KR20160045656A - Reverse Transcriptase Having Improved Thermostability - Google Patents

Abstract

The present invention relates to reverse transcriptase having improved thermostability, and more particularly, to mutant reverse transcriptase with improved thermostability by a substitution of one or more amino acids selected from the group consisting of the 63rd glutamine (Q63), the 264th lysine (K264), the 295th lysine (K295), the 306th threonine (T306), the 346th glutamic acid (E346), the 408th proline (P408), the 438th histidine (H438), and the 454th asparagin (N454) of the amino acid sequence of M-MLV originated reverse transcriptase represented by SEQ. ID. NO: 1 with other amino acids. The mutant reverse transcriptase of the present invention demonstrates excellent thermostability, compared with the wild type reverse transcriptase. Therefore, it is advantageous to obtain a target cDNA with stable reverse transcription activity even when it fails to efficiently obtain a reverse transcription product due to a specific RNA that can form a stable secondary structure at a high temperature.

Description

Reverse Transcriptase Having Improved Thermostability}

The present invention relates to a reverse transcriptase, and more particularly, to a reverse transcriptase having increased thermal stability by mutating a specific amino acid residue constituting a conventional reverse transcriptase.

Reverse transcriptase derived from RNA tumor viruses, especially Moloney-Murine Leukemia Virus (M-MLV), Human Acquired Immunodeficiency Virus (HIV), and Avian Myeloblastosis Virus (AMV). ), many studies have been made, and various functions and properties have been known. Due to its characteristic property that RNA can be used as a template to synthesize DNA (cDNA) complementary to it, reverse transcriptase is used in many molecular biological methods, such as construction of cDNA libraries, reverse transcription and polymerase chain reactions. reaction, PCR), etc.

Three prototype types of retroviral reverse transcriptase are being studied extensively. The reverse transcriptase of Moloney rodent leukemia virus contains a single subunit of 78 kDa with RNA-dependent DNA polymerase and RNase H activity. The enzyme is Escherichia coli ) in a fully active form. The reverse transcriptase of HIV is a heterodimer of the subunits of p66 and p51, and the p51 subunit is produced by proteolytic cleavage of the p66 subunit. The p66 subunit has both an RNA-dependent DNA polymerase and an RNase H domain, whereas the p51 subunit has only a DNA polymerase domain. The p66/p51 reverse transcriptase of activated HIV is Escherichia coli ) has been cloned and expressed in a number of expression hosts. Within the HIV p66/p51 heterodimer, the 51 kDa subunit is catalytically inactive and the 66 kDa subunit has both DNA polymerase and RNase H activities. In addition, reverse transcriptase of Rous Sarcoma Virus (RSV), reverse transcriptase of avian myeloblastosis virus, reverse transcriptase of Avian Erythroblastosis Virus (AEV) helper virus MCAV, and avian myelocytosis virus (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 Avian Sarcoma-Leukosis Virus (Avian Sarcoma-Leukosis Virus), such as reverse transcriptase of YAV's helper virus, reverse transcriptase of Rous Associated Virus (RAV) and reverse transcriptase of Myeloblastosis Associated Virus (MAV). ASLV) is also a heterodimer of two subunits alpha (approximately 62 kDa) and beta (approximately 94 kDa), which alpha is produced by proteolytic cleavage from beta. ASLV reverse transcriptase can exist in two catalytically active structural forms, alpha-beta and beta-beta. By sedimentation analysis, it is known that the alpha-beta and beta-beta structures are dimers, and that the alpha exists in equilibrium between the monomer form and the dimer form. The alpha-beta and beta-beta reverse transcriptases of ASLV are the only conventionally known to have three different activities: DNA polymerase activity, RNase H activity and DNA endonuclease (integrase) activity within the same protein complex. It is a retroviral reverse transcriptase. The alpha form has no integrase domain and activity.

The conversion from mRNA to cDNA by reverse transcriptase-mediated reverse transcription is important for many gene expression studies. However, the use of an unmodified reverse transcriptase to catalyze reverse transcription is undesirable in several respects. First, reverse transcriptase may degrade the RNA template before the first strand reaction is initiated or completed by RNase H activity present in reverse transcriptase. In addition, errors may occur in the first strand of cDNA due to mis-priming of the mRNA template molecule. In fact, in the cDNA synthesis process, an error occurs at 1 base per 3,000 to 6,000 nucleotides in the case of HIV reverse transcriptase and 1 base per 10,000 nucleotides in the case of AMV reverse transcriptase.

Another factor that influences the efficacy of reverse transcriptase is whether RNA can form secondary structures. Such secondary structures can be formed when, for example, regions of RNA molecules are sufficiently complementary to hybridize to form double-stranded RNA. In general, the formation of RNA secondary structures can be reduced by raising the temperature of the solution containing the RNA molecule. Therefore, in many cases, it is desirable to reverse transcription of RNA at a temperature of 37°C or higher. However, reverse transcriptase known in the art generally loses its activity only when incubated at a temperature significantly higher than 37°C (eg, 50°C).

Various methods are known in the art for processing thermostable reverse transcriptases. The above methods include a method of using a heat-stable DNA polymerase having reverse transcriptase activity, a method of inducing a mutation in a heat-stable DNA polymerase to increase its reverse transcriptase activity, a step of inducing a mutation in a heat-labile reverse transcriptase, Taq / in the presence of the Tth DNA polymerase with the method, heat labile reverse transcriptase using a Mn ^{+ 2} in place of Mg ^{2 +,} and the like method using an additive such as trehalose.

In addition, those skilled in the art have used various enzyme compositions and methods to increase fidelity when polymerizing DNA or RNA templates. For example, Shevelev et al. ( Nature Rev. Mol . Cell Biol . 3:364 (2002)) provides a review paper on 3'-5' exonucleases. Perrino et al., PNAS USA, 86:3085 (1989)), to increase the accuracy of calf thymus DNA polymerase alpha, Escherichia coli ) The epsilon subunit of DNA polymerase III is used. Bakhanashvili ( Eur . J. Biochem . 268:2047 (2001)) discloses the proofreading activity of p53 protein, Huang et al., Oncogene , 17:261 (1998) ) Discloses the use of p53 to enhance the accuracy of DNA replication. In addition, U.S. Patent Publication No. 2003/0198944A1 and U.S. Patent No. 6,518,019 contain two or more reverse transcriptases (e.g., each reverse transcriptase has a different transcription stop site) and, if necessary, one or more DNA polymerases. Enzyme mixtures are disclosed. US 2002/0119465A1 discloses a composition comprising a variant thermostable DNA polymerase and a variant reverse transcriptase (eg, variant Taq DNA polymerase and variant MMLV-RT). U.S. Patent No. 6,485,917B1, U.S. Patent Publication No. 2003/0077762 and European Patent Publication EP1132470 disclose cDNA in the presence of an enzyme having a reverse transcriptase activity and an α-type DNA polymerase having a 3'-5' exonuclease activity. A method of synthesis is disclosed.

If the RNase H activity of the reverse transcriptase is removed, the problem of RNA degradation of the RNA template can be excluded, and the reverse transcription efficiency can be improved. However, this reverse transcriptase ('RNase H-' type) has a disadvantage in that it cannot improve problems such as miss-priming and mRNA secondary structure. In addition, conventional reverse transcriptase generally has low thermal stability, and thus reverse transcription reaction proceeds only at a relatively low temperature. In some cases, it is due to structural interference with RNA progression that forms a stable secondary structure even at a high temperature of 65°C or higher. Therefore, it has a limitation in that it cannot efficiently obtain reverse transcription products. This limitation acts as a major limiting factor in various biochemical experiments that require reverse transcription, such as RNA detection and profiling, as well as the process of producing cDNA from total RNA. Accordingly, there is an increasing need for a novel reverse transcriptase having a stable reverse transcription activity even at a higher temperature.

US Patent Publication No. 2003/0198944A1 U.S. Patent No. 6,518,019 US Patent Publication No. 2002/0119465A1 U.S. Patent No. 6,485,917B1 US Patent Publication No. 2003/0077762 European Patent Publication EP1132470

Shevelev et al., Nature Rev. Mol. Cell Biol. 3:364 (2002) Perrino et al., PNAS USA, 86:3085 (1989) Bakhanashvili, Eur. J. Biochem. 268:2047 (2001) Huang et al., Oncogene, 17:261 (1998)

The present invention was derived to improve the problems of the conventional reverse transcriptase as described above, and by mutating a specific amino acid residue of the M-MLV-derived reverse transcriptase, it exhibits excellent reverse transcription activity even at a high temperature above the temperature at which a structural change of RNA can occur. It is an object of the present invention to provide a reverse transcriptase with increased thermal stability that can stably produce cDNA.

In order to achieve the above object, the present invention is a wild-type M-MLV (Moloney-Murine Leukemia Virus, M-MLV) derived from SEQ ID NO: 1, the 306 th threonine of the amino acid sequence of the reverse transcriptase is substituted with leucine (T306L). Provides reverse transcriptase with increased thermal stability.

According to one embodiment of the present invention, the reverse transcriptase of the present invention is a substitution of leucine at the 63rd glutamine in the amino acid sequence of the wild-type M-MLV-derived reverse transcriptase represented by SEQ ID NO: 1 (Q63L), leucine at the 264th lysine. Substitution with god (K264L), lysine at position 295 to glutamine (K295Q), glutamic acid at position 346 to methionine (E346M), proline at position 408 to glutamic acid (P408E), histidine at position 438 to tyrosine It may further include one or more amino acid substitutions selected from the group consisting of a substitution of (H438Y) and a substitution of phenylalanine at the 454th asparagine (N454F). According to a preferred embodiment of the present invention, the reverse transcriptase of the present invention may consist of an amino acid sequence represented by SEQ ID NO: 5.

According to another embodiment of the present invention, the reverse transcriptase of the present invention may exhibit excellent reverse transcription activity compared to the wild-type M-MLV reverse transcriptase consisting of the amino acid sequence represented by SEQ ID NO: 1 at a temperature of 60°C to 70°C.

In addition, the present invention provides a gene encoding the reverse transcriptase having increased thermal stability.

According to an embodiment of the present invention, the gene is a wild-type reverse transcriptase represented by SEQ ID NO: 1 at the 63rd glutamine to leucine substitution (Q63L), the 264th lysine to leucine substitution (K264L), 295th Lysine to glutamine (K295Q), glutamic acid at 346 to methionine (E346M), proline at 408 to glutamic acid (P408E), histidine at 438 to tyrosine (H438Y) and asparagine at 454 It is possible to encode a mutant reverse transcriptase further comprising one or more amino acid substitutions selected from the group consisting of substitution with phenylalanine (N454F). According to a preferred embodiment of the present invention, the gene may consist of a nucleotide sequence represented by SEQ ID NO: 14.

In addition, the present invention provides an expression vector containing the gene.

In addition, the present invention provides a transformant transformed with the expression vector.

In addition, the present invention provides a composition for RT-PCR comprising a reverse transcriptase, a primer pair, dNTP, and a buffer solution having increased thermal stability.

In addition, the present invention provides a kit for a reverse transcription reaction comprising a reverse transcriptase having increased thermal stability.

The mutant reverse transcriptase of the present invention exhibits excellent thermal stability compared to the wild-type reverse transcriptase, so even when the reverse transcription product cannot be efficiently obtained due to structural interference of a specific RNA that can form a stable secondary structure even at high temperature, it is stable reverse transcription. It shows activity and can obtain a desired cDNA.

1 shows the location and type of amino acids mutated in the mutant reverse transcriptase of the present invention.
2A to 2B show the positions and sequences of mutated bases in the gene encoding the mutant reverse transcriptase of the present invention.
3 is an electrophoretic photograph showing the results of reverse transcription reaction at 42, 50, 60, 65 and 70°C using wild-type reverse transcriptase (SEQ ID NO: 1) and mutant reverse transcriptase of the present invention (SEQ ID NO: 2 to SEQ ID NO: 9) to be.
Figure 4 is an electrophoresis photograph showing the results of reverse transcription reaction at 37, 42, 50, 60, 65 and 70°C using wild-type reverse transcriptase (SEQ ID NO: 1) and T306L mutant reverse transcriptase of the present invention (SEQ ID NO: 5). .

Unless otherwise stated, terms, techniques, and the like described in the present invention have the meanings generally used in the technical field to which the present invention belongs. In addition, all of the documents disclosed in the present specification are included in the present specification as documents for explaining the present invention.

In the present invention, the term "reverse transcriptase activity" or "reverse transcription" refers to the ability of an enzyme to synthesize a DNA chain (ie, complementary DNA or cDNA) using an RNA chain as a template. In the present invention, "reverse transcriptase" refers to any enzyme that exhibits reverse transcriptase activity when measured according to any method disclosed herein or known in the art.

In the present invention, the term "reverse transcriptase activity" or "reverse transcriptase activity" refers to the ability of an enzyme (eg, reverse transcriptase or DNA polymerase) to synthesize a DNA chain (ie, cDNA) using an RNA chain as a template. It is used interchangeably to indicate

In the present invention, the term "mutation" or "mutation" refers to a change introduced into a wild-type DNA sequence to change the amino acid sequence encoded by the DNA. These include, but are not limited to, substitutions, insertions, deletions, and point mutations.

In the present invention, the term "wild type" refers to a gene or gene product having the same characteristics as when a gene or gene product is isolated from a natural source. In contrast, the term "modified" or "mutant" refers to a gene or gene product that has characteristics that are altered compared to a wild-type gene or gene product.

In the present invention, the term "expression vector" refers to a collection (expression cassette) in which one or more transcription control regions necessarily including a coding sequence and a promoter are operably linked, Or it means a vector containing the aggregate.

In the present invention, the term "coding sequence" refers to a DNA sequence encoding a specific amino acid or functional RNA.

In the present invention, the term "promoter" refers to a site that induces a transcription process of a coding sequence into RNA, and generally, a promoter refers to a site where a polymerase and transcription factors bind.

In the present invention, amino acid residues constituting reverse transcriptase are represented by three or one alphabetic abbreviations shown in Table 1.

Alanine A Ala Cysteine C Cys Aspartic acid D Asp Glutamic acid E Glu Phenylalanine F Phe Glycine G Gly Histidine H His Isoleucine I Ile Lee Sin K Lys Leucine L Leu Methionine M Met Asparagine N Asn Proline P Pro Glutamine Q Gln Arginine R Arg Serine S Ser Threonine T Thr Selenocysteine U Sec Valine V Val Tryptophan W Trp Tyrosine Y Tyr

Hereinafter, the present invention will be described in detail.

The present invention is the amino acid sequence of the M-MLV derived reverse transcriptase described in SEQ ID NO: 1 at the 63rd glutamine (Q63), the 264th lysine (K264), the 295th lysine (K295), and the 306th threonine (T306). , At least one amino acid selected from the group consisting of glutamic acid at position 346 (E346), proline at position 408 (P408), histidine at position 438 (H438) and asparagine at position 454 (N454) is substituted with another amino acid to provide thermal stability. Provides increased reverse transcriptase.

Substitution of amino acids as described above is a substitution from glutamine at position 63 to leucine (Q63L), lysine at position 264 to leucine (K264L), lysine at position 295 to glutamine (K295Q), threonine at position 306 to Lewis Substitution with god (T306L), glutamic acid at position 346 to methionine (E346M), proline at position 408 to glutamic acid (P408E), histidine at position 438 to tyrosine (H438Y) and asparagine at position 454 to phenylalanine A substitution of one or more amino acids selected from the group consisting of (N454F) is included, but is not limited thereto (see FIG. 1 ). According to one embodiment of the present invention, the heat stability effect of eight mutant reverse transcriptases having an amino acid sequence represented by SEQ ID NO: 2 to SEQ ID NO: 9 in which the amino acids at the 8 positions are substituted, respectively, was confirmed, but at least two positions Reverse transcriptases in which mutations have occurred at the same time may also be included within the scope of the present invention within the scope of the present invention as long as it has thermal stability at high temperatures.

According to a preferred embodiment of the present invention, the mutant reverse transcriptase of the present invention has heat stability compared to the wild-type reverse transcriptase, and among them, K295Q, T306L and P408E mutant reverse transcriptases exhibit excellent heat stability (see FIG. 3). In particular, in the case of the T306L mutant reverse transcriptase exhibiting the most excellent thermal stability, cDNA can be generated by exhibiting superior thermal stability than the wild-type M-MLV-derived reverse transcriptase even at 60°C, 65°C, and 70°C (see FIG. 4).

Those skilled in the art will generally recognize that amino acids substituted as described above may have similar physiological and biochemical properties even if they are replaced with other amino acids of relatively similar properties (i.e., conservative amino acid substitutions). . The effect of amino acid substitution on the properties and protein structure and function of various amino acids has been the subject of extensive research and knowledge in the art.

For example, the hydropathic index of amino acids can be considered (Kyte & Doolittle, 1982, J. Mol . Biol ., 157:105-132). The relative hydropathic properties of amino acids contribute to the secondary structure of the resulting protein, which in turn defines the protein's interaction with other molecules. Each amino acid is assigned a hydropathic index based on its hydrophobicity and charge properties (Kyte & Doolittle, 1982, J. Mol . Biol ., 157:105-132), which are as follows: isoleucine (+) 4.5); Valine (+4.2); Leucine (+3.8); Phenylalanine (+2.8); Cysteine/cystine (+2.5); Methionine (+1.9); Alanine (+1.8); Glycine (-0.4); Threonine (-0.7); Serine (-0.8); Tryptophan (-0.9); Tyrosine (-1.3); Proline (-1.6); Histidine (-3.2); Glutamic acid (-3.5); Glutamine (-3.5); Aspartic acid (-3.5); Asparagine (-3.5); Lysine (-3.9); And arginine (-4.5). In making conservative substitutions, it is preferable to use an amino acid whose hydropathic index is within ±2, more preferably within ±1, and even more preferably within ±0.5.

Amino acid substitutions can also take into account the hydrophilicity of amino acid residues (see, eg, US Pat. No. 4,554,101). Hydrophilicity values are assigned for each amino acid residue, which are as follows: arginine (+3.0); Lysine (+3.0); Aspartic acid (+3.0); Glutamic acid (+3.0); Serine (+0.3); Asparagine (+0.2); Glutamine (+0.2); Glycine (0); Threonine (-0.4); Proline (-0.5 .+-.1); Alanine (-0.5); Histidine (-0.5); Cysteine (-1.0); Methionine (-1.3); Valine (-1.5); Leucine (-1.8); Isoleucine (-1.8); Tyrosine (-2.3); Phenylalanine (-2.5); Tryptophan (-3.4). It is desirable, but not necessary, to replace the amino acid with another amino acid having similar hydrophilicity.

Another consideration may be the size of the amino acid side chains. For example, it would generally not be desirable to replace an amino acid having a dense side chain such as glycine or serine with an amino acid having a large side chain such as tryptophan or tyrosine. The influence of various amino acid residues on protein secondary structure is also a consideration. Through empirical studies, the influence of different amino acid residues on the tendency of protein domains to adopt an alpha-helix, beta-sheet or reverse turn secondary structure has been determined and is known in the art ( See, e.g., Chou & Fasman, 1974, Biochemistry , 13:222-245; 1978, Ann. Rev. Biochem ., 47:251-276; 1979, Biophys. J. , 26:367-384).

Based on these considerations and extensive empirical studies, tables of conservative amino acid substitutions have been created and are known in the art. For example arginine and lysine; Glutamic acid and aspartic acid; Serine and threonine; Glutamine and asparagine; And valine, leucine and isoleucine. On the other hand, Ala(A) leu, ile, val; Arg(R) gln, asn, lys; Asn(N) his, asp, lys, arg, gln; Asp(D) asn, glu; Cys(C) ala, ser; Gln(Q) glu, asn; Glu(E) gln, asp; Gly(G) ala; His(H) asn, gln, lys, arg; Ile(I) val, met, ala, phe, leu; Leu(L) val, met, ala, phe, ile; Lys(K) gln, asn, arg; Met(M) phe, ile, leu; Phe(F) leu, val, ile, ala, tyr; Pro(P) ala; Ser(S), thr; Thr(T) ser; Trp(W) phe, tyr; Tyr(Y) trp, phe, thr, ser; Val(V) ile, leu, met, phe, ala.

Other considerations for amino acid substitution include whether the residue is located inside the protein or whether it is exposed to a solvent. For internal residues, conservative substitutions may include: Asp and Asn; Ser and Thr; Ser and Ala; Thr and Ala; Ala and Gly; Ile and Val; Val and Leu; Leu and Ile; Leu and Met; Phe and Tyr; Tyr and Trp. For solvent exposed residues, conservative substitutions may include: Asp and Asn; Asp and Glu; Glu and Gin; Glu and Ala; Gly and Asn; Ala and Pro; Ala and Gly; Ala and Ser; Ala and Lys; Ser and Thr; Lys and Arg; Val and Leu; Leu and Ile; Ile and Val; Phe and Tyr.

In determining amino acid substitution, ionic bonds (salt bridges) between positively charged residues (e.g., His, Arg, Lys) and negatively charged residues (e.g., Asp, Glu) or disulfide bonds between adjacent cysteine residues It is desirable to take into account the presence of intermolecular or intramolecular bonds, such as the formation of.

In addition, the present invention provides a gene encoding the reverse transcriptase having increased thermal stability.

The gene is a wild-type reverse transcriptase represented by SEQ ID NO: 1 at 63 glutamine to leucine substitution (Q63L), 264 lysine to leucine substitution (K264L), 295 lysine to glutamine substitution (K295Q) , Threonine at position 306 to leucine (T306L), glutamic acid at position 346 to methionine (E346M), proline at position 408 to glutamic acid (P408E), histidine at position 438 to tyrosine (H438Y), and It is a gene encoding a mutant reverse transcriptase containing one or more amino acid substitutions selected from the group consisting of substitution of asparagine at position 454 to phenylalanine (N454F). According to one embodiment of the present invention, the gene of the present invention is SEQ ID NO: 11 to encode eight mutant reverse transcriptases having an amino acid sequence represented by SEQ ID NO: 2 to SEQ ID NO: 9 in which the amino acids at the 8 positions are respectively substituted. It is preferably a gene having a nucleotide sequence represented by SEQ ID NO: 18, but is not limited thereto. For example, glutamine at the 63rd position (Q63), lysine at the 264th position (K264), lysine at the 295th position (K295), threonine at the 306th position in the amino acid sequence of the reverse transcriptase derived from M-MLV shown in SEQ ID NO: 1, Any gene encoding a reverse transcriptase in which two or more of glutamic acid at position 346 (E346), proline at position 408 (P408), histidine at position 438 (H438), and asparagine at position 454 (N454) are mutated can also be used in the present invention. It may be included within the scope of rights (see FIGS. 2A to 2D).

In addition, polynucleotides that are substantially identical and functionally equivalent to the genes mentioned in the present invention are also included in the scope of the genes of the present invention. The term'substantially identical and functionally equivalent polynucleotides' means that two polynucleotides are at least 70%, preferably at least 80%, more preferably when properly arranged by computerized execution of known algorithms or by investigation. It is meant to share at least 90%, most preferably at least 95%, sequence homology.

In addition, the present invention provides an expression vector containing the gene.

There is no particular limitation on the parent vector that can be used to prepare the expression vector of the present invention, and a vector for transformation of a conventional prokaryote or eukaryote may be used. In a preferred embodiment of the present invention, a recombinant expression vector was prepared in which each mutant gene represented by SEQ ID NO: 11 to SEQ ID NO: 18 was inserted into the extranuclear gene vector PET 22b(+).

In addition, the present invention provides a transformant transformed with the expression vector.

The transformant of the present invention can be easily prepared by introducing the expression vector into any prokaryotic or eukaryotic cell, and a method of introducing a specific vector into a cell is well known to those skilled in the art. Examples of such methods include the lipofectamine method. In a preferred embodiment of the present invention, a transformant was prepared by introducing the mutant gene-inserted PET 22b(+) vector into E. coli DH5α host cells.

In addition, the present invention provides a reverse transcription reaction kit comprising the reverse transcriptase.

In addition to the reverse transcriptase, the kit for reverse transcription of the present invention is a primer pair that specifically binds to a target gene or oligonucleotide that is desired to be amplified, such as a primer pair, dNTP, a reaction buffer, optionally It may further include a DNA polymerase and the like.

Hereinafter, the present invention will be described in detail by examples.

However, the following examples are for illustrative purposes only, and the contents of the present invention are not limited by the following examples.

Example 1. Preparation of gene coating mutant reverse transcriptase

In order to introduce a mutation into the M-MLV-derived reverse transcriptase having the amino acid sequence shown in SEQ ID NO: 1, the nucleotide sequence on the gene encoding the M-MLV-derived reverse transcriptase was substituted. Specifically, M-MLV-derived reverse transcriptase having the amino acid sequence shown in SEQ ID NO: 1 at the 63rd glutamine to leucine (Q63L), the 264th lysine to leucine (K264L), at the 295th lysine Glutamine mutation (K295Q), threonine to leucine at position 306 (T306L), glutamic acid to methionine at position 346 (E346M), proline to glutamic acid at position 408 (P408E), histidine to tyrosine at position 438 Eight mutant reverse transcriptases in which mutations to (H438Y mutation) and 454th asparagine to phenylalanine (N454F) were introduced were prepared (see FIG. 1), and for this purpose, reverse transcriptase having the amino acid sequence of SEQ ID NO: 1 was used. A mutation was introduced into the gene represented by the coding SEQ ID NO: 10 (see FIGS. 2A to 2D). Each of the mutant genes has a nucleotide sequence represented by SEQ ID NO: 11 to SEQ ID NO: 18. The manufacturing process of the mutant gene is as follows:

In order to increase the protein expression level in Escherichia coli , the gene sequence of the M-MLV reverse transcriptase described in SEQ ID NO: 10 was optimized according to the codon usage of Escherichia coli to obtain the nucleotide sequence shown in SEQ ID NO: 19. . Based on the optimized nucleotide sequence, a wild-type gene and a K295Q mutant gene were synthesized. First, in order to obtain the nucleotide sequence of the wild-type gene represented by SEQ ID NO: 19, the sense strand and the antisense strand were designed to each have an annealing temperature of about 60°C (20 to 40 bases) based on the entire nucleotide sequence. , Designed oligonucleotides were synthesized (Bionia, KOREA). The synthesized oligonucleotides each have a nucleotide sequence represented by SEQ ID NO: 20 to SEQ ID NO: 149. The base described in SEQ ID NO: 19 by repeatedly reacting at 40°C and 60°C using the synthesized oligonucleotides by the Kination-LCR method (Korean Patent Application No. 2008-0025050). The entire double-stranded gene having the sequence was synthesized. In addition, the K295Q mutant gene was synthesized using the same method as the wild-type gene, except that the nucleotide sequences shown in SEQ ID NO: 150 and SEQ ID NO: 151 were used instead of the nucleotide sequences shown in SEQ ID NO: 77 and SEQ ID NO: 78. After pre-heating the synthesized gene at 95°C for 5 minutes, using a primer pair described in SEQ ID NO: 152 and SEQ ID NO: 153 for 1 minute at 95°C, 1 minute at 65°C, 2 minutes 30 seconds at 72°C The reaction was carried out, and after repeating this process 30 times, the gene was increased by reacting for an additional 10 minutes at 72°C. The increased synthetic gene was ligated into a pGEM-T easy vector (Promega, USA), and then transformed into E. coli DH5a (RBC, USA) to obtain a large amount of clones of wild-type and K295Q mutant genes.

Using the thus synthesized wild-type gene sequence as a template, Q63L, K264L, T306L, E346M, P408E, H438Y and N454F were mutated using site-directed mutagenesis. The position-directed mutation method was carried out as follows: First, oligonucleotides having nucleotide sequences represented by SEQ ID NO: 154 to SEQ ID NO: 167 were synthesized for the mutant gene. After heat treatment at 95° C. for 5 minutes, the synthesized oligonucleotide was reacted for 1 minute at 95° C., 1 minute at 65° C., 5 minutes 30 seconds at 72° C. using pfu DNA polymerase as a primer. After repeating a total of 30 times, a mutant gene product was generated by further reacting at the next 72°C. The mutant gene products thus generated were subjected to kination-self ligation and then transformed into E. coli DH5a (RBC, USA) to secure large amounts of clones of the Q63L, K264L, T306L, E346M, P408E, H438Y and N454F mutant genes. I did.

Example 2. Transformation of mutant gene

Eight mutant reverse transcriptase genes prepared in Example 1 were cloned using the extranuclear gene PET 22b(+) (NOVAGEN CO.) as a recombinant vector. To this end, the mutant genes (Q63L, K264L, T306L and E346M) synthesized by being inserted into the pGEM-T easy vector (Promega) were subjected to PCR using the following primer pairs.

Sense strand: 5'-GCG CGC CAT ATG CTG AAC ATC GAA GAC GAA CAC CGT CTG CAC GAA AC-3' (Nde I) (SEQ ID NO: 168)

Antisense strand: 5'-GCG CGC GCG GCC GCT TAG ATC AGC AGG GTA GAG GTG TCC GGG GTT TC-3' (Not I) (SEQ ID NO: 169)

In the case of other mutant genes (K295Q, P408E, H438Y, N454F), PCR was performed using the following primer pairs.

Sense strand: 5'-GCG CGC CAT ATG CTG AAC ATC GAA GAC GAA CAC CGT CTG CAC GAA AC-3' (Nde I) (SEQ ID NO: 170)

Antisense strand: 5'-GCG CGC GCG GCC GCG ATC AGC AGG GTA GAG GTG TCC GGG GTT TC-3' (Not I) (SEQ ID NO: 171)

For each M-MLV reverse transcription gene fragment obtained through the extranuclear gene vector PET 22b(+) and PCR, the 5'end was treated with Nde I and the 3'end was cut with Not I, and then a gel extraction kit (BIONEER CO.) was used. It was purified using. Thereafter, the cut fragments were reacted at 16° C. for 2 hours using T4 DNA ligase (TAKARA CO.), and each mutant gene was inserted into the extranuclear gene vector PET 22b(+).

PET 22b (+) vector into which the mutant gene was inserted was transferred to Escherichia coli) DH5α host cells were transformed to generate recombinant host cells. The transformation was carried out in the following order: DH5α host cells in a frozen state at -70°C were thawed. The thawed host cells and 1 µg/µl of the DNA recombinant vector were mixed and left on ice for 30 minutes, followed by heat treatment at 42°C for 90 seconds. The heat-treated host cells were plated on an LB cell culture medium plate containing ampicillin (50 mg/ml) and cultured overnight at 37°C to obtain clones. The obtained clone was confirmed for each gene sequence using a primer pair having the following nucleotide sequence, and the confirmed clone was transformed into a BL21 (DE3) host cell again to obtain an expression strain.

T7 promoter sense strand: 5'-TAA TAC GAC TCA CTA TAG GG-3' (SEQ ID NO: 172)

T7 terminator antisense strand: 5'-GCT AGT TAT TGC TCA GCG G-3' (SEQ ID NO: 173)

Example 3. Expression of mutant reverse transcriptase

After taking a single colony of the expression strain transformed into BL21(DE3) host cell, put it in 15 ml of LB (Luria-Bertani medium) cell culture solution containing ampicillin (50 mg/ml) and incubated overnight at 37°C to seed. (seed) strain cells were obtained. The seed strain cells were added to 1 ℓ of LB cell culture solution containing ampicillin and cultured at 37° C. for 2 hours, and then 0.5 mM IPTG (Isopropyl β-D-1-thiogalactopyranoside) was added and expressed for 2 hours. After centrifuging the cultured cells of each strain, the obtained pellet was stored at -50°C until purification.

Example 4. Purification of mutant reverse transcriptase

After crushing 5 g of the frozen cell mass obtained in Example 3, lysis buffer (25 mM Tris hydrochloric acid (Tris-HCl pH 7.6), 1 mM EDTA, 10% (v/v) β-mergatto) under stirring at 4° C. It was suspended in ethanol and phenylmethylsulfonyl fluoride (PMSF) (20 ml) for 30 minutes. Then, the cells were lysed with a fisher sonicator. Then, the cell lysate was centrifuged at 11,000 rpm for 1 hour at 4° C., and the pellet was decanted. The clear lysate was immediately dialyzed against a buffer solution (40 mM Tris hydrochloric acid (Tris-HCl pH 7.6), 2 mM EDTA, 28.58 mM β-mercaptoethanol, 100 mM KCl).

Example 5. Chromatography of mutant reverse transcriptase

To purify the mutant reverse transcriptase, it was carried out using an Amsiam FPLC apparatus. As the primary column, flaxiam XK16 (1.6 cm x 30.0 cm) was used. The column was charged with ion exchange resin column chromatography DEAE hyper-D (pall co.), washed with 250 ml of a column buffer containing 1 M KCl, and then equilibrated with a buffer solution to obtain a 60 ml bed. Then, 20 ml of clear cell lysate was applied to the column at a flow rate of 5 ml/min. Among the applied lysates, lysates that were not adsorbed on the column were obtained and applied to the next column. The obtained lysate was subjected to secondary chromatography. After washing and equilibrating the column flaxiam XK16 (1.6 cm×30.0 cm), affinity chromatography heparin hyper-D (pall co.) was added to obtain a 50 ml bed. Then, it was equilibrated with a buffer solution. After the lysate was applied to the column, it was eluted using a 0.2 M to 0.6 M KCl linear salt gradient of 100 to 150 ml in a column buffer. The eluted lysate was applied to a column 26X60 S-200 gel filteration (GE healthcare) to obtain a high-purity reverse transcriptase. The column fraction was subjected to SDS-polyacrylamide gel electrophoresis (SDS-PAGE), followed by staining (coomassie brilliant blue staining) to analyze. The 10 μl fractions taken from each fraction were analyzed in each gel lane. A protein with a molecular weight (74 kDa) similar to that of the wild type was taken, and to ensure the stability of the protein, a preservative solution (20 mM Tris-HCl (pH 7.6), 0.1 mM EDTA, 150 mM NaCl, 0.1% IGEPAL CA-630 (Polyethoxyethanol)) ) Dialysis was performed with 1 mM DTT (Dithiothreitol) and 50% glycerol to obtain a mutant enzyme and stored at -20°C.

Example 6. RT-PCR performance using mutant reverse transcriptase

Reverse transcription PCR was performed to confirm the activity of the isolated reverse transcriptase. Materials used for reverse transcription PCR were as follows, and total RNA was used at concentrations of 5 ng/µl to 500, 50, and 5 pg/µl.

5X reaction buffer 4 µl (Bioneer co.)

10 mM dNTP 2 μl

dT(18) 10 pmol 1 μl

100 mM DTT 2 μl

1 μl of RNase inhibitor (50 ng/μl)

Distilled water 7 μl

Total RNA 2 μl

As a comparative example, wild-type reverse transcriptase was used, and reverse transcription PCR was performed for 1 hour at 42°C, 50°C, 60°C, 65°C and 70°C (T306L mutation added the reaction at 37°C). PCR was performed using the cDNA generated above. GAPDH was used as a gene to be amplified (gene size 500 bp), and a primer pair having the following nucleotide sequence was used.

Sense strand: 5'-GAAGGTGAAGGTCGGAGTCAACG-3' (SEQ ID NO: 174)

Antisense strand: 5'-AGTCCTTCCACGATACCAAAGTTG-3' (SEQ ID NO: 175)

As the preparation, those of the following composition were used:

PCR premix type (Bionia)

Sense Primer: 5 p㏖, 1 µl

Antisense Primer: 5 p㏖, 1 µl

Distilled water 13 μl

cDNA 5 μl

PCR was performed for 30 cycles under reaction conditions of 95°C for 30 seconds, 57°C for 30 seconds, and 72°C for 30 seconds, and the PCR result was subjected to electrophoresis on an agarose gel to confirm the amplification product.

As a result, the mutant reverse transcriptase of the present invention derived from wild-type M-MLV has superior thermal stability compared to the wild-type reverse transcriptase, and among them, K295Q, T306L and P408E mutant reverse transcriptases exhibited particularly excellent thermal stability. In particular, wild-type M-MLV reverse transcriptase did not have reverse transcription activity at a reaction temperature of 60° C., but K295Q, T306L and P408E mutant reverse transcriptases maintained reverse transcription activity (FIG. 3). In the case of the T306L mutant reverse transcriptase, which exhibits the best thermal stability, at 37°C, 42°C and 50°C, the same thermal stability as the wild-type M-MLV-derived reverse transcriptase was shown, but at 60°C, 65°C and 70°C, the wild-type M-MLV It showed superior thermal stability than the derived reverse transcriptase (FIG. 4).

<110> BIONEER CORPORATION <120> Reverse Transcriptase Having Improved Thermostability <130> 2016-dpa-1367d <160> 175 <170> KopatentIn 2.0 <210> 1 <211> 672 <212> PRT <213> Moloney Murine Leukemia Virus <400> 1 Met 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 Ile 660 665 670 <210> 2 <211> 672 <212> PRT <213> Moloney Murine Leukemia Virus <220> <221> MUTAGEN <222> (63) <223> mutant reverse transcriptase having leucine residue instead of glutamine residue at 63 position of SEQ. ID. No: 1 <400> 2 Met 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 Leu 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 Ile 660 665 670 <210> 3 <211> 672 <212> PRT <213> Moloney Murine Leukemia Virus <220> <221> MUTAGEN <222> (264) <223> mutant reverse transcriptase having leucine residue instead of lysine residue at 264 position of SEQ. ID. No: 1 <400> 3 Met 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 Leu 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 Ile 660 665 670 <210> 4 <211> 672 <212> PRT <213> Moloney Murine Leukemia Virus <220> <221> MUTAGEN <222> (295) <223> mutant reverse transcriptase having glutamine residue instead of lysine residue at 295 position of SEQ. ID. No: 1 <400> 4 Met 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 Gln 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 Ile 660 665 670 <210> 5 <211> 672 <212> PRT <213> Moloney Murine Leukemia Virus <220> <221> MUTAGEN <222> (306) <223> mutant reverse transcriptase having leucine residue instead of threonine residue at 306 position of SEQ. ID. No: 1 <400> 5 Met 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 Leu 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 Ile 660 665 670 <210> 6 <211> 672 <212> PRT <213> Moloney Murine Leukemia Virus <220> <221> MUTAGEN <222> (346) <223> mutant reverse transcriptase having methionine residue instead of glutamic acid residue at 346 position of SEQ. ID. No: 1 <400> 6 Met 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 Met 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 Ile 660 665 670 <210> 7 <211> 672 <212> PRT <213> Moloney Murine Leukemia Virus <220> <221> MUTAGEN <222> (408) <223> mutant reverse transcriptase having glutamic acid residue instead of proline residue at 408 position of SEQ. ID. No: 1 <400> 7 Met 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 Glu 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 Ile 660 665 670 <210> 8 <211> 672 <212> PRT <213> Moloney Murine Leukemia Virus <220> <221> MUTAGEN <222> (438) <223> mutant reverse transcriptase having tyrosine residue instead of histidine residue at 438 position of SEQ. ID. No: 1 <400> 8 Met 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 Tyr 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 Ile 660 665 670 <210> 9 <211> 672 <212> PRT <213> Moloney Murine Leukemia Virus <220> <221> MUTAGEN <222> (454) <223> mutant reverse transcriptase having phenylalanine residue instead of asparagine residue at 454 position of SEQ. ID. No: 1 <400> 9 Met 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 Phe 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 Ile 660 665 670 <210> 10 <211> 2016 <212> DNA <213> Moloney Murine Leukemia Virus <220> <221> mat_peptide <222> (1)..(2016) <223> nucleotide sequence coding for the amino acid sequence of SEQ. ID. No: 1 <400> 10 atgctaaata tagaagatga gcatcggcta catgagacct caaaagagcc agatgtttct 60 ctagggtcca catggctgtc tgattttcct caggcctggg cggaaaccgg gggcatggga 120 ctggcagttc gccaagctcc tctgatcata cctctgaaag caacctctac ccccgtgtcc 180 ataaaacaat accccatgtc acaagaagcc agactgggga tcaagcccca catacagaga 240 ctgttggacc agggaatact ggtaccctgc cagtccccct ggaacacgcc cctgctaccc 300 gttaagaaac cagggactaa tgattatagg cctgtccagg atctgagaga agtcaacaag 360 cgggtggaag acatccaccc caccgtgccc aacccttaca acctcttgag cgggctccca 420 ccgtcccacc agtggtacac tgtgcttgat ttaaaggatg cctttttctg cctgagactc 480 caccccacca gtcagcctct cttcgccttt gagtggagag atccagagat gggaatctca 540 ggacaattga cctggaccag actcccacag ggtttcaaaa acagtcccac cctgtttgat 600 gaggcactgc acagagacct agcagacttc cggatccagc acccagactt gatcctgcta 660 cagtacgtgg atgacttact gctggccgcc acttctgagc tagactgcca acaaggtact 720 cgggccctgt tacaaaccct agggaacctc gggtatcggg cctcggccaa gaaagcccaa 780 atttgccaga aacaggtcaa gtatctgggg tatcttctaa aagagggtca gagatggctg 840 actgaggcca gaaaagagac tgtgatgggg cagcctactc cgaagacccc tcgacaacta 900 agggagttcc tagggacggc aggcttctgt cgcctctgga tccctgggtt tgcagaaatg 960 gcagccccct tgtaccctct caccaaaacg gggactctgt ttaattgggg cccagaccaa 1020 caaaaggcct atcaagaaat caagcaagct cttctaactg ccccagccct ggggttgcca 1080 gatttgacta agccctttga actctttgtc gacgagaagc agggctacgc caaaggtgtc 1140 ctaacgcaaa aactgggacc ttggcgtcgg ccggtggcct acctgtccaa aaagctagac 1200 ccagtagcag ctgggtggcc cccttgccta cggatggtag cagccattgc cgtactgaca 1260 aaggatgcag gcaagctaac catgggacag ccactagtca ttctggcccc ccatgcagta 1320 gaggcactag tcaaacaacc ccccgaccgc tggctttcca acgcccggat gactcactat 1380 caggccttgc ttttggacac ggaccgggtc cagttcggac cggtggtagc cctgaacccg 1440 gctacgctgc tcccactgcc tgaggaaggg ctgcaacaca actgccttga tatcctggcc 1500 gaagcccacg gaacccgacc cgacctaacg gaccagccgc tcccagacgc cgaccacacc 1560 tggtacacgg atggaagcag tctcttacaa gagggacagc gtaaggcggg agctgcggtg 1620 accaccgaga ccgaggtaat ctgggctaaa gccctgccag ccgggacatc cgctcagcgg 1680 gctgaactga tagcactcac ccaggcccta aagatggcag aaggtaagaa gctaaatgtt 1740 tatactgata gccgttatgc ttttgctact gcccatatcc atggagaaat atacagaagg 1800 cgtgggttgc tcacatcaga aggcaaagag atcaaaaata aagacgagat cttggcccta 1860 ctaaaagccc tctttctgcc caaaagactt agcataatcc attgtccagg acatcaaaag 1920 ggacacagcg ccgaggctag aggcaaccgg atggctgacc aagcggcccg aaaggcagcc 1980 atcacagaga ctccagacac ctctaccctc ctcata 2016 <210> 11 <211> 2016 <212> DNA <213> Moloney Murine Leukemia Virus <220> <221> mat_peptide <222> (1)..(2016) <223> nucleotide sequence coding for the amino acid sequence of SEQ. ID. No: 2 <220> <221> mutation <222> (187)..(189) <223> substitution of ctt instead of caa at 187-189 positions of SEQ. ID. No: 10 <400> 11 atgctaaata tagaagatga gcatcggcta catgagacct caaaagagcc agatgtttct 60 ctagggtcca catggctgtc tgattttcct caggcctggg cggaaaccgg gggcatggga 120 ctggcagttc gccaagctcc tctgatcata cctctgaaag caacctctac ccccgtgtcc 180 ataaaacttt accccatgtc acaagaagcc agactgggga tcaagcccca catacagaga 240 ctgttggacc agggaatact ggtaccctgc cagtccccct ggaacacgcc cctgctaccc 300 gttaagaaac cagggactaa tgattatagg cctgtccagg atctgagaga agtcaacaag 360 cgggtggaag acatccaccc caccgtgccc aacccttaca acctcttgag cgggctccca 420 ccgtcccacc agtggtacac tgtgcttgat ttaaaggatg cctttttctg cctgagactc 480 caccccacca gtcagcctct cttcgccttt gagtggagag atccagagat gggaatctca 540 ggacaattga cctggaccag actcccacag ggtttcaaaa acagtcccac cctgtttgat 600 gaggcactgc acagagacct agcagacttc cggatccagc acccagactt gatcctgcta 660 cagtacgtgg atgacttact gctggccgcc acttctgagc tagactgcca acaaggtact 720 cgggccctgt tacaaaccct agggaacctc gggtatcggg cctcggccaa gaaagcccaa 780 atttgccaga aacaggtcaa gtatctgggg tatcttctaa aagagggtca gagatggctg 840 actgaggcca gaaaagagac tgtgatgggg cagcctactc cgaagacccc tcgacaacta 900 agggagttcc tagggacggc aggcttctgt cgcctctgga tccctgggtt tgcagaaatg 960 gcagccccct tgtaccctct caccaaaacg gggactctgt ttaattgggg cccagaccaa 1020 caaaaggcct atcaagaaat caagcaagct cttctaactg ccccagccct ggggttgcca 1080 gatttgacta agccctttga actctttgtc gacgagaagc agggctacgc caaaggtgtc 1140 ctaacgcaaa aactgggacc ttggcgtcgg ccggtggcct acctgtccaa aaagctagac 1200 ccagtagcag ctgggtggcc cccttgccta cggatggtag cagccattgc cgtactgaca 1260 aaggatgcag gcaagctaac catgggacag ccactagtca ttctggcccc ccatgcagta 1320 gaggcactag tcaaacaacc ccccgaccgc tggctttcca acgcccggat gactcactat 1380 caggccttgc ttttggacac ggaccgggtc cagttcggac cggtggtagc cctgaacccg 1440 gctacgctgc tcccactgcc tgaggaaggg ctgcaacaca actgccttga tatcctggcc 1500 gaagcccacg gaacccgacc cgacctaacg gaccagccgc tcccagacgc cgaccacacc 1560 tggtacacgg atggaagcag tctcttacaa gagggacagc gtaaggcggg agctgcggtg 1620 accaccgaga ccgaggtaat ctgggctaaa gccctgccag ccgggacatc cgctcagcgg 1680 gctgaactga tagcactcac ccaggcccta aagatggcag aaggtaagaa gctaaatgtt 1740 tatactgata gccgttatgc ttttgctact gcccatatcc atggagaaat atacagaagg 1800 cgtgggttgc tcacatcaga aggcaaagag atcaaaaata aagacgagat cttggcccta 1860 ctaaaagccc tctttctgcc caaaagactt agcataatcc attgtccagg acatcaaaag 1920 ggacacagcg ccgaggctag aggcaaccgg atggctgacc aagcggcccg aaaggcagcc 1980 atcacagaga ctccagacac ctctaccctc ctcata 2016 <210> 12 <211> 2016 <212> DNA <213> Moloney Murine Leukemia Virus <220> <221> mat_peptide <222> (1)..(2016) <223> nucleotide sequence coding for the amino acid sequence of SEQ. ID. No: 3 <220> <221> mutation <222> (790)..(792) <223> substitution of ctt instead of aaa at 790-792 positions of SEQ. ID. No: 10 <400> 12 atgctaaata tagaagatga gcatcggcta catgagacct caaaagagcc agatgtttct 60 ctagggtcca catggctgtc tgattttcct caggcctggg cggaaaccgg gggcatggga 120 ctggcagttc gccaagctcc tctgatcata cctctgaaag caacctctac ccccgtgtcc 180 ataaaacaat accccatgtc acaagaagcc agactgggga tcaagcccca catacagaga 240 ctgttggacc agggaatact ggtaccctgc cagtccccct ggaacacgcc cctgctaccc 300 gttaagaaac cagggactaa tgattatagg cctgtccagg atctgagaga agtcaacaag 360 cgggtggaag acatccaccc caccgtgccc aacccttaca acctcttgag cgggctccca 420 ccgtcccacc agtggtacac tgtgcttgat ttaaaggatg cctttttctg cctgagactc 480 caccccacca gtcagcctct cttcgccttt gagtggagag atccagagat gggaatctca 540 ggacaattga cctggaccag actcccacag ggtttcaaaa acagtcccac cctgtttgat 600 gaggcactgc acagagacct agcagacttc cggatccagc acccagactt gatcctgcta 660 cagtacgtgg atgacttact gctggccgcc acttctgagc tagactgcca acaaggtact 720 cgggccctgt tacaaaccct agggaacctc gggtatcggg cctcggccaa gaaagcccaa 780 atttgccagc ttcaggtcaa gtatctgggg tatcttctaa aagagggtca gagatggctg 840 actgaggcca gaaaagagac tgtgatgggg cagcctactc cgaagacccc tcgacaacta 900 agggagttcc tagggacggc aggcttctgt cgcctctgga tccctgggtt tgcagaaatg 960 gcagccccct tgtaccctct caccaaaacg gggactctgt ttaattgggg cccagaccaa 1020 caaaaggcct atcaagaaat caagcaagct cttctaactg ccccagccct ggggttgcca 1080 gatttgacta agccctttga actctttgtc gacgagaagc agggctacgc caaaggtgtc 1140 ctaacgcaaa aactgggacc ttggcgtcgg ccggtggcct acctgtccaa aaagctagac 1200 ccagtagcag ctgggtggcc cccttgccta cggatggtag cagccattgc cgtactgaca 1260 aaggatgcag gcaagctaac catgggacag ccactagtca ttctggcccc ccatgcagta 1320 gaggcactag tcaaacaacc ccccgaccgc tggctttcca acgcccggat gactcactat 1380 caggccttgc ttttggacac ggaccgggtc cagttcggac cggtggtagc cctgaacccg 1440 gctacgctgc tcccactgcc tgaggaaggg ctgcaacaca actgccttga tatcctggcc 1500 gaagcccacg gaacccgacc cgacctaacg gaccagccgc tcccagacgc cgaccacacc 1560 tggtacacgg atggaagcag tctcttacaa gagggacagc gtaaggcggg agctgcggtg 1620 accaccgaga ccgaggtaat ctgggctaaa gccctgccag ccgggacatc cgctcagcgg 1680 gctgaactga tagcactcac ccaggcccta aagatggcag aaggtaagaa gctaaatgtt 1740 tatactgata gccgttatgc ttttgctact gcccatatcc atggagaaat atacagaagg 1800 cgtgggttgc tcacatcaga aggcaaagag atcaaaaata aagacgagat cttggcccta 1860 ctaaaagccc tctttctgcc caaaagactt agcataatcc attgtccagg acatcaaaag 1920 ggacacagcg ccgaggctag aggcaaccgg atggctgacc aagcggcccg aaaggcagcc 1980 atcacagaga ctccagacac ctctaccctc ctcata 2016 <210> 13 <211> 2016 <212> DNA <213> Moloney Murine Leukemia Virus <220> <221> mat_peptide <222> (1)..(2016) <223> nucleotide sequence coding for the amino acid sequence of SEQ. ID. No: 4 <220> <221> mutation <222> (883)..(885) <223> substitution of caa instead of aag at 883-885 positions of SEQ. ID. No: 10 <400> 13 atgctaaata tagaagatga gcatcggcta catgagacct caaaagagcc agatgtttct 60 ctagggtcca catggctgtc tgattttcct caggcctggg cggaaaccgg gggcatggga 120 ctggcagttc gccaagctcc tctgatcata cctctgaaag caacctctac ccccgtgtcc 180 ataaaacaat accccatgtc acaagaagcc agactgggga tcaagcccca catacagaga 240 ctgttggacc agggaatact ggtaccctgc cagtccccct ggaacacgcc cctgctaccc 300 gttaagaaac cagggactaa tgattatagg cctgtccagg atctgagaga agtcaacaag 360 cgggtggaag acatccaccc caccgtgccc aacccttaca acctcttgag cgggctccca 420 ccgtcccacc agtggtacac tgtgcttgat ttaaaggatg cctttttctg cctgagactc 480 caccccacca gtcagcctct cttcgccttt gagtggagag atccagagat gggaatctca 540 ggacaattga cctggaccag actcccacag ggtttcaaaa acagtcccac cctgtttgat 600 gaggcactgc acagagacct agcagacttc cggatccagc acccagactt gatcctgcta 660 cagtacgtgg atgacttact gctggccgcc acttctgagc tagactgcca acaaggtact 720 cgggccctgt tacaaaccct agggaacctc gggtatcggg cctcggccaa gaaagcccaa 780 atttgccaga aacaggtcaa gtatctgggg tatcttctaa aagagggtca gagatggctg 840 actgaggcca gaaaagagac tgtgatgggg cagcctactc cgcaaacccc tcgacaacta 900 agggagttcc tagggacggc aggcttctgt cgcctctgga tccctgggtt tgcagaaatg 960 gcagccccct tgtaccctct caccaaaacg gggactctgt ttaattgggg cccagaccaa 1020 caaaaggcct atcaagaaat caagcaagct cttctaactg ccccagccct ggggttgcca 1080 gatttgacta agccctttga actctttgtc gacgagaagc agggctacgc caaaggtgtc 1140 ctaacgcaaa aactgggacc ttggcgtcgg ccggtggcct acctgtccaa aaagctagac 1200 ccagtagcag ctgggtggcc cccttgccta cggatggtag cagccattgc cgtactgaca 1260 aaggatgcag gcaagctaac catgggacag ccactagtca ttctggcccc ccatgcagta 1320 gaggcactag tcaaacaacc ccccgaccgc tggctttcca acgcccggat gactcactat 1380 caggccttgc ttttggacac ggaccgggtc cagttcggac cggtggtagc cctgaacccg 1440 gctacgctgc tcccactgcc tgaggaaggg ctgcaacaca actgccttga tatcctggcc 1500 gaagcccacg gaacccgacc cgacctaacg gaccagccgc tcccagacgc cgaccacacc 1560 tggtacacgg atggaagcag tctcttacaa gagggacagc gtaaggcggg agctgcggtg 1620 accaccgaga ccgaggtaat ctgggctaaa gccctgccag ccgggacatc cgctcagcgg 1680 gctgaactga tagcactcac ccaggcccta aagatggcag aaggtaagaa gctaaatgtt 1740 tatactgata gccgttatgc ttttgctact gcccatatcc atggagaaat atacagaagg 1800 cgtgggttgc tcacatcaga aggcaaagag atcaaaaata aagacgagat cttggcccta 1860 ctaaaagccc tctttctgcc caaaagactt agcataatcc attgtccagg acatcaaaag 1920 ggacacagcg ccgaggctag aggcaaccgg atggctgacc aagcggcccg aaaggcagcc 1980 atcacagaga ctccagacac ctctaccctc ctcata 2016 <210> 14 <211> 2016 <212> DNA <213> Moloney Murine Leukemia Virus <220> <221> mat_peptide <222> (1)..(2016) <223> nucleotide sequence coding for the amino acid sequence of SEQ. ID. No: 5 <220> <221> mutation <222> (916)..(918) <223> substitution of ctt instead of acg at 916-918 positions of SEQ. ID. No: 10 <400> 14 atgctaaata tagaagatga gcatcggcta catgagacct caaaagagcc agatgtttct 60 ctagggtcca catggctgtc tgattttcct caggcctggg cggaaaccgg gggcatggga 120 ctggcagttc gccaagctcc tctgatcata cctctgaaag caacctctac ccccgtgtcc 180 ataaaacaat accccatgtc acaagaagcc agactgggga tcaagcccca catacagaga 240 ctgttggacc agggaatact ggtaccctgc cagtccccct ggaacacgcc cctgctaccc 300 gttaagaaac cagggactaa tgattatagg cctgtccagg atctgagaga agtcaacaag 360 cgggtggaag acatccaccc caccgtgccc aacccttaca acctcttgag cgggctccca 420 ccgtcccacc agtggtacac tgtgcttgat ttaaaggatg cctttttctg cctgagactc 480 caccccacca gtcagcctct cttcgccttt gagtggagag atccagagat gggaatctca 540 ggacaattga cctggaccag actcccacag ggtttcaaaa acagtcccac cctgtttgat 600 gaggcactgc acagagacct agcagacttc cggatccagc acccagactt gatcctgcta 660 cagtacgtgg atgacttact gctggccgcc acttctgagc tagactgcca acaaggtact 720 cgggccctgt tacaaaccct agggaacctc gggtatcggg cctcggccaa gaaagcccaa 780 atttgccaga aacaggtcaa gtatctgggg tatcttctaa aagagggtca gagatggctg 840 actgaggcca gaaaagagac tgtgatgggg cagcctactc cgaagacccc tcgacaacta 900 agggagttcc tagggcttgc aggcttctgt cgcctctgga tccctgggtt tgcagaaatg 960 gcagccccct tgtaccctct caccaaaacg gggactctgt ttaattgggg cccagaccaa 1020 caaaaggcct atcaagaaat caagcaagct cttctaactg ccccagccct ggggttgcca 1080 gatttgacta agccctttga actctttgtc gacgagaagc agggctacgc caaaggtgtc 1140 ctaacgcaaa aactgggacc ttggcgtcgg ccggtggcct acctgtccaa aaagctagac 1200 ccagtagcag ctgggtggcc cccttgccta cggatggtag cagccattgc cgtactgaca 1260 aaggatgcag gcaagctaac catgggacag ccactagtca ttctggcccc ccatgcagta 1320 gaggcactag tcaaacaacc ccccgaccgc tggctttcca acgcccggat gactcactat 1380 caggccttgc ttttggacac ggaccgggtc cagttcggac cggtggtagc cctgaacccg 1440 gctacgctgc tcccactgcc tgaggaaggg ctgcaacaca actgccttga tatcctggcc 1500 gaagcccacg gaacccgacc cgacctaacg gaccagccgc tcccagacgc cgaccacacc 1560 tggtacacgg atggaagcag tctcttacaa gagggacagc gtaaggcggg agctgcggtg 1620 accaccgaga ccgaggtaat ctgggctaaa gccctgccag ccgggacatc cgctcagcgg 1680 gctgaactga tagcactcac ccaggcccta aagatggcag aaggtaagaa gctaaatgtt 1740 tatactgata gccgttatgc ttttgctact gcccatatcc atggagaaat atacagaagg 1800 cgtgggttgc tcacatcaga aggcaaagag atcaaaaata aagacgagat cttggcccta 1860 ctaaaagccc tctttctgcc caaaagactt agcataatcc attgtccagg acatcaaaag 1920 ggacacagcg ccgaggctag aggcaaccgg atggctgacc aagcggcccg aaaggcagcc 1980 atcacagaga ctccagacac ctctaccctc ctcata 2016 <210> 15 <211> 2016 <212> DNA <213> Moloney Murine Leukemia Virus <220> <221> mat_peptide <222> (1)..(2016) <223> nucleotide sequence coding for the amino acid sequence of SEQ. ID. No: 6 <220> <221> mutation <222> (1036)..(1038) <223> substitution of atg instead of gaa at 1036-1038 positions of SEQ. ID. No: 10 <400> 15 atgctaaata tagaagatga gcatcggcta catgagacct caaaagagcc agatgtttct 60 ctagggtcca catggctgtc tgattttcct caggcctggg cggaaaccgg gggcatggga 120 ctggcagttc gccaagctcc tctgatcata cctctgaaag caacctctac ccccgtgtcc 180 ataaaacaat accccatgtc acaagaagcc agactgggga tcaagcccca catacagaga 240 ctgttggacc agggaatact ggtaccctgc cagtccccct ggaacacgcc cctgctaccc 300 gttaagaaac cagggactaa tgattatagg cctgtccagg atctgagaga agtcaacaag 360 cgggtggaag acatccaccc caccgtgccc aacccttaca acctcttgag cgggctccca 420 ccgtcccacc agtggtacac tgtgcttgat ttaaaggatg cctttttctg cctgagactc 480 caccccacca gtcagcctct cttcgccttt gagtggagag atccagagat gggaatctca 540 ggacaattga cctggaccag actcccacag ggtttcaaaa acagtcccac cctgtttgat 600 gaggcactgc acagagacct agcagacttc cggatccagc acccagactt gatcctgcta 660 cagtacgtgg atgacttact gctggccgcc acttctgagc tagactgcca acaaggtact 720 cgggccctgt tacaaaccct agggaacctc gggtatcggg cctcggccaa gaaagcccaa 780 atttgccaga aacaggtcaa gtatctgggg tatcttctaa aagagggtca gagatggctg 840 actgaggcca gaaaagagac tgtgatgggg cagcctactc cgaagacccc tcgacaacta 900 agggagttcc tagggacggc aggcttctgt cgcctctgga tccctgggtt tgcagaaatg 960 gcagccccct tgtaccctct caccaaaacg gggactctgt ttaattgggg cccagaccaa 1020 caaaaggcct atcaaatgat caagcaagct cttctaactg ccccagccct ggggttgcca 1080 gatttgacta agccctttga actctttgtc gacgagaagc agggctacgc caaaggtgtc 1140 ctaacgcaaa aactgggacc ttggcgtcgg ccggtggcct acctgtccaa aaagctagac 1200 ccagtagcag ctgggtggcc cccttgccta cggatggtag cagccattgc cgtactgaca 1260 aaggatgcag gcaagctaac catgggacag ccactagtca ttctggcccc ccatgcagta 1320 gaggcactag tcaaacaacc ccccgaccgc tggctttcca acgcccggat gactcactat 1380 caggccttgc ttttggacac ggaccgggtc cagttcggac cggtggtagc cctgaacccg 1440 gctacgctgc tcccactgcc tgaggaaggg ctgcaacaca actgccttga tatcctggcc 1500 gaagcccacg gaacccgacc cgacctaacg gaccagccgc tcccagacgc cgaccacacc 1560 tggtacacgg atggaagcag tctcttacaa gagggacagc gtaaggcggg agctgcggtg 1620 accaccgaga ccgaggtaat ctgggctaaa gccctgccag ccgggacatc cgctcagcgg 1680 gctgaactga tagcactcac ccaggcccta aagatggcag aaggtaagaa gctaaatgtt 1740 tatactgata gccgttatgc ttttgctact gcccatatcc atggagaaat atacagaagg 1800 cgtgggttgc tcacatcaga aggcaaagag atcaaaaata aagacgagat cttggcccta 1860 ctaaaagccc tctttctgcc caaaagactt agcataatcc attgtccagg acatcaaaag 1920 ggacacagcg ccgaggctag aggcaaccgg atggctgacc aagcggcccg aaaggcagcc 1980 atcacagaga ctccagacac ctctaccctc ctcata 2016 <210> 16 <211> 2016 <212> DNA <213> Moloney Murine Leukemia Virus <220> <221> mat_peptide <222> (1)..(2016) <223> nucleotide sequence coding for the amino acid sequence of SEQ. ID. No: 7 <220> <221> mutation <222> (1222)..(1224) <223> substitution of gaa instead of cct at 1222-1224 positions of SEQ. ID. No: 10 <400> 16 atgctaaata tagaagatga gcatcggcta catgagacct caaaagagcc agatgtttct 60 ctagggtcca catggctgtc tgattttcct caggcctggg cggaaaccgg gggcatggga 120 ctggcagttc gccaagctcc tctgatcata cctctgaaag caacctctac ccccgtgtcc 180 ataaaacaat accccatgtc acaagaagcc agactgggga tcaagcccca catacagaga 240 ctgttggacc agggaatact ggtaccctgc cagtccccct ggaacacgcc cctgctaccc 300 gttaagaaac cagggactaa tgattatagg cctgtccagg atctgagaga agtcaacaag 360 cgggtggaag acatccaccc caccgtgccc aacccttaca acctcttgag cgggctccca 420 ccgtcccacc agtggtacac tgtgcttgat ttaaaggatg cctttttctg cctgagactc 480 caccccacca gtcagcctct cttcgccttt gagtggagag atccagagat gggaatctca 540 ggacaattga cctggaccag actcccacag ggtttcaaaa acagtcccac cctgtttgat 600 gaggcactgc acagagacct agcagacttc cggatccagc acccagactt gatcctgcta 660 cagtacgtgg atgacttact gctggccgcc acttctgagc tagactgcca acaaggtact 720 cgggccctgt tacaaaccct agggaacctc gggtatcggg cctcggccaa gaaagcccaa 780 atttgccaga aacaggtcaa gtatctgggg tatcttctaa aagagggtca gagatggctg 840 actgaggcca gaaaagagac tgtgatgggg cagcctactc cgaagacccc tcgacaacta 900 agggagttcc tagggacggc aggcttctgt cgcctctgga tccctgggtt tgcagaaatg 960 gcagccccct tgtaccctct caccaaaacg gggactctgt ttaattgggg cccagaccaa 1020 caaaaggcct atcaagaaat caagcaagct cttctaactg ccccagccct ggggttgcca 1080 gatttgacta agccctttga actctttgtc gacgagaagc agggctacgc caaaggtgtc 1140 ctaacgcaaa aactgggacc ttggcgtcgg ccggtggcct acctgtccaa aaagctagac 1200 ccagtagcag ctgggtggcc cgaatgccta cggatggtag cagccattgc cgtactgaca 1260 aaggatgcag gcaagctaac catgggacag ccactagtca ttctggcccc ccatgcagta 1320 gaggcactag tcaaacaacc ccccgaccgc tggctttcca acgcccggat gactcactat 1380 caggccttgc ttttggacac ggaccgggtc cagttcggac cggtggtagc cctgaacccg 1440 gctacgctgc tcccactgcc tgaggaaggg ctgcaacaca actgccttga tatcctggcc 1500 gaagcccacg gaacccgacc cgacctaacg gaccagccgc tcccagacgc cgaccacacc 1560 tggtacacgg atggaagcag tctcttacaa gagggacagc gtaaggcggg agctgcggtg 1620 accaccgaga ccgaggtaat ctgggctaaa gccctgccag ccgggacatc cgctcagcgg 1680 gctgaactga tagcactcac ccaggcccta aagatggcag aaggtaagaa gctaaatgtt 1740 tatactgata gccgttatgc ttttgctact gcccatatcc atggagaaat atacagaagg 1800 cgtgggttgc tcacatcaga aggcaaagag atcaaaaata aagacgagat cttggcccta 1860 ctaaaagccc tctttctgcc caaaagactt agcataatcc attgtccagg acatcaaaag 1920 ggacacagcg ccgaggctag aggcaaccgg atggctgacc aagcggcccg aaaggcagcc 1980 atcacagaga ctccagacac ctctaccctc ctcata 2016 <210> 17 <211> 2016 <212> DNA <213> Moloney Murine Leukemia Virus <220> <221> mat_peptide <222> (1)..(2016) <223> nucleotide sequence coding for the amino acid sequence of SEQ. ID. No: 8 <220> <221> mutation <222> (1312)..(1314) <223> substitution of tac instead of cat at 1312-1314 positions of SEQ. ID. No: 10 <400> 17 atgctaaata tagaagatga gcatcggcta catgagacct caaaagagcc agatgtttct 60 ctagggtcca catggctgtc tgattttcct caggcctggg cggaaaccgg gggcatggga 120 ctggcagttc gccaagctcc tctgatcata cctctgaaag caacctctac ccccgtgtcc 180 ataaaacaat accccatgtc acaagaagcc agactgggga tcaagcccca catacagaga 240 ctgttggacc agggaatact ggtaccctgc cagtccccct ggaacacgcc cctgctaccc 300 gttaagaaac cagggactaa tgattatagg cctgtccagg atctgagaga agtcaacaag 360 cgggtggaag acatccaccc caccgtgccc aacccttaca acctcttgag cgggctccca 420 ccgtcccacc agtggtacac tgtgcttgat ttaaaggatg cctttttctg cctgagactc 480 caccccacca gtcagcctct cttcgccttt gagtggagag atccagagat gggaatctca 540 ggacaattga cctggaccag actcccacag ggtttcaaaa acagtcccac cctgtttgat 600 gaggcactgc acagagacct agcagacttc cggatccagc acccagactt gatcctgcta 660 cagtacgtgg atgacttact gctggccgcc acttctgagc tagactgcca acaaggtact 720 cgggccctgt tacaaaccct agggaacctc gggtatcggg cctcggccaa gaaagcccaa 780 atttgccaga aacaggtcaa gtatctgggg tatcttctaa aagagggtca gagatggctg 840 actgaggcca gaaaagagac tgtgatgggg cagcctactc cgaagacccc tcgacaacta 900 agggagttcc tagggacggc aggcttctgt cgcctctgga tccctgggtt tgcagaaatg 960 gcagccccct tgtaccctct caccaaaacg gggactctgt ttaattgggg cccagaccaa 1020 caaaaggcct atcaagaaat caagcaagct cttctaactg ccccagccct ggggttgcca 1080 gatttgacta agccctttga actctttgtc gacgagaagc agggctacgc caaaggtgtc 1140 ctaacgcaaa aactgggacc ttggcgtcgg ccggtggcct acctgtccaa aaagctagac 1200 ccagtagcag ctgggtggcc cccttgccta cggatggtag cagccattgc cgtactgaca 1260 aaggatgcag gcaagctaac catgggacag ccactagtca ttctggcccc ctacgcagta 1320 gaggcactag tcaaacaacc ccccgaccgc tggctttcca acgcccggat gactcactat 1380 caggccttgc ttttggacac ggaccgggtc cagttcggac cggtggtagc cctgaacccg 1440 gctacgctgc tcccactgcc tgaggaaggg ctgcaacaca actgccttga tatcctggcc 1500 gaagcccacg gaacccgacc cgacctaacg gaccagccgc tcccagacgc cgaccacacc 1560 tggtacacgg atggaagcag tctcttacaa gagggacagc gtaaggcggg agctgcggtg 1620 accaccgaga ccgaggtaat ctgggctaaa gccctgccag ccgggacatc cgctcagcgg 1680 gctgaactga tagcactcac ccaggcccta aagatggcag aaggtaagaa gctaaatgtt 1740 tatactgata gccgttatgc ttttgctact gcccatatcc atggagaaat atacagaagg 1800 cgtgggttgc tcacatcaga aggcaaagag atcaaaaata aagacgagat cttggcccta 1860 ctaaaagccc tctttctgcc caaaagactt agcataatcc attgtccagg acatcaaaag 1920 ggacacagcg ccgaggctag aggcaaccgg atggctgacc aagcggcccg aaaggcagcc 1980 atcacagaga ctccagacac ctctaccctc ctcata 2016 <210> 18 <211> 2016 <212> DNA <213> Moloney Murine Leukemia Virus <220> <221> mat_peptide <222> (1)..(2016) <223> nucleotide sequence coding for the amino acid sequence of SEQ. ID. No: 9 <220> <221> mutation <222> (1360)..(1362) <223> substitution of ttc instead of aac at 1360-1362 positions of SEQ. ID. No: 10 <400> 18 atgctaaata tagaagatga gcatcggcta catgagacct caaaagagcc agatgtttct 60 ctagggtcca catggctgtc tgattttcct caggcctggg cggaaaccgg gggcatggga 120 ctggcagttc gccaagctcc tctgatcata cctctgaaag caacctctac ccccgtgtcc 180 ataaaacaat accccatgtc acaagaagcc agactgggga tcaagcccca catacagaga 240 ctgttggacc agggaatact ggtaccctgc cagtccccct ggaacacgcc cctgctaccc 300 gttaagaaac cagggactaa tgattatagg cctgtccagg atctgagaga agtcaacaag 360 cgggtggaag acatccaccc caccgtgccc aacccttaca acctcttgag cgggctccca 420 ccgtcccacc agtggtacac tgtgcttgat ttaaaggatg cctttttctg cctgagactc 480 caccccacca gtcagcctct cttcgccttt gagtggagag atccagagat gggaatctca 540 ggacaattga cctggaccag actcccacag ggtttcaaaa acagtcccac cctgtttgat 600 gaggcactgc acagagacct agcagacttc cggatccagc acccagactt gatcctgcta 660 cagtacgtgg atgacttact gctggccgcc acttctgagc tagactgcca acaaggtact 720 cgggccctgt tacaaaccct agggaacctc gggtatcggg cctcggccaa gaaagcccaa 780 atttgccaga aacaggtcaa gtatctgggg tatcttctaa aagagggtca gagatggctg 840 actgaggcca gaaaagagac tgtgatgggg cagcctactc cgaagacccc tcgacaacta 900 agggagttcc tagggacggc aggcttctgt cgcctctgga tccctgggtt tgcagaaatg 960 gcagccccct tgtaccctct caccaaaacg gggactctgt ttaattgggg cccagaccaa 1020 caaaaggcct atcaagaaat caagcaagct cttctaactg ccccagccct ggggttgcca 1080 gatttgacta agccctttga actctttgtc gacgagaagc agggctacgc caaaggtgtc 1140 ctaacgcaaa aactgggacc ttggcgtcgg ccggtggcct acctgtccaa aaagctagac 1200 ccagtagcag ctgggtggcc cccttgccta cggatggtag cagccattgc cgtactgaca 1260 aaggatgcag gcaagctaac catgggacag ccactagtca ttctggcccc ccatgcagta 1320 gaggcactag tcaaacaacc ccccgaccgc tggctttcct tcgcccggat gactcactat 1380 caggccttgc ttttggacac ggaccgggtc cagttcggac cggtggtagc cctgaacccg 1440 gctacgctgc tcccactgcc tgaggaaggg ctgcaacaca actgccttga tatcctggcc 1500 gaagcccacg gaacccgacc cgacctaacg gaccagccgc tcccagacgc cgaccacacc 1560 tggtacacgg atggaagcag tctcttacaa gagggacagc gtaaggcggg agctgcggtg 1620 accaccgaga ccgaggtaat ctgggctaaa gccctgccag ccgggacatc cgctcagcgg 1680 gctgaactga tagcactcac ccaggcccta aagatggcag aaggtaagaa gctaaatgtt 1740 tatactgata gccgttatgc ttttgctact gcccatatcc atggagaaat atacagaagg 1800 cgtgggttgc tcacatcaga aggcaaagag atcaaaaata aagacgagat cttggcccta 1860 ctaaaagccc tctttctgcc caaaagactt agcataatcc attgtccagg acatcaaaag 1920 ggacacagcg ccgaggctag aggcaaccgg atggctgacc aagcggcccg aaaggcagcc 1980 atcacagaga ctccagacac ctctaccctc ctcata 2016 <210> 19 <211> 2019 <212> DNA <213> Artificial Sequence <220> <223> Optimized Moloney Murine Leukemia Virus reverse transcriptase in accordance with codon usage <220> <221> mat_peptide <222> (1)..(2016) <223> nucleotide sequence coding for the amino acid sequence of SEQ. ID. No: 1 <400> 19 atgctgaaca tcgaagacga acaccgtctg cacgaaacct ctaaagaacc ggacgtttct 60 ctgggttcta cctggctgtc tgacttcccg caggcttggg ctgaaaccgg tggtatgggt 120 ctggctgttc gtcaggctcc gctgatcatc ccgctgaaag ctacctctac cccggtttct 180 atcaaacagt acccgatgtc tcaggaagct cgtctgggta tcaaaccgca catccagcgt 240 ctgctggacc agggtatcct ggttccgtgc cagtctccgt ggaacacccc gctgctgccg 300 gttaaaaaac cgggtaccaa cgactaccgt ccggttcagg acctgcgtga agttaacaaa 360 cgtgttgaag acatccaccc gaccgttccg aacccgtaca acctgctgtc tggtctgccg 420 ccgtctcacc agtggtacac cgttctggac ctgaaagacg ctttcttctg cctgcgtctg 480 cacccgacct ctcagccgct gttcgctttc gaatggcgtg acccggaaat gggtatctct 540 ggtcagctga cctggacccg tctgccgcag ggtttcaaaa actctccgac cctgttcgac 600 gaagctctgc accgtgacct ggctgacttc cgtatccagc acccggacct gatcctgctg 660 cagtacgttg acgacctgct gctggctgct acctctgaac tggactgcca gcagggtacc 720 cgtgctctgc tgcagaccct gggtaacctg ggttaccgtg cttctgctaa aaaagctcag 780 atctgccaga aacaggttaa atacctgggt tacctgctga aagaaggtca gcgttggctg 840 accgaagctc gtaaagaaac cgttatgggt cagccgaccc cgaaaacccc gcgtcagctg 900 cgtgaattcc tgggtaccgc tggtttctgc cgtctgtgga taccgggttt cgctgaaatg 960 gctgctccgc tgtacccgct gaccaaaacc ggtaccctgt tcaactgggg tccggaccag 1020 cagaaagcgt accaggaaat caaacaggct ctgctgaccg ctccggctct gggtctgccg 1080 gacctgacca aaccgttcga actgttcgtt gacgaaaaac agggttacgc taaaggtgtt 1140 ctgacccaga aactgggtcc gtggcgtcgt ccggttgctt acctgtctaa aaaactggac 1200 ccggttgctg ctggttggcc gccgtgcctg cgtatggttg ctgctatcgc tgttctgacc 1260 aaagacgctg gtaaactgac catgggtcag ccgctggtta tcctggctcc gcacgctgtt 1320 gaagctctgg ttaaacagcc gccggaccgt tggctgtcta acgctcgtat gacccactac 1380 caggctctgc tgctggacac cgaccgtgtt cagttcggtc cggttgttgc tctgaacccg 1440 gctaccctgc tgccgctgcc ggaagaaggt ctgcagcaca actgcctgga catcctggct 1500 gaagctcacg gtacccgtcc ggacctgacc gaccagccgc tgccggacgc tgaccacacc 1560 tggtacaccg acggttcttc tctgctgcag gaaggtcagc gtaaagctgg tgctgctgtt 1620 accaccgaaa ccgaagttat ctgggctaaa gctctgccgg ctggtacctc tgctcagcgt 1680 gctgaactga tcgctctgac ccaggctctg aaaatggctg aaggtaaaaa actgaacgtt 1740 tacaccgact ctcgttacgc tttcgctacc gctcacatcc acggtgaaat ctaccgtcgt 1800 cgtggtctgc tgacctctga aggtaaagaa atcaaaaaca aagacgaaat cctggctctg 1860 ctgaaagctc tgttcctgcc gaaacgtctg tctatcatcc actgcccggg tcaccagaaa 1920 ggtcactctg ctgaagctcg tggtaaccgt atggctgacc aggctgctcg taaagctgct 1980 atcaccgaaa ccccggacac ctctaccctg ctgatctaa 2019 <210> 20 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> R0 oligonucleotide <400> 20 ttcagggata gagggagta 19 <210> 21 <211> 36 <212> DNA <213> Artificial Sequence <220> <223> F0 oligonucleotide <400> 21 tactccctct atccctgaac atcgaagacg aacacc 36 <210> 22 <211> 33 <212> DNA <213> Artificial Sequence <220> <223> R19 oligonucleotide <400> 22 gaggtttcgt gcagacggtg ttcgtcttcg atg 33 <210> 23 <211> 34 <212> DNA <213> Artificial Sequence <220> <223> F36 oligonucleotide <400> 23 gtctgcacga aacctctaaa gaaccggacg tttc 34 <210> 24 <211> 34 <212> DNA <213> Artificial Sequence <220> <223> R52 oligonucleotide <400> 24 ccaggtagaa cccagagaaa cgtccggttc ttta 34 <210> 25 <211> 32 <212> DNA <213> Artificial Sequence <220> <223> F70 oligonucleotide <400> 25 tctgggttct acctggctgt ctgacttccc gc 32 <210> 26 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> R86 oligonucleotide <400> 26 ttcagcccaa gcctgcggga agtcagacag 30 <210> 27 <211> 29 <212> DNA <213> Artificial Sequence <220> <223> F102 oligonucleotide <400> 27 aggcttgggc tgaaaccggt ggtatgggt 29 <210> 28 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> R116 oligonucleotide <400> 28 ctgacgaaca gccagaccca taccaccggt 30 <210> 29 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> F131 oligonucleotide <400> 29 ctggctgttc gtcaggctcc gctgatcatc 30 <210> 30 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> R146 oligonucleotide <400> 30 ggtagctttc agcgggatga tcagcggagc 30 <210> 31 <211> 33 <212> DNA <213> Artificial Sequence <220> <223> F161 oligonucleotide <400> 31 ccgctgaaag ctacctctac cccggtttct atc 33 <210> 32 <211> 35 <212> DNA <213> Artificial Sequence <220> <223> R176 oligonucleotide <400> 32 gacatcgggt actgtttgat agaaaccggg gtaga 35 <210> 33 <211> 33 <212> DNA <213> Artificial Sequence <220> <223> F194 oligonucleotide <400> 33 aaacagtacc cgatgtctca ggaagctcgt ctg 33 <210> 34 <211> 32 <212> DNA <213> Artificial Sequence <220> <223> R211 oligonucleotide <400> 34 tgtgcggttt gatacccaga cgagcttcct ga 32 <210> 35 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> F227 oligonucleotide <400> 35 ggtatcaaac cgcacatcca gcgtctgctg 30 <210> 36 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> R243 oligonucleotide <400> 36 ccaggatacc ctggtccagc agacgctgga 30 <210> 37 <211> 31 <212> DNA <213> Artificial Sequence <220> <223> F257 oligonucleotide <400> 37 gaccagggta tcctggttcc gtgccagtct c 31 <210> 38 <211> 29 <212> DNA <213> Artificial Sequence <220> <223> R273 oligonucleotide <400> 38 cggggtgttc cacggagact ggcacggaa 29 <210> 39 <211> 29 <212> DNA <213> Artificial Sequence <220> <223> F288 oligonucleotide <400> 39 cgtggaacac cccgctgctg ccggttaaa 29 <210> 40 <211> 31 <212> DNA <213> Artificial Sequence <220> <223> R302 oligonucleotide <400> 40 cgttggtacc cggtttttta accggcagca g 31 <210> 41 <211> 31 <212> DNA <213> Artificial Sequence <220> <223> F317 oligonucleotide <400> 41 aaaccgggta ccaacgacta ccgtccggtt c 31 <210> 42 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> R333 oligonucleotide <400> 42 cttcacgcag gtcctgaacc ggacggtagt 30 <210> 43 <211> 35 <212> DNA <213> Artificial Sequence <220> <223> F348 oligonucleotide <400> 43 aggacctgcg tgaagttaac aaacgtgttg aagac 35 <210> 44 <211> 34 <212> DNA <213> Artificial Sequence <220> <223> R363 oligonucleotide <400> 44 acggtcgggt ggatgtcttc aacacgtttg ttaa 34 <210> 45 <211> 29 <212> DNA <213> Artificial Sequence <220> <223> F383 oligonucleotide <400> 45 atccacccga ccgttccgaa cccgtacaa 29 <210> 46 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> R397 oligonucleotide <400> 46 agaccagaca gcaggttgta cgggttcgga 30 <210> 47 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> F412 oligonucleotide <400> 47 cctgctgtct ggtctgccgc cgtctca 27 <210> 48 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> R427 oligonucleotide <400> 48 acggtgtacc actggtgaga cggcggc 27 <210> 49 <211> 32 <212> DNA <213> Artificial Sequence <220> <223> F439 oligonucleotide <400> 49 ccagtggtac accgttctgg acctgaaaga cg 32 <210> 50 <211> 33 <212> DNA <213> Artificial Sequence <220> <223> R454 oligonucleotide <400> 50 cgcaggcaga agaaagcgtc tttcaggtcc aga 33 <210> 51 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> F471 oligonucleotide <400> 51 ctttcttctg cctgcgtctg cacccgacct 30 <210> 52 <211> 29 <212> DNA <213> Artificial Sequence <220> <223> R487 oligonucleotide <400> 52 cgaacagcgg ctgagaggtc gggtgcaga 29 <210> 53 <211> 31 <212> DNA <213> Artificial Sequence <220> <223> F501 oligonucleotide <400> 53 ctcagccgct gttcgctttc gaatggcgtg a 31 <210> 54 <211> 33 <212> DNA <213> Artificial Sequence <220> <223> R516 oligonucleotide <400> 54 agatacccat ttccgggtca cgccattcga aag 33 <210> 55 <211> 32 <212> DNA <213> Artificial Sequence <220> <223> F532 oligonucleotide <400> 55 cccggaaatg ggtatctctg gtcagctgac ct 32 <210> 56 <211> 28 <212> DNA <213> Artificial Sequence <220> <223> R549 oligonucleotide <400> 56 ggcagacggg tccaggtcag ctgaccag 28 <210> 57 <211> 31 <212> DNA <213> Artificial Sequence <220> <223> F564 oligonucleotide <400> 57 ggacccgtct gccgcagggt ttcaaaaact c 31 <210> 58 <211> 32 <212> DNA <213> Artificial Sequence <220> <223> R577 oligonucleotide <400> 58 cgaacagggt cggagagttt ttgaaaccct gc 32 <210> 59 <211> 29 <212> DNA <213> Artificial Sequence <220> <223> F595 oligonucleotide <400> 59 tccgaccctg ttcgacgaag ctctgcacc 29 <210> 60 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> R609 oligonucleotide <400> 60 agtcagccag gtcacggtgc agagcttcgt 30 <210> 61 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> F624 oligonucleotide <400> 61 gtgacctggc tgacttccgt atccagcacc 30 <210> 62 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> R639 oligonucleotide <400> 62 gcaggatcag gtccgggtgc tggatacgga 30 <210> 63 <211> 31 <212> DNA <213> Artificial Sequence <220> <223> F654 oligonucleotide <400> 63 cggacctgat cctgctgcag tacgttgacg a 31 <210> 64 <211> 29 <212> DNA <213> Artificial Sequence <220> <223> R669 oligonucleotide <400> 64 agccagcagc aggtcgtcaa cgtactgca 29 <210> 65 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> F685 oligonucleotide <400> 65 cctgctgctg gctgctacct ctgaactgga 30 <210> 66 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> R698 oligonucleotide <400> 66 accctgctgg cagtccagtt cagaggtagc 30 <210> 67 <211> 26 <212> DNA <213> Artificial Sequence <220> <223> F715 oligonucleotide <400> 67 ctgccagcag ggtacccgtg ctctgc 26 <210> 68 <211> 28 <212> DNA <213> Artificial Sequence <220> <223> R728 oligonucleotide <400> 68 tacccagggt ctgcagcaga gcacgggt 28 <210> 69 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> F741 oligonucleotide <400> 69 tgcagaccct gggtaacctg ggttaccgtg 30 <210> 70 <211> 35 <212> DNA <213> Artificial Sequence <220> <223> R756 oligonucleotide <400> 70 ctgagctttt ttagcagaag cacggtaacc caggt 35 <210> 71 <211> 37 <212> DNA <213> Artificial Sequence <220> <223> F771 oligonucleotide <400> 71 cttctgctaa aaaagctcag atctgccaga aacaggt 37 <210> 72 <211> 37 <212> DNA <213> Artificial Sequence <220> <223> R791 oligonucleotide <400> 72 gcaggtaacc caggtattta acctgtttct ggcagat 37 <210> 73 <211> 37 <212> DNA <213> Artificial Sequence <220> <223> F808 oligonucleotide <400> 73 taaatacctg ggttacctgc tgaaagaagg tcagcgt 37 <210> 74 <211> 31 <212> DNA <213> Artificial Sequence <220> <223> R828 oligonucleotide <400> 74 gcttcggtca gccaacgctg accttctttc a 31 <210> 75 <211> 34 <212> DNA <213> Artificial Sequence <220> <223> F845 oligonucleotide <400> 75 tggctgaccg aagctcgtaa agaaaccgtt atgg 34 <210> 76 <211> 33 <212> DNA <213> Artificial Sequence <220> <223> R859 oligonucleotide <400> 76 ggggtcggct gacccataac ggtttcttta cga 33 <210> 77 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> F879 oligonucleotide <400> 77 gtcagccgac cccgaaaacc ccgcgtc 27 <210> 78 <211> 29 <212> DNA <213> Artificial Sequence <220> <223> R892 oligonucleotide <400> 78 ggaattcacg cagctgacgc ggggttttc 29 <210> 79 <211> 29 <212> DNA <213> Artificial Sequence <220> <223> F906 oligonucleotide <400> 79 agctgcgtga attcctgggt accgctggt 29 <210> 80 <211> 29 <212> DNA <213> Artificial Sequence <220> <223> R921 oligonucleotide <400> 80 ccacagacgg cagaaaccag cggtaccca 29 <210> 81 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> F935 oligonucleotide <400> 81 ttctgccgtc tgtggatacc gggtttcgct 30 <210> 82 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> R950 oligonucleotide <400> 82 cggagcagcc atttcagcga aacccggtat 30 <210> 83 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> F965 oligonucleotide <400> 83 gaaatggctg ctccgctgta cccgctgacc 30 <210> 84 <211> 31 <212> DNA <213> Artificial Sequence <220> <223> R980 oligonucleotide <400> 84 acagggtacc ggttttggtc agcgggtaca g 31 <210> 85 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> F995 oligonucleotide <400> 85 aaaaccggta ccctgttcaa ctggggtccg 30 <210> 86 <211> 29 <212> DNA <213> Artificial Sequence <220> <223> R1011 oligonucleotide <400> 86 cgctttctgc tggtccggac cccagttga 29 <210> 87 <211> 34 <212> DNA <213> Artificial Sequence <220> <223> F1025 oligonucleotide <400> 87 gaccagcaga aagcgtacca ggaaatcaaa cagg 34 <210> 88 <211> 33 <212> DNA <213> Artificial Sequence <220> <223> R1040 oligonucleotide <400> 88 agcggtcagc agagcctgtt tgatttcctg gta 33 <210> 89 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> F1059 oligonucleotide <400> 89 ctctgctgac cgctccggct ctgggtc 27 <210> 90 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> R1073 oligonucleotide <400> 90 ggtcaggtcc ggcagaccca gagccgg 27 <210> 91 <211> 31 <212> DNA <213> Artificial Sequence <220> <223> F1086 oligonucleotide <400> 91 tgccggacct gaccaaaccg ttcgaactgt t 31 <210> 92 <211> 34 <212> DNA <213> Artificial Sequence <220> <223> R1100 oligonucleotide <400> 92 cctgtttttc gtcaacgaac agttcgaacg gttt 34 <210> 93 <211> 36 <212> DNA <213> Artificial Sequence <220> <223> F1117 oligonucleotide <400> 93 cgttgacgaa aaacagggtt acgctaaagg tgttct 36 <210> 94 <211> 35 <212> DNA <213> Artificial Sequence <220> <223> R1134 oligonucleotide <400> 94 acccagtttc tgggtcagaa cacctttagc gtaac 35 <210> 95 <211> 28 <212> DNA <213> Artificial Sequence <220> <223> F1153 oligonucleotide <400> 95 gacccagaaa ctgggtccgt ggcgtcgt 28 <210> 96 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> R1169 oligonucleotide <400> 96 caggtaagca accggacgac gccacgg 27 <210> 97 <211> 33 <212> DNA <213> Artificial Sequence <220> <223> F1181 oligonucleotide <400> 97 ccggttgctt acctgtctaa aaaactggac ccg 33 <210> 98 <211> 33 <212> DNA <213> Artificial Sequence <220> <223> R1196 oligonucleotide <400> 98 ccaaccagca gcaaccgggt ccagtttttt aga 33 <210> 99 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> F1214 oligonucleotide <400> 99 gttgctgctg gttggccgcc gtgcctg 27 <210> 100 <211> 29 <212> DNA <213> Artificial Sequence <220> <223> R1229 oligonucleotide <400> 100 atagcagcaa ccatacgcag gcacggcgg 29 <210> 101 <211> 33 <212> DNA <213> Artificial Sequence <220> <223> F1241 oligonucleotide <400> 101 cgtatggttg ctgctatcgc tgttctgacc aaa 33 <210> 102 <211> 33 <212> DNA <213> Artificial Sequence <220> <223> R1258 oligonucleotide <400> 102 gtcagtttac cagcgtcttt ggtcagaaca gcg 33 <210> 103 <211> 31 <212> DNA <213> Artificial Sequence <220> <223> F1274 oligonucleotide <400> 103 gacgctggta aactgaccat gggtcagccg c 31 <210> 104 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> R1291 oligonucleotide <400> 104 ggagccagga taaccagcgg ctgacccatg 30 <210> 105 <211> 31 <212> DNA <213> Artificial Sequence <220> <223> F1305 oligonucleotide <400> 105 tggttatcct ggctccgcac gctgttgaag c 31 <210> 106 <211> 32 <212> DNA <213> Artificial Sequence <220> <223> R1321 oligonucleotide <400> 106 gcggctgttt aaccagagct tcaacagcgt gc 32 <210> 107 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> F1336 oligonucleotide <400> 107 tctggttaaa cagccgccgg accgttggct 30 <210> 108 <211> 31 <212> DNA <213> Artificial Sequence <220> <223> R1353 oligonucleotide <400> 108 gtcatacgag cgttagacag ccaacggtcc g 31 <210> 109 <211> 34 <212> DNA <213> Artificial Sequence <220> <223> F1366 oligonucleotide <400> 109 gtctaacgct cgtatgaccc actaccaggc tctg 34 <210> 110 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> R1384 oligonucleotide <400> 110 tcggtgtcca gcagcagagc ctggtagtgg 30 <210> 111 <211> 29 <212> DNA <213> Artificial Sequence <220> <223> F1400 oligonucleotide <400> 111 ctgctggaca ccgaccgtgt tcagttcgg 29 <210> 112 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> R1414 oligonucleotide <400> 112 agagcaacaa ccggaccgaa ctgaacacgg 30 <210> 113 <211> 29 <212> DNA <213> Artificial Sequence <220> <223> F1429 oligonucleotide <400> 113 tccggttgtt gctctgaacc cggctaccc 29 <210> 114 <211> 26 <212> DNA <213> Artificial Sequence <220> <223> R1444 oligonucleotide <400> 114 gcagcggcag cagggtagcc gggttc 26 <210> 115 <211> 28 <212> DNA <213> Artificial Sequence <220> <223> F1458 oligonucleotide <400> 115 tgctgccgct gccggaagaa ggtctgca 28 <210> 116 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> R1470 oligonucleotide <400> 116 ccaggcagtt gtgctgcaga ccttcttccg 30 <210> 117 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> F1486 oligonucleotide <400> 117 gcacaactgc ctggacatcc tggctgaagc 30 <210> 118 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> R1500 oligonucleotide <400> 118 gacgggtacc gtgagcttca gccaggatgt 30 <210> 119 <211> 28 <212> DNA <213> Artificial Sequence <220> <223> F1516 oligonucleotide <400> 119 tcacggtacc cgtccggacc tgaccgac 28 <210> 120 <211> 26 <212> DNA <213> Artificial Sequence <220> <223> R1530 oligonucleotide <400> 120 cggcagcggc tggtcggtca ggtccg 26 <210> 121 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> F1544 oligonucleotide <400> 121 cagccgctgc cggacgctga ccacacc 27 <210> 122 <211> 29 <212> DNA <213> Artificial Sequence <220> <223> R1556 oligonucleotide <400> 122 ccgtcggtgt accaggtgtg gtcagcgtc 29 <210> 123 <211> 31 <212> DNA <213> Artificial Sequence <220> <223> F1571 oligonucleotide <400> 123 tggtacaccg acggttcttc tctgctgcag g 31 <210> 124 <211> 33 <212> DNA <213> Artificial Sequence <220> <223> R1585 oligonucleotide <400> 124 gctttacgct gaccttcctg cagcagagaa gaa 33 <210> 125 <211> 33 <212> DNA <213> Artificial Sequence <220> <223> F1602 oligonucleotide <400> 125 aaggtcagcg taaagctggt gctgctgtta cca 33 <210> 126 <211> 36 <212> DNA <213> Artificial Sequence <220> <223> R1618 oligonucleotide <400> 126 cagataactt cggtttcggt ggtaacagca gcacca 36 <210> 127 <211> 35 <212> DNA <213> Artificial Sequence <220> <223> F1635 oligonucleotide <400> 127 ccgaaaccga agttatctgg gctaaagctc tgccg 35 <210> 128 <211> 32 <212> DNA <213> Artificial Sequence <220> <223> R1654 oligonucleotide <400> 128 gagcagaggt accagccggc agagctttag cc 32 <210> 129 <211> 32 <212> DNA <213> Artificial Sequence <220> <223> F1670 oligonucleotide <400> 129 gctggtacct ctgctcagcg tgctgaactg at 32 <210> 130 <211> 31 <212> DNA <213> Artificial Sequence <220> <223> R1686 oligonucleotide <400> 130 gcctgggtca gagcgatcag ttcagcacgc t 31 <210> 131 <211> 35 <212> DNA <213> Artificial Sequence <220> <223> F1702 oligonucleotide <400> 131 cgctctgacc caggctctga aaatggctga aggta 35 <210> 132 <211> 40 <212> DNA <213> Artificial Sequence <220> <223> R1717 oligonucleotide <400> 132 ggtgtaaacg ttcagttttt taccttcagc cattttcaga 40 <210> 133 <211> 38 <212> DNA <213> Artificial Sequence <220> <223> F1737 oligonucleotide <400> 133 aaaaactgaa cgtttacacc gactctcgtt acgctttc 38 <210> 134 <211> 34 <212> DNA <213> Artificial Sequence <220> <223> R1757 oligonucleotide <400> 134 ggatgtgagc ggtagcgaaa gcgtaacgag agtc 34 <210> 135 <211> 33 <212> DNA <213> Artificial Sequence <220> <223> F1775 oligonucleotide <400> 135 gctaccgctc acatccacgg tgaaatctac cgt 33 <210> 136 <211> 32 <212> DNA <213> Artificial Sequence <220> <223> R1791 oligonucleotide <400> 136 cagcagacca cgacgacggt agatttcacc gt 32 <210> 137 <211> 35 <212> DNA <213> Artificial Sequence <220> <223> F1808 oligonucleotide <400> 137 cgtcgtggtc tgctgacctc tgaaggtaaa gaaat 35 <210> 138 <211> 40 <212> DNA <213> Artificial Sequence <220> <223> R1823 oligonucleotide <400> 138 ggatttcgtc tttgtttttg atttctttac cttcagaggt 40 <210> 139 <211> 37 <212> DNA <213> Artificial Sequence <220> <223> F1843 oligonucleotide <400> 139 caaaaacaaa gacgaaatcc tggctctgct gaaagct 37 <210> 140 <211> 33 <212> DNA <213> Artificial Sequence <220> <223> R1863 oligonucleotide <400> 140 gtttcggcag gaacagagct ttcagcagag cca 33 <210> 141 <211> 36 <212> DNA <213> Artificial Sequence <220> <223> F1880 oligonucleotide <400> 141 ctgttcctgc cgaaacgtct gtctatcatc cactgc 36 <210> 142 <211> 34 <212> DNA <213> Artificial Sequence <220> <223> R1896 oligonucleotide <400> 142 ttctggtgac ccgggcagtg gatgatagac agac 34 <210> 143 <211> 31 <212> DNA <213> Artificial Sequence <220> <223> F1916 oligonucleotide <400> 143 ccgggtcacc agaaaggtca ctctgctgaa g 31 <210> 144 <211> 34 <212> DNA <213> Artificial Sequence <220> <223> R1930 oligonucleotide <400> 144 catacggtta ccacgagctt cagcagagtg acct 34 <210> 145 <211> 31 <212> DNA <213> Artificial Sequence <220> <223> F1947 oligonucleotide <400> 145 ctcgtggtaa ccgtatggct gaccaggctg c 31 <210> 146 <211> 33 <212> DNA <213> Artificial Sequence <220> <223> R1964 oligonucleotide <400> 146 ggtgatagca gctttacgag cagcctggtc agc 33 <210> 147 <211> 34 <212> DNA <213> Artificial Sequence <220> <223> F1978 oligonucleotide <400> 147 tcgtaaagct gctatcaccg aaaccccgga cacc 34 <210> 148 <211> 33 <212> DNA <213> Artificial Sequence <220> <223> R1997 oligonucleotide <400> 148 ttagatcagc agggtagagg tgtccggggt ttc 33 <210> 149 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> F2012 oligonucleotide <400> 149 tctaccctgc tgatctaa 18 <210> 150 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> forward primer for the synthesis of K295Q mutant <400> 150 gtcagccgac cccgcaaacc ccgcgtc 27 <210> 151 <211> 29 <212> DNA <213> Artificial Sequence <220> <223> reverse primer for the synthesis of K295Q mutant <400> 151 ggaattcacg cagctgacgc ggggtttgc 29 <210> 152 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> forward primer for the amplification of synthesized gene <400> 152 ctgaacatcg aagacgaa 18 <210> 153 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> reverse primer for the amplification of synthesized gene <400> 153 ttagatcagc agggtaga 18 <210> 154 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> forward primer for site-directed mutagenesis of Q63L <400> 154 caaactttac ccgatgtctc aggaagctcg 30 <210> 155 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> reverse primer for site-directed mutagenesis of Q63L <400> 155 atagaaaccg gggtagaggt agctttcagc 30 <210> 156 <211> 31 <212> DNA <213> Artificial Sequence <220> <223> forward primer for site-directed mutagenesis of K264L <400> 156 ccagcttcag gttaaatacc tgggttacct g 31 <210> 157 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> reverse primer for site-directed mutagenesis of K264L <400> 157 cagatctgag cttttttagc agaagcacgg 30 <210> 158 <211> 26 <212> DNA <213> Artificial Sequence <220> <223> forward primer for site-directed mutagenesis of T306L <400> 158 ggtcttgctg gtttctgccg tctgtg 26 <210> 159 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> reverse primer for site-directed mutagenesis of T306L <400> 159 caggaattca cgcagctgac gcgg 24 <210> 160 <211> 28 <212> DNA <213> Artificial Sequence <220> <223> forward primer for site-directed mutagenesis of E346M <400> 160 cagatgatca aacaggctct gctgaccg 28 <210> 161 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> reverse primer for site-directed mutagenesis of E346M <400> 161 gtacgctttc tgctggtccg gacc 24 <210> 162 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> forward primer for site-directed mutagenesis of P408E <400> 162 ccggaatgcc tgcgtatggt tgctg 25 <210> 163 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> reverse primer for site-directed mutagenesis of P408E <400> 163 ccaaccagca gcaaccgggt cc 22 <210> 164 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> forward primer for site-directed mutagenesis of H438Y <400> 164 cgtacgctgt tgaagctctg gttaaacagc 30 <210> 165 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> reverse primer for site-directed mutagenesis of H438Y <400> 165 gagccaggat aaccagcggc tgacc 25 <210> 166 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> forward primer for site-directed mutagenesis of N454F <400> 166 ctgtctttcg ctcgtatgac ccactaccag 30 <210> 167 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> reverse primer for site-directed mutagenesis of N454F <400> 167 ccaacggtcc ggcggctgtt taac 24 <210> 168 <211> 47 <212> DNA <213> Artificial Sequence <220> <223> primer for sense strand of Q63L, K264L, T306L and E346M mutant genes <400> 168 gcgcgccata tgctgaacat cgaagacgaa caccgtctgc acgaaac 47 <210> 169 <211> 47 <212> DNA <213> Artificial Sequence <220> <223> primer for antisense strand of Q63L, K264L, T306L and E346M mutant genes <400> 169 gcgcgcgcgg ccgcttagat cagcagggta gaggtgtccg gggtttc 47 <210> 170 <211> 47 <212> DNA <213> Artificial Sequence <220> <223> primer for sense strand of K295Q, P408E, H438Y and N454F mutant genes <400> 170 gcgcgccata tgctgaacat cgaagacgaa caccgtctgc acgaaac 47 <210> 171 <211> 44 <212> DNA <213> Artificial Sequence <220> <223> primer for antisense strand of K295Q, P408E, H438Y and N454F mutant genes <400> 171 gcgcgcgcgg ccgcgatcag cagggtagag gtgtccgggg tttc 44 <210> 172 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> primer for sense strand of T7 promoter <400> 172 taatacgact cactataggg 20 <210> 173 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> primer for antisense strand of T7 promoter <400> 173 gctagttatt gctcagcgg 19 <210> 174 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> primer for sense strand of GAPDH gene <400> 174 gaaggtgaag gtcggagtca acg 23 <210> 175 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> primer for antisense strand of GAPDH gene <400> 175 agtccttcca cgataccaaa gttg 24

Claims (12)

A reverse transcriptase in which the 306th threonine of the amino acid sequence of a Moloney-Murine Leukemia Virus (M-MLV) reverse transcriptase described in SEQ ID NO: 1 is substituted with a lysine (T306L) to increase thermal stability. The method according to claim 1,
Substitution (Q63L) from 63rd glutamine to lucine in the amino acid sequence of M-MLV derived reverse transcriptase described in SEQ ID NO: 1, substitution with lysine (K264L) from 264th lysine, substitution with glutamine from 295th lysine (K295Q), substitution with methionine at 346th glutamic acid (E346M), substitution with glutamic acid at 408th proline (P408E), substitution at tyrosine at 438th (H438Y) and substitution with phenylalanine at 454th asparagine (N454F ). &Lt; / RTI &gt; In another embodiment, the reverse transcriptase further comprises at least one amino acid substitution selected from the group consisting of: The method according to claim 1,
A reverse transcriptase comprising an amino acid sequence as set forth in SEQ ID NO: 5. The method according to any one of claims 1 to 3,
A reverse transcriptase exhibiting excellent reverse transcription activity as compared to an M-MLV-derived reverse transcriptase consisting of the amino acid sequence shown in SEQ ID NO: 1 at a temperature of 60 ° C to 70 ° C. A gene encoding a reverse transcriptase with increased thermostability of claim 1. The method of claim 5,
(Q63L) of the reverse transcriptase of SEQ ID NO: 1 (Q63L), substitution of leucine to lysine (K264L), substitution of glutamine to 295th lysine (K295Q), substitution of 346th glutamic acid Substitution with methionine (E346M), substitution with glutamic acid at position 408 proline (P408E), substitution with tyrosine at position 438 (H438Y), and substitution with phenylalanine at position 454 asparagine (N454F) A gene encoding a mutant reverse transcriptase further comprising one or more amino acid substitutions. The method of claim 5,
A gene consisting of the nucleotide sequence shown in SEQ ID NO: 14. An expression vector comprising the gene of claim 5. A transformant transformed with the expression vector of claim 8. A composition for RT-PCR comprising the thermostable increased reverse transcriptase, primer pair, dNTP and buffer solution of any one of claims 1 to 3. The method of claim 10,
Wherein the reverse transcriptase exhibits excellent reverse transcription activity as compared to an M-MLV-derived reverse transcriptase comprising the amino acid sequence of SEQ ID NO: 1 at a temperature of 60 ° C to 70 ° C. A kit for a reverse reaction comprising the thermostable-enhanced reverse transcriptase of any one of claims 1 to 3.

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