H., and
Berg, P., Mol. Cell. Biol. 2:161-170 (1982); Gubler, U., and
Hoffman, B. J., Gene 25:263-269 (1983)). The nucleic acid molecules
thus isolated may then be used to prepare cDNA molecules and cDNA
libraries in accordance with the present invention.
[0197] In the practice of the invention, cDNA molecules or cDNA
libraries may be produced by mixing one or more nucleic acid
molecules obtained as described above, which is preferably one or
more mRNA molecules such as a population of mRNA molecules, with
the composition of the invention, under conditions favoring the
reverse transcription of the nucleic acid molecule by the action of
the enzymes of the compositions to form a cDNA molecule
(single-stranded or double-stranded). Thus, the method of the
invention comprises (a) mixing one or more nucleic acid templates
(preferably one or more RNA or mRNA templates, such as a population
of mRNA molecules) with a mutant RT of the invention and (b)
incubating the mixture under conditions sufficient to permit cDNA
synthesis, e.g., to all or a portion of the one or more
templates.
[0198] The compositions of the present invention may be used in
conjunction with methods of cDNA synthesis such as those described
in the Examples below, or others that are well-known in the art
(see, e.g., Gubler, U., and Hoffman, B. J., Gene 25:263-269 (1983);
Krug, M. S., and Berger, S. L., Meth. Enzymol. 152:316-325 (1987);
Sambrook, J., et al., Molecular Cloning: A Laboratory Manual, 2nd
ed., Cold Spring Harbor, N.Y.: Cold Spring Harbor Laboratory Press,
pp. 8.60-8.63 (1989)), to produce cDNA molecules or libraries.
[0199] The invention is directed to such methods which further
produce a first strand and a second strand cDNA, as known in the
art. According to the invention, the first and second strand cDNAs
produced by the methods may form a double stranded DNA molecule
which may be a full length cDNA molecule.
[0200] Other methods of cDNA synthesis which may advantageously use
the present invention will be readily apparent to one of ordinary
skill in the art.
[0201] Subsequent Manipulation of Synthesized cDNA
[0202] Having obtained cDNA molecules or libraries according to the
present methods, these cDNAs may be isolated or the reaction
mixture containing the cDNAs may be directly used for further
analysis or manipulation. Detailed methodologies for purification
of cDNAs are taught in the GENETRAPPER.TM. manual (Invitrogen, Inc.
Carlsbad, Calif.), which is incorporated herein by reference in its
entirety, although alternative standard techniques of cDNA
isolation such as those described in the Examples below or others
that are known in the art (see, e.g., Sambrook, J., et al.,
Molecular Cloning: A Laboratory Manual, 2nd ed., Cold Spring
Harbor, N.Y.: Cold Spring Harbor Laboratory Press, pp. 8.60-8.63
(1989)) may also be used.
[0203] In other aspects of the invention, the invention may be used
in methods for amplifying nucleic acid molecules. Nucleic acid
amplification methods according to this aspect of the invention may
be one-step (e.g., one-step RT-PCR) or two-step (e.g., two-step
RT-PCR) reactions. According to the invention, one-step RT-PCR type
reactions may be accomplished in one tube thereby lowering the
possibility of contamination. Such one-step reactions comprise (a)
mixing a nucleic acid template (e.g., mRNA) with an enzyme of the
present invention and (b) incubating the mixture under conditions
sufficient to permit amplification. Two-step RT-PCR reactions may
be accomplished in two separate steps. Such a method comprises (a)
mixing a nucleic acid template (e.g., mRNA) with an enzyme of the
present invention, (b) incubating the mixture under conditions
sufficient to permit cDNA synthesis, (c) mixing the reaction
mixture in (b) with one or more DNA polymerases and (d) incubating
the mixture of step (c) under conditions sufficient to permit
amplification. For amplification of long nucleic acid molecules
(i.e., greater than about 3-5 Kb in length), a combination of DNA
polymerases may be used, such as one DNA polymerase having 3'-5'
exonuclease activity and another DNA polymerase being reduced in
3'-5' exonuclease activity.
[0204] Amplification methods which may be used in accordance with
the present invention include PCR (e.g., U.S. Pat. Nos. 4,683,195
and 4,683,202), Strand Displacement Amplification (SDA; e.g., U.S.
Pat. No. 5,455,166; EP 0 684 315), and Nucleic Acid Sequence-Based
Amplification (NASBA; e.g., U.S. Pat. No. 5,409,818; EP 0 329 822).
In a particularly preferred aspects, the invention may be used in
methods of amplifying nucleic acid molecule comprising one or more
polymerase chain reactions (PCRs), such as any of the PCR-based
methods described above. All references are entirely incorporated
by reference.
[0205] Various specific PCR amplification applications are
available in the art (for reviews, see for example, Erlich, 1999,
Rev Immunogenet., 1:127-34; Prediger 2001, Methods Mol. Biol.
160:49-63; Jurecic et al., 2000, Curr. Opin. Microbiol. 3:316-21;
Triglia, 2000, Methods Mol. Biol. 130:79-83; MaClelland et al.,
1994, PCR Methods Appl. 4:S66-81; Abramson and Myers, 1993, Current
Opinion in Biotechnology 4:41-47; each of which is incorporated
herein by references).
[0206] The subject invention can be used in PCR applications
including, but not limited to, i) hot-start PCR which reduces
non-specific amplification; ii) touch-down PCR which starts at high
annealing temperature, then decreases annealing temperature in
steps to reduce non-specific PCR product; iii) nested PCR which
synthesizes more reliable product using an outer set of primers and
an inner set of primers; iv) inverse PCR for amplification of
regions flanking a known sequence. In this method, DNA is digested,
the desired fragment is circularized by ligation, then PCR using
primer complementary to the known sequence extending outwards; v)
AP-PCR (arbitrary primed)/RAPD (random amplified polymorphic DNA).
These methods create genomic fingerprints from species with
little-known target sequences by amplifying using arbitrary
oligonucleotides; vi) RT-PCR which uses RNA-directed DNA polymerase
(e.g., reverse transcriptase) to synthesize cDNAs which is then
used for PCR. This method is extremely sensitive for detecting the
expression of a specific sequence in a tissue or cells. It may also
be used to quantify mRNA transcripts; vii) RACE (rapid
amplification of cDNA ends). This is used where information about
DNA/protein sequence is limited. The method amplifies 3' or 5' ends
of cDNAs generating fragments of cDNA with only one specific primer
each (plus one adaptor primer). Overlapping RACE products can then
be combined to produce full length cDNA; viii) DD-PCR (differential
display PCR) which is used to identify differentially expressed
genes in different tissues. The first step in DD-PCR involves
RT-PCR, then amplification is performed using short, intentionally
nonspecific primers; ix) Multiplex-PCR in which two or more unique
targets of DNA sequences in the same specimen are amplified
simultaneously. One DNA sequence can be used as control to verify
the quality of PCR; x) Q/C-PCR (Quantitative comparative) which
uses an internal control DNA sequence (but of different size) which
competes with the target DNA (competitive PCR) for the same set of
primers; xi) Recusive PCR which is used to synthesize genes.
Oligonucleotides used in this method are complementary to stretches
of a gene (>80 bases), alternately to the sense and to the
antisense strands with ends overlapping (.about.20 bases); xii)
Asymmetric PCR; xiii) In Situ PCR; xiv) Site-directed PCR
Mutagenesis.
[0207] It should be understood that this invention is not limited
to any particular amplification system. As other systems are
developed, those systems may benefit by practice of this
invention.
[0208] The primer used for synthesizing a cDNA from an RNA as a
template in the present invention is not limited to a specific one
as long as it is an oligonucleotide that has a nucleotide sequence
complementary to that of the template RNA and that can anneal to
the template RNA under reaction conditions used. The primer may be
an oligonucleotide such as an oligo(dT) or an oligonucleotide
having a random sequence (a random primer) or a gene-specific
primer.
[0209] The nucleic acid molecules (e.g., synthesized cDNA or
amplified product) or cDNA libraries prepared by the methods of the
present invention may be further characterized, for example by
cloning and sequencing (i.e., determining the nucleotide sequence
of the nucleic acid molecule), by the sequencing methods of the
invention or by others that are standard in the art (see, e.g.,
U.S. Pat. Nos. 4,962,022 and 5,498,523, which are directed to
methods of DNA sequencing). Alternatively, these nucleic acid
molecules may be used for the manufacture of various materials in
industrial processes, such as hybridization probes by methods that
are well-known in the art. Production of hybridization probes from
cDNAs will, for example, provide the ability for those in the
medical field to examine a patient's cells or tissues for the
presence of a particular genetic marker such as a marker of cancer,
of an infectious or genetic disease, or a marker of embryonic
development. Furthermore, such hybridization probes can be used to
isolate DNA fragments from genomic DNA or cDNA libraries prepared
from a different cell, tissue or organism for further
characterization.
[0210] It is understood that the amplified product produced using
the subject enzyme can be cloned by any method known in the art. In
one embodiment, the invention provides a composition which allows
direct cloning of PCR amplified product.
[0211] The most common method for cloning PCR products involves
incorporation of flanking restriction sites onto the ends of primer
molecules. The PCR cycling is carried out and the amplified DNA is
then purified, restricted with an appropriate endonuclease(s) and
ligated to a compatible vector preparation.
[0212] A method for directly cloning PCR products eliminates the
need for preparing primers having restriction recognition sequences
and it would eliminate the need for a restriction step to prepare
the PCR product for cloning. Additionally, such method would
preferably allow cloning PCR products directly without an
intervening purification step.
[0213] U.S. Pat. Nos. 5,827,657 and 5,487,993 (hereby incorporated
by their entirety) disclose methods for direct cloning of PCR
products using a DNA polymerase which takes advantage of the single
3'-deoxy-adenosine monophosphate (dAMP) residues attached to the 3'
termini of PCR generated nucleic acids. Vectors are prepared with
recognition sequences that afford single 3'-terminal
deoxy-thymidine monophosphate (dTMP) residues upon reaction with a
suitable restriction enzyme. Thus, PCR generated copies of genes
can be directly cloned into the vectors without need for preparing
primers having suitable restriction sites therein.
[0214] Taq DNA polymerase exhibits terminal transferase activity
that adds a single dATP to the 3' ends of PCR products in the
absence of template. This activity is the basis for the TA cloning
method in which PCR products amplified with Taq are directly
ligated into vectors containing single 3'dT overhangs. Pfu DNA
polymerase, on the other hand, lacks terminal transferase activity,
and thus produces blunt-ended PCR products that are efficiently
cloned into blunt-ended vectors.
[0215] In one embodiment, the invention provides for a PCR product,
generated in the presence of a mutant DNA polymerase with reduced
uracil detection activity, that is subsequently incubated with Taq
DNA polymerase in the presence of dATP at 72.degree. C. for 15-30
minutes. Addition of 3'-dAMP to the ends of the amplified DNA
product then permits cloning into TA cloning vectors according to
methods that are well known to a person skilled in the art.
[0216] The nucleic acid molecules (e.g., synthesized cDNA or
amplified product) of the present invention may also be used to
prepare compositions for use in recombinant DNA methodologies.
Accordingly, the present invention relates to recombinant vectors
which comprise the cDNA or amplified nucleic acid molecules of the
present invention, to host cells which are genetically engineered
with the recombinant vectors, to methods for the production of a
recombinant polypeptide using these vectors and host cells, and to
recombinant polypeptides produced using these methods.
[0217] Recombinant vectors may be produced according to this aspect
of the invention by inserting, using methods that are well-known in
the art, one or more of the cDNA molecules or amplified nucleic
acid molecules prepared according to the present methods into a
vector. The vector used in this aspect of the invention may be, for
example, a phage or a plasmid, and is preferably a plasmid.
Preferred are vectors comprising cis-acting control regions to the
nucleic acid encoding the polypeptide of interest. Appropriate
trans-acting factors may be supplied by the host, supplied by a
complementing vector or supplied by the vector itself upon
introduction into the host.
[0218] In certain preferred embodiments in this regard, the vectors
provide for specific expression (and are therefore termed
"expression vectors"), which may be inducible and/or cell
type-specific. Particularly preferred among such vectors are those
inducible by environmental factors that are easy to manipulate,
such as temperature and nutrient additives.
[0219] Expression vectors useful in the present invention include
chromosomal-, episomal- and virus-derived vectors, e.g., vectors
derived from bacterial plasmids or bacteriophages, and vectors
derived from combinations thereof, such as cosmids and phagemids,
and will preferably include at least one selectable marker such as
a tetracycline or ampicillin resistance gene for culturing in a
bacterial host cell. Prior to insertion into such an expression
vector, the cDNA or amplified nucleic acid molecules of the
invention should be operatively linked to an appropriate promoter,
such as the phage lambda P.sub.L promoter, the E. coli lac, trp and
tac promoters. Other suitable promoters will be known to the
skilled artisan.
[0220] Among vectors preferred for use in the present invention
include pQE70, pQE60 and pQE-9, available from Qiagen; pBS vectors,
Phagescript vectors, Bluescript vectors, pNH8A, pNH16a, pNH18A,
pNH46A, available from Stratagene; pcDNA3 available from
Invitrogen; pGEX, pTrxfus, pTrc99a, pET-5, pET-9, pKK223-3,
pKK233-3, pDR540, pRIT5 available from Pharmacia; and pSPORT1,
pSPORT2 and pSV.multidot.SPORT1, available from Life Technologies,
Inc. Other suitable vectors will be readily apparent to the skilled
artisan.
[0221] The invention also provides methods of producing a
recombinant host cell comprising the cDNA molecules, amplified
nucleic acid molecules or recombinant vectors of the invention, as
well as host cells produced by such methods. Representative host
cells (prokaryotic or eukaryotic) that may be produced according to
the invention include, but are not limited to, bacterial cells,
yeast cells, plant cells and animal cells. Preferred bacterial host
cells include Escherichia coli cells (most particularly E. coli
strains DH1OB and Stb12, which are available commercially (Life
Technologies, Inc; Rockville, Md.)), Bacillus subtilis cells,
Bacillus megaterium cells, Streptomyces spp. cells, Erwinia spp.
cells, Klebsiella spp. cells and Salmonella typhimurium cells.
Preferred animal host cells include insect cells (most particularly
Spodoptera frugiperda 5.19 and Sf21 cells and Trichoplusa High-Five
cells) and mammalian cells (most particularly CHO, COS, VERO, BHK
and human cells). Such host cells may be prepared by well-known
transformation, electroporation or transfection techniques that
will be familiar to one of ordinary skill in the art.
[0222] In addition, the invention provides methods for producing a
recombinant polypeptide, and polypeptides produced by these
methods. According to this aspect of the invention, a recombinant
polypeptide may be produced by culturing any of the above
recombinant host cells under conditions favoring production of a
polypeptide therefrom, and isolation of the polypeptide. Methods
for culturing recombinant host cells, and for production and
isolation of polypeptides therefrom, are well-known to one of
ordinary skill in the art.
[0223] It will be readily apparent to one of ordinary skill in the
relevant arts that other suitable modifications and adaptations to
the methods and applications described herein are obvious and may
be made without departing from the scope of the invention or any
embodiment thereof. Having now described the present invention in
detail, the same will be more clearly understood by reference to
the following examples, which are included herewith for purposes of
illustration only and are not intended to be limiting of the
invention.
VI. Kits
[0224] The present compositions may be assembled into kits for use
in reverse transcription, cloning or amplification of a nucleic
acid molecule. Kits according to this aspect of the invention
comprise a carrier means, such as a box, carton, tube or the like,
having in close confinement therein one or more container means,
such as vials, tubes, ampules, bottles and the like. The kits of
the invention may also comprise (in the same or separate
containers) one or more reverse transcriptases, a suitable buffer,
one or more nucleotides and/or one or more primers or any other
reagents described for compositions of the present invention.
[0225] The kit of the present invention may include reagents
facilitating the subsequent manipulation of cDNA synthesized as
known in the art.
VII. EXAMPLES
Example 1
Generating RNase H Minus MMLV-RT Point Mutant Library for
Thermostability Screen
[0226] RNase H minus MMLV-RT (D524N) gene (2 kb) was mutagenized
using the GeneMorph Random Mutagenesis Kit (Stratagene Catalog
#200550 or 600550) and primers pSTRAT-F and pSTRAT-R (Table 2)
according to the manufacturer's recommendations. Mutated PCR
products "Mega primers" were used to replace the wild type RNase H
minus MMLV-RT gene using the QuikChange Site-directed Mutagenesis
Kit (Stratagene Catalog #200518) according to the manufacturer's
recommendations. The resulting plasmids were cloned into XL-10 Gold
competent cells (Stratagene Catalog #200317). The library size was
5.times.10.sup.4 (containing 1-6 mutations/kb). DNA was extracted
from the entire library using StrataPrep Plasmid Miniprep Kit
(Stratagene Catalog #400761). A portion of the DNA was then
transformed into BL21-DE3-RIL cells (Stratagene Catalog #230240) to
generate a library with a size of 5.times.10.sup.4.
[0227] Results: The clones in this library contained 1-6
mutations/kb.
Example 2
Generating RNase H Minus MMLV-RT Random C-Terminal Extension
Library for Thermostability Screen
[0228] Primers RTSSC12AXhoI and RTSSEI-vecF (Table 2) were used to
amplify RNase H minus MMLV-RT gene using Herculase DNA polymerase
(Stratagene Catalog #600260). The PCR products were then digested
with EcoRI and Xhol and cloned into pCal-n-FLAG (Stratagene Catalog
#214311) that is missing the Calmodulin binding unit and the FLAG
sequence. The resulting C-terminal extension library was cloned
into XL-10 Gold competent cells (Stratagene Catalog #200317). DNA
was extracted from the entire library using StrataPrep Plasmid
Miniprep Kit (Stratagene Catalog #400761) and transformed into
BL21-DE3-RIL cells (Stratagene Catalog #230240).
[0229] Results:
[0230] The library size was 10.sup.4. From 17 clones sequenced, 12
had 7-14 amino acid additions, 2 had 1-2 amino acid additions, 1
had 18 amino acid additions, 1 had 30 amino acid additions, and one
had no additions.
Example 3
RT Thermostability Screen Assay
[0231] Mutant colonies from the BL21-DE3-RIL libraries (both point
mutant and C-terminal extension libraries) were inoculated into 120
.mu.I LB media containing 100 .mu.g/ml Ampicillin and 35 .mu.g/ml
Chloramphenicol (Costar 96 well plate (29444-102)) and grown over
night at 37.degree. C. 10 .mu.I of these cultures were inoculated
into 110 .mu.l LB media containing 100 .mu.g/ml Ampicillin, 35
.mu.g/ml Chloramphenicol, and 1 mM IPTG (Costar 96 well plate
(29444-102)) and grown over night. Cells were lysed using 30 .mu.l
lysis buffer (125 mM Tris pH 8, 4.5% glucose, 50 mM EDTA, 2.5%
Triton, 5 mg/ml lysozyme, and 50 mM DTT). 10 .mu.l of lysates were
used in a 50 .mu.l assay containing 50 mM Tris pH 8.3, 75 ml KCl, 8
mM MgCl.sub.2, 2 .mu.g poly(rC), 0.5 .mu.g oligo(dG), 10 mM DTT, 50
mM dGTP, and 0.5 .mu.Ci .alpha..sup.33pdGTP. Reactions were
incubated at 42.degree. C. or 55.degree. C. for 60 minutes (FIG.
1). 4 .mu.l of these reactions were spotted on DE-81 filters, and
dried. The filters were then washed 5 times with 2.times.SSC and
dried. The filters were then exposed to Kodak BioMax MR-1 films
(VWR IB8941114)) over night.
[0232] Results:
[0233] 3400 clones from the point mutation library were screened
using the thermostability assay described above. The mutants that
showed higher activity at 55.degree. C. compared to the WT enzyme
(FIG. 1) were selected and re-screened using the same RT activity
assay three more times. The best mutants were selected, sequenced,
and His-tag purified (as in example 6). Mutations E69K, L435M,
N454K, and M651L were discovered and their RT activity at
52.degree. C./42.degree. C. (as in example 4) were compared to the
WT enzyme (FIG. 2). All His-tagged purified mutants showed higher
activity at 52.degree. C./42.degree. C. compared to the WT
enzyme.
[0234] 4000 clones from the C-terminal extension library were also
screened using the thermostability assay described above. The
mutants that showed higher activity at 55.degree. C. compared to
the WT enzyme were selected and re-screened using the same RT
activity assay three more times. The best mutants were selected,
sequenced, and His-tag purified (as in example 6). Multiple peptide
tails increased the activity of RT at 52.degree. C./42.degree. C.
(assayed as in example 4) compared to the WT enzyme (FIG. 3).
Example 4
RT Activity Assay
[0235] The RNA dependent DNA polymerization assays for His-tagged
purified WT and mutants were performed as follows. .about.5 units
of each enzyme (equivalent amount of protein on a SDS-PAGE gel)
were used in a 50 .mu.l assay containing 50 mM Tris pH 8.3, 75 mM
KCl, 8 mM MgCl.sub.2, 2 .mu.g poly(rC), 0.5 .mu.g oligo(dG), 10 mM
DTT, 50 mM dGTP, and 0.5 .mu.Ci .alpha..sup.33pdGTP. Reactions were
incubated at 42.degree. C. or 52.degree. C. for 30 minutes. 5 .mu.l
of these reactions were spotted on DE-81 filters, and dried. The
filters were then washed 5 times with 2.times.SSC, followed by a
brief wash with 100% ethanol. The filters were then dried.
Incorporated radioactivity was measured by scintillation counting.
Reactions that lacked enzyme were set up along with sample
incubations to determine "total cpms" (omit filter wash steps) and
"minimum cpms" (wash filters as above). Minimum cpms were
subtracted from sample cpms to determine "corrected cpms".
Example 5
Saturation Mutagenesis at Putative "Thermostability" Residues to
Identify Best Mutation at Each Site Independently
[0236] Saturation mutagenesis was performed using QuikChange
Site-directed Mutagenesis Kit (Stratagene Catalog #200518) and
primers containing degenerate site (NNG/T) at E69, E302, F303,
G305, W313, L435, N454, M651 (Table 2) according to the
manufacturer's recommendations. 200 clones from every library were
screened (as in example 3). The mutants with the highest activity
at 55.degree. C. were selected, and sequenced.
[0237] Results:
[0238] The following mutations show the highest activity at
55.degree. C.:
[0239] E69K, E302K, E302R, W313F, L435M, L435G, N454K, N454R,
M651L
Example 6
Combination of Thermostable Mutations
[0240] The QuikChange Multi Site-directed Mutagenesis Kit
(Stratagene Catalog #200514) with four primers (Table 2) was used
to introduce the mutations E69K, W313F, L435G, and N454K into an
RNase H minus MMLV-RT gene that already contained the E302R
mutation.
[0241] Ten clones were sequenced.
Results: The following combinations were obtained:
TABLE-US-00002 Clone 1: E302R/E69K/W313F/L435G/ RKFGK N454K (SEQ ID
NO: 36) Clone 2: E302R/W313F/L435G/N454K RFGK (SEQ ID NO: 37) Clone
3: E302R/W313F/L435G RFG Clone 4: E302R/E69K/N454K RKK Clone 5:
E302R/W313F RF
[0242] Activity assays (as in example 3--using DE-81 filters and
poly(rC):oligo(dG).sub.18) (SEQ ID NO:62) were performed at
42.degree. C. and 57.degree. C. and the results (FIG. 4) indicate
higher activity at 57.degree. C. for clones containing single or
multiple mutations as compared to the wild type enzyme.
Example 7
Activity Assay Using Poly(A) RNA Ladder
[0243] Full length cDNA profiling was performed for WT RT versus
RKFGK (SEQ ID NO:36) mutant RT (His-tagged proteins) using a
poly(A)-tailed RNA ladder (Ambion #7150). Reactions contained 2
.mu.g RNA ladder, 0.5 .mu.s oligo(dT).sub.18 (SEQ ID NO:63), 3.2 mM
dNTPs and .about.100 units of enzyme (equivalent protein amount on
a SDS-PAGE gel) in 1.times. StrataScript buffer containing 3 or 6
mM Mg.sup.2+. Reactions were incubated at 42.degree. C., 50.degree.
C., and 52.degree. C. for 60 minutes, run on a 1% alkaline agarose
gel and stained with SYBR Gold.
[0244] Results:
[0245] RKFGK (SEQ ID NO: 36) mutant RT generates longer cDNA
ladders at higher temperature (52.degree. C.) compared to the WT
enzyme (FIG. 5).
Example 8
Purification and Thermostability Comparison of Final Constructs
[0246] Three His tagged constructs including RNase H minus MMLV-RT
(D524N), RNase H minus MMLV-RT (D524N, E302R, E69K, W313F, L435G,
N454K), and RNase H minus MMLV-RT (D524N, E302R, E69K, W313F,
L435G, N454K) plus the C-terminal extension (RDRNKNNDRRKAKENE) (SEQ
ID NO: 1) were expressed and purified according to the
QIAexpressionist (Qiagen). An RT activity assay using
Poly(rC):poly(dG) was performed similar to as in Example 4.
[0247] Results: The RNase H minus MMLV-RT (D524N, E302R, E69K,
W313F, L435G, N454K) with the C-terminal extension
(RDRNKNNDRRKAKENE) (SEQ ID NO: 1) shows the highest activity at
55.degree. C. and 60.degree. C. (FIG. 6).
Example 9
Half Life Determination
[0248] Half-lives of mutant reverse transcriptase enzymes of the
invention were determined as follows.
[0249] Three non-His tagged constructs including RNase H minus
MMLV-RT (D524N)
[0250] (FIG. 7A, plot 1), RNase H minus MMLV-RT (D524N, E302R,
E69K, W313F, L435G, N454K) (FIG. 7B, plot 2), and RNase H minus
MMLV-RT(D524N, E302R, E69K, W313F, L435G, N454K) plus the
C-terminal extension (RDRNKNNDRRKAKENE) (SEQ ID NO: 1) (FIG. 7C,
plot 3) were assayed as follows. Mixtures containing 0.5 pmol of
each enzyme in the presence of 2 .mu.g poly(rC), 0.5 .mu.g
oligo(dG).sub.18 (SEQ ID NO:62) were incubated at 55.degree. C. for
various times as indicated in the plots. Incubation was stopped by
placing the tubes on ice. An aliquot was assayed for residual
activity in 50 mM Tris pH 8.3, 75 mM KCl, 3 mM MgCl.sub.2, 10 mM
DTT, 50 mM dGTP, and 0.5 .mu.Ci .alpha..sup.33pdGTP. Reactions were
incubated at 42.degree. C. for 30 minutes. 5 .mu.l of these
reactions were spotted on DE-81 filters, and dried. The filters
were then washed 5 times with 2.times.SSC, followed by a brief wash
with 100% ethanol. The filters were then dried. Incorporated
radioactivity was measured by scintillation counting. Reactions
that lacked enzyme were set up along with sample incubations to
determine "total cpms" (omit filter wash steps) and "minimum cpms"
(wash filters as above). Minimum cpms were subtracted from sample
cpms to determine "corrected cpms".
Results:
[0251] The half life of RNase H minus MMLV-RT (D524N) (FIG. 7A,
plot 1) is <5 minutes where the half life of RNase H minus
MMLV-RT (D524N, E302R, E69K, W313F, L435G, N454K) (FIG. 7B, plot 2)
is >30 minutes, and the half life of RNase H minus MMLV-RT
(D524N, E302R, E69K, W313F, L435G, N454K) plus the C-terminal
extension (RDRNKNNDRRKAKENE) (SEQ ID NO: 1) (FIG. 7C, plot 3) if
.about.30 minutes at 55.degree. C.
TABLE-US-00003 TABLE 1 Primer sequences pSTRAT-F:
5'-ACCCTAAATATAGAAGATGAGCATCG (SEQ ID NO: 38) pSTRAT-R:
5'-GAGGAGGGTAGAGGTGTCTGGAGTC (SEQ ID NO: 39) RTSSC12AXhoI:
5'-CTTGGCCAAGGATCCGCTCGAGCTACTTACTTAN
NNNNNNNNNNNNNNNNNNNNGAGGAGGGTAGAGGTGT C (SEQ ID NO. 40)
RTSSEI-vecF: 5'-AGCGGATAACAATTCCCCTCTAGAATTCGA (SEQ ID NO: 41)
pE69X-F: 5'-CCCATGTCACAANNKGCCAGACTGGG K = G or T (SEQ ID NO: 42)
pE302X-F: 5'-GACAACTAAGGNNKTTCCTAGGGACG (SEQ ID NO: 43) pF303X-F:
5'-CAACTAAGGGAGNNKCTAGGGACGGC (SEQ ID NO: 44) pG305X-F:
5'-GGAGTTCCTANNKACGGCAGGCTTC (SEQ ID NO: 45) pW313X-F:
5'-TCTGTCGCCTCNNKATCCCTGGGTTTG (SEQ ED NO: 46) pL435X-F:
5'-CCACTAGTCATTNNKGCCCCCCATGCAG (SEQ ID NO: 47) pN454X-F:
5'-GCTGGCTTTCCNNKGCCCGGATGACTC (SEQ ID NO: 48) pM651X-F
5'-GAGGCAACCGGNNKGCTGACCAAGCG (SEQ ID NO: 49) pE69X-R:
5'-CCCAGTCTGGCMNNTTGTGACATGGG M = A or C (SEQ ID NO: 50) pE302X-R:
5'-CGTCCCTAGGAAMNNCCTTAGTTGTC (SEQ ID NO: 51) pF303X-R:
5'-GCCGTCCCTAGMNNCTCCCTTAGTTG (SEQ ID NO: 52) pG305X-R:
5'-GAAGCCTGCCGTMNNTAGGAACTCC (SEQ ID NO: 53) pW313X-R: 5
CAAACCCAGGGATMNNGAGGCGACAGA (SEQ ID NO: 54) pL435X-R:
5'-CTGCATGGGGGGCMNNAATGACTAGTGG (SEQ ID NO: 55) pN454X-R:
5'-GAGTCATCCGGGCMNNGGAAAGCCAGC (SEQ ID NO: 56) pM651X-R: 5
CGCTTGGTCAGCMNNCCGGTTGCCTC (SEQ ID NO: 57) pE69K:
5'-TACCCCATGTCACAAAAAGCCAGACTGGGGATCA AG (SEQ ID NO: 58) pW313F:
5'-GGCTTCTGTCGCCTCTTTATCCCTGGGTTTGC (SEQ ID NO: 59) pL435G:
5'-CAGCCACTAGTCATTGGGGCCCCCCATGCAGTAG (SEQ ID NO: 60) pN454K:
5'-GACCGCTGGCTTTCCAAGGCCCGGATGACTCAC (SEQ ID NO: 61)
[0252] All patents, patent applications, and published references
cited herein are hereby incorporated by reference in their
entirety. While this invention has been particularly shown and
described with references to preferred embodiments thereof, it will
be understood by those skilled in the art that various changes in
form and details may be made therein without departing from the
scope of the invention encompassed by the appended claims.
Sequence CWU 1
1
70116PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 1Arg Asp Arg Asn Lys Asn Asn Asp Arg Arg Lys Ala
Lys Glu Asn Glu 1 5 10 15 22013DNAMoloney murine leukemia virus
2accctaaata tagaagatga gcatcggcta catgagacct caaaagagcc agatgtttct
60ctagggtcca catggctgtc tgattttcct caggcctggg cggaaaccgg gggcatggga
120ctggcagttc gccaagctcc tctgatcata cctctgaaag caacctctac
ccccgtgtcc 180ataaaacaat accccatgtc acaagaagcc agactgggga
tcaagcccca catacagaga 240ctgttggacc agggaatact ggtaccctgc
cagtccccct ggaacacgcc cctgctaccc 300gttaagaaac cagggactaa
tgattatagg cctgtccagg atctgagaga agtcaacaag 360cgggtggaag
acatccaccc caccgtgccc aacccttaca acctcttgag cgggctccca
420ccgtcccacc agtggtacac tgtgcttgat ttaaaggatg cctttttctg
cctgagactc 480caccccacca gtcagcctct cttcgccttt gagtggagag
atccagagat gggaatctca 540ggacaattga cctggaccag actcccacag
ggtttcaaaa acagtcccac cctgtttgat 600gaggcactgc acagagacct
agcagacttc cggatccagc acccagactt gatcctgcta 660cagtacgtgg
atgacttact gctggccgcc acttctgagc tagactgcca acaaggtact
720cgggccctgt tacaaaccct agggaacctc gggtatcggg cctcggccaa
gaaagcccaa 780atttgccaga aacaggtcaa gtatctgggg tatcttctaa
aagagggtca gagatggctg 840actgaggcca gaaaagagac tgtgatgggg
cagcctactc cgaagacccc tcgacaacta 900agggagttcc tagggacggc
aggcttctgt cgcctctgga tccctgggtt tgcagaaatg 960gcagccccct
tgtaccctct caccaaaacg gggactctgt ttaattgggg cccagaccaa
1020caaaaggcct atcaagaaat caagcaagct cttctaactg ccccagccct
ggggttgcca 1080gatttgacta agccctttga actctttgtc gacgagaagc
agggctacgc caaaggtgtc 1140ctaacgcaaa aactgggacc ttggcgtcgg
ccggtggcct acctgtccaa aaagctagac 1200ccagtagcag ctgggtggcc
cccttgccta cggatggtag cagccattgc cgtactgaca 1260aaggatgcag
gcaagctaac catgggacag ccactagtca ttctggcccc ccatgcagta
1320gaggcactag tcaaacaacc ccccgaccgc tggctttcca acgcccggat
gactcactat 1380caggccttgc ttttggacac ggaccgggtc cagttcggac
cggtggtagc cctgaacccg 1440gctacgctgc tcccactgcc tgaggaaggg
ctgcaacaca actgccttga tatcctggcc 1500gaagcccacg gaacccgacc
cgacctaacg gaccagccgc tcccagacgc cgaccacacc 1560tggtacacgg
atggaagcag tctcttacaa gagggacagc gtaaggcggg agctgcggtg
1620accaccgaga ccgaggtaat ctgggctaaa gccctgccag ccgggacatc
cgctcagcgg 1680gctgaactga tagcactcac ccaggcccta aagatggcag
aaggtaagaa gctaaatgtt 1740tatactgata gccgttatgc ttttgctact
gcccatatcc atggagaaat atacagaagg 1800cgtgggttgc tcacatcaga
aggcaaagag atcaaaaata aagacgagat cttggcccta 1860ctaaaagccc
tctttctgcc caaaagactt agcataatcc attgtccagg acatcaaaag
1920ggacacagcg ccgaggctag aggcaaccgg atggctgacc aagcggcccg
aaaggcagcc 1980atcacagaga ctccagacac ctctaccctc ctc
201332013DNAMoloney murine leukemia
virusmodified_base(205)..(207)This region may encompass 'aaa' or
'aag' 3accctaaata tagaagatga gcatcggcta catgagacct caaaagagcc
agatgtttct 60ctagggtcca catggctgtc tgattttcct caggcctggg cggaaaccgg
gggcatggga 120ctggcagttc gccaagctcc tctgatcata cctctgaaag
caacctctac ccccgtgtcc 180ataaaacaat accccatgtc acaannngcc
agactgggga tcaagcccca catacagaga 240ctgttggacc agggaatact
ggtaccctgc cagtccccct ggaacacgcc cctgctaccc 300gttaagaaac
cagggactaa tgattatagg cctgtccagg atctgagaga agtcaacaag
360cgggtggaag acatccaccc caccgtgccc aacccttaca acctcttgag
cgggctccca 420ccgtcccacc agtggtacac tgtgcttgat ttaaaggatg
cctttttctg cctgagactc 480caccccacca gtcagcctct cttcgccttt
gagtggagag atccagagat gggaatctca 540ggacaattga cctggaccag
actcccacag ggtttcaaaa acagtcccac cctgtttgat 600gaggcactgc
acagagacct agcagacttc cggatccagc acccagactt gatcctgcta
660cagtacgtgg atgacttact gctggccgcc acttctgagc tagactgcca
acaaggtact 720cgggccctgt tacaaaccct agggaacctc gggtatcggg
cctcggccaa gaaagcccaa 780atttgccaga aacaggtcaa gtatctgggg
tatcttctaa aagagggtca gagatggctg 840actgaggcca gaaaagagac
tgtgatgggg cagcctactc cgaagacccc tcgacaacta 900agggagttcc
tagggacggc aggcttctgt cgcctctgga tccctgggtt tgcagaaatg
960gcagccccct tgtaccctct caccaaaacg gggactctgt ttaattgggg
cccagaccaa 1020caaaaggcct atcaagaaat caagcaagct cttctaactg
ccccagccct ggggttgcca 1080gatttgacta agccctttga actctttgtc
gacgagaagc agggctacgc caaaggtgtc 1140ctaacgcaaa aactgggacc
ttggcgtcgg ccggtggcct acctgtccaa aaagctagac 1200ccagtagcag
ctgggtggcc cccttgccta cggatggtag cagccattgc cgtactgaca
1260aaggatgcag gcaagctaac catgggacag ccactagtca ttctggcccc
ccatgcagta 1320gaggcactag tcaaacaacc ccccgaccgc tggctttcca
acgcccggat gactcactat 1380caggccttgc ttttggacac ggaccgggtc
cagttcggac cggtggtagc cctgaacccg 1440gctacgctgc tcccactgcc
tgaggaaggg ctgcaacaca actgccttga tatcctggcc 1500gaagcccacg
gaacccgacc cgacctaacg gaccagccgc tcccagacgc cgaccacacc
1560tggtacacgg atggaagcag tctcttacaa gagggacagc gtaaggcggg
agctgcggtg 1620accaccgaga ccgaggtaat ctgggctaaa gccctgccag
ccgggacatc cgctcagcgg 1680gctgaactga tagcactcac ccaggcccta
aagatggcag aaggtaagaa gctaaatgtt 1740tatactgata gccgttatgc
ttttgctact gcccatatcc atggagaaat atacagaagg 1800cgtgggttgc
tcacatcaga aggcaaagag atcaaaaata aagacgagat cttggcccta
1860ctaaaagccc tctttctgcc caaaagactt agcataatcc attgtccagg
acatcaaaag 1920ggacacagcg ccgaggctag aggcaaccgg atggctgacc
aagcggcccg aaaggcagcc 1980atcacagaga ctccagacac ctctaccctc ctc
201342013DNAMoloney murine leukemia
virusmodified_base(904)..(906)This region may encompass 'aaa' or
'aag' 4accctaaata tagaagatga gcatcggcta catgagacct caaaagagcc
agatgtttct 60ctagggtcca catggctgtc tgattttcct caggcctggg cggaaaccgg
gggcatggga 120ctggcagttc gccaagctcc tctgatcata cctctgaaag
caacctctac ccccgtgtcc 180ataaaacaat accccatgtc acaagaagcc
agactgggga tcaagcccca catacagaga 240ctgttggacc agggaatact
ggtaccctgc cagtccccct ggaacacgcc cctgctaccc 300gttaagaaac
cagggactaa tgattatagg cctgtccagg atctgagaga agtcaacaag
360cgggtggaag acatccaccc caccgtgccc aacccttaca acctcttgag
cgggctccca 420ccgtcccacc agtggtacac tgtgcttgat ttaaaggatg
cctttttctg cctgagactc 480caccccacca gtcagcctct cttcgccttt
gagtggagag atccagagat gggaatctca 540ggacaattga cctggaccag
actcccacag ggtttcaaaa acagtcccac cctgtttgat 600gaggcactgc
acagagacct agcagacttc cggatccagc acccagactt gatcctgcta
660cagtacgtgg atgacttact gctggccgcc acttctgagc tagactgcca
acaaggtact 720cgggccctgt tacaaaccct agggaacctc gggtatcggg
cctcggccaa gaaagcccaa 780atttgccaga aacaggtcaa gtatctgggg
tatcttctaa aagagggtca gagatggctg 840actgaggcca gaaaagagac
tgtgatgggg cagcctactc cgaagacccc tcgacaacta 900aggnnnttcc
tagggacggc aggcttctgt cgcctctgga tccctgggtt tgcagaaatg
960gcagccccct tgtaccctct caccaaaacg gggactctgt ttaattgggg
cccagaccaa 1020caaaaggcct atcaagaaat caagcaagct cttctaactg
ccccagccct ggggttgcca 1080gatttgacta agccctttga actctttgtc
gacgagaagc agggctacgc caaaggtgtc 1140ctaacgcaaa aactgggacc
ttggcgtcgg ccggtggcct acctgtccaa aaagctagac 1200ccagtagcag
ctgggtggcc cccttgccta cggatggtag cagccattgc cgtactgaca
1260aaggatgcag gcaagctaac catgggacag ccactagtca ttctggcccc
ccatgcagta 1320gaggcactag tcaaacaacc ccccgaccgc tggctttcca
acgcccggat gactcactat 1380caggccttgc ttttggacac ggaccgggtc
cagttcggac cggtggtagc cctgaacccg 1440gctacgctgc tcccactgcc
tgaggaaggg ctgcaacaca actgccttga tatcctggcc 1500gaagcccacg
gaacccgacc cgacctaacg gaccagccgc tcccagacgc cgaccacacc
1560tggtacacgg atggaagcag tctcttacaa gagggacagc gtaaggcggg
agctgcggtg 1620accaccgaga ccgaggtaat ctgggctaaa gccctgccag
ccgggacatc cgctcagcgg 1680gctgaactga tagcactcac ccaggcccta
aagatggcag aaggtaagaa gctaaatgtt 1740tatactgata gccgttatgc
ttttgctact gcccatatcc atggagaaat atacagaagg 1800cgtgggttgc
tcacatcaga aggcaaagag atcaaaaata aagacgagat cttggcccta
1860ctaaaagccc tctttctgcc caaaagactt agcataatcc attgtccagg
acatcaaaag 1920ggacacagcg ccgaggctag aggcaaccgg atggctgacc
aagcggcccg aaaggcagcc 1980atcacagaga ctccagacac ctctaccctc ctc
201352013DNAMoloney murine leukemia
virusmodified_base(904)..(906)This region may encompass 'cgt',
'cgc', 'cga', 'cgg', 'aga' or 'agg' 5accctaaata tagaagatga
gcatcggcta catgagacct caaaagagcc agatgtttct 60ctagggtcca catggctgtc
tgattttcct caggcctggg cggaaaccgg gggcatggga 120ctggcagttc
gccaagctcc tctgatcata cctctgaaag caacctctac ccccgtgtcc
180ataaaacaat accccatgtc acaagaagcc agactgggga tcaagcccca
catacagaga 240ctgttggacc agggaatact ggtaccctgc cagtccccct
ggaacacgcc cctgctaccc 300gttaagaaac cagggactaa tgattatagg
cctgtccagg atctgagaga agtcaacaag 360cgggtggaag acatccaccc
caccgtgccc aacccttaca acctcttgag cgggctccca 420ccgtcccacc
agtggtacac tgtgcttgat ttaaaggatg cctttttctg cctgagactc
480caccccacca gtcagcctct cttcgccttt gagtggagag atccagagat
gggaatctca 540ggacaattga cctggaccag actcccacag ggtttcaaaa
acagtcccac cctgtttgat 600gaggcactgc acagagacct agcagacttc
cggatccagc acccagactt gatcctgcta 660cagtacgtgg atgacttact
gctggccgcc acttctgagc tagactgcca acaaggtact 720cgggccctgt
tacaaaccct agggaacctc gggtatcggg cctcggccaa gaaagcccaa
780atttgccaga aacaggtcaa gtatctgggg tatcttctaa aagagggtca
gagatggctg 840actgaggcca gaaaagagac tgtgatgggg cagcctactc
cgaagacccc tcgacaacta 900aggnnnttcc tagggacggc aggcttctgt
cgcctctgga tccctgggtt tgcagaaatg 960gcagccccct tgtaccctct
caccaaaacg gggactctgt ttaattgggg cccagaccaa 1020caaaaggcct
atcaagaaat caagcaagct cttctaactg ccccagccct ggggttgcca
1080gatttgacta agccctttga actctttgtc gacgagaagc agggctacgc
caaaggtgtc 1140ctaacgcaaa aactgggacc ttggcgtcgg ccggtggcct
acctgtccaa aaagctagac 1200ccagtagcag ctgggtggcc cccttgccta
cggatggtag cagccattgc cgtactgaca 1260aaggatgcag gcaagctaac
catgggacag ccactagtca ttctggcccc ccatgcagta 1320gaggcactag
tcaaacaacc ccccgaccgc tggctttcca acgcccggat gactcactat
1380caggccttgc ttttggacac ggaccgggtc cagttcggac cggtggtagc
cctgaacccg 1440gctacgctgc tcccactgcc tgaggaaggg ctgcaacaca
actgccttga tatcctggcc 1500gaagcccacg gaacccgacc cgacctaacg
gaccagccgc tcccagacgc cgaccacacc 1560tggtacacgg atggaagcag
tctcttacaa gagggacagc gtaaggcggg agctgcggtg 1620accaccgaga
ccgaggtaat ctgggctaaa gccctgccag ccgggacatc cgctcagcgg
1680gctgaactga tagcactcac ccaggcccta aagatggcag aaggtaagaa
gctaaatgtt 1740tatactgata gccgttatgc ttttgctact gcccatatcc
atggagaaat atacagaagg 1800cgtgggttgc tcacatcaga aggcaaagag
atcaaaaata aagacgagat cttggcccta 1860ctaaaagccc tctttctgcc
caaaagactt agcataatcc attgtccagg acatcaaaag 1920ggacacagcg
ccgaggctag aggcaaccgg atggctgacc aagcggcccg aaaggcagcc
1980atcacagaga ctccagacac ctctaccctc ctc 201362013DNAMoloney murine
leukemia virusmodified_base(937)..(939)This region may encompass
'ttt' or 'ttc' 6accctaaata tagaagatga gcatcggcta catgagacct
caaaagagcc agatgtttct 60ctagggtcca catggctgtc tgattttcct caggcctggg
cggaaaccgg gggcatggga 120ctggcagttc gccaagctcc tctgatcata
cctctgaaag caacctctac ccccgtgtcc 180ataaaacaat accccatgtc
acaagaagcc agactgggga tcaagcccca catacagaga 240ctgttggacc
agggaatact ggtaccctgc cagtccccct ggaacacgcc cctgctaccc
300gttaagaaac cagggactaa tgattatagg cctgtccagg atctgagaga
agtcaacaag 360cgggtggaag acatccaccc caccgtgccc aacccttaca
acctcttgag cgggctccca 420ccgtcccacc agtggtacac tgtgcttgat
ttaaaggatg cctttttctg cctgagactc 480caccccacca gtcagcctct
cttcgccttt gagtggagag atccagagat gggaatctca 540ggacaattga
cctggaccag actcccacag ggtttcaaaa acagtcccac cctgtttgat
600gaggcactgc acagagacct agcagacttc cggatccagc acccagactt
gatcctgcta 660cagtacgtgg atgacttact gctggccgcc acttctgagc
tagactgcca acaaggtact 720cgggccctgt tacaaaccct agggaacctc
gggtatcggg cctcggccaa gaaagcccaa 780atttgccaga aacaggtcaa
gtatctgggg tatcttctaa aagagggtca gagatggctg 840actgaggcca
gaaaagagac tgtgatgggg cagcctactc cgaagacccc tcgacaacta
900agggagttcc tagggacggc aggcttctgt cgcctcnnna tccctgggtt
tgcagaaatg 960gcagccccct tgtaccctct caccaaaacg gggactctgt
ttaattgggg cccagaccaa 1020caaaaggcct atcaagaaat caagcaagct
cttctaactg ccccagccct ggggttgcca 1080gatttgacta agccctttga
actctttgtc gacgagaagc agggctacgc caaaggtgtc 1140ctaacgcaaa
aactgggacc ttggcgtcgg ccggtggcct acctgtccaa aaagctagac
1200ccagtagcag ctgggtggcc cccttgccta cggatggtag cagccattgc
cgtactgaca 1260aaggatgcag gcaagctaac catgggacag ccactagtca
ttctggcccc ccatgcagta 1320gaggcactag tcaaacaacc ccccgaccgc
tggctttcca acgcccggat gactcactat 1380caggccttgc ttttggacac
ggaccgggtc cagttcggac cggtggtagc cctgaacccg 1440gctacgctgc
tcccactgcc tgaggaaggg ctgcaacaca actgccttga tatcctggcc
1500gaagcccacg gaacccgacc cgacctaacg gaccagccgc tcccagacgc
cgaccacacc 1560tggtacacgg atggaagcag tctcttacaa gagggacagc
gtaaggcggg agctgcggtg 1620accaccgaga ccgaggtaat ctgggctaaa
gccctgccag ccgggacatc cgctcagcgg 1680gctgaactga tagcactcac
ccaggcccta aagatggcag aaggtaagaa gctaaatgtt 1740tatactgata
gccgttatgc ttttgctact gcccatatcc atggagaaat atacagaagg
1800cgtgggttgc tcacatcaga aggcaaagag atcaaaaata aagacgagat
cttggcccta 1860ctaaaagccc tctttctgcc caaaagactt agcataatcc
attgtccagg acatcaaaag 1920ggacacagcg ccgaggctag aggcaaccgg
atggctgacc aagcggcccg aaaggcagcc 1980atcacagaga ctccagacac
ctctaccctc ctc 201372013DNAMoloney murine leukemia
virusmodified_base(1303)..(1305)This region may encompass 'ggt',
'ggc', 'gga' or 'ggg' 7accctaaata tagaagatga gcatcggcta catgagacct
caaaagagcc agatgtttct 60ctagggtcca catggctgtc tgattttcct caggcctggg
cggaaaccgg gggcatggga 120ctggcagttc gccaagctcc tctgatcata
cctctgaaag caacctctac ccccgtgtcc 180ataaaacaat accccatgtc
acaagaagcc agactgggga tcaagcccca catacagaga 240ctgttggacc
agggaatact ggtaccctgc cagtccccct ggaacacgcc cctgctaccc
300gttaagaaac cagggactaa tgattatagg cctgtccagg atctgagaga
agtcaacaag 360cgggtggaag acatccaccc caccgtgccc aacccttaca
acctcttgag cgggctccca 420ccgtcccacc agtggtacac tgtgcttgat
ttaaaggatg cctttttctg cctgagactc 480caccccacca gtcagcctct
cttcgccttt gagtggagag atccagagat gggaatctca 540ggacaattga
cctggaccag actcccacag ggtttcaaaa acagtcccac cctgtttgat
600gaggcactgc acagagacct agcagacttc cggatccagc acccagactt
gatcctgcta 660cagtacgtgg atgacttact gctggccgcc acttctgagc
tagactgcca acaaggtact 720cgggccctgt tacaaaccct agggaacctc
gggtatcggg cctcggccaa gaaagcccaa 780atttgccaga aacaggtcaa
gtatctgggg tatcttctaa aagagggtca gagatggctg 840actgaggcca
gaaaagagac tgtgatgggg cagcctactc cgaagacccc tcgacaacta
900agggagttcc tagggacggc aggcttctgt cgcctctgga tccctgggtt
tgcagaaatg 960gcagccccct tgtaccctct caccaaaacg gggactctgt
ttaattgggg cccagaccaa 1020caaaaggcct atcaagaaat caagcaagct
cttctaactg ccccagccct ggggttgcca 1080gatttgacta agccctttga
actctttgtc gacgagaagc agggctacgc caaaggtgtc 1140ctaacgcaaa
aactgggacc ttggcgtcgg ccggtggcct acctgtccaa aaagctagac
1200ccagtagcag ctgggtggcc cccttgccta cggatggtag cagccattgc
cgtactgaca 1260aaggatgcag gcaagctaac catgggacag ccactagtca
ttnnngcccc ccatgcagta 1320gaggcactag tcaaacaacc ccccgaccgc
tggctttcca acgcccggat gactcactat 1380caggccttgc ttttggacac
ggaccgggtc cagttcggac cggtggtagc cctgaacccg 1440gctacgctgc
tcccactgcc tgaggaaggg ctgcaacaca actgccttga tatcctggcc
1500gaagcccacg gaacccgacc cgacctaacg gaccagccgc tcccagacgc
cgaccacacc 1560tggtacacgg atggaagcag tctcttacaa gagggacagc
gtaaggcggg agctgcggtg 1620accaccgaga ccgaggtaat ctgggctaaa
gccctgccag ccgggacatc cgctcagcgg 1680gctgaactga tagcactcac
ccaggcccta aagatggcag aaggtaagaa gctaaatgtt 1740tatactgata
gccgttatgc ttttgctact gcccatatcc atggagaaat atacagaagg
1800cgtgggttgc tcacatcaga aggcaaagag atcaaaaata aagacgagat
cttggcccta 1860ctaaaagccc tctttctgcc caaaagactt agcataatcc
attgtccagg acatcaaaag 1920ggacacagcg ccgaggctag aggcaaccgg
atggctgacc aagcggcccg aaaggcagcc 1980atcacagaga ctccagacac
ctctaccctc ctc 201382013DNAMoloney murine leukemia virus
8accctaaata tagaagatga gcatcggcta catgagacct caaaagagcc agatgtttct
60ctagggtcca catggctgtc tgattttcct caggcctggg cggaaaccgg gggcatggga
120ctggcagttc gccaagctcc tctgatcata cctctgaaag caacctctac
ccccgtgtcc 180ataaaacaat accccatgtc acaagaagcc agactgggga
tcaagcccca catacagaga 240ctgttggacc agggaatact ggtaccctgc
cagtccccct ggaacacgcc cctgctaccc 300gttaagaaac cagggactaa
tgattatagg cctgtccagg atctgagaga agtcaacaag 360cgggtggaag
acatccaccc caccgtgccc aacccttaca acctcttgag cgggctccca
420ccgtcccacc agtggtacac tgtgcttgat ttaaaggatg cctttttctg
cctgagactc 480caccccacca gtcagcctct cttcgccttt gagtggagag
atccagagat gggaatctca 540ggacaattga cctggaccag actcccacag
ggtttcaaaa acagtcccac cctgtttgat 600gaggcactgc acagagacct
agcagacttc cggatccagc acccagactt gatcctgcta 660cagtacgtgg
atgacttact gctggccgcc acttctgagc tagactgcca acaaggtact
720cgggccctgt tacaaaccct agggaacctc gggtatcggg cctcggccaa
gaaagcccaa 780atttgccaga aacaggtcaa gtatctgggg tatcttctaa
aagagggtca gagatggctg 840actgaggcca gaaaagagac tgtgatgggg
cagcctactc cgaagacccc tcgacaacta 900agggagttcc tagggacggc
aggcttctgt cgcctctgga tccctgggtt tgcagaaatg 960gcagccccct
tgtaccctct caccaaaacg gggactctgt ttaattgggg cccagaccaa
1020caaaaggcct atcaagaaat caagcaagct cttctaactg ccccagccct
ggggttgcca 1080gatttgacta agccctttga actctttgtc gacgagaagc
agggctacgc caaaggtgtc 1140ctaacgcaaa aactgggacc ttggcgtcgg
ccggtggcct acctgtccaa aaagctagac 1200ccagtagcag ctgggtggcc
cccttgccta cggatggtag cagccattgc cgtactgaca 1260aaggatgcag
gcaagctaac catgggacag ccactagtca ttatggcccc ccatgcagta
1320gaggcactag tcaaacaacc ccccgaccgc tggctttcca acgcccggat
gactcactat 1380caggccttgc ttttggacac ggaccgggtc cagttcggac
cggtggtagc cctgaacccg 1440gctacgctgc tcccactgcc tgaggaaggg
ctgcaacaca actgccttga tatcctggcc 1500gaagcccacg gaacccgacc
cgacctaacg gaccagccgc tcccagacgc cgaccacacc 1560tggtacacgg
atggaagcag tctcttacaa gagggacagc gtaaggcggg agctgcggtg
1620accaccgaga ccgaggtaat ctgggctaaa gccctgccag ccgggacatc
cgctcagcgg 1680gctgaactga tagcactcac ccaggcccta aagatggcag
aaggtaagaa gctaaatgtt 1740tatactgata gccgttatgc ttttgctact
gcccatatcc atggagaaat atacagaagg 1800cgtgggttgc tcacatcaga
aggcaaagag atcaaaaata aagacgagat cttggcccta 1860ctaaaagccc
tctttctgcc caaaagactt agcataatcc attgtccagg acatcaaaag
1920ggacacagcg ccgaggctag aggcaaccgg atggctgacc aagcggcccg
aaaggcagcc 1980atcacagaga ctccagacac ctctaccctc ctc
201392013DNAMoloney murine
leukemia virusmodified_base(1360)..(1362)This region may encompass
'aaa' or 'aag' 9accctaaata tagaagatga gcatcggcta catgagacct
caaaagagcc agatgtttct 60ctagggtcca catggctgtc tgattttcct caggcctggg
cggaaaccgg gggcatggga 120ctggcagttc gccaagctcc tctgatcata
cctctgaaag caacctctac ccccgtgtcc 180ataaaacaat accccatgtc
acaagaagcc agactgggga tcaagcccca catacagaga 240ctgttggacc
agggaatact ggtaccctgc cagtccccct ggaacacgcc cctgctaccc
300gttaagaaac cagggactaa tgattatagg cctgtccagg atctgagaga
agtcaacaag 360cgggtggaag acatccaccc caccgtgccc aacccttaca
acctcttgag cgggctccca 420ccgtcccacc agtggtacac tgtgcttgat
ttaaaggatg cctttttctg cctgagactc 480caccccacca gtcagcctct
cttcgccttt gagtggagag atccagagat gggaatctca 540ggacaattga
cctggaccag actcccacag ggtttcaaaa acagtcccac cctgtttgat
600gaggcactgc acagagacct agcagacttc cggatccagc acccagactt
gatcctgcta 660cagtacgtgg atgacttact gctggccgcc acttctgagc
tagactgcca acaaggtact 720cgggccctgt tacaaaccct agggaacctc
gggtatcggg cctcggccaa gaaagcccaa 780atttgccaga aacaggtcaa
gtatctgggg tatcttctaa aagagggtca gagatggctg 840actgaggcca
gaaaagagac tgtgatgggg cagcctactc cgaagacccc tcgacaacta
900agggagttcc tagggacggc aggcttctgt cgcctctgga tccctgggtt
tgcagaaatg 960gcagccccct tgtaccctct caccaaaacg gggactctgt
ttaattgggg cccagaccaa 1020caaaaggcct atcaagaaat caagcaagct
cttctaactg ccccagccct ggggttgcca 1080gatttgacta agccctttga
actctttgtc gacgagaagc agggctacgc caaaggtgtc 1140ctaacgcaaa
aactgggacc ttggcgtcgg ccggtggcct acctgtccaa aaagctagac
1200ccagtagcag ctgggtggcc cccttgccta cggatggtag cagccattgc
cgtactgaca 1260aaggatgcag gcaagctaac catgggacag ccactagtca
ttctggcccc ccatgcagta 1320gaggcactag tcaaacaacc ccccgaccgc
tggctttccn nngcccggat gactcactat 1380caggccttgc ttttggacac
ggaccgggtc cagttcggac cggtggtagc cctgaacccg 1440gctacgctgc
tcccactgcc tgaggaaggg ctgcaacaca actgccttga tatcctggcc
1500gaagcccacg gaacccgacc cgacctaacg gaccagccgc tcccagacgc
cgaccacacc 1560tggtacacgg atggaagcag tctcttacaa gagggacagc
gtaaggcggg agctgcggtg 1620accaccgaga ccgaggtaat ctgggctaaa
gccctgccag ccgggacatc cgctcagcgg 1680gctgaactga tagcactcac
ccaggcccta aagatggcag aaggtaagaa gctaaatgtt 1740tatactgata
gccgttatgc ttttgctact gcccatatcc atggagaaat atacagaagg
1800cgtgggttgc tcacatcaga aggcaaagag atcaaaaata aagacgagat
cttggcccta 1860ctaaaagccc tctttctgcc caaaagactt agcataatcc
attgtccagg acatcaaaag 1920ggacacagcg ccgaggctag aggcaaccgg
atggctgacc aagcggcccg aaaggcagcc 1980atcacagaga ctccagacac
ctctaccctc ctc 2013102013DNAMoloney murine leukemia
virusmodified_base(1360)..(1362)This region may encompass 'cgt',
'cgc', 'cga', 'cgg', 'aga' or 'agg' 10accctaaata tagaagatga
gcatcggcta catgagacct caaaagagcc agatgtttct 60ctagggtcca catggctgtc
tgattttcct caggcctggg cggaaaccgg gggcatggga 120ctggcagttc
gccaagctcc tctgatcata cctctgaaag caacctctac ccccgtgtcc
180ataaaacaat accccatgtc acaagaagcc agactgggga tcaagcccca
catacagaga 240ctgttggacc agggaatact ggtaccctgc cagtccccct
ggaacacgcc cctgctaccc 300gttaagaaac cagggactaa tgattatagg
cctgtccagg atctgagaga agtcaacaag 360cgggtggaag acatccaccc
caccgtgccc aacccttaca acctcttgag cgggctccca 420ccgtcccacc
agtggtacac tgtgcttgat ttaaaggatg cctttttctg cctgagactc
480caccccacca gtcagcctct cttcgccttt gagtggagag atccagagat
gggaatctca 540ggacaattga cctggaccag actcccacag ggtttcaaaa
acagtcccac cctgtttgat 600gaggcactgc acagagacct agcagacttc
cggatccagc acccagactt gatcctgcta 660cagtacgtgg atgacttact
gctggccgcc acttctgagc tagactgcca acaaggtact 720cgggccctgt
tacaaaccct agggaacctc gggtatcggg cctcggccaa gaaagcccaa
780atttgccaga aacaggtcaa gtatctgggg tatcttctaa aagagggtca
gagatggctg 840actgaggcca gaaaagagac tgtgatgggg cagcctactc
cgaagacccc tcgacaacta 900agggagttcc tagggacggc aggcttctgt
cgcctctgga tccctgggtt tgcagaaatg 960gcagccccct tgtaccctct
caccaaaacg gggactctgt ttaattgggg cccagaccaa 1020caaaaggcct
atcaagaaat caagcaagct cttctaactg ccccagccct ggggttgcca
1080gatttgacta agccctttga actctttgtc gacgagaagc agggctacgc
caaaggtgtc 1140ctaacgcaaa aactgggacc ttggcgtcgg ccggtggcct
acctgtccaa aaagctagac 1200ccagtagcag ctgggtggcc cccttgccta
cggatggtag cagccattgc cgtactgaca 1260aaggatgcag gcaagctaac
catgggacag ccactagtca ttctggcccc ccatgcagta 1320gaggcactag
tcaaacaacc ccccgaccgc tggctttccn nngcccggat gactcactat
1380caggccttgc ttttggacac ggaccgggtc cagttcggac cggtggtagc
cctgaacccg 1440gctacgctgc tcccactgcc tgaggaaggg ctgcaacaca
actgccttga tatcctggcc 1500gaagcccacg gaacccgacc cgacctaacg
gaccagccgc tcccagacgc cgaccacacc 1560tggtacacgg atggaagcag
tctcttacaa gagggacagc gtaaggcggg agctgcggtg 1620accaccgaga
ccgaggtaat ctgggctaaa gccctgccag ccgggacatc cgctcagcgg
1680gctgaactga tagcactcac ccaggcccta aagatggcag aaggtaagaa
gctaaatgtt 1740tatactgata gccgttatgc ttttgctact gcccatatcc
atggagaaat atacagaagg 1800cgtgggttgc tcacatcaga aggcaaagag
atcaaaaata aagacgagat cttggcccta 1860ctaaaagccc tctttctgcc
caaaagactt agcataatcc attgtccagg acatcaaaag 1920ggacacagcg
ccgaggctag aggcaaccgg atggctgacc aagcggcccg aaaggcagcc
1980atcacagaga ctccagacac ctctaccctc ctc 2013112013DNAMoloney
murine leukemia virusmodified_base(1570)..(1572)This region may
encompass 'aat' or 'aac' 11accctaaata tagaagatga gcatcggcta
catgagacct caaaagagcc agatgtttct 60ctagggtcca catggctgtc tgattttcct
caggcctggg cggaaaccgg gggcatggga 120ctggcagttc gccaagctcc
tctgatcata cctctgaaag caacctctac ccccgtgtcc 180ataaaacaat
accccatgtc acaagaagcc agactgggga tcaagcccca catacagaga
240ctgttggacc agggaatact ggtaccctgc cagtccccct ggaacacgcc
cctgctaccc 300gttaagaaac cagggactaa tgattatagg cctgtccagg
atctgagaga agtcaacaag 360cgggtggaag acatccaccc caccgtgccc
aacccttaca acctcttgag cgggctccca 420ccgtcccacc agtggtacac
tgtgcttgat ttaaaggatg cctttttctg cctgagactc 480caccccacca
gtcagcctct cttcgccttt gagtggagag atccagagat gggaatctca
540ggacaattga cctggaccag actcccacag ggtttcaaaa acagtcccac
cctgtttgat 600gaggcactgc acagagacct agcagacttc cggatccagc
acccagactt gatcctgcta 660cagtacgtgg atgacttact gctggccgcc
acttctgagc tagactgcca acaaggtact 720cgggccctgt tacaaaccct
agggaacctc gggtatcggg cctcggccaa gaaagcccaa 780atttgccaga
aacaggtcaa gtatctgggg tatcttctaa aagagggtca gagatggctg
840actgaggcca gaaaagagac tgtgatgggg cagcctactc cgaagacccc
tcgacaacta 900agggagttcc tagggacggc aggcttctgt cgcctctgga
tccctgggtt tgcagaaatg 960gcagccccct tgtaccctct caccaaaacg
gggactctgt ttaattgggg cccagaccaa 1020caaaaggcct atcaagaaat
caagcaagct cttctaactg ccccagccct ggggttgcca 1080gatttgacta
agccctttga actctttgtc gacgagaagc agggctacgc caaaggtgtc
1140ctaacgcaaa aactgggacc ttggcgtcgg ccggtggcct acctgtccaa
aaagctagac 1200ccagtagcag ctgggtggcc cccttgccta cggatggtag
cagccattgc cgtactgaca 1260aaggatgcag gcaagctaac catgggacag
ccactagtca ttctggcccc ccatgcagta 1320gaggcactag tcaaacaacc
ccccgaccgc tggctttcca acgcccggat gactcactat 1380caggccttgc
ttttggacac ggaccgggtc cagttcggac cggtggtagc cctgaacccg
1440gctacgctgc tcccactgcc tgaggaaggg ctgcaacaca actgccttga
tatcctggcc 1500gaagcccacg gaacccgacc cgacctaacg gaccagccgc
tcccagacgc cgaccacacc 1560tggtacacgn nnggaagcag tctcttacaa
gagggacagc gtaaggcggg agctgcggtg 1620accaccgaga ccgaggtaat
ctgggctaaa gccctgccag ccgggacatc cgctcagcgg 1680gctgaactga
tagcactcac ccaggcccta aagatggcag aaggtaagaa gctaaatgtt
1740tatactgata gccgttatgc ttttgctact gcccatatcc atggagaaat
atacagaagg 1800cgtgggttgc tcacatcaga aggcaaagag atcaaaaata
aagacgagat cttggcccta 1860ctaaaagccc tctttctgcc caaaagactt
agcataatcc attgtccagg acatcaaaag 1920ggacacagcg ccgaggctag
aggcaaccgg atggctgacc aagcggcccg aaaggcagcc 1980atcacagaga
ctccagacac ctctaccctc ctc 2013122013DNAMoloney murine leukemia
virusmodified_base(1951)..(1953)This region may encompass 'ctt',
'ctc', 'cta', 'ctg', 'tta' or 'ttg' 12accctaaata tagaagatga
gcatcggcta catgagacct caaaagagcc agatgtttct 60ctagggtcca catggctgtc
tgattttcct caggcctggg cggaaaccgg gggcatggga 120ctggcagttc
gccaagctcc tctgatcata cctctgaaag caacctctac ccccgtgtcc
180ataaaacaat accccatgtc acaagaagcc agactgggga tcaagcccca
catacagaga 240ctgttggacc agggaatact ggtaccctgc cagtccccct
ggaacacgcc cctgctaccc 300gttaagaaac cagggactaa tgattatagg
cctgtccagg atctgagaga agtcaacaag 360cgggtggaag acatccaccc
caccgtgccc aacccttaca acctcttgag cgggctccca 420ccgtcccacc
agtggtacac tgtgcttgat ttaaaggatg cctttttctg cctgagactc
480caccccacca gtcagcctct cttcgccttt gagtggagag atccagagat
gggaatctca 540ggacaattga cctggaccag actcccacag ggtttcaaaa
acagtcccac cctgtttgat 600gaggcactgc acagagacct agcagacttc
cggatccagc acccagactt gatcctgcta 660cagtacgtgg atgacttact
gctggccgcc acttctgagc tagactgcca acaaggtact 720cgggccctgt
tacaaaccct agggaacctc gggtatcggg cctcggccaa gaaagcccaa
780atttgccaga aacaggtcaa gtatctgggg tatcttctaa aagagggtca
gagatggctg 840actgaggcca gaaaagagac tgtgatgggg cagcctactc
cgaagacccc tcgacaacta 900agggagttcc tagggacggc aggcttctgt
cgcctctgga tccctgggtt tgcagaaatg 960gcagccccct tgtaccctct
caccaaaacg gggactctgt ttaattgggg cccagaccaa 1020caaaaggcct
atcaagaaat caagcaagct cttctaactg ccccagccct ggggttgcca
1080gatttgacta agccctttga actctttgtc gacgagaagc agggctacgc
caaaggtgtc 1140ctaacgcaaa aactgggacc ttggcgtcgg ccggtggcct
acctgtccaa aaagctagac 1200ccagtagcag ctgggtggcc cccttgccta
cggatggtag cagccattgc cgtactgaca 1260aaggatgcag gcaagctaac
catgggacag ccactagtca ttctggcccc ccatgcagta 1320gaggcactag
tcaaacaacc ccccgaccgc tggctttcca acgcccggat gactcactat
1380caggccttgc ttttggacac ggaccgggtc cagttcggac cggtggtagc
cctgaacccg 1440gctacgctgc tcccactgcc tgaggaaggg ctgcaacaca
actgccttga tatcctggcc 1500gaagcccacg gaacccgacc cgacctaacg
gaccagccgc tcccagacgc cgaccacacc 1560tggtacacgg atggaagcag
tctcttacaa gagggacagc gtaaggcggg agctgcggtg 1620accaccgaga
ccgaggtaat ctgggctaaa gccctgccag ccgggacatc cgctcagcgg
1680gctgaactga tagcactcac ccaggcccta aagatggcag aaggtaagaa
gctaaatgtt 1740tatactgata gccgttatgc ttttgctact gcccatatcc
atggagaaat atacagaagg 1800cgtgggttgc tcacatcaga aggcaaagag
atcaaaaata aagacgagat cttggcccta 1860ctaaaagccc tctttctgcc
caaaagactt agcataatcc attgtccagg acatcaaaag 1920ggacacagcg
ccgaggctag aggcaaccgg nnngctgacc aagcggcccg aaaggcagcc
1980atcacagaga ctccagacac ctctaccctc ctc 2013132013DNAMoloney
murine leukemia virusmodified_base(205)..(207)This region may
encompass 'aaa' or 'aag' 13accctaaata tagaagatga gcatcggcta
catgagacct caaaagagcc agatgtttct 60ctagggtcca catggctgtc tgattttcct
caggcctggg cggaaaccgg gggcatggga 120ctggcagttc gccaagctcc
tctgatcata cctctgaaag caacctctac ccccgtgtcc 180ataaaacaat
accccatgtc acaannngcc agactgggga tcaagcccca catacagaga
240ctgttggacc agggaatact ggtaccctgc cagtccccct ggaacacgcc
cctgctaccc 300gttaagaaac cagggactaa tgattatagg cctgtccagg
atctgagaga agtcaacaag 360cgggtggaag acatccaccc caccgtgccc
aacccttaca acctcttgag cgggctccca 420ccgtcccacc agtggtacac
tgtgcttgat ttaaaggatg cctttttctg cctgagactc 480caccccacca
gtcagcctct cttcgccttt gagtggagag atccagagat gggaatctca
540ggacaattga cctggaccag actcccacag ggtttcaaaa acagtcccac
cctgtttgat 600gaggcactgc acagagacct agcagacttc cggatccagc
acccagactt gatcctgcta 660cagtacgtgg atgacttact gctggccgcc
acttctgagc tagactgcca acaaggtact 720cgggccctgt tacaaaccct
agggaacctc gggtatcggg cctcggccaa gaaagcccaa 780atttgccaga
aacaggtcaa gtatctgggg tatcttctaa aagagggtca gagatggctg
840actgaggcca gaaaagagac tgtgatgggg cagcctactc cgaagacccc
tcgacaacta 900aggnnnttcc tagggacggc aggcttctgt cgcctcnnna
tccctgggtt tgcagaaatg 960gcagccccct tgtaccctct caccaaaacg
gggactctgt ttaattgggg cccagaccaa 1020caaaaggcct atcaagaaat
caagcaagct cttctaactg ccccagccct ggggttgcca 1080gatttgacta
agccctttga actctttgtc gacgagaagc agggctacgc caaaggtgtc
1140ctaacgcaaa aactgggacc ttggcgtcgg ccggtggcct acctgtccaa
aaagctagac 1200ccagtagcag ctgggtggcc cccttgccta cggatggtag
cagccattgc cgtactgaca 1260aaggatgcag gcaagctaac catgggacag
ccactagtca ttnnngcccc ccatgcagta 1320gaggcactag tcaaacaacc
ccccgaccgc tggctttccn nngcccggat gactcactat 1380caggccttgc
ttttggacac ggaccgggtc cagttcggac cggtggtagc cctgaacccg
1440gctacgctgc tcccactgcc tgaggaaggg ctgcaacaca actgccttga
tatcctggcc 1500gaagcccacg gaacccgacc cgacctaacg gaccagccgc
tcccagacgc cgaccacacc 1560tggtacacgg atggaagcag tctcttacaa
gagggacagc gtaaggcggg agctgcggtg 1620accaccgaga ccgaggtaat
ctgggctaaa gccctgccag ccgggacatc cgctcagcgg 1680gctgaactga
tagcactcac ccaggcccta aagatggcag aaggtaagaa gctaaatgtt
1740tatactgata gccgttatgc ttttgctact gcccatatcc atggagaaat
atacagaagg 1800cgtgggttgc tcacatcaga aggcaaagag atcaaaaata
aagacgagat cttggcccta 1860ctaaaagccc tctttctgcc caaaagactt
agcataatcc attgtccagg acatcaaaag 1920ggacacagcg ccgaggctag
aggcaaccgg atggctgacc aagcggcccg aaaggcagcc 1980atcacagaga
ctccagacac ctctaccctc ctc 2013142013DNAMoloney murine leukemia
virusmodified_base(904)..(906)This region may encompass 'aaa' or
'aag' 14accctaaata tagaagatga gcatcggcta catgagacct caaaagagcc
agatgtttct 60ctagggtcca catggctgtc tgattttcct caggcctggg cggaaaccgg
gggcatggga 120ctggcagttc gccaagctcc tctgatcata cctctgaaag
caacctctac ccccgtgtcc 180ataaaacaat accccatgtc acaagaagcc
agactgggga tcaagcccca catacagaga 240ctgttggacc agggaatact
ggtaccctgc cagtccccct ggaacacgcc cctgctaccc 300gttaagaaac
cagggactaa tgattatagg cctgtccagg atctgagaga agtcaacaag
360cgggtggaag acatccaccc caccgtgccc aacccttaca acctcttgag
cgggctccca 420ccgtcccacc agtggtacac tgtgcttgat ttaaaggatg
cctttttctg cctgagactc 480caccccacca gtcagcctct cttcgccttt
gagtggagag atccagagat gggaatctca 540ggacaattga cctggaccag
actcccacag ggtttcaaaa acagtcccac cctgtttgat 600gaggcactgc
acagagacct agcagacttc cggatccagc acccagactt gatcctgcta
660cagtacgtgg atgacttact gctggccgcc acttctgagc tagactgcca
acaaggtact 720cgggccctgt tacaaaccct agggaacctc gggtatcggg
cctcggccaa gaaagcccaa 780atttgccaga aacaggtcaa gtatctgggg
tatcttctaa aagagggtca gagatggctg 840actgaggcca gaaaagagac
tgtgatgggg cagcctactc cgaagacccc tcgacaacta 900aggnnnttcc
tagggacggc aggcttctgt cgcctcnnna tccctgggtt tgcagaaatg
960gcagccccct tgtaccctct caccaaaacg gggactctgt ttaattgggg
cccagaccaa 1020caaaaggcct atcaagaaat caagcaagct cttctaactg
ccccagccct ggggttgcca 1080gatttgacta agccctttga actctttgtc
gacgagaagc agggctacgc caaaggtgtc 1140ctaacgcaaa aactgggacc
ttggcgtcgg ccggtggcct acctgtccaa aaagctagac 1200ccagtagcag
ctgggtggcc cccttgccta cggatggtag cagccattgc cgtactgaca
1260aaggatgcag gcaagctaac catgggacag ccactagtca ttnnngcccc
ccatgcagta 1320gaggcactag tcaaacaacc ccccgaccgc tggctttccn
nngcccggat gactcactat 1380caggccttgc ttttggacac ggaccgggtc
cagttcggac cggtggtagc cctgaacccg 1440gctacgctgc tcccactgcc
tgaggaaggg ctgcaacaca actgccttga tatcctggcc 1500gaagcccacg
gaacccgacc cgacctaacg gaccagccgc tcccagacgc cgaccacacc
1560tggtacacgg atggaagcag tctcttacaa gagggacagc gtaaggcggg
agctgcggtg 1620accaccgaga ccgaggtaat ctgggctaaa gccctgccag
ccgggacatc cgctcagcgg 1680gctgaactga tagcactcac ccaggcccta
aagatggcag aaggtaagaa gctaaatgtt 1740tatactgata gccgttatgc
ttttgctact gcccatatcc atggagaaat atacagaagg 1800cgtgggttgc
tcacatcaga aggcaaagag atcaaaaata aagacgagat cttggcccta
1860ctaaaagccc tctttctgcc caaaagactt agcataatcc attgtccagg
acatcaaaag 1920ggacacagcg ccgaggctag aggcaaccgg atggctgacc
aagcggcccg aaaggcagcc 1980atcacagaga ctccagacac ctctaccctc ctc
2013152013DNAMoloney murine leukemia
virusmodified_base(904)..(906)This region may encompass 'aaa' or
'aag' 15accctaaata tagaagatga gcatcggcta catgagacct caaaagagcc
agatgtttct 60ctagggtcca catggctgtc tgattttcct caggcctggg cggaaaccgg
gggcatggga 120ctggcagttc gccaagctcc tctgatcata cctctgaaag
caacctctac ccccgtgtcc 180ataaaacaat accccatgtc acaagaagcc
agactgggga tcaagcccca catacagaga 240ctgttggacc agggaatact
ggtaccctgc cagtccccct ggaacacgcc cctgctaccc 300gttaagaaac
cagggactaa tgattatagg cctgtccagg atctgagaga agtcaacaag
360cgggtggaag acatccaccc caccgtgccc aacccttaca acctcttgag
cgggctccca 420ccgtcccacc agtggtacac tgtgcttgat ttaaaggatg
cctttttctg cctgagactc 480caccccacca gtcagcctct cttcgccttt
gagtggagag atccagagat gggaatctca 540ggacaattga cctggaccag
actcccacag ggtttcaaaa acagtcccac cctgtttgat 600gaggcactgc
acagagacct agcagacttc cggatccagc acccagactt gatcctgcta
660cagtacgtgg atgacttact gctggccgcc acttctgagc tagactgcca
acaaggtact 720cgggccctgt tacaaaccct agggaacctc gggtatcggg
cctcggccaa gaaagcccaa 780atttgccaga aacaggtcaa gtatctgggg
tatcttctaa aagagggtca gagatggctg 840actgaggcca gaaaagagac
tgtgatgggg cagcctactc cgaagacccc tcgacaacta 900aggnnnttcc
tagggacggc aggcttctgt cgcctcnnna tccctgggtt tgcagaaatg
960gcagccccct tgtaccctct caccaaaacg gggactctgt ttaattgggg
cccagaccaa 1020caaaaggcct atcaagaaat caagcaagct cttctaactg
ccccagccct ggggttgcca 1080gatttgacta agccctttga actctttgtc
gacgagaagc agggctacgc caaaggtgtc 1140ctaacgcaaa aactgggacc
ttggcgtcgg ccggtggcct acctgtccaa aaagctagac 1200ccagtagcag
ctgggtggcc cccttgccta cggatggtag cagccattgc cgtactgaca
1260aaggatgcag gcaagctaac catgggacag ccactagtca ttnnngcccc
ccatgcagta 1320gaggcactag tcaaacaacc ccccgaccgc tggctttcca
acgcccggat gactcactat 1380caggccttgc ttttggacac ggaccgggtc
cagttcggac cggtggtagc cctgaacccg 1440gctacgctgc tcccactgcc
tgaggaaggg ctgcaacaca actgccttga tatcctggcc 1500gaagcccacg
gaacccgacc cgacctaacg gaccagccgc tcccagacgc cgaccacacc
1560tggtacacgg atggaagcag tctcttacaa gagggacagc gtaaggcggg
agctgcggtg 1620accaccgaga ccgaggtaat ctgggctaaa gccctgccag
ccgggacatc cgctcagcgg 1680gctgaactga tagcactcac ccaggcccta
aagatggcag aaggtaagaa gctaaatgtt 1740tatactgata gccgttatgc
ttttgctact gcccatatcc atggagaaat atacagaagg 1800cgtgggttgc
tcacatcaga aggcaaagag atcaaaaata aagacgagat cttggcccta
1860ctaaaagccc tctttctgcc caaaagactt agcataatcc attgtccagg
acatcaaaag 1920ggacacagcg ccgaggctag aggcaaccgg atggctgacc
aagcggcccg aaaggcagcc 1980atcacagaga ctccagacac ctctaccctc ctc
2013162013DNAMoloney murine leukemia
virusmodified_base(205)..(207)This region may encompass 'aaa' or
'aag'
16accctaaata tagaagatga gcatcggcta catgagacct caaaagagcc agatgtttct
60ctagggtcca catggctgtc tgattttcct caggcctggg cggaaaccgg gggcatggga
120ctggcagttc gccaagctcc tctgatcata cctctgaaag caacctctac
ccccgtgtcc 180ataaaacaat accccatgtc acaannngcc agactgggga
tcaagcccca catacagaga 240ctgttggacc agggaatact ggtaccctgc
cagtccccct ggaacacgcc cctgctaccc 300gttaagaaac cagggactaa
tgattatagg cctgtccagg atctgagaga agtcaacaag 360cgggtggaag
acatccaccc caccgtgccc aacccttaca acctcttgag cgggctccca
420ccgtcccacc agtggtacac tgtgcttgat ttaaaggatg cctttttctg
cctgagactc 480caccccacca gtcagcctct cttcgccttt gagtggagag
atccagagat gggaatctca 540ggacaattga cctggaccag actcccacag
ggtttcaaaa acagtcccac cctgtttgat 600gaggcactgc acagagacct
agcagacttc cggatccagc acccagactt gatcctgcta 660cagtacgtgg
atgacttact gctggccgcc acttctgagc tagactgcca acaaggtact
720cgggccctgt tacaaaccct agggaacctc gggtatcggg cctcggccaa
gaaagcccaa 780atttgccaga aacaggtcaa gtatctgggg tatcttctaa
aagagggtca gagatggctg 840actgaggcca gaaaagagac tgtgatgggg
cagcctactc cgaagacccc tcgacaacta 900aggnnnttcc tagggacggc
aggcttctgt cgcctctgga tccctgggtt tgcagaaatg 960gcagccccct
tgtaccctct caccaaaacg gggactctgt ttaattgggg cccagaccaa
1020caaaaggcct atcaagaaat caagcaagct cttctaactg ccccagccct
ggggttgcca 1080gatttgacta agccctttga actctttgtc gacgagaagc
agggctacgc caaaggtgtc 1140ctaacgcaaa aactgggacc ttggcgtcgg
ccggtggcct acctgtccaa aaagctagac 1200ccagtagcag ctgggtggcc
cccttgccta cggatggtag cagccattgc cgtactgaca 1260aaggatgcag
gcaagctaac catgggacag ccactagtca ttctggcccc ccatgcagta
1320gaggcactag tcaaacaacc ccccgaccgc tggctttccn nngcccggat
gactcactat 1380caggccttgc ttttggacac ggaccgggtc cagttcggac
cggtggtagc cctgaacccg 1440gctacgctgc tcccactgcc tgaggaaggg
ctgcaacaca actgccttga tatcctggcc 1500gaagcccacg gaacccgacc
cgacctaacg gaccagccgc tcccagacgc cgaccacacc 1560tggtacacgg
atggaagcag tctcttacaa gagggacagc gtaaggcggg agctgcggtg
1620accaccgaga ccgaggtaat ctgggctaaa gccctgccag ccgggacatc
cgctcagcgg 1680gctgaactga tagcactcac ccaggcccta aagatggcag
aaggtaagaa gctaaatgtt 1740tatactgata gccgttatgc ttttgctact
gcccatatcc atggagaaat atacagaagg 1800cgtgggttgc tcacatcaga
aggcaaagag atcaaaaata aagacgagat cttggcccta 1860ctaaaagccc
tctttctgcc caaaagactt agcataatcc attgtccagg acatcaaaag
1920ggacacagcg ccgaggctag aggcaaccgg atggctgacc aagcggcccg
aaaggcagcc 1980atcacagaga ctccagacac ctctaccctc ctc
2013172013DNAMoloney murine leukemia
virusmodified_base(904)..(906)This region may encompass 'aaa' or
'aag' 17accctaaata tagaagatga gcatcggcta catgagacct caaaagagcc
agatgtttct 60ctagggtcca catggctgtc tgattttcct caggcctggg cggaaaccgg
gggcatggga 120ctggcagttc gccaagctcc tctgatcata cctctgaaag
caacctctac ccccgtgtcc 180ataaaacaat accccatgtc acaagaagcc
agactgggga tcaagcccca catacagaga 240ctgttggacc agggaatact
ggtaccctgc cagtccccct ggaacacgcc cctgctaccc 300gttaagaaac
cagggactaa tgattatagg cctgtccagg atctgagaga agtcaacaag
360cgggtggaag acatccaccc caccgtgccc aacccttaca acctcttgag
cgggctccca 420ccgtcccacc agtggtacac tgtgcttgat ttaaaggatg
cctttttctg cctgagactc 480caccccacca gtcagcctct cttcgccttt
gagtggagag atccagagat gggaatctca 540ggacaattga cctggaccag
actcccacag ggtttcaaaa acagtcccac cctgtttgat 600gaggcactgc
acagagacct agcagacttc cggatccagc acccagactt gatcctgcta
660cagtacgtgg atgacttact gctggccgcc acttctgagc tagactgcca
acaaggtact 720cgggccctgt tacaaaccct agggaacctc gggtatcggg
cctcggccaa gaaagcccaa 780atttgccaga aacaggtcaa gtatctgggg
tatcttctaa aagagggtca gagatggctg 840actgaggcca gaaaagagac
tgtgatgggg cagcctactc cgaagacccc tcgacaacta 900aggnnnttcc
tagggacggc aggcttctgt cgcctcnnna tccctgggtt tgcagaaatg
960gcagccccct tgtaccctct caccaaaacg gggactctgt ttaattgggg
cccagaccaa 1020caaaaggcct atcaagaaat caagcaagct cttctaactg
ccccagccct ggggttgcca 1080gatttgacta agccctttga actctttgtc
gacgagaagc agggctacgc caaaggtgtc 1140ctaacgcaaa aactgggacc
ttggcgtcgg ccggtggcct acctgtccaa aaagctagac 1200ccagtagcag
ctgggtggcc cccttgccta cggatggtag cagccattgc cgtactgaca
1260aaggatgcag gcaagctaac catgggacag ccactagtca ttctggcccc
ccatgcagta 1320gaggcactag tcaaacaacc ccccgaccgc tggctttcca
acgcccggat gactcactat 1380caggccttgc ttttggacac ggaccgggtc
cagttcggac cggtggtagc cctgaacccg 1440gctacgctgc tcccactgcc
tgaggaaggg ctgcaacaca actgccttga tatcctggcc 1500gaagcccacg
gaacccgacc cgacctaacg gaccagccgc tcccagacgc cgaccacacc
1560tggtacacgg atggaagcag tctcttacaa gagggacagc gtaaggcggg
agctgcggtg 1620accaccgaga ccgaggtaat ctgggctaaa gccctgccag
ccgggacatc cgctcagcgg 1680gctgaactga tagcactcac ccaggcccta
aagatggcag aaggtaagaa gctaaatgtt 1740tatactgata gccgttatgc
ttttgctact gcccatatcc atggagaaat atacagaagg 1800cgtgggttgc
tcacatcaga aggcaaagag atcaaaaata aagacgagat cttggcccta
1860ctaaaagccc tctttctgcc caaaagactt agcataatcc attgtccagg
acatcaaaag 1920ggacacagcg ccgaggctag aggcaaccgg atggctgacc
aagcggcccg aaaggcagcc 1980atcacagaga ctccagacac ctctaccctc ctc
2013182013DNAMoloney murine leukemia
virusmodified_base(205)..(207)This region may encompass 'aaa' or
'aag' 18accctaaata tagaagatga gcatcggcta catgagacct caaaagagcc
agatgtttct 60ctagggtcca catggctgtc tgattttcct caggcctggg cggaaaccgg
gggcatggga 120ctggcagttc gccaagctcc tctgatcata cctctgaaag
caacctctac ccccgtgtcc 180ataaaacaat accccatgtc acaannngcc
agactgggga tcaagcccca catacagaga 240ctgttggacc agggaatact
ggtaccctgc cagtccccct ggaacacgcc cctgctaccc 300gttaagaaac
cagggactaa tgattatagg cctgtccagg atctgagaga agtcaacaag
360cgggtggaag acatccaccc caccgtgccc aacccttaca acctcttgag
cgggctccca 420ccgtcccacc agtggtacac tgtgcttgat ttaaaggatg
cctttttctg cctgagactc 480caccccacca gtcagcctct cttcgccttt
gagtggagag atccagagat gggaatctca 540ggacaattga cctggaccag
actcccacag ggtttcaaaa acagtcccac cctgtttgat 600gaggcactgc
acagagacct agcagacttc cggatccagc acccagactt gatcctgcta
660cagtacgtgg atgacttact gctggccgcc acttctgagc tagactgcca
acaaggtact 720cgggccctgt tacaaaccct agggaacctc gggtatcggg
cctcggccaa gaaagcccaa 780atttgccaga aacaggtcaa gtatctgggg
tatcttctaa aagagggtca gagatggctg 840actgaggcca gaaaagagac
tgtgatgggg cagcctactc cgaagacccc tcgacaacta 900aggnnnttcc
tagggacggc aggcttctgt cgcctcnnna tccctgggtt tgcagaaatg
960gcagccccct tgtaccctct caccaaaacg gggactctgt ttaattgggg
cccagaccaa 1020caaaaggcct atcaagaaat caagcaagct cttctaactg
ccccagccct ggggttgcca 1080gatttgacta agccctttga actctttgtc
gacgagaagc agggctacgc caaaggtgtc 1140ctaacgcaaa aactgggacc
ttggcgtcgg ccggtggcct acctgtccaa aaagctagac 1200ccagtagcag
ctgggtggcc cccttgccta cggatggtag cagccattgc cgtactgaca
1260aaggatgcag gcaagctaac catgggacag ccactagtca ttnnngcccc
ccatgcagta 1320gaggcactag tcaaacaacc ccccgaccgc tggctttccn
nngcccggat gactcactat 1380caggccttgc ttttggacac ggaccgggtc
cagttcggac cggtggtagc cctgaacccg 1440gctacgctgc tcccactgcc
tgaggaaggg ctgcaacaca actgccttga tatcctggcc 1500gaagcccacg
gaacccgacc cgacctaacg gaccagccgc tcccagacgc cgaccacacc
1560tggtacacgn nnggaagcag tctcttacaa gagggacagc gtaaggcggg
agctgcggtg 1620accaccgaga ccgaggtaat ctgggctaaa gccctgccag
ccgggacatc cgctcagcgg 1680gctgaactga tagcactcac ccaggcccta
aagatggcag aaggtaagaa gctaaatgtt 1740tatactgata gccgttatgc
ttttgctact gcccatatcc atggagaaat atacagaagg 1800cgtgggttgc
tcacatcaga aggcaaagag atcaaaaata aagacgagat cttggcccta
1860ctaaaagccc tctttctgcc caaaagactt agcataatcc attgtccagg
acatcaaaag 1920ggacacagcg ccgaggctag aggcaaccgg atggctgacc
aagcggcccg aaaggcagcc 1980atcacagaga ctccagacac ctctaccctc ctc
201319671PRTMoloney murine leukemia virus 19Thr Leu Asn Ile Glu Asp
Glu His Arg Leu His Glu Thr Ser Lys Glu 1 5 10 15 Pro Asp Val Ser
Leu Gly Ser Thr Trp Leu Ser Asp Phe Pro Gln Ala 20 25 30 Trp Ala
Glu Thr Gly Gly Met Gly Leu Ala Val Arg Gln Ala Pro Leu 35 40 45
Ile Ile Pro Leu Lys Ala Thr Ser Thr Pro Val Ser Ile Lys Gln Tyr 50
55 60 Pro Met Ser Gln Glu Ala Arg Leu Gly Ile Lys Pro His Ile Gln
Arg 65 70 75 80 Leu Leu Asp Gln Gly Ile Leu Val Pro Cys Gln Ser Pro
Trp Asn Thr 85 90 95 Pro Leu Leu Pro Val Lys Lys Pro Gly Thr Asn
Asp Tyr Arg Pro Val 100 105 110 Gln Asp Leu Arg Glu Val Asn Lys Arg
Val Glu Asp Ile His Pro Thr 115 120 125 Val Pro Asn Pro Tyr Asn Leu
Leu Ser Gly Leu Pro Pro Ser His Gln 130 135 140 Trp Tyr Thr Val Leu
Asp Leu Lys Asp Ala Phe Phe Cys Leu Arg Leu 145 150 155 160 His Pro
Thr Ser Gln Pro Leu Phe Ala Phe Glu Trp Arg Asp Pro Glu 165 170 175
Met Gly Ile Ser Gly Gln Leu Thr Trp Thr Arg Leu Pro Gln Gly Phe 180
185 190 Lys Asn Ser Pro Thr Leu Phe Asp Glu Ala Leu His Arg Asp Leu
Ala 195 200 205 Asp Phe Arg Ile Gln His Pro Asp Leu Ile Leu Leu Gln
Tyr Val Asp 210 215 220 Asp Leu Leu Leu Ala Ala Thr Ser Glu Leu Asp
Cys Gln Gln Gly Thr 225 230 235 240 Arg Ala Leu Leu Gln Thr Leu Gly
Asn Leu Gly Tyr Arg Ala Ser Ala 245 250 255 Lys Lys Ala Gln Ile Cys
Gln Lys Gln Val Lys Tyr Leu Gly Tyr Leu 260 265 270 Leu Lys Glu Gly
Gln Arg Trp Leu Thr Glu Ala Arg Lys Glu Thr Val 275 280 285 Met Gly
Gln Pro Thr Pro Lys Thr Pro Arg Gln Leu Arg Glu Phe Leu 290 295 300
Gly Thr Ala Gly Phe Cys Arg Leu Trp Ile Pro Gly Phe Ala Glu Met 305
310 315 320 Ala Ala Pro Leu Tyr Pro Leu Thr Lys Thr Gly Thr Leu Phe
Asn Trp 325 330 335 Gly Pro Asp Gln Gln Lys Ala Tyr Gln Glu Ile Lys
Gln Ala Leu Leu 340 345 350 Thr Ala Pro Ala Leu Gly Leu Pro Asp Leu
Thr Lys Pro Phe Glu Leu 355 360 365 Phe Val Asp Glu Lys Gln Gly Tyr
Ala Lys Gly Val Leu Thr Gln Lys 370 375 380 Leu Gly Pro Trp Arg Arg
Pro Val Ala Tyr Leu Ser Lys Lys Leu Asp 385 390 395 400 Pro Val Ala
Ala Gly Trp Pro Pro Cys Leu Arg Met Val Ala Ala Ile 405 410 415 Ala
Val Leu Thr Lys Asp Ala Gly Lys Leu Thr Met Gly Gln Pro Leu 420 425
430 Val Ile Leu Ala Pro His Ala Val Glu Ala Leu Val Lys Gln Pro Pro
435 440 445 Asp Arg Trp Leu Ser Asn Ala Arg Met Thr His Tyr Gln Ala
Leu Leu 450 455 460 Leu Asp Thr Asp Arg Val Gln Phe Gly Pro Val Val
Ala Leu Asn Pro 465 470 475 480 Ala Thr Leu Leu Pro Leu Pro Glu Glu
Gly Leu Gln His Asn Cys Leu 485 490 495 Asp Ile Leu Ala Glu Ala His
Gly Thr Arg Pro Asp Leu Thr Asp Gln 500 505 510 Pro Leu Pro Asp Ala
Asp His Thr Trp Tyr Thr Asp Gly Ser Ser Leu 515 520 525 Leu Gln Glu
Gly Gln Arg Lys Ala Gly Ala Ala Val Thr Thr Glu Thr 530 535 540 Glu
Val Ile Trp Ala Lys Ala Leu Pro Ala Gly Thr Ser Ala Gln Arg 545 550
555 560 Ala Glu Leu Ile Ala Leu Thr Gln Ala Leu Lys Met Ala Glu Gly
Lys 565 570 575 Lys Leu Asn Val Tyr Thr Asp Ser Arg Tyr Ala Phe Ala
Thr Ala His 580 585 590 Ile His Gly Glu Ile Tyr Arg Arg Arg Gly Leu
Leu Thr Ser Glu Gly 595 600 605 Lys Glu Ile Lys Asn Lys Asp Glu Ile
Leu Ala Leu Leu Lys Ala Leu 610 615 620 Phe Leu Pro Lys Arg Leu Ser
Ile Ile His Cys Pro Gly His Gln Lys 625 630 635 640 Gly His Ser Ala
Glu Ala Arg Gly Asn Arg Met Ala Asp Gln Ala Ala 645 650 655 Arg Lys
Ala Ala Ile Thr Glu Thr Pro Asp Thr Ser Thr Leu Leu 660 665 670
20671PRTMoloney murine leukemia virus 20Thr Leu Asn Ile Glu Asp Glu
His Arg Leu His Glu Thr Ser Lys Glu 1 5 10 15 Pro Asp Val Ser Leu
Gly Ser Thr Trp Leu Ser Asp Phe Pro Gln Ala 20 25 30 Trp Ala Glu
Thr Gly Gly Met Gly Leu Ala Val Arg Gln Ala Pro Leu 35 40 45 Ile
Ile Pro Leu Lys Ala Thr Ser Thr Pro Val Ser Ile Lys Gln Tyr 50 55
60 Pro Met Ser Gln Lys Ala Arg Leu Gly Ile Lys Pro His Ile Gln Arg
65 70 75 80 Leu Leu Asp Gln Gly Ile Leu Val Pro Cys Gln Ser Pro Trp
Asn Thr 85 90 95 Pro Leu Leu Pro Val Lys Lys Pro Gly Thr Asn Asp
Tyr Arg Pro Val 100 105 110 Gln Asp Leu Arg Glu Val Asn Lys Arg Val
Glu Asp Ile His Pro Thr 115 120 125 Val Pro Asn Pro Tyr Asn Leu Leu
Ser Gly Leu Pro Pro Ser His Gln 130 135 140 Trp Tyr Thr Val Leu Asp
Leu Lys Asp Ala Phe Phe Cys Leu Arg Leu 145 150 155 160 His Pro Thr
Ser Gln Pro Leu Phe Ala Phe Glu Trp Arg Asp Pro Glu 165 170 175 Met
Gly Ile Ser Gly Gln Leu Thr Trp Thr Arg Leu Pro Gln Gly Phe 180 185
190 Lys Asn Ser Pro Thr Leu Phe Asp Glu Ala Leu His Arg Asp Leu Ala
195 200 205 Asp Phe Arg Ile Gln His Pro Asp Leu Ile Leu Leu Gln Tyr
Val Asp 210 215 220 Asp Leu Leu Leu Ala Ala Thr Ser Glu Leu Asp Cys
Gln Gln Gly Thr 225 230 235 240 Arg Ala Leu Leu Gln Thr Leu Gly Asn
Leu Gly Tyr Arg Ala Ser Ala 245 250 255 Lys Lys Ala Gln Ile Cys Gln
Lys Gln Val Lys Tyr Leu Gly Tyr Leu 260 265 270 Leu Lys Glu Gly Gln
Arg Trp Leu Thr Glu Ala Arg Lys Glu Thr Val 275 280 285 Met Gly Gln
Pro Thr Pro Lys Thr Pro Arg Gln Leu Arg Glu Phe Leu 290 295 300 Gly
Thr Ala Gly Phe Cys Arg Leu Trp Ile Pro Gly Phe Ala Glu Met 305 310
315 320 Ala Ala Pro Leu Tyr Pro Leu Thr Lys Thr Gly Thr Leu Phe Asn
Trp 325 330 335 Gly Pro Asp Gln Gln Lys Ala Tyr Gln Glu Ile Lys Gln
Ala Leu Leu 340 345 350 Thr Ala Pro Ala Leu Gly Leu Pro Asp Leu Thr
Lys Pro Phe Glu Leu 355 360 365 Phe Val Asp Glu Lys Gln Gly Tyr Ala
Lys Gly Val Leu Thr Gln Lys 370 375 380 Leu Gly Pro Trp Arg Arg Pro
Val Ala Tyr Leu Ser Lys Lys Leu Asp 385 390 395 400 Pro Val Ala Ala
Gly Trp Pro Pro Cys Leu Arg Met Val Ala Ala Ile 405 410 415 Ala Val
Leu Thr Lys Asp Ala Gly Lys Leu Thr Met Gly Gln Pro Leu 420 425 430
Val Ile Leu Ala Pro His Ala Val Glu Ala Leu Val Lys Gln Pro Pro 435
440 445 Asp Arg Trp Leu Ser Asn Ala Arg Met Thr His Tyr Gln Ala Leu
Leu 450 455 460 Leu Asp Thr Asp Arg Val Gln Phe Gly Pro Val Val Ala
Leu Asn Pro 465 470 475 480 Ala Thr Leu Leu Pro Leu Pro Glu Glu Gly
Leu Gln His Asn Cys Leu 485 490 495 Asp Ile Leu Ala Glu Ala His Gly
Thr Arg Pro Asp Leu Thr Asp Gln 500 505 510 Pro Leu Pro Asp Ala Asp
His Thr Trp Tyr Thr Asp Gly Ser Ser Leu 515 520 525 Leu Gln Glu Gly
Gln Arg Lys Ala Gly Ala Ala Val Thr Thr Glu Thr 530 535 540 Glu Val
Ile Trp Ala Lys Ala Leu Pro Ala Gly Thr Ser Ala Gln Arg 545 550 555
560 Ala Glu Leu Ile Ala Leu Thr Gln Ala Leu Lys Met Ala Glu Gly Lys
565 570 575 Lys Leu Asn Val Tyr Thr Asp Ser Arg Tyr Ala Phe Ala Thr
Ala His 580 585 590 Ile His Gly Glu Ile Tyr Arg Arg Arg Gly Leu Leu
Thr Ser Glu Gly 595 600 605 Lys Glu Ile Lys Asn Lys Asp Glu Ile Leu
Ala Leu Leu Lys Ala Leu 610 615 620 Phe Leu Pro Lys Arg Leu Ser Ile
Ile His Cys Pro Gly His Gln Lys 625 630 635 640 Gly His Ser Ala Glu
Ala Arg Gly Asn Arg Met Ala Asp Gln Ala Ala 645 650 655 Arg Lys Ala
Ala Ile Thr Glu Thr
Pro Asp Thr Ser Thr Leu Leu 660 665 670 21671PRTMoloney murine
leukemia virus 21Thr Leu Asn Ile Glu Asp Glu His Arg Leu His Glu
Thr Ser Lys Glu 1 5 10 15 Pro Asp Val Ser Leu Gly Ser Thr Trp Leu
Ser Asp Phe Pro Gln Ala 20 25 30 Trp Ala Glu Thr Gly Gly Met Gly
Leu Ala Val Arg Gln Ala Pro Leu 35 40 45 Ile Ile Pro Leu Lys Ala
Thr Ser Thr Pro Val Ser Ile Lys Gln Tyr 50 55 60 Pro Met Ser Gln
Glu Ala Arg Leu Gly Ile Lys Pro His Ile Gln Arg 65 70 75 80 Leu Leu
Asp Gln Gly Ile Leu Val Pro Cys Gln Ser Pro Trp Asn Thr 85 90 95
Pro Leu Leu Pro Val Lys Lys Pro Gly Thr Asn Asp Tyr Arg Pro Val 100
105 110 Gln Asp Leu Arg Glu Val Asn Lys Arg Val Glu Asp Ile His Pro
Thr 115 120 125 Val Pro Asn Pro Tyr Asn Leu Leu Ser Gly Leu Pro Pro
Ser His Gln 130 135 140 Trp Tyr Thr Val Leu Asp Leu Lys Asp Ala Phe
Phe Cys Leu Arg Leu 145 150 155 160 His Pro Thr Ser Gln Pro Leu Phe
Ala Phe Glu Trp Arg Asp Pro Glu 165 170 175 Met Gly Ile Ser Gly Gln
Leu Thr Trp Thr Arg Leu Pro Gln Gly Phe 180 185 190 Lys Asn Ser Pro
Thr Leu Phe Asp Glu Ala Leu His Arg Asp Leu Ala 195 200 205 Asp Phe
Arg Ile Gln His Pro Asp Leu Ile Leu Leu Gln Tyr Val Asp 210 215 220
Asp Leu Leu Leu Ala Ala Thr Ser Glu Leu Asp Cys Gln Gln Gly Thr 225
230 235 240 Arg Ala Leu Leu Gln Thr Leu Gly Asn Leu Gly Tyr Arg Ala
Ser Ala 245 250 255 Lys Lys Ala Gln Ile Cys Gln Lys Gln Val Lys Tyr
Leu Gly Tyr Leu 260 265 270 Leu Lys Glu Gly Gln Arg Trp Leu Thr Glu
Ala Arg Lys Glu Thr Val 275 280 285 Met Gly Gln Pro Thr Pro Lys Thr
Pro Arg Gln Leu Arg Lys Phe Leu 290 295 300 Gly Thr Ala Gly Phe Cys
Arg Leu Trp Ile Pro Gly Phe Ala Glu Met 305 310 315 320 Ala Ala Pro
Leu Tyr Pro Leu Thr Lys Thr Gly Thr Leu Phe Asn Trp 325 330 335 Gly
Pro Asp Gln Gln Lys Ala Tyr Gln Glu Ile Lys Gln Ala Leu Leu 340 345
350 Thr Ala Pro Ala Leu Gly Leu Pro Asp Leu Thr Lys Pro Phe Glu Leu
355 360 365 Phe Val Asp Glu Lys Gln Gly Tyr Ala Lys Gly Val Leu Thr
Gln Lys 370 375 380 Leu Gly Pro Trp Arg Arg Pro Val Ala Tyr Leu Ser
Lys Lys Leu Asp 385 390 395 400 Pro Val Ala Ala Gly Trp Pro Pro Cys
Leu Arg Met Val Ala Ala Ile 405 410 415 Ala Val Leu Thr Lys Asp Ala
Gly Lys Leu Thr Met Gly Gln Pro Leu 420 425 430 Val Ile Leu Ala Pro
His Ala Val Glu Ala Leu Val Lys Gln Pro Pro 435 440 445 Asp Arg Trp
Leu Ser Asn Ala Arg Met Thr His Tyr Gln Ala Leu Leu 450 455 460 Leu
Asp Thr Asp Arg Val Gln Phe Gly Pro Val Val Ala Leu Asn Pro 465 470
475 480 Ala Thr Leu Leu Pro Leu Pro Glu Glu Gly Leu Gln His Asn Cys
Leu 485 490 495 Asp Ile Leu Ala Glu Ala His Gly Thr Arg Pro Asp Leu
Thr Asp Gln 500 505 510 Pro Leu Pro Asp Ala Asp His Thr Trp Tyr Thr
Asp Gly Ser Ser Leu 515 520 525 Leu Gln Glu Gly Gln Arg Lys Ala Gly
Ala Ala Val Thr Thr Glu Thr 530 535 540 Glu Val Ile Trp Ala Lys Ala
Leu Pro Ala Gly Thr Ser Ala Gln Arg 545 550 555 560 Ala Glu Leu Ile
Ala Leu Thr Gln Ala Leu Lys Met Ala Glu Gly Lys 565 570 575 Lys Leu
Asn Val Tyr Thr Asp Ser Arg Tyr Ala Phe Ala Thr Ala His 580 585 590
Ile His Gly Glu Ile Tyr Arg Arg Arg Gly Leu Leu Thr Ser Glu Gly 595
600 605 Lys Glu Ile Lys Asn Lys Asp Glu Ile Leu Ala Leu Leu Lys Ala
Leu 610 615 620 Phe Leu Pro Lys Arg Leu Ser Ile Ile His Cys Pro Gly
His Gln Lys 625 630 635 640 Gly His Ser Ala Glu Ala Arg Gly Asn Arg
Met Ala Asp Gln Ala Ala 645 650 655 Arg Lys Ala Ala Ile Thr Glu Thr
Pro Asp Thr Ser Thr Leu Leu 660 665 670 22671PRTMoloney murine
leukemia virus 22Thr Leu Asn Ile Glu Asp Glu His Arg Leu His Glu
Thr Ser Lys Glu 1 5 10 15 Pro Asp Val Ser Leu Gly Ser Thr Trp Leu
Ser Asp Phe Pro Gln Ala 20 25 30 Trp Ala Glu Thr Gly Gly Met Gly
Leu Ala Val Arg Gln Ala Pro Leu 35 40 45 Ile Ile Pro Leu Lys Ala
Thr Ser Thr Pro Val Ser Ile Lys Gln Tyr 50 55 60 Pro Met Ser Gln
Glu Ala Arg Leu Gly Ile Lys Pro His Ile Gln Arg 65 70 75 80 Leu Leu
Asp Gln Gly Ile Leu Val Pro Cys Gln Ser Pro Trp Asn Thr 85 90 95
Pro Leu Leu Pro Val Lys Lys Pro Gly Thr Asn Asp Tyr Arg Pro Val 100
105 110 Gln Asp Leu Arg Glu Val Asn Lys Arg Val Glu Asp Ile His Pro
Thr 115 120 125 Val Pro Asn Pro Tyr Asn Leu Leu Ser Gly Leu Pro Pro
Ser His Gln 130 135 140 Trp Tyr Thr Val Leu Asp Leu Lys Asp Ala Phe
Phe Cys Leu Arg Leu 145 150 155 160 His Pro Thr Ser Gln Pro Leu Phe
Ala Phe Glu Trp Arg Asp Pro Glu 165 170 175 Met Gly Ile Ser Gly Gln
Leu Thr Trp Thr Arg Leu Pro Gln Gly Phe 180 185 190 Lys Asn Ser Pro
Thr Leu Phe Asp Glu Ala Leu His Arg Asp Leu Ala 195 200 205 Asp Phe
Arg Ile Gln His Pro Asp Leu Ile Leu Leu Gln Tyr Val Asp 210 215 220
Asp Leu Leu Leu Ala Ala Thr Ser Glu Leu Asp Cys Gln Gln Gly Thr 225
230 235 240 Arg Ala Leu Leu Gln Thr Leu Gly Asn Leu Gly Tyr Arg Ala
Ser Ala 245 250 255 Lys Lys Ala Gln Ile Cys Gln Lys Gln Val Lys Tyr
Leu Gly Tyr Leu 260 265 270 Leu Lys Glu Gly Gln Arg Trp Leu Thr Glu
Ala Arg Lys Glu Thr Val 275 280 285 Met Gly Gln Pro Thr Pro Lys Thr
Pro Arg Gln Leu Arg Arg Phe Leu 290 295 300 Gly Thr Ala Gly Phe Cys
Arg Leu Trp Ile Pro Gly Phe Ala Glu Met 305 310 315 320 Ala Ala Pro
Leu Tyr Pro Leu Thr Lys Thr Gly Thr Leu Phe Asn Trp 325 330 335 Gly
Pro Asp Gln Gln Lys Ala Tyr Gln Glu Ile Lys Gln Ala Leu Leu 340 345
350 Thr Ala Pro Ala Leu Gly Leu Pro Asp Leu Thr Lys Pro Phe Glu Leu
355 360 365 Phe Val Asp Glu Lys Gln Gly Tyr Ala Lys Gly Val Leu Thr
Gln Lys 370 375 380 Leu Gly Pro Trp Arg Arg Pro Val Ala Tyr Leu Ser
Lys Lys Leu Asp 385 390 395 400 Pro Val Ala Ala Gly Trp Pro Pro Cys
Leu Arg Met Val Ala Ala Ile 405 410 415 Ala Val Leu Thr Lys Asp Ala
Gly Lys Leu Thr Met Gly Gln Pro Leu 420 425 430 Val Ile Leu Ala Pro
His Ala Val Glu Ala Leu Val Lys Gln Pro Pro 435 440 445 Asp Arg Trp
Leu Ser Asn Ala Arg Met Thr His Tyr Gln Ala Leu Leu 450 455 460 Leu
Asp Thr Asp Arg Val Gln Phe Gly Pro Val Val Ala Leu Asn Pro 465 470
475 480 Ala Thr Leu Leu Pro Leu Pro Glu Glu Gly Leu Gln His Asn Cys
Leu 485 490 495 Asp Ile Leu Ala Glu Ala His Gly Thr Arg Pro Asp Leu
Thr Asp Gln 500 505 510 Pro Leu Pro Asp Ala Asp His Thr Trp Tyr Thr
Asp Gly Ser Ser Leu 515 520 525 Leu Gln Glu Gly Gln Arg Lys Ala Gly
Ala Ala Val Thr Thr Glu Thr 530 535 540 Glu Val Ile Trp Ala Lys Ala
Leu Pro Ala Gly Thr Ser Ala Gln Arg 545 550 555 560 Ala Glu Leu Ile
Ala Leu Thr Gln Ala Leu Lys Met Ala Glu Gly Lys 565 570 575 Lys Leu
Asn Val Tyr Thr Asp Ser Arg Tyr Ala Phe Ala Thr Ala His 580 585 590
Ile His Gly Glu Ile Tyr Arg Arg Arg Gly Leu Leu Thr Ser Glu Gly 595
600 605 Lys Glu Ile Lys Asn Lys Asp Glu Ile Leu Ala Leu Leu Lys Ala
Leu 610 615 620 Phe Leu Pro Lys Arg Leu Ser Ile Ile His Cys Pro Gly
His Gln Lys 625 630 635 640 Gly His Ser Ala Glu Ala Arg Gly Asn Arg
Met Ala Asp Gln Ala Ala 645 650 655 Arg Lys Ala Ala Ile Thr Glu Thr
Pro Asp Thr Ser Thr Leu Leu 660 665 670 23671PRTMoloney murine
leukemia virus 23Thr Leu Asn Ile Glu Asp Glu His Arg Leu His Glu
Thr Ser Lys Glu 1 5 10 15 Pro Asp Val Ser Leu Gly Ser Thr Trp Leu
Ser Asp Phe Pro Gln Ala 20 25 30 Trp Ala Glu Thr Gly Gly Met Gly
Leu Ala Val Arg Gln Ala Pro Leu 35 40 45 Ile Ile Pro Leu Lys Ala
Thr Ser Thr Pro Val Ser Ile Lys Gln Tyr 50 55 60 Pro Met Ser Gln
Glu Ala Arg Leu Gly Ile Lys Pro His Ile Gln Arg 65 70 75 80 Leu Leu
Asp Gln Gly Ile Leu Val Pro Cys Gln Ser Pro Trp Asn Thr 85 90 95
Pro Leu Leu Pro Val Lys Lys Pro Gly Thr Asn Asp Tyr Arg Pro Val 100
105 110 Gln Asp Leu Arg Glu Val Asn Lys Arg Val Glu Asp Ile His Pro
Thr 115 120 125 Val Pro Asn Pro Tyr Asn Leu Leu Ser Gly Leu Pro Pro
Ser His Gln 130 135 140 Trp Tyr Thr Val Leu Asp Leu Lys Asp Ala Phe
Phe Cys Leu Arg Leu 145 150 155 160 His Pro Thr Ser Gln Pro Leu Phe
Ala Phe Glu Trp Arg Asp Pro Glu 165 170 175 Met Gly Ile Ser Gly Gln
Leu Thr Trp Thr Arg Leu Pro Gln Gly Phe 180 185 190 Lys Asn Ser Pro
Thr Leu Phe Asp Glu Ala Leu His Arg Asp Leu Ala 195 200 205 Asp Phe
Arg Ile Gln His Pro Asp Leu Ile Leu Leu Gln Tyr Val Asp 210 215 220
Asp Leu Leu Leu Ala Ala Thr Ser Glu Leu Asp Cys Gln Gln Gly Thr 225
230 235 240 Arg Ala Leu Leu Gln Thr Leu Gly Asn Leu Gly Tyr Arg Ala
Ser Ala 245 250 255 Lys Lys Ala Gln Ile Cys Gln Lys Gln Val Lys Tyr
Leu Gly Tyr Leu 260 265 270 Leu Lys Glu Gly Gln Arg Trp Leu Thr Glu
Ala Arg Lys Glu Thr Val 275 280 285 Met Gly Gln Pro Thr Pro Lys Thr
Pro Arg Gln Leu Arg Glu Phe Leu 290 295 300 Gly Thr Ala Gly Phe Cys
Arg Leu Phe Ile Pro Gly Phe Ala Glu Met 305 310 315 320 Ala Ala Pro
Leu Tyr Pro Leu Thr Lys Thr Gly Thr Leu Phe Asn Trp 325 330 335 Gly
Pro Asp Gln Gln Lys Ala Tyr Gln Glu Ile Lys Gln Ala Leu Leu 340 345
350 Thr Ala Pro Ala Leu Gly Leu Pro Asp Leu Thr Lys Pro Phe Glu Leu
355 360 365 Phe Val Asp Glu Lys Gln Gly Tyr Ala Lys Gly Val Leu Thr
Gln Lys 370 375 380 Leu Gly Pro Trp Arg Arg Pro Val Ala Tyr Leu Ser
Lys Lys Leu Asp 385 390 395 400 Pro Val Ala Ala Gly Trp Pro Pro Cys
Leu Arg Met Val Ala Ala Ile 405 410 415 Ala Val Leu Thr Lys Asp Ala
Gly Lys Leu Thr Met Gly Gln Pro Leu 420 425 430 Val Ile Leu Ala Pro
His Ala Val Glu Ala Leu Val Lys Gln Pro Pro 435 440 445 Asp Arg Trp
Leu Ser Asn Ala Arg Met Thr His Tyr Gln Ala Leu Leu 450 455 460 Leu
Asp Thr Asp Arg Val Gln Phe Gly Pro Val Val Ala Leu Asn Pro 465 470
475 480 Ala Thr Leu Leu Pro Leu Pro Glu Glu Gly Leu Gln His Asn Cys
Leu 485 490 495 Asp Ile Leu Ala Glu Ala His Gly Thr Arg Pro Asp Leu
Thr Asp Gln 500 505 510 Pro Leu Pro Asp Ala Asp His Thr Trp Tyr Thr
Asp Gly Ser Ser Leu 515 520 525 Leu Gln Glu Gly Gln Arg Lys Ala Gly
Ala Ala Val Thr Thr Glu Thr 530 535 540 Glu Val Ile Trp Ala Lys Ala
Leu Pro Ala Gly Thr Ser Ala Gln Arg 545 550 555 560 Ala Glu Leu Ile
Ala Leu Thr Gln Ala Leu Lys Met Ala Glu Gly Lys 565 570 575 Lys Leu
Asn Val Tyr Thr Asp Ser Arg Tyr Ala Phe Ala Thr Ala His 580 585 590
Ile His Gly Glu Ile Tyr Arg Arg Arg Gly Leu Leu Thr Ser Glu Gly 595
600 605 Lys Glu Ile Lys Asn Lys Asp Glu Ile Leu Ala Leu Leu Lys Ala
Leu 610 615 620 Phe Leu Pro Lys Arg Leu Ser Ile Ile His Cys Pro Gly
His Gln Lys 625 630 635 640 Gly His Ser Ala Glu Ala Arg Gly Asn Arg
Met Ala Asp Gln Ala Ala 645 650 655 Arg Lys Ala Ala Ile Thr Glu Thr
Pro Asp Thr Ser Thr Leu Leu 660 665 670 24671PRTMoloney murine
leukemia virus 24Thr Leu Asn Ile Glu Asp Glu His Arg Leu His Glu
Thr Ser Lys Glu 1 5 10 15 Pro Asp Val Ser Leu Gly Ser Thr Trp Leu
Ser Asp Phe Pro Gln Ala 20 25 30 Trp Ala Glu Thr Gly Gly Met Gly
Leu Ala Val Arg Gln Ala Pro Leu 35 40 45 Ile Ile Pro Leu Lys Ala
Thr Ser Thr Pro Val Ser Ile Lys Gln Tyr 50 55 60 Pro Met Ser Gln
Glu Ala Arg Leu Gly Ile Lys Pro His Ile Gln Arg 65 70 75 80 Leu Leu
Asp Gln Gly Ile Leu Val Pro Cys Gln Ser Pro Trp Asn Thr 85 90 95
Pro Leu Leu Pro Val Lys Lys Pro Gly Thr Asn Asp Tyr Arg Pro Val 100
105 110 Gln Asp Leu Arg Glu Val Asn Lys Arg Val Glu Asp Ile His Pro
Thr 115 120 125 Val Pro Asn Pro Tyr Asn Leu Leu Ser Gly Leu Pro Pro
Ser His Gln 130 135 140 Trp Tyr Thr Val Leu Asp Leu Lys Asp Ala Phe
Phe Cys Leu Arg Leu 145 150 155 160 His Pro Thr Ser Gln Pro Leu Phe
Ala Phe Glu Trp Arg Asp Pro Glu 165 170 175 Met Gly Ile Ser Gly Gln
Leu Thr Trp Thr Arg Leu Pro Gln Gly Phe 180 185 190 Lys Asn Ser Pro
Thr Leu Phe Asp Glu Ala Leu His Arg Asp Leu Ala 195 200 205 Asp Phe
Arg Ile Gln His Pro Asp Leu Ile Leu Leu Gln Tyr Val Asp 210 215 220
Asp Leu Leu Leu Ala Ala Thr Ser Glu Leu Asp Cys Gln Gln Gly Thr 225
230 235 240 Arg Ala Leu Leu Gln Thr Leu Gly Asn Leu Gly Tyr Arg Ala
Ser Ala 245 250 255 Lys Lys Ala Gln Ile Cys Gln Lys Gln Val Lys Tyr
Leu Gly Tyr Leu
260 265 270 Leu Lys Glu Gly Gln Arg Trp Leu Thr Glu Ala Arg Lys Glu
Thr Val 275 280 285 Met Gly Gln Pro Thr Pro Lys Thr Pro Arg Gln Leu
Arg Glu Phe Leu 290 295 300 Gly Thr Ala Gly Phe Cys Arg Leu Trp Ile
Pro Gly Phe Ala Glu Met 305 310 315 320 Ala Ala Pro Leu Tyr Pro Leu
Thr Lys Thr Gly Thr Leu Phe Asn Trp 325 330 335 Gly Pro Asp Gln Gln
Lys Ala Tyr Gln Glu Ile Lys Gln Ala Leu Leu 340 345 350 Thr Ala Pro
Ala Leu Gly Leu Pro Asp Leu Thr Lys Pro Phe Glu Leu 355 360 365 Phe
Val Asp Glu Lys Gln Gly Tyr Ala Lys Gly Val Leu Thr Gln Lys 370 375
380 Leu Gly Pro Trp Arg Arg Pro Val Ala Tyr Leu Ser Lys Lys Leu Asp
385 390 395 400 Pro Val Ala Ala Gly Trp Pro Pro Cys Leu Arg Met Val
Ala Ala Ile 405 410 415 Ala Val Leu Thr Lys Asp Ala Gly Lys Leu Thr
Met Gly Gln Pro Leu 420 425 430 Val Ile Gly Ala Pro His Ala Val Glu
Ala Leu Val Lys Gln Pro Pro 435 440 445 Asp Arg Trp Leu Ser Asn Ala
Arg Met Thr His Tyr Gln Ala Leu Leu 450 455 460 Leu Asp Thr Asp Arg
Val Gln Phe Gly Pro Val Val Ala Leu Asn Pro 465 470 475 480 Ala Thr
Leu Leu Pro Leu Pro Glu Glu Gly Leu Gln His Asn Cys Leu 485 490 495
Asp Ile Leu Ala Glu Ala His Gly Thr Arg Pro Asp Leu Thr Asp Gln 500
505 510 Pro Leu Pro Asp Ala Asp His Thr Trp Tyr Thr Asp Gly Ser Ser
Leu 515 520 525 Leu Gln Glu Gly Gln Arg Lys Ala Gly Ala Ala Val Thr
Thr Glu Thr 530 535 540 Glu Val Ile Trp Ala Lys Ala Leu Pro Ala Gly
Thr Ser Ala Gln Arg 545 550 555 560 Ala Glu Leu Ile Ala Leu Thr Gln
Ala Leu Lys Met Ala Glu Gly Lys 565 570 575 Lys Leu Asn Val Tyr Thr
Asp Ser Arg Tyr Ala Phe Ala Thr Ala His 580 585 590 Ile His Gly Glu
Ile Tyr Arg Arg Arg Gly Leu Leu Thr Ser Glu Gly 595 600 605 Lys Glu
Ile Lys Asn Lys Asp Glu Ile Leu Ala Leu Leu Lys Ala Leu 610 615 620
Phe Leu Pro Lys Arg Leu Ser Ile Ile His Cys Pro Gly His Gln Lys 625
630 635 640 Gly His Ser Ala Glu Ala Arg Gly Asn Arg Met Ala Asp Gln
Ala Ala 645 650 655 Arg Lys Ala Ala Ile Thr Glu Thr Pro Asp Thr Ser
Thr Leu Leu 660 665 670 25671PRTMoloney murine leukemia virus 25Thr
Leu Asn Ile Glu Asp Glu His Arg Leu His Glu Thr Ser Lys Glu 1 5 10
15 Pro Asp Val Ser Leu Gly Ser Thr Trp Leu Ser Asp Phe Pro Gln Ala
20 25 30 Trp Ala Glu Thr Gly Gly Met Gly Leu Ala Val Arg Gln Ala
Pro Leu 35 40 45 Ile Ile Pro Leu Lys Ala Thr Ser Thr Pro Val Ser
Ile Lys Gln Tyr 50 55 60 Pro Met Ser Gln Glu Ala Arg Leu Gly Ile
Lys Pro His Ile Gln Arg 65 70 75 80 Leu Leu Asp Gln Gly Ile Leu Val
Pro Cys Gln Ser Pro Trp Asn Thr 85 90 95 Pro Leu Leu Pro Val Lys
Lys Pro Gly Thr Asn Asp Tyr Arg Pro Val 100 105 110 Gln Asp Leu Arg
Glu Val Asn Lys Arg Val Glu Asp Ile His Pro Thr 115 120 125 Val Pro
Asn Pro Tyr Asn Leu Leu Ser Gly Leu Pro Pro Ser His Gln 130 135 140
Trp Tyr Thr Val Leu Asp Leu Lys Asp Ala Phe Phe Cys Leu Arg Leu 145
150 155 160 His Pro Thr Ser Gln Pro Leu Phe Ala Phe Glu Trp Arg Asp
Pro Glu 165 170 175 Met Gly Ile Ser Gly Gln Leu Thr Trp Thr Arg Leu
Pro Gln Gly Phe 180 185 190 Lys Asn Ser Pro Thr Leu Phe Asp Glu Ala
Leu His Arg Asp Leu Ala 195 200 205 Asp Phe Arg Ile Gln His Pro Asp
Leu Ile Leu Leu Gln Tyr Val Asp 210 215 220 Asp Leu Leu Leu Ala Ala
Thr Ser Glu Leu Asp Cys Gln Gln Gly Thr 225 230 235 240 Arg Ala Leu
Leu Gln Thr Leu Gly Asn Leu Gly Tyr Arg Ala Ser Ala 245 250 255 Lys
Lys Ala Gln Ile Cys Gln Lys Gln Val Lys Tyr Leu Gly Tyr Leu 260 265
270 Leu Lys Glu Gly Gln Arg Trp Leu Thr Glu Ala Arg Lys Glu Thr Val
275 280 285 Met Gly Gln Pro Thr Pro Lys Thr Pro Arg Gln Leu Arg Glu
Phe Leu 290 295 300 Gly Thr Ala Gly Phe Cys Arg Leu Trp Ile Pro Gly
Phe Ala Glu Met 305 310 315 320 Ala Ala Pro Leu Tyr Pro Leu Thr Lys
Thr Gly Thr Leu Phe Asn Trp 325 330 335 Gly Pro Asp Gln Gln Lys Ala
Tyr Gln Glu Ile Lys Gln Ala Leu Leu 340 345 350 Thr Ala Pro Ala Leu
Gly Leu Pro Asp Leu Thr Lys Pro Phe Glu Leu 355 360 365 Phe Val Asp
Glu Lys Gln Gly Tyr Ala Lys Gly Val Leu Thr Gln Lys 370 375 380 Leu
Gly Pro Trp Arg Arg Pro Val Ala Tyr Leu Ser Lys Lys Leu Asp 385 390
395 400 Pro Val Ala Ala Gly Trp Pro Pro Cys Leu Arg Met Val Ala Ala
Ile 405 410 415 Ala Val Leu Thr Lys Asp Ala Gly Lys Leu Thr Met Gly
Gln Pro Leu 420 425 430 Val Ile Met Ala Pro His Ala Val Glu Ala Leu
Val Lys Gln Pro Pro 435 440 445 Asp Arg Trp Leu Ser Asn Ala Arg Met
Thr His Tyr Gln Ala Leu Leu 450 455 460 Leu Asp Thr Asp Arg Val Gln
Phe Gly Pro Val Val Ala Leu Asn Pro 465 470 475 480 Ala Thr Leu Leu
Pro Leu Pro Glu Glu Gly Leu Gln His Asn Cys Leu 485 490 495 Asp Ile
Leu Ala Glu Ala His Gly Thr Arg Pro Asp Leu Thr Asp Gln 500 505 510
Pro Leu Pro Asp Ala Asp His Thr Trp Tyr Thr Asp Gly Ser Ser Leu 515
520 525 Leu Gln Glu Gly Gln Arg Lys Ala Gly Ala Ala Val Thr Thr Glu
Thr 530 535 540 Glu Val Ile Trp Ala Lys Ala Leu Pro Ala Gly Thr Ser
Ala Gln Arg 545 550 555 560 Ala Glu Leu Ile Ala Leu Thr Gln Ala Leu
Lys Met Ala Glu Gly Lys 565 570 575 Lys Leu Asn Val Tyr Thr Asp Ser
Arg Tyr Ala Phe Ala Thr Ala His 580 585 590 Ile His Gly Glu Ile Tyr
Arg Arg Arg Gly Leu Leu Thr Ser Glu Gly 595 600 605 Lys Glu Ile Lys
Asn Lys Asp Glu Ile Leu Ala Leu Leu Lys Ala Leu 610 615 620 Phe Leu
Pro Lys Arg Leu Ser Ile Ile His Cys Pro Gly His Gln Lys 625 630 635
640 Gly His Ser Ala Glu Ala Arg Gly Asn Arg Met Ala Asp Gln Ala Ala
645 650 655 Arg Lys Ala Ala Ile Thr Glu Thr Pro Asp Thr Ser Thr Leu
Leu 660 665 670 26671PRTMoloney murine leukemia virus 26Thr Leu Asn
Ile Glu Asp Glu His Arg Leu His Glu Thr Ser Lys Glu 1 5 10 15 Pro
Asp Val Ser Leu Gly Ser Thr Trp Leu Ser Asp Phe Pro Gln Ala 20 25
30 Trp Ala Glu Thr Gly Gly Met Gly Leu Ala Val Arg Gln Ala Pro Leu
35 40 45 Ile Ile Pro Leu Lys Ala Thr Ser Thr Pro Val Ser Ile Lys
Gln Tyr 50 55 60 Pro Met Ser Gln Glu Ala Arg Leu Gly Ile Lys Pro
His Ile Gln Arg 65 70 75 80 Leu Leu Asp Gln Gly Ile Leu Val Pro Cys
Gln Ser Pro Trp Asn Thr 85 90 95 Pro Leu Leu Pro Val Lys Lys Pro
Gly Thr Asn Asp Tyr Arg Pro Val 100 105 110 Gln Asp Leu Arg Glu Val
Asn Lys Arg Val Glu Asp Ile His Pro Thr 115 120 125 Val Pro Asn Pro
Tyr Asn Leu Leu Ser Gly Leu Pro Pro Ser His Gln 130 135 140 Trp Tyr
Thr Val Leu Asp Leu Lys Asp Ala Phe Phe Cys Leu Arg Leu 145 150 155
160 His Pro Thr Ser Gln Pro Leu Phe Ala Phe Glu Trp Arg Asp Pro Glu
165 170 175 Met Gly Ile Ser Gly Gln Leu Thr Trp Thr Arg Leu Pro Gln
Gly Phe 180 185 190 Lys Asn Ser Pro Thr Leu Phe Asp Glu Ala Leu His
Arg Asp Leu Ala 195 200 205 Asp Phe Arg Ile Gln His Pro Asp Leu Ile
Leu Leu Gln Tyr Val Asp 210 215 220 Asp Leu Leu Leu Ala Ala Thr Ser
Glu Leu Asp Cys Gln Gln Gly Thr 225 230 235 240 Arg Ala Leu Leu Gln
Thr Leu Gly Asn Leu Gly Tyr Arg Ala Ser Ala 245 250 255 Lys Lys Ala
Gln Ile Cys Gln Lys Gln Val Lys Tyr Leu Gly Tyr Leu 260 265 270 Leu
Lys Glu Gly Gln Arg Trp Leu Thr Glu Ala Arg Lys Glu Thr Val 275 280
285 Met Gly Gln Pro Thr Pro Lys Thr Pro Arg Gln Leu Arg Glu Phe Leu
290 295 300 Gly Thr Ala Gly Phe Cys Arg Leu Trp Ile Pro Gly Phe Ala
Glu Met 305 310 315 320 Ala Ala Pro Leu Tyr Pro Leu Thr Lys Thr Gly
Thr Leu Phe Asn Trp 325 330 335 Gly Pro Asp Gln Gln Lys Ala Tyr Gln
Glu Ile Lys Gln Ala Leu Leu 340 345 350 Thr Ala Pro Ala Leu Gly Leu
Pro Asp Leu Thr Lys Pro Phe Glu Leu 355 360 365 Phe Val Asp Glu Lys
Gln Gly Tyr Ala Lys Gly Val Leu Thr Gln Lys 370 375 380 Leu Gly Pro
Trp Arg Arg Pro Val Ala Tyr Leu Ser Lys Lys Leu Asp 385 390 395 400
Pro Val Ala Ala Gly Trp Pro Pro Cys Leu Arg Met Val Ala Ala Ile 405
410 415 Ala Val Leu Thr Lys Asp Ala Gly Lys Leu Thr Met Gly Gln Pro
Leu 420 425 430 Val Ile Leu Ala Pro His Ala Val Glu Ala Leu Val Lys
Gln Pro Pro 435 440 445 Asp Arg Trp Leu Ser Lys Ala Arg Met Thr His
Tyr Gln Ala Leu Leu 450 455 460 Leu Asp Thr Asp Arg Val Gln Phe Gly
Pro Val Val Ala Leu Asn Pro 465 470 475 480 Ala Thr Leu Leu Pro Leu
Pro Glu Glu Gly Leu Gln His Asn Cys Leu 485 490 495 Asp Ile Leu Ala
Glu Ala His Gly Thr Arg Pro Asp Leu Thr Asp Gln 500 505 510 Pro Leu
Pro Asp Ala Asp His Thr Trp Tyr Thr Asp Gly Ser Ser Leu 515 520 525
Leu Gln Glu Gly Gln Arg Lys Ala Gly Ala Ala Val Thr Thr Glu Thr 530
535 540 Glu Val Ile Trp Ala Lys Ala Leu Pro Ala Gly Thr Ser Ala Gln
Arg 545 550 555 560 Ala Glu Leu Ile Ala Leu Thr Gln Ala Leu Lys Met
Ala Glu Gly Lys 565 570 575 Lys Leu Asn Val Tyr Thr Asp Ser Arg Tyr
Ala Phe Ala Thr Ala His 580 585 590 Ile His Gly Glu Ile Tyr Arg Arg
Arg Gly Leu Leu Thr Ser Glu Gly 595 600 605 Lys Glu Ile Lys Asn Lys
Asp Glu Ile Leu Ala Leu Leu Lys Ala Leu 610 615 620 Phe Leu Pro Lys
Arg Leu Ser Ile Ile His Cys Pro Gly His Gln Lys 625 630 635 640 Gly
His Ser Ala Glu Ala Arg Gly Asn Arg Met Ala Asp Gln Ala Ala 645 650
655 Arg Lys Ala Ala Ile Thr Glu Thr Pro Asp Thr Ser Thr Leu Leu 660
665 670 27671PRTMoloney murine leukemia virus 27Thr Leu Asn Ile Glu
Asp Glu His Arg Leu His Glu Thr Ser Lys Glu 1 5 10 15 Pro Asp Val
Ser Leu Gly Ser Thr Trp Leu Ser Asp Phe Pro Gln Ala 20 25 30 Trp
Ala Glu Thr Gly Gly Met Gly Leu Ala Val Arg Gln Ala Pro Leu 35 40
45 Ile Ile Pro Leu Lys Ala Thr Ser Thr Pro Val Ser Ile Lys Gln Tyr
50 55 60 Pro Met Ser Gln Glu Ala Arg Leu Gly Ile Lys Pro His Ile
Gln Arg 65 70 75 80 Leu Leu Asp Gln Gly Ile Leu Val Pro Cys Gln Ser
Pro Trp Asn Thr 85 90 95 Pro Leu Leu Pro Val Lys Lys Pro Gly Thr
Asn Asp Tyr Arg Pro Val 100 105 110 Gln Asp Leu Arg Glu Val Asn Lys
Arg Val Glu Asp Ile His Pro Thr 115 120 125 Val Pro Asn Pro Tyr Asn
Leu Leu Ser Gly Leu Pro Pro Ser His Gln 130 135 140 Trp Tyr Thr Val
Leu Asp Leu Lys Asp Ala Phe Phe Cys Leu Arg Leu 145 150 155 160 His
Pro Thr Ser Gln Pro Leu Phe Ala Phe Glu Trp Arg Asp Pro Glu 165 170
175 Met Gly Ile Ser Gly Gln Leu Thr Trp Thr Arg Leu Pro Gln Gly Phe
180 185 190 Lys Asn Ser Pro Thr Leu Phe Asp Glu Ala Leu His Arg Asp
Leu Ala 195 200 205 Asp Phe Arg Ile Gln His Pro Asp Leu Ile Leu Leu
Gln Tyr Val Asp 210 215 220 Asp Leu Leu Leu Ala Ala Thr Ser Glu Leu
Asp Cys Gln Gln Gly Thr 225 230 235 240 Arg Ala Leu Leu Gln Thr Leu
Gly Asn Leu Gly Tyr Arg Ala Ser Ala 245 250 255 Lys Lys Ala Gln Ile
Cys Gln Lys Gln Val Lys Tyr Leu Gly Tyr Leu 260 265 270 Leu Lys Glu
Gly Gln Arg Trp Leu Thr Glu Ala Arg Lys Glu Thr Val 275 280 285 Met
Gly Gln Pro Thr Pro Lys Thr Pro Arg Gln Leu Arg Glu Phe Leu 290 295
300 Gly Thr Ala Gly Phe Cys Arg Leu Trp Ile Pro Gly Phe Ala Glu Met
305 310 315 320 Ala Ala Pro Leu Tyr Pro Leu Thr Lys Thr Gly Thr Leu
Phe Asn Trp 325 330 335 Gly Pro Asp Gln Gln Lys Ala Tyr Gln Glu Ile
Lys Gln Ala Leu Leu 340 345 350 Thr Ala Pro Ala Leu Gly Leu Pro Asp
Leu Thr Lys Pro Phe Glu Leu 355 360 365 Phe Val Asp Glu Lys Gln Gly
Tyr Ala Lys Gly Val Leu Thr Gln Lys 370 375 380 Leu Gly Pro Trp Arg
Arg Pro Val Ala Tyr Leu Ser Lys Lys Leu Asp 385 390 395 400 Pro Val
Ala Ala Gly Trp Pro Pro Cys Leu Arg Met Val Ala Ala Ile 405 410 415
Ala Val Leu Thr Lys Asp Ala Gly Lys Leu Thr Met Gly Gln Pro Leu 420
425 430 Val Ile Leu Ala Pro His Ala Val Glu Ala Leu Val Lys Gln Pro
Pro 435 440 445 Asp Arg Trp Leu Ser Arg Ala Arg Met Thr His Tyr Gln
Ala Leu Leu 450 455 460 Leu Asp Thr Asp Arg Val Gln Phe Gly Pro Val
Val Ala Leu Asn Pro 465 470 475 480 Ala Thr Leu Leu Pro Leu Pro Glu
Glu Gly Leu Gln His Asn Cys Leu 485 490 495 Asp Ile Leu Ala Glu Ala
His Gly Thr Arg Pro Asp Leu Thr Asp Gln 500 505 510 Pro Leu Pro Asp
Ala Asp His Thr Trp Tyr Thr Asp Gly Ser Ser Leu 515 520 525 Leu Gln
Glu Gly Gln Arg Lys Ala Gly Ala Ala Val Thr Thr Glu Thr 530 535
540
Glu Val Ile Trp Ala Lys Ala Leu Pro Ala Gly Thr Ser Ala Gln Arg 545
550 555 560 Ala Glu Leu Ile Ala Leu Thr Gln Ala Leu Lys Met Ala Glu
Gly Lys 565 570 575 Lys Leu Asn Val Tyr Thr Asp Ser Arg Tyr Ala Phe
Ala Thr Ala His 580 585 590 Ile His Gly Glu Ile Tyr Arg Arg Arg Gly
Leu Leu Thr Ser Glu Gly 595 600 605 Lys Glu Ile Lys Asn Lys Asp Glu
Ile Leu Ala Leu Leu Lys Ala Leu 610 615 620 Phe Leu Pro Lys Arg Leu
Ser Ile Ile His Cys Pro Gly His Gln Lys 625 630 635 640 Gly His Ser
Ala Glu Ala Arg Gly Asn Arg Met Ala Asp Gln Ala Ala 645 650 655 Arg
Lys Ala Ala Ile Thr Glu Thr Pro Asp Thr Ser Thr Leu Leu 660 665 670
28671PRTMoloney murine leukemia virus 28Thr Leu Asn Ile Glu Asp Glu
His Arg Leu His Glu Thr Ser Lys Glu 1 5 10 15 Pro Asp Val Ser Leu
Gly Ser Thr Trp Leu Ser Asp Phe Pro Gln Ala 20 25 30 Trp Ala Glu
Thr Gly Gly Met Gly Leu Ala Val Arg Gln Ala Pro Leu 35 40 45 Ile
Ile Pro Leu Lys Ala Thr Ser Thr Pro Val Ser Ile Lys Gln Tyr 50 55
60 Pro Met Ser Gln Glu Ala Arg Leu Gly Ile Lys Pro His Ile Gln Arg
65 70 75 80 Leu Leu Asp Gln Gly Ile Leu Val Pro Cys Gln Ser Pro Trp
Asn Thr 85 90 95 Pro Leu Leu Pro Val Lys Lys Pro Gly Thr Asn Asp
Tyr Arg Pro Val 100 105 110 Gln Asp Leu Arg Glu Val Asn Lys Arg Val
Glu Asp Ile His Pro Thr 115 120 125 Val Pro Asn Pro Tyr Asn Leu Leu
Ser Gly Leu Pro Pro Ser His Gln 130 135 140 Trp Tyr Thr Val Leu Asp
Leu Lys Asp Ala Phe Phe Cys Leu Arg Leu 145 150 155 160 His Pro Thr
Ser Gln Pro Leu Phe Ala Phe Glu Trp Arg Asp Pro Glu 165 170 175 Met
Gly Ile Ser Gly Gln Leu Thr Trp Thr Arg Leu Pro Gln Gly Phe 180 185
190 Lys Asn Ser Pro Thr Leu Phe Asp Glu Ala Leu His Arg Asp Leu Ala
195 200 205 Asp Phe Arg Ile Gln His Pro Asp Leu Ile Leu Leu Gln Tyr
Val Asp 210 215 220 Asp Leu Leu Leu Ala Ala Thr Ser Glu Leu Asp Cys
Gln Gln Gly Thr 225 230 235 240 Arg Ala Leu Leu Gln Thr Leu Gly Asn
Leu Gly Tyr Arg Ala Ser Ala 245 250 255 Lys Lys Ala Gln Ile Cys Gln
Lys Gln Val Lys Tyr Leu Gly Tyr Leu 260 265 270 Leu Lys Glu Gly Gln
Arg Trp Leu Thr Glu Ala Arg Lys Glu Thr Val 275 280 285 Met Gly Gln
Pro Thr Pro Lys Thr Pro Arg Gln Leu Arg Glu Phe Leu 290 295 300 Gly
Thr Ala Gly Phe Cys Arg Leu Trp Ile Pro Gly Phe Ala Glu Met 305 310
315 320 Ala Ala Pro Leu Tyr Pro Leu Thr Lys Thr Gly Thr Leu Phe Asn
Trp 325 330 335 Gly Pro Asp Gln Gln Lys Ala Tyr Gln Glu Ile Lys Gln
Ala Leu Leu 340 345 350 Thr Ala Pro Ala Leu Gly Leu Pro Asp Leu Thr
Lys Pro Phe Glu Leu 355 360 365 Phe Val Asp Glu Lys Gln Gly Tyr Ala
Lys Gly Val Leu Thr Gln Lys 370 375 380 Leu Gly Pro Trp Arg Arg Pro
Val Ala Tyr Leu Ser Lys Lys Leu Asp 385 390 395 400 Pro Val Ala Ala
Gly Trp Pro Pro Cys Leu Arg Met Val Ala Ala Ile 405 410 415 Ala Val
Leu Thr Lys Asp Ala Gly Lys Leu Thr Met Gly Gln Pro Leu 420 425 430
Val Ile Leu Ala Pro His Ala Val Glu Ala Leu Val Lys Gln Pro Pro 435
440 445 Asp Arg Trp Leu Ser Asn Ala Arg Met Thr His Tyr Gln Ala Leu
Leu 450 455 460 Leu Asp Thr Asp Arg Val Gln Phe Gly Pro Val Val Ala
Leu Asn Pro 465 470 475 480 Ala Thr Leu Leu Pro Leu Pro Glu Glu Gly
Leu Gln His Asn Cys Leu 485 490 495 Asp Ile Leu Ala Glu Ala His Gly
Thr Arg Pro Asp Leu Thr Asp Gln 500 505 510 Pro Leu Pro Asp Ala Asp
His Thr Trp Tyr Thr Asn Gly Ser Ser Leu 515 520 525 Leu Gln Glu Gly
Gln Arg Lys Ala Gly Ala Ala Val Thr Thr Glu Thr 530 535 540 Glu Val
Ile Trp Ala Lys Ala Leu Pro Ala Gly Thr Ser Ala Gln Arg 545 550 555
560 Ala Glu Leu Ile Ala Leu Thr Gln Ala Leu Lys Met Ala Glu Gly Lys
565 570 575 Lys Leu Asn Val Tyr Thr Asp Ser Arg Tyr Ala Phe Ala Thr
Ala His 580 585 590 Ile His Gly Glu Ile Tyr Arg Arg Arg Gly Leu Leu
Thr Ser Glu Gly 595 600 605 Lys Glu Ile Lys Asn Lys Asp Glu Ile Leu
Ala Leu Leu Lys Ala Leu 610 615 620 Phe Leu Pro Lys Arg Leu Ser Ile
Ile His Cys Pro Gly His Gln Lys 625 630 635 640 Gly His Ser Ala Glu
Ala Arg Gly Asn Arg Met Ala Asp Gln Ala Ala 645 650 655 Arg Lys Ala
Ala Ile Thr Glu Thr Pro Asp Thr Ser Thr Leu Leu 660 665 670
29671PRTMoloney murine leukemia virus 29Thr Leu Asn Ile Glu Asp Glu
His Arg Leu His Glu Thr Ser Lys Glu 1 5 10 15 Pro Asp Val Ser Leu
Gly Ser Thr Trp Leu Ser Asp Phe Pro Gln Ala 20 25 30 Trp Ala Glu
Thr Gly Gly Met Gly Leu Ala Val Arg Gln Ala Pro Leu 35 40 45 Ile
Ile Pro Leu Lys Ala Thr Ser Thr Pro Val Ser Ile Lys Gln Tyr 50 55
60 Pro Met Ser Gln Glu Ala Arg Leu Gly Ile Lys Pro His Ile Gln Arg
65 70 75 80 Leu Leu Asp Gln Gly Ile Leu Val Pro Cys Gln Ser Pro Trp
Asn Thr 85 90 95 Pro Leu Leu Pro Val Lys Lys Pro Gly Thr Asn Asp
Tyr Arg Pro Val 100 105 110 Gln Asp Leu Arg Glu Val Asn Lys Arg Val
Glu Asp Ile His Pro Thr 115 120 125 Val Pro Asn Pro Tyr Asn Leu Leu
Ser Gly Leu Pro Pro Ser His Gln 130 135 140 Trp Tyr Thr Val Leu Asp
Leu Lys Asp Ala Phe Phe Cys Leu Arg Leu 145 150 155 160 His Pro Thr
Ser Gln Pro Leu Phe Ala Phe Glu Trp Arg Asp Pro Glu 165 170 175 Met
Gly Ile Ser Gly Gln Leu Thr Trp Thr Arg Leu Pro Gln Gly Phe 180 185
190 Lys Asn Ser Pro Thr Leu Phe Asp Glu Ala Leu His Arg Asp Leu Ala
195 200 205 Asp Phe Arg Ile Gln His Pro Asp Leu Ile Leu Leu Gln Tyr
Val Asp 210 215 220 Asp Leu Leu Leu Ala Ala Thr Ser Glu Leu Asp Cys
Gln Gln Gly Thr 225 230 235 240 Arg Ala Leu Leu Gln Thr Leu Gly Asn
Leu Gly Tyr Arg Ala Ser Ala 245 250 255 Lys Lys Ala Gln Ile Cys Gln
Lys Gln Val Lys Tyr Leu Gly Tyr Leu 260 265 270 Leu Lys Glu Gly Gln
Arg Trp Leu Thr Glu Ala Arg Lys Glu Thr Val 275 280 285 Met Gly Gln
Pro Thr Pro Lys Thr Pro Arg Gln Leu Arg Glu Phe Leu 290 295 300 Gly
Thr Ala Gly Phe Cys Arg Leu Trp Ile Pro Gly Phe Ala Glu Met 305 310
315 320 Ala Ala Pro Leu Tyr Pro Leu Thr Lys Thr Gly Thr Leu Phe Asn
Trp 325 330 335 Gly Pro Asp Gln Gln Lys Ala Tyr Gln Glu Ile Lys Gln
Ala Leu Leu 340 345 350 Thr Ala Pro Ala Leu Gly Leu Pro Asp Leu Thr
Lys Pro Phe Glu Leu 355 360 365 Phe Val Asp Glu Lys Gln Gly Tyr Ala
Lys Gly Val Leu Thr Gln Lys 370 375 380 Leu Gly Pro Trp Arg Arg Pro
Val Ala Tyr Leu Ser Lys Lys Leu Asp 385 390 395 400 Pro Val Ala Ala
Gly Trp Pro Pro Cys Leu Arg Met Val Ala Ala Ile 405 410 415 Ala Val
Leu Thr Lys Asp Ala Gly Lys Leu Thr Met Gly Gln Pro Leu 420 425 430
Val Ile Leu Ala Pro His Ala Val Glu Ala Leu Val Lys Gln Pro Pro 435
440 445 Asp Arg Trp Leu Ser Asn Ala Arg Met Thr His Tyr Gln Ala Leu
Leu 450 455 460 Leu Asp Thr Asp Arg Val Gln Phe Gly Pro Val Val Ala
Leu Asn Pro 465 470 475 480 Ala Thr Leu Leu Pro Leu Pro Glu Glu Gly
Leu Gln His Asn Cys Leu 485 490 495 Asp Ile Leu Ala Glu Ala His Gly
Thr Arg Pro Asp Leu Thr Asp Gln 500 505 510 Pro Leu Pro Asp Ala Asp
His Thr Trp Tyr Thr Asp Gly Ser Ser Leu 515 520 525 Leu Gln Glu Gly
Gln Arg Lys Ala Gly Ala Ala Val Thr Thr Glu Thr 530 535 540 Glu Val
Ile Trp Ala Lys Ala Leu Pro Ala Gly Thr Ser Ala Gln Arg 545 550 555
560 Ala Glu Leu Ile Ala Leu Thr Gln Ala Leu Lys Met Ala Glu Gly Lys
565 570 575 Lys Leu Asn Val Tyr Thr Asp Ser Arg Tyr Ala Phe Ala Thr
Ala His 580 585 590 Ile His Gly Glu Ile Tyr Arg Arg Arg Gly Leu Leu
Thr Ser Glu Gly 595 600 605 Lys Glu Ile Lys Asn Lys Asp Glu Ile Leu
Ala Leu Leu Lys Ala Leu 610 615 620 Phe Leu Pro Lys Arg Leu Ser Ile
Ile His Cys Pro Gly His Gln Lys 625 630 635 640 Gly His Ser Ala Glu
Ala Arg Gly Asn Arg Leu Ala Asp Gln Ala Ala 645 650 655 Arg Lys Ala
Ala Ile Thr Glu Thr Pro Asp Thr Ser Thr Leu Leu 660 665 670
30671PRTMoloney murine leukemia virus 30Thr Leu Asn Ile Glu Asp Glu
His Arg Leu His Glu Thr Ser Lys Glu 1 5 10 15 Pro Asp Val Ser Leu
Gly Ser Thr Trp Leu Ser Asp Phe Pro Gln Ala 20 25 30 Trp Ala Glu
Thr Gly Gly Met Gly Leu Ala Val Arg Gln Ala Pro Leu 35 40 45 Ile
Ile Pro Leu Lys Ala Thr Ser Thr Pro Val Ser Ile Lys Gln Tyr 50 55
60 Pro Met Ser Gln Lys Ala Arg Leu Gly Ile Lys Pro His Ile Gln Arg
65 70 75 80 Leu Leu Asp Gln Gly Ile Leu Val Pro Cys Gln Ser Pro Trp
Asn Thr 85 90 95 Pro Leu Leu Pro Val Lys Lys Pro Gly Thr Asn Asp
Tyr Arg Pro Val 100 105 110 Gln Asp Leu Arg Glu Val Asn Lys Arg Val
Glu Asp Ile His Pro Thr 115 120 125 Val Pro Asn Pro Tyr Asn Leu Leu
Ser Gly Leu Pro Pro Ser His Gln 130 135 140 Trp Tyr Thr Val Leu Asp
Leu Lys Asp Ala Phe Phe Cys Leu Arg Leu 145 150 155 160 His Pro Thr
Ser Gln Pro Leu Phe Ala Phe Glu Trp Arg Asp Pro Glu 165 170 175 Met
Gly Ile Ser Gly Gln Leu Thr Trp Thr Arg Leu Pro Gln Gly Phe 180 185
190 Lys Asn Ser Pro Thr Leu Phe Asp Glu Ala Leu His Arg Asp Leu Ala
195 200 205 Asp Phe Arg Ile Gln His Pro Asp Leu Ile Leu Leu Gln Tyr
Val Asp 210 215 220 Asp Leu Leu Leu Ala Ala Thr Ser Glu Leu Asp Cys
Gln Gln Gly Thr 225 230 235 240 Arg Ala Leu Leu Gln Thr Leu Gly Asn
Leu Gly Tyr Arg Ala Ser Ala 245 250 255 Lys Lys Ala Gln Ile Cys Gln
Lys Gln Val Lys Tyr Leu Gly Tyr Leu 260 265 270 Leu Lys Glu Gly Gln
Arg Trp Leu Thr Glu Ala Arg Lys Glu Thr Val 275 280 285 Met Gly Gln
Pro Thr Pro Lys Thr Pro Arg Gln Leu Arg Arg Phe Leu 290 295 300 Gly
Thr Ala Gly Phe Cys Arg Leu Phe Ile Pro Gly Phe Ala Glu Met 305 310
315 320 Ala Ala Pro Leu Tyr Pro Leu Thr Lys Thr Gly Thr Leu Phe Asn
Trp 325 330 335 Gly Pro Asp Gln Gln Lys Ala Tyr Gln Glu Ile Lys Gln
Ala Leu Leu 340 345 350 Thr Ala Pro Ala Leu Gly Leu Pro Asp Leu Thr
Lys Pro Phe Glu Leu 355 360 365 Phe Val Asp Glu Lys Gln Gly Tyr Ala
Lys Gly Val Leu Thr Gln Lys 370 375 380 Leu Gly Pro Trp Arg Arg Pro
Val Ala Tyr Leu Ser Lys Lys Leu Asp 385 390 395 400 Pro Val Ala Ala
Gly Trp Pro Pro Cys Leu Arg Met Val Ala Ala Ile 405 410 415 Ala Val
Leu Thr Lys Asp Ala Gly Lys Leu Thr Met Gly Gln Pro Leu 420 425 430
Val Ile Gly Ala Pro His Ala Val Glu Ala Leu Val Lys Gln Pro Pro 435
440 445 Asp Arg Trp Leu Ser Lys Ala Arg Met Thr His Tyr Gln Ala Leu
Leu 450 455 460 Leu Asp Thr Asp Arg Val Gln Phe Gly Pro Val Val Ala
Leu Asn Pro 465 470 475 480 Ala Thr Leu Leu Pro Leu Pro Glu Glu Gly
Leu Gln His Asn Cys Leu 485 490 495 Asp Ile Leu Ala Glu Ala His Gly
Thr Arg Pro Asp Leu Thr Asp Gln 500 505 510 Pro Leu Pro Asp Ala Asp
His Thr Trp Tyr Thr Asp Gly Ser Ser Leu 515 520 525 Leu Gln Glu Gly
Gln Arg Lys Ala Gly Ala Ala Val Thr Thr Glu Thr 530 535 540 Glu Val
Ile Trp Ala Lys Ala Leu Pro Ala Gly Thr Ser Ala Gln Arg 545 550 555
560 Ala Glu Leu Ile Ala Leu Thr Gln Ala Leu Lys Met Ala Glu Gly Lys
565 570 575 Lys Leu Asn Val Tyr Thr Asp Ser Arg Tyr Ala Phe Ala Thr
Ala His 580 585 590 Ile His Gly Glu Ile Tyr Arg Arg Arg Gly Leu Leu
Thr Ser Glu Gly 595 600 605 Lys Glu Ile Lys Asn Lys Asp Glu Ile Leu
Ala Leu Leu Lys Ala Leu 610 615 620 Phe Leu Pro Lys Arg Leu Ser Ile
Ile His Cys Pro Gly His Gln Lys 625 630 635 640 Gly His Ser Ala Glu
Ala Arg Gly Asn Arg Met Ala Asp Gln Ala Ala 645 650 655 Arg Lys Ala
Ala Ile Thr Glu Thr Pro Asp Thr Ser Thr Leu Leu 660 665 670
31671PRTMoloney murine leukemia virus 31Thr Leu Asn Ile Glu Asp Glu
His Arg Leu His Glu Thr Ser Lys Glu 1 5 10 15 Pro Asp Val Ser Leu
Gly Ser Thr Trp Leu Ser Asp Phe Pro Gln Ala 20 25 30 Trp Ala Glu
Thr Gly Gly Met Gly Leu Ala Val Arg Gln Ala Pro Leu 35 40 45 Ile
Ile Pro Leu Lys Ala Thr Ser Thr Pro Val Ser Ile Lys Gln Tyr 50 55
60 Pro Met Ser Gln Glu Ala Arg Leu Gly Ile Lys Pro His Ile Gln Arg
65 70 75 80 Leu Leu Asp Gln Gly Ile Leu Val Pro Cys Gln Ser Pro Trp
Asn Thr 85 90 95 Pro Leu Leu Pro Val Lys Lys Pro Gly Thr Asn Asp
Tyr Arg Pro Val 100 105 110 Gln Asp Leu Arg Glu Val Asn Lys Arg Val
Glu Asp Ile His Pro Thr 115 120 125 Val Pro Asn Pro Tyr Asn Leu Leu
Ser Gly Leu Pro Pro Ser His Gln 130 135 140
Trp Tyr Thr Val Leu Asp Leu Lys Asp Ala Phe Phe Cys Leu Arg Leu 145
150 155 160 His Pro Thr Ser Gln Pro Leu Phe Ala Phe Glu Trp Arg Asp
Pro Glu 165 170 175 Met Gly Ile Ser Gly Gln Leu Thr Trp Thr Arg Leu
Pro Gln Gly Phe 180 185 190 Lys Asn Ser Pro Thr Leu Phe Asp Glu Ala
Leu His Arg Asp Leu Ala 195 200 205 Asp Phe Arg Ile Gln His Pro Asp
Leu Ile Leu Leu Gln Tyr Val Asp 210 215 220 Asp Leu Leu Leu Ala Ala
Thr Ser Glu Leu Asp Cys Gln Gln Gly Thr 225 230 235 240 Arg Ala Leu
Leu Gln Thr Leu Gly Asn Leu Gly Tyr Arg Ala Ser Ala 245 250 255 Lys
Lys Ala Gln Ile Cys Gln Lys Gln Val Lys Tyr Leu Gly Tyr Leu 260 265
270 Leu Lys Glu Gly Gln Arg Trp Leu Thr Glu Ala Arg Lys Glu Thr Val
275 280 285 Met Gly Gln Pro Thr Pro Lys Thr Pro Arg Gln Leu Arg Arg
Phe Leu 290 295 300 Gly Thr Ala Gly Phe Cys Arg Leu Phe Ile Pro Gly
Phe Ala Glu Met 305 310 315 320 Ala Ala Pro Leu Tyr Pro Leu Thr Lys
Thr Gly Thr Leu Phe Asn Trp 325 330 335 Gly Pro Asp Gln Gln Lys Ala
Tyr Gln Glu Ile Lys Gln Ala Leu Leu 340 345 350 Thr Ala Pro Ala Leu
Gly Leu Pro Asp Leu Thr Lys Pro Phe Glu Leu 355 360 365 Phe Val Asp
Glu Lys Gln Gly Tyr Ala Lys Gly Val Leu Thr Gln Lys 370 375 380 Leu
Gly Pro Trp Arg Arg Pro Val Ala Tyr Leu Ser Lys Lys Leu Asp 385 390
395 400 Pro Val Ala Ala Gly Trp Pro Pro Cys Leu Arg Met Val Ala Ala
Ile 405 410 415 Ala Val Leu Thr Lys Asp Ala Gly Lys Leu Thr Met Gly
Gln Pro Leu 420 425 430 Val Ile Gly Ala Pro His Ala Val Glu Ala Leu
Val Lys Gln Pro Pro 435 440 445 Asp Arg Trp Leu Ser Lys Ala Arg Met
Thr His Tyr Gln Ala Leu Leu 450 455 460 Leu Asp Thr Asp Arg Val Gln
Phe Gly Pro Val Val Ala Leu Asn Pro 465 470 475 480 Ala Thr Leu Leu
Pro Leu Pro Glu Glu Gly Leu Gln His Asn Cys Leu 485 490 495 Asp Ile
Leu Ala Glu Ala His Gly Thr Arg Pro Asp Leu Thr Asp Gln 500 505 510
Pro Leu Pro Asp Ala Asp His Thr Trp Tyr Thr Asp Gly Ser Ser Leu 515
520 525 Leu Gln Glu Gly Gln Arg Lys Ala Gly Ala Ala Val Thr Thr Glu
Thr 530 535 540 Glu Val Ile Trp Ala Lys Ala Leu Pro Ala Gly Thr Ser
Ala Gln Arg 545 550 555 560 Ala Glu Leu Ile Ala Leu Thr Gln Ala Leu
Lys Met Ala Glu Gly Lys 565 570 575 Lys Leu Asn Val Tyr Thr Asp Ser
Arg Tyr Ala Phe Ala Thr Ala His 580 585 590 Ile His Gly Glu Ile Tyr
Arg Arg Arg Gly Leu Leu Thr Ser Glu Gly 595 600 605 Lys Glu Ile Lys
Asn Lys Asp Glu Ile Leu Ala Leu Leu Lys Ala Leu 610 615 620 Phe Leu
Pro Lys Arg Leu Ser Ile Ile His Cys Pro Gly His Gln Lys 625 630 635
640 Gly His Ser Ala Glu Ala Arg Gly Asn Arg Met Ala Asp Gln Ala Ala
645 650 655 Arg Lys Ala Ala Ile Thr Glu Thr Pro Asp Thr Ser Thr Leu
Leu 660 665 670 32671PRTMoloney murine leukemia virus 32Thr Leu Asn
Ile Glu Asp Glu His Arg Leu His Glu Thr Ser Lys Glu 1 5 10 15 Pro
Asp Val Ser Leu Gly Ser Thr Trp Leu Ser Asp Phe Pro Gln Ala 20 25
30 Trp Ala Glu Thr Gly Gly Met Gly Leu Ala Val Arg Gln Ala Pro Leu
35 40 45 Ile Ile Pro Leu Lys Ala Thr Ser Thr Pro Val Ser Ile Lys
Gln Tyr 50 55 60 Pro Met Ser Gln Glu Ala Arg Leu Gly Ile Lys Pro
His Ile Gln Arg 65 70 75 80 Leu Leu Asp Gln Gly Ile Leu Val Pro Cys
Gln Ser Pro Trp Asn Thr 85 90 95 Pro Leu Leu Pro Val Lys Lys Pro
Gly Thr Asn Asp Tyr Arg Pro Val 100 105 110 Gln Asp Leu Arg Glu Val
Asn Lys Arg Val Glu Asp Ile His Pro Thr 115 120 125 Val Pro Asn Pro
Tyr Asn Leu Leu Ser Gly Leu Pro Pro Ser His Gln 130 135 140 Trp Tyr
Thr Val Leu Asp Leu Lys Asp Ala Phe Phe Cys Leu Arg Leu 145 150 155
160 His Pro Thr Ser Gln Pro Leu Phe Ala Phe Glu Trp Arg Asp Pro Glu
165 170 175 Met Gly Ile Ser Gly Gln Leu Thr Trp Thr Arg Leu Pro Gln
Gly Phe 180 185 190 Lys Asn Ser Pro Thr Leu Phe Asp Glu Ala Leu His
Arg Asp Leu Ala 195 200 205 Asp Phe Arg Ile Gln His Pro Asp Leu Ile
Leu Leu Gln Tyr Val Asp 210 215 220 Asp Leu Leu Leu Ala Ala Thr Ser
Glu Leu Asp Cys Gln Gln Gly Thr 225 230 235 240 Arg Ala Leu Leu Gln
Thr Leu Gly Asn Leu Gly Tyr Arg Ala Ser Ala 245 250 255 Lys Lys Ala
Gln Ile Cys Gln Lys Gln Val Lys Tyr Leu Gly Tyr Leu 260 265 270 Leu
Lys Glu Gly Gln Arg Trp Leu Thr Glu Ala Arg Lys Glu Thr Val 275 280
285 Met Gly Gln Pro Thr Pro Lys Thr Pro Arg Gln Leu Arg Arg Phe Leu
290 295 300 Gly Thr Ala Gly Phe Cys Arg Leu Phe Ile Pro Gly Phe Ala
Glu Met 305 310 315 320 Ala Ala Pro Leu Tyr Pro Leu Thr Lys Thr Gly
Thr Leu Phe Asn Trp 325 330 335 Gly Pro Asp Gln Gln Lys Ala Tyr Gln
Glu Ile Lys Gln Ala Leu Leu 340 345 350 Thr Ala Pro Ala Leu Gly Leu
Pro Asp Leu Thr Lys Pro Phe Glu Leu 355 360 365 Phe Val Asp Glu Lys
Gln Gly Tyr Ala Lys Gly Val Leu Thr Gln Lys 370 375 380 Leu Gly Pro
Trp Arg Arg Pro Val Ala Tyr Leu Ser Lys Lys Leu Asp 385 390 395 400
Pro Val Ala Ala Gly Trp Pro Pro Cys Leu Arg Met Val Ala Ala Ile 405
410 415 Ala Val Leu Thr Lys Asp Ala Gly Lys Leu Thr Met Gly Gln Pro
Leu 420 425 430 Val Ile Gly Ala Pro His Ala Val Glu Ala Leu Val Lys
Gln Pro Pro 435 440 445 Asp Arg Trp Leu Ser Asn Ala Arg Met Thr His
Tyr Gln Ala Leu Leu 450 455 460 Leu Asp Thr Asp Arg Val Gln Phe Gly
Pro Val Val Ala Leu Asn Pro 465 470 475 480 Ala Thr Leu Leu Pro Leu
Pro Glu Glu Gly Leu Gln His Asn Cys Leu 485 490 495 Asp Ile Leu Ala
Glu Ala His Gly Thr Arg Pro Asp Leu Thr Asp Gln 500 505 510 Pro Leu
Pro Asp Ala Asp His Thr Trp Tyr Thr Asp Gly Ser Ser Leu 515 520 525
Leu Gln Glu Gly Gln Arg Lys Ala Gly Ala Ala Val Thr Thr Glu Thr 530
535 540 Glu Val Ile Trp Ala Lys Ala Leu Pro Ala Gly Thr Ser Ala Gln
Arg 545 550 555 560 Ala Glu Leu Ile Ala Leu Thr Gln Ala Leu Lys Met
Ala Glu Gly Lys 565 570 575 Lys Leu Asn Val Tyr Thr Asp Ser Arg Tyr
Ala Phe Ala Thr Ala His 580 585 590 Ile His Gly Glu Ile Tyr Arg Arg
Arg Gly Leu Leu Thr Ser Glu Gly 595 600 605 Lys Glu Ile Lys Asn Lys
Asp Glu Ile Leu Ala Leu Leu Lys Ala Leu 610 615 620 Phe Leu Pro Lys
Arg Leu Ser Ile Ile His Cys Pro Gly His Gln Lys 625 630 635 640 Gly
His Ser Ala Glu Ala Arg Gly Asn Arg Met Ala Asp Gln Ala Ala 645 650
655 Arg Lys Ala Ala Ile Thr Glu Thr Pro Asp Thr Ser Thr Leu Leu 660
665 670 33671PRTMoloney murine leukemia virus 33Thr Leu Asn Ile Glu
Asp Glu His Arg Leu His Glu Thr Ser Lys Glu 1 5 10 15 Pro Asp Val
Ser Leu Gly Ser Thr Trp Leu Ser Asp Phe Pro Gln Ala 20 25 30 Trp
Ala Glu Thr Gly Gly Met Gly Leu Ala Val Arg Gln Ala Pro Leu 35 40
45 Ile Ile Pro Leu Lys Ala Thr Ser Thr Pro Val Ser Ile Lys Gln Tyr
50 55 60 Pro Met Ser Gln Lys Ala Arg Leu Gly Ile Lys Pro His Ile
Gln Arg 65 70 75 80 Leu Leu Asp Gln Gly Ile Leu Val Pro Cys Gln Ser
Pro Trp Asn Thr 85 90 95 Pro Leu Leu Pro Val Lys Lys Pro Gly Thr
Asn Asp Tyr Arg Pro Val 100 105 110 Gln Asp Leu Arg Glu Val Asn Lys
Arg Val Glu Asp Ile His Pro Thr 115 120 125 Val Pro Asn Pro Tyr Asn
Leu Leu Ser Gly Leu Pro Pro Ser His Gln 130 135 140 Trp Tyr Thr Val
Leu Asp Leu Lys Asp Ala Phe Phe Cys Leu Arg Leu 145 150 155 160 His
Pro Thr Ser Gln Pro Leu Phe Ala Phe Glu Trp Arg Asp Pro Glu 165 170
175 Met Gly Ile Ser Gly Gln Leu Thr Trp Thr Arg Leu Pro Gln Gly Phe
180 185 190 Lys Asn Ser Pro Thr Leu Phe Asp Glu Ala Leu His Arg Asp
Leu Ala 195 200 205 Asp Phe Arg Ile Gln His Pro Asp Leu Ile Leu Leu
Gln Tyr Val Asp 210 215 220 Asp Leu Leu Leu Ala Ala Thr Ser Glu Leu
Asp Cys Gln Gln Gly Thr 225 230 235 240 Arg Ala Leu Leu Gln Thr Leu
Gly Asn Leu Gly Tyr Arg Ala Ser Ala 245 250 255 Lys Lys Ala Gln Ile
Cys Gln Lys Gln Val Lys Tyr Leu Gly Tyr Leu 260 265 270 Leu Lys Glu
Gly Gln Arg Trp Leu Thr Glu Ala Arg Lys Glu Thr Val 275 280 285 Met
Gly Gln Pro Thr Pro Lys Thr Pro Arg Gln Leu Arg Arg Phe Leu 290 295
300 Gly Thr Ala Gly Phe Cys Arg Leu Trp Ile Pro Gly Phe Ala Glu Met
305 310 315 320 Ala Ala Pro Leu Tyr Pro Leu Thr Lys Thr Gly Thr Leu
Phe Asn Trp 325 330 335 Gly Pro Asp Gln Gln Lys Ala Tyr Gln Glu Ile
Lys Gln Ala Leu Leu 340 345 350 Thr Ala Pro Ala Leu Gly Leu Pro Asp
Leu Thr Lys Pro Phe Glu Leu 355 360 365 Phe Val Asp Glu Lys Gln Gly
Tyr Ala Lys Gly Val Leu Thr Gln Lys 370 375 380 Leu Gly Pro Trp Arg
Arg Pro Val Ala Tyr Leu Ser Lys Lys Leu Asp 385 390 395 400 Pro Val
Ala Ala Gly Trp Pro Pro Cys Leu Arg Met Val Ala Ala Ile 405 410 415
Ala Val Leu Thr Lys Asp Ala Gly Lys Leu Thr Met Gly Gln Pro Leu 420
425 430 Val Ile Leu Ala Pro His Ala Val Glu Ala Leu Val Lys Gln Pro
Pro 435 440 445 Asp Arg Trp Leu Ser Lys Ala Arg Met Thr His Tyr Gln
Ala Leu Leu 450 455 460 Leu Asp Thr Asp Arg Val Gln Phe Gly Pro Val
Val Ala Leu Asn Pro 465 470 475 480 Ala Thr Leu Leu Pro Leu Pro Glu
Glu Gly Leu Gln His Asn Cys Leu 485 490 495 Asp Ile Leu Ala Glu Ala
His Gly Thr Arg Pro Asp Leu Thr Asp Gln 500 505 510 Pro Leu Pro Asp
Ala Asp His Thr Trp Tyr Thr Asp Gly Ser Ser Leu 515 520 525 Leu Gln
Glu Gly Gln Arg Lys Ala Gly Ala Ala Val Thr Thr Glu Thr 530 535 540
Glu Val Ile Trp Ala Lys Ala Leu Pro Ala Gly Thr Ser Ala Gln Arg 545
550 555 560 Ala Glu Leu Ile Ala Leu Thr Gln Ala Leu Lys Met Ala Glu
Gly Lys 565 570 575 Lys Leu Asn Val Tyr Thr Asp Ser Arg Tyr Ala Phe
Ala Thr Ala His 580 585 590 Ile His Gly Glu Ile Tyr Arg Arg Arg Gly
Leu Leu Thr Ser Glu Gly 595 600 605 Lys Glu Ile Lys Asn Lys Asp Glu
Ile Leu Ala Leu Leu Lys Ala Leu 610 615 620 Phe Leu Pro Lys Arg Leu
Ser Ile Ile His Cys Pro Gly His Gln Lys 625 630 635 640 Gly His Ser
Ala Glu Ala Arg Gly Asn Arg Met Ala Asp Gln Ala Ala 645 650 655 Arg
Lys Ala Ala Ile Thr Glu Thr Pro Asp Thr Ser Thr Leu Leu 660 665 670
34671PRTMoloney murine leukemia virus 34Thr Leu Asn Ile Glu Asp Glu
His Arg Leu His Glu Thr Ser Lys Glu 1 5 10 15 Pro Asp Val Ser Leu
Gly Ser Thr Trp Leu Ser Asp Phe Pro Gln Ala 20 25 30 Trp Ala Glu
Thr Gly Gly Met Gly Leu Ala Val Arg Gln Ala Pro Leu 35 40 45 Ile
Ile Pro Leu Lys Ala Thr Ser Thr Pro Val Ser Ile Lys Gln Tyr 50 55
60 Pro Met Ser Gln Glu Ala Arg Leu Gly Ile Lys Pro His Ile Gln Arg
65 70 75 80 Leu Leu Asp Gln Gly Ile Leu Val Pro Cys Gln Ser Pro Trp
Asn Thr 85 90 95 Pro Leu Leu Pro Val Lys Lys Pro Gly Thr Asn Asp
Tyr Arg Pro Val 100 105 110 Gln Asp Leu Arg Glu Val Asn Lys Arg Val
Glu Asp Ile His Pro Thr 115 120 125 Val Pro Asn Pro Tyr Asn Leu Leu
Ser Gly Leu Pro Pro Ser His Gln 130 135 140 Trp Tyr Thr Val Leu Asp
Leu Lys Asp Ala Phe Phe Cys Leu Arg Leu 145 150 155 160 His Pro Thr
Ser Gln Pro Leu Phe Ala Phe Glu Trp Arg Asp Pro Glu 165 170 175 Met
Gly Ile Ser Gly Gln Leu Thr Trp Thr Arg Leu Pro Gln Gly Phe 180 185
190 Lys Asn Ser Pro Thr Leu Phe Asp Glu Ala Leu His Arg Asp Leu Ala
195 200 205 Asp Phe Arg Ile Gln His Pro Asp Leu Ile Leu Leu Gln Tyr
Val Asp 210 215 220 Asp Leu Leu Leu Ala Ala Thr Ser Glu Leu Asp Cys
Gln Gln Gly Thr 225 230 235 240 Arg Ala Leu Leu Gln Thr Leu Gly Asn
Leu Gly Tyr Arg Ala Ser Ala 245 250 255 Lys Lys Ala Gln Ile Cys Gln
Lys Gln Val Lys Tyr Leu Gly Tyr Leu 260 265 270 Leu Lys Glu Gly Gln
Arg Trp Leu Thr Glu Ala Arg Lys Glu Thr Val 275 280 285 Met Gly Gln
Pro Thr Pro Lys Thr Pro Arg Gln Leu Arg Arg Phe Leu 290 295 300 Gly
Thr Ala Gly Phe Cys Arg Leu Phe Ile Pro Gly Phe Ala Glu Met 305 310
315 320 Ala Ala Pro Leu Tyr Pro Leu Thr Lys Thr Gly Thr Leu Phe Asn
Trp 325 330 335 Gly Pro Asp Gln Gln Lys Ala Tyr Gln Glu Ile Lys Gln
Ala Leu Leu 340 345 350 Thr Ala Pro Ala Leu Gly Leu Pro Asp Leu Thr
Lys Pro Phe Glu Leu 355 360 365 Phe Val Asp Glu Lys Gln Gly Tyr Ala
Lys Gly Val Leu Thr Gln Lys 370 375 380 Leu Gly Pro Trp Arg Arg Pro
Val Ala Tyr Leu Ser Lys Lys Leu Asp 385 390 395 400 Pro Val Ala Ala
Gly Trp Pro Pro Cys Leu Arg Met Val Ala Ala Ile 405 410 415 Ala Val
Leu Thr Lys Asp Ala Gly Lys Leu Thr Met Gly Gln Pro Leu 420
425 430 Val Ile Leu Ala Pro His Ala Val Glu Ala Leu Val Lys Gln Pro
Pro 435 440 445 Asp Arg Trp Leu Ser Asn Ala Arg Met Thr His Tyr Gln
Ala Leu Leu 450 455 460 Leu Asp Thr Asp Arg Val Gln Phe Gly Pro Val
Val Ala Leu Asn Pro 465 470 475 480 Ala Thr Leu Leu Pro Leu Pro Glu
Glu Gly Leu Gln His Asn Cys Leu 485 490 495 Asp Ile Leu Ala Glu Ala
His Gly Thr Arg Pro Asp Leu Thr Asp Gln 500 505 510 Pro Leu Pro Asp
Ala Asp His Thr Trp Tyr Thr Asp Gly Ser Ser Leu 515 520 525 Leu Gln
Glu Gly Gln Arg Lys Ala Gly Ala Ala Val Thr Thr Glu Thr 530 535 540
Glu Val Ile Trp Ala Lys Ala Leu Pro Ala Gly Thr Ser Ala Gln Arg 545
550 555 560 Ala Glu Leu Ile Ala Leu Thr Gln Ala Leu Lys Met Ala Glu
Gly Lys 565 570 575 Lys Leu Asn Val Tyr Thr Asp Ser Arg Tyr Ala Phe
Ala Thr Ala His 580 585 590 Ile His Gly Glu Ile Tyr Arg Arg Arg Gly
Leu Leu Thr Ser Glu Gly 595 600 605 Lys Glu Ile Lys Asn Lys Asp Glu
Ile Leu Ala Leu Leu Lys Ala Leu 610 615 620 Phe Leu Pro Lys Arg Leu
Ser Ile Ile His Cys Pro Gly His Gln Lys 625 630 635 640 Gly His Ser
Ala Glu Ala Arg Gly Asn Arg Met Ala Asp Gln Ala Ala 645 650 655 Arg
Lys Ala Ala Ile Thr Glu Thr Pro Asp Thr Ser Thr Leu Leu 660 665 670
35671PRTMoloney murine leukemia virus 35Thr Leu Asn Ile Glu Asp Glu
His Arg Leu His Glu Thr Ser Lys Glu 1 5 10 15 Pro Asp Val Ser Leu
Gly Ser Thr Trp Leu Ser Asp Phe Pro Gln Ala 20 25 30 Trp Ala Glu
Thr Gly Gly Met Gly Leu Ala Val Arg Gln Ala Pro Leu 35 40 45 Ile
Ile Pro Leu Lys Ala Thr Ser Thr Pro Val Ser Ile Lys Gln Tyr 50 55
60 Pro Met Ser Gln Lys Ala Arg Leu Gly Ile Lys Pro His Ile Gln Arg
65 70 75 80 Leu Leu Asp Gln Gly Ile Leu Val Pro Cys Gln Ser Pro Trp
Asn Thr 85 90 95 Pro Leu Leu Pro Val Lys Lys Pro Gly Thr Asn Asp
Tyr Arg Pro Val 100 105 110 Gln Asp Leu Arg Glu Val Asn Lys Arg Val
Glu Asp Ile His Pro Thr 115 120 125 Val Pro Asn Pro Tyr Asn Leu Leu
Ser Gly Leu Pro Pro Ser His Gln 130 135 140 Trp Tyr Thr Val Leu Asp
Leu Lys Asp Ala Phe Phe Cys Leu Arg Leu 145 150 155 160 His Pro Thr
Ser Gln Pro Leu Phe Ala Phe Glu Trp Arg Asp Pro Glu 165 170 175 Met
Gly Ile Ser Gly Gln Leu Thr Trp Thr Arg Leu Pro Gln Gly Phe 180 185
190 Lys Asn Ser Pro Thr Leu Phe Asp Glu Ala Leu His Arg Asp Leu Ala
195 200 205 Asp Phe Arg Ile Gln His Pro Asp Leu Ile Leu Leu Gln Tyr
Val Asp 210 215 220 Asp Leu Leu Leu Ala Ala Thr Ser Glu Leu Asp Cys
Gln Gln Gly Thr 225 230 235 240 Arg Ala Leu Leu Gln Thr Leu Gly Asn
Leu Gly Tyr Arg Ala Ser Ala 245 250 255 Lys Lys Ala Gln Ile Cys Gln
Lys Gln Val Lys Tyr Leu Gly Tyr Leu 260 265 270 Leu Lys Glu Gly Gln
Arg Trp Leu Thr Glu Ala Arg Lys Glu Thr Val 275 280 285 Met Gly Gln
Pro Thr Pro Lys Thr Pro Arg Gln Leu Arg Arg Phe Leu 290 295 300 Gly
Thr Ala Gly Phe Cys Arg Leu Phe Ile Pro Gly Phe Ala Glu Met 305 310
315 320 Ala Ala Pro Leu Tyr Pro Leu Thr Lys Thr Gly Thr Leu Phe Asn
Trp 325 330 335 Gly Pro Asp Gln Gln Lys Ala Tyr Gln Glu Ile Lys Gln
Ala Leu Leu 340 345 350 Thr Ala Pro Ala Leu Gly Leu Pro Asp Leu Thr
Lys Pro Phe Glu Leu 355 360 365 Phe Val Asp Glu Lys Gln Gly Tyr Ala
Lys Gly Val Leu Thr Gln Lys 370 375 380 Leu Gly Pro Trp Arg Arg Pro
Val Ala Tyr Leu Ser Lys Lys Leu Asp 385 390 395 400 Pro Val Ala Ala
Gly Trp Pro Pro Cys Leu Arg Met Val Ala Ala Ile 405 410 415 Ala Val
Leu Thr Lys Asp Ala Gly Lys Leu Thr Met Gly Gln Pro Leu 420 425 430
Val Ile Gly Ala Pro His Ala Val Glu Ala Leu Val Lys Gln Pro Pro 435
440 445 Asp Arg Trp Leu Ser Lys Ala Arg Met Thr His Tyr Gln Ala Leu
Leu 450 455 460 Leu Asp Thr Asp Arg Val Gln Phe Gly Pro Val Val Ala
Leu Asn Pro 465 470 475 480 Ala Thr Leu Leu Pro Leu Pro Glu Glu Gly
Leu Gln His Asn Cys Leu 485 490 495 Asp Ile Leu Ala Glu Ala His Gly
Thr Arg Pro Asp Leu Thr Asp Gln 500 505 510 Pro Leu Pro Asp Ala Asp
His Thr Trp Tyr Thr Asn Gly Ser Ser Leu 515 520 525 Leu Gln Glu Gly
Gln Arg Lys Ala Gly Ala Ala Val Thr Thr Glu Thr 530 535 540 Glu Val
Ile Trp Ala Lys Ala Leu Pro Ala Gly Thr Ser Ala Gln Arg 545 550 555
560 Ala Glu Leu Ile Ala Leu Thr Gln Ala Leu Lys Met Ala Glu Gly Lys
565 570 575 Lys Leu Asn Val Tyr Thr Asp Ser Arg Tyr Ala Phe Ala Thr
Ala His 580 585 590 Ile His Gly Glu Ile Tyr Arg Arg Arg Gly Leu Leu
Thr Ser Glu Gly 595 600 605 Lys Glu Ile Lys Asn Lys Asp Glu Ile Leu
Ala Leu Leu Lys Ala Leu 610 615 620 Phe Leu Pro Lys Arg Leu Ser Ile
Ile His Cys Pro Gly His Gln Lys 625 630 635 640 Gly His Ser Ala Glu
Ala Arg Gly Asn Arg Met Ala Asp Gln Ala Ala 645 650 655 Arg Lys Ala
Ala Ile Thr Glu Thr Pro Asp Thr Ser Thr Leu Leu 660 665 670
365PRTMoloney murine leukemia virus 36Arg Lys Phe Gly Lys 1 5
374PRTMoloney murine leukemia virus 37Arg Phe Gly Lys 1
3826DNAArtificial SequenceDescription of Artificial Sequence
Synthetic primer 38accctaaata tagaagatga gcatcg 263925DNAArtificial
SequenceDescription of Artificial Sequence Synthetic primer
39gaggagggta gaggtgtctg gagtc 254095DNAArtificial
SequenceDescription of Artificial Sequence Synthetic primer
40cttggccaag gatccgctcg agctacttac ttannnnnnn nnnnnnnnnn nnnnnnnnnn
60nnnnnnnnng aggagggtag aggtgtctgg agtct 954130DNAArtificial
SequenceDescription of Artificial Sequence Synthetic primer
41agcggataac aattcccctc tagaattcga 304226DNAArtificial
SequenceDescription of Artificial Sequence Synthetic primer
42cccatgtcac aannkgccag actggg 264326DNAArtificial
SequenceDescription of Artificial Sequence Synthetic primer
43gacaactaag gnnkttccta gggacg 264426DNAArtificial
SequenceDescription of Artificial Sequence Synthetic primer
44caactaaggg agnnkctagg gacggc 264525DNAArtificial
SequenceDescription of Artificial Sequence Synthetic primer
45ggagttccta nnkacggcag gcttc 254627DNAArtificial
SequenceDescription of Artificial Sequence Synthetic primer
46tctgtcgcct cnnkatccct gggtttg 274728DNAArtificial
SequenceDescription of Artificial Sequence Synthetic primer
47ccactagtca ttnnkgcccc ccatgcag 284827DNAArtificial
SequenceDescription of Artificial Sequence Synthetic primer
48gctggctttc cnnkgcccgg atgactc 274926DNAArtificial
SequenceDescription of Artificial Sequence Synthetic primer
49gaggcaaccg gnnkgctgac caagcg 265026DNAArtificial
SequenceDescription of Artificial Sequence Synthetic primer
50cccagtctgg cmnnttgtga catggg 265126DNAArtificial
SequenceDescription of Artificial Sequence Synthetic primer
51cgtccctagg aamnncctta gttgtc 265226DNAArtificial
SequenceDescription of Artificial Sequence Synthetic primer
52gccgtcccta gmnnctccct tagttg 265325DNAArtificial
SequenceDescription of Artificial Sequence Synthetic primer
53gaagcctgcc gtmnntagga actcc 255427DNAArtificial
SequenceDescription of Artificial Sequence Synthetic primer
54caaacccagg gatmnngagg cgacaga 275528DNAArtificial
SequenceDescription of Artificial Sequence Synthetic primer
55ctgcatgggg ggcmnnaatg actagtgg 285627DNAArtificial
SequenceDescription of Artificial Sequence Synthetic primer
56gagtcatccg ggcmnnggaa agccagc 275726DNAArtificial
SequenceDescription of Artificial Sequence Synthetic primer
57cgcttggtca gcmnnccggt tgcctc 265836DNAArtificial
SequenceDescription of Artificial Sequence Synthetic primer
58taccccatgt cacaaaaagc cagactgggg atcaag 365932DNAArtificial
SequenceDescription of Artificial Sequence Synthetic primer
59ggcttctgtc gcctctttat ccctgggttt gc 326034DNAArtificial
SequenceDescription of Artificial Sequence Synthetic primer
60cagccactag tcattggggc cccccatgca gtag 346133DNAArtificial
SequenceDescription of Artificial Sequence Synthetic primer
61gaccgctggc tttccaaggc ccggatgact cac 336218DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 62gggggggggg gggggggg 186318DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 63tttttttttt tttttttt 186412PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 64His
Ser Arg Arg Arg Leu Lys Arg His Ile Phe Asn 1 5 10
6513PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 65Ser Lys Arg Thr Asn Pro Ile Asn Ile His Thr Asn
Lys 1 5 10 6612PRTArtificial SequenceDescription of Artificial
Sequence Synthetic peptide 66Gln Glu Gly Lys Asn Arg Gln Gly Glu
Gly Gln Thr 1 5 10 6726PRTArtificial SequenceDescription of
Artificial Sequence Synthetic peptide 67Arg Asp Arg Asn Lys Asn Asn
Asp Arg Arg Lys Ala Lys Arg Asp Arg 1 5 10 15 Asn Lys Asn Asn Asp
Arg Arg Lys Ala Lys 20 25 6828PRTArtificial SequenceDescription of
Artificial Sequence Synthetic peptide 68Arg Asp Arg Asn Lys Asn Asn
Asp Arg Arg Lys Ala Lys Glu Asn Glu 1 5 10 15 Glu Asn Glu Glu Asn
Glu Glu Asn Glu Glu Asn Glu 20 25 69243PRTEscherichia coli 69Met
Ser Thr Ala Ile Thr Arg Gln Ile Val Leu Asp Thr Glu Thr Thr 1 5 10
15 Gly Met Asn Gln Ile Gly Ala His Tyr Glu Gly His Lys Ile Ile Glu
20 25 30 Ile Gly Ala Val Glu Val Val Asn Arg Arg Leu Thr Gly Asn
Asn Phe 35 40 45 His Val Tyr Leu Lys Pro Asp Arg Leu Val Asp Pro
Glu Ala Phe Gly 50 55 60 Val His Gly Ile Ala Asp Glu Phe Leu Leu
Asp Lys Pro Thr Phe Ala 65 70 75 80 Glu Val Ala Asp Glu Phe Met Asp
Tyr Ile Arg Gly Ala Glu Leu Val 85 90 95 Ile His Asn Ala Ala Phe
Asp Ile Gly Phe Met Asp Tyr Glu Phe Ser 100 105 110 Leu Leu Lys Arg
Asp Ile Pro Lys Thr Asn Thr Phe Cys Lys Val Thr 115 120 125 Asp Ser
Leu Ala Val Ala Arg Lys Met Phe Pro Gly Lys Arg Asn Ser 130 135 140
Leu Asp Ala Leu Cys Ala Arg Tyr Glu Ile Asp Asn Ser Lys Arg Thr 145
150 155 160 Leu His Gly Ala Leu Leu Asp Ala Gln Ile Leu Ala Glu Val
Tyr Leu 165 170 175 Ala Met Thr Gly Gly Gln Thr Ser Met Ala Phe Ala
Met Glu Gly Glu 180 185 190 Thr Gln Gln Gln Gln Gly Glu Ala Thr Ile
Gln Arg Ile Val Arg Gln 195 200 205 Ala Ser Lys Leu Arg Val Val Phe
Ala Thr Asp Glu Glu Ile Ala Ala 210 215 220 His Glu Ala Arg Leu Asp
Leu Val Gln Lys Lys Gly Gly Ser Cys Leu 225 230 235 240 Trp Arg Ala
70750DNAEscherichia coli 70cacaggtatt tatgctcgcc agaggcaact
tccgcctttc ttctgcacca gatcgagacg 60ggcttcatga gctgcaatct cttcatctgt
cgcaaaaaca acgcgtaact tacttgcctg 120acgtacaatg cgctgaattg
ttgcttcacc ttgttgctgt tgtgtctctc cttccatcgc 180aaaagccatc
gacgtttgac caccggtcat cgccagataa acttccgcaa ggatctgggc
240atcgagtaat gccccgtgca gcgttcgttt actgttatct atttcgtagc
gagcacataa 300cgcatcgagg ctgttgcgct taccgggaaa cattttcctc
gccaccgcaa ggctatcggt 360gaccttacag aaagtattgg tcttcggaat
atcgcgctta agcaacgaaa actcgtagtc 420cataaagccg atatcgaacg
ctgcgttatg gatcaccaac tccgcgccgc gaatatagtc 480catgaactca
tcggctactt cggcaaacgt gggcttatcg agcaaaaatt catcggcaat
540accatgtacg ccaaaggctt ccggatccac cagccgatcg ggtttgagat
aaacatggaa 600gttattgccc gtcaggcgac ggttcaccac ttcaacggca
ccaatctcaa tgatcttgtg 660gccttcatag tgcgcaccaa tctggttcat
accggtggtt tcggtatcga gaacgatctg 720gcgtgtaatt gcagtgctca
tagcggtcat 750
* * * * *