WO2001049839A2 - Method for expressing gene and use thereof - Google Patents
Method for expressing gene and use thereof Download PDFInfo
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- WO2001049839A2 WO2001049839A2 PCT/JP2001/000048 JP0100048W WO0149839A2 WO 2001049839 A2 WO2001049839 A2 WO 2001049839A2 JP 0100048 W JP0100048 W JP 0100048W WO 0149839 A2 WO0149839 A2 WO 0149839A2
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P21/00—Preparation of peptides or proteins
- C12P21/02—Preparation of peptides or proteins having a known sequence of two or more amino acids, e.g. glutathione
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/10—Processes for the isolation, preparation or purification of DNA or RNA
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/10—Processes for the isolation, preparation or purification of DNA or RNA
- C12N15/1096—Processes for the isolation, preparation or purification of DNA or RNA cDNA Synthesis; Subtracted cDNA library construction, e.g. RT, RT-PCR
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P19/00—Preparation of compounds containing saccharide radicals
- C12P19/26—Preparation of nitrogen-containing carbohydrates
- C12P19/28—N-glycosides
- C12P19/30—Nucleotides
- C12P19/34—Polynucleotides, e.g. nucleic acids, oligoribonucleotides
Definitions
- the present invention relates to a novel method for expressing gene and use thereof, which are based on novel gene expression routes (reversible non-chromosomal gene expression routes) differing from a conventionally known gene expression route (Irreversible chromosomal gene expression route). More particularly, the present invention relates to a method of converting one of DNA, RNA and protein to any one of DNA, RNA and protein by t e novel gene expression routes which have not been known, a method of producing DNA, RNA or protein by the gene expression routes, a reagent having an action of promoting each reaction of the gene expression routes, and a medicament for expressing an objective protein in vivo in the body of human or animal .
- a synthetic DNA plasmid containing a part of genes of a pathogenic prgan is administered to a body of human or animal by injection and the like to express a protein (antigen) encoded by the DNA in the cells, thereby inducing an immune reaction in vivo.
- marker molecules are expressed in the cells by employing a gene vaccine, thereby making it possible to selectively attack infected cells.
- the gene vaccine targets the infected cells directly, a high effect thereof can be expected; in addition, since the gene vaccine can be produced employing DNA as a material, the production of a highly homogeneous vaccine can be expected.
- the reverse transcription was interpreted to be added to a basic route by a specific route; in fact, the reverse transcription leads to a recombinant DNA as a basic technique forming a cDNA, thereby expressing individual genes as an mRNA-DNA-RNA-protein, which becomes a basic technique supporting present biotechnology.
- it is one of targets for a gene remedy how efficiently an objective gene could be developed to a protein in vivo for a remedy for hereditary diseases.
- the above is performed according to the above central dogma from minute technical skill of all gene operation techniques to a gene amplification device (PCR) applied at a high frequency.
- PCR gene amplification device
- the present inventors have engaged in various studies upon a gene expression route in vivo, and as a result have found that a new gene expression route differing from a conventionally known gene expression route (Irreversible chromosomal gene expression route) is present in vivo. Since this gene expression route is a reversible reaction system while the conventional, classical chromosomal gene expression route is an irreversible reaction system, the present inventors have defined the former as a reversible non-chromosomal gene expression route.
- This gene expression route essentially consists of new gene expression systems which comprises processes of conversion from protein to RNA (reverse translation) , conversion from RNA to DNA (reverse transcription), conversion from protein to DNA (reverse direct expression)., direct conversion from DNA to protein (direct expression) and replication of DNA, along with conventional processes of transcription from DNA to RNA and translation from mRNA to protein.
- RNA (mRNA) DNA
- an individual reaction occurs reversibly both in normal direction gene expression [DNA — RNA (mRNA) — protein] and reverse direction gene expression (see Fig. 1).
- the present inventors have found that these reactions are all promoted by CaM (which means substances containing calmodulin and substances having the properties as same as that of calumodulin or calmodulin-like function).
- the calmodulin is universally present inside and outside cells of from vegetables to animals and humans as a protein for adjusting calcium ions that a gene expression mechanism according to the reversible reaction is present in vivo.
- this new gene expression route is to be an alternative gene expression route for responding to the outer circumstances and maintaining the homeostasis in vivo to stresses, obstacles, diseases and the like.
- this new gene expression route has named this new gene expression route as “Eastern route”, and also named the method of converting genes according to the present invention as “Eastern method”.
- the present invention for solving the above subjects comprises the following technical means.
- a method of converting one of DNA, RNA and a protein to any one of corresponding DNA, RNA or protein according to the following reactions: 1) conversion in a system containing no reverse transcriptase from an RNA to a DNA (reverse transcription) , 2) conversion from a protein to an RNA (reverse translation ) ,
- a CaM-containing reaction-promoting reagent which has an action of promoting a reaction of converting one of a DNA, an RNA and a protein to any one of a DNA, an RNA and a proteinaccording to above (1) or (2).
- a reagent according to above (8) which has an action of promoting a reaction of reverse-transcribing an mRNA or an RNA to a DNA.
- a reagent according to above (8) which has an action of promoting a reaction of reverse-translating a protein to an mRNA or an RNA.
- a reagent according to above (8) which has an action of promoting a reaction of directly reverse- expressing a protein to a DNA.
- a reagent according to above (8) which has an action of promoting a reaction of directly expressing a DNA to a protein.
- a reagent according to above (8) which has an action of promoting a reaction of replicating a DNA.
- a medicament for expressing an objective protein in vivo by the administration thereof to a human or an animal which comprises one or more selected from a DNA, an RNA and a protein and CaM.
- a medicament for expressing an objective protein in vivo by the administration thereof to a human or an animal which comprises one or more selected from a true naked DNA, RNA and protein not integrated in a vector.
- a method of expressing an objective protein in vivo by administering a medicament according to any one of above (14) to (16) to a human or an animal by topical, oral administration or injection.
- a method of producing DNA from protein by reverse direct expression which comprises incubating the protein in the solution containing tRNA, dNTP with calmodulin to prepare the corresponding DNA, and then isolating the DNA from the solution.
- a method of producing protein from DNA by direct expression which comprises incubating the DNA in the solution containing 1) tRNA, ribosome, amynoacyl-tRNA synthetase and amino acids, or 2) reticulocyte lysate and amino acids to prepare the corresponding protein, and then isolating the protein from the solution.
- a method of producing DNA from RNA by reverse transcription which comprises incubating the RNA in the solution containing calmodulin, dNTP to prepare the corresponding DNA, and then isolating the DNA from the solution.
- RNA from DNA by transcription comprises incubating the DNA in the solution containing calmodulin, NTP to prepare the corresponding RNA, and then isolating the RNA from the solution.
- RNA from protein by reverse translation comprises incubating the protein in the solution containing calmodulin, tRNA and NTP to prepare the RNA, and then isolating the RNA from the solution.
- a method of producing DNA from protein by using reverse direct expression route which comprises 1) incubating the protein in the solution containing calumodulin, tRNA and dNTP to prepare the corresponding DNA, 2) subjecting the DNA to a DNA amplifying reaction solution, and
- a method of characterizing profiles and/or biological and chemical activity of protein produced from DNA by direct expression which comprises cross-linking known or unknown gene DNA to a membrane , incubating the DNA in the reaction .solution containing reticulocyte lysate, tRNA and amino acids to prepare the corresponding protein, washing the product on the membrane.
- a method of characterizing profiles of DNA produced from protein by reverse direct expression which comprises transfer known or unknown protein to positively charged membrane, incubating the protein* in the reaction solution containing tRNA, dNTPs and calmodulin to prepare the corresponding DNA, washing the product on the membrane, and extracting the DNA to characterize profiles thereof .
- a method of testing gene DNA hybridized with DNA on a DNA chip which comprises subjecting a sample containing known or unknown gene DNA to the DNA chip, incubating the gene DNA with calmodulin to amplify the DNA, and detecting the DNA hybridized with DNA on the chip.
- FIG. 1 A new gene expression route found by the present inventors is shown in Fig. 1 in contrast with a conventionally known gene expression route.
- the new gene expression route is shown in the upper part, and the conventional gene expression route is shown in the lower part.
- the new gene expression route comprises reversible expression systems of non- chromosomal genes.
- DNA genomic, genome type
- DNA genomic, gene type
- CaM shows calmodulin or substances having the properties as same as that of calmodulin or calmodulin-like function.
- - CaM is defined as to mean substances containing calmodulin and substances having the above calmodulin-like function.
- the gene expression route comprises, in additon to processes of transcription from DNA to RNA/mRNA and translation from RNA/ RNA to protein, processes of conversion from RNA to DNA (reverse transcription), conversion from protein to RNA (reverse translation), conversion from protein to DNA (reverse direct expression), direct conversion from DNA to protein (direct expression) and replication of DNA.
- the present inventors have confirmed that one of DNA, RNA and protein is converted to any one of corresponding DNA, RNA or a protein by the gene expression route..
- each reaction of the gene expression route is promoted by the presence of CaM.
- calmodulin or substances having a calmodulin-like function is preferable and important in the present invention.
- a concentration and reaction conditions are properly adjusted according to each reaction system containing DNA, RNA or protein, as will be shown in examples to be described later.
- Fig. 25 is shown the dissociation of a bovine brain calmodulin in an isotonic phosphoric acid buffer (pH: 7.0) [PBS(+)] containing Ca2+ and Mg2+.
- CaM calmodulin and substances having the calmodulin-like function
- CaM means calmodulin, a fragment or a molecule containing a part or all of the calmodulin protein as a constituent, a molecule with amino acid deleted, substituted or added in a part of the amino acid sequence of the calmodulin protein or a derivative of calmodulin, and those having the calmodulin function can be employed preferably.
- such substances having the calmodulin-like function and an action of promoting the reactions of the above gene expression systems can be employed similarly.
- a substance having the calmodulin-like function means a substance having a function equal to that of "calmodulin which has a function of accepting phosphoric acid residue to be bound to organic radical (namely, phosphate pocket) and an action of activating the reactions of the above gene expression systems.
- such substances are generally referred to as CaM.
- Fig. 26 is generally shown the activation mechanism of the reactions of the gene expression systems of the present invention based on the above calmodulin-like function (phosphate pocket) of CaM.
- the gene expression route of the present invention is useful as a method of converting one of DNA, RNA and protein to any one of corresponding DNA, RNA or protein in a reaction system containing CaM.
- the method is useful, for example, as a method of producing DNA, RNA or protein in vitro by the above gene expression route.
- DNA, RNA and protein may be of a proper sequence, and neither kind nor length of the sequence is particularly restricted. It is possible, for example, to convert a specific protein to DNA coding the protein in a reaction system containing CaM by this method.
- a specific DNA coding the protein can be formed, for example, by employing a specific protein as the protein in the method of the present invention.
- a proper reaction product corresponding to a proper DNA, RNA or protein can be obtained without any restriction of the kind of DNA, RNA and protein.
- the M2 gene is a gene (single-stranded RNA) coding a matrix protein of an IHN virus (infectious he atopoietic necrosis virus) (see Fig. 2).
- IHN virus infectious he atopoietic necrosis virus
- the CaM is useful as a reaction-promoting reagent having an action of promoting the reactions.
- reagents of the present invention can be exemplified CaM-containing products, and products (reagent kits) with other proper components, for example, tRNA, NTP, dNTP, amino acid mixture, a calcium ion and a phosphoric acid ion combined therewith.
- the reagents are useful in particular as a reagent for performing a method of synthesizing DNA from protein and RNA in a reaction system in vitro, and a method of replicating DNA.
- the medicament of the present invention is useful as a medicament for expressing any of an objective DNA, RNA or protein in vivo by administering one of DNA, RNA and protein to human or animal body by a proper method.
- T e medicament preferably contains DNA, RNA or protein , and the CaM as constituents.
- proper subsidiary components can be incorporated in addition to these components.
- the method of administration may be topical, oral administration or injection; however, since the present invention is characterized in that an objective protein is expressed in vivo by administering the above medicament and that a true naked DNA, RNA or protein is administered without ligating it to a vector such as a plasmid, for example, a method of topical administration to the skin and a method of oral administration are desired from the viewpoint of the stability of the medicament in vivo after administration. . *
- an objective protein can be expressed in vivo by this method, it can be realized that by administering a specific DNA a specific antibody encoded by the DNA is generated in vivo (DNA vaccine), a specific protein (various physiologically active substances, enzymes and the like), and by administering a specific RNA and protein an objective protein is generated.
- This method can be utilized for the expression of a useful gene in vivo.
- cDNA DNA
- mRNA RNA
- proteins encoded by the genes can be formed in vivo and their effect can be imparted to human or animal body.
- proteins and genes can be formed in these cases, the effect can further be improved by adding CaM together with the genes.
- a gene DNA needs no vector with a promoter, and a sufficient effect can be exhibited with only an open reading frame (ORF).
- This method is useful for realizing, for example, the administration of genes of various physiologically active substances by topical administration to the skin, the topical administration of a carcinogenesis inhibitory gene to the skin, a so-called simple method for a gene remedy (topical administration of a gene to the skin and oral administration thereof), the production of an ORF-DNA vaccine and its simple administration.
- this method can be utilized for synthesizing a gene from a specific protein and obtaining thereof.
- a gene ORF-DNA can be obtained from a protein at a stroke. If at least the C-terminal and the N-terminal of an objective protein are known, the whole gene ORF can optionally be amplified employing them as an upper primer and a lower primer; hence, this method, combined with a PCR, is useful as a method of amplifying the whole gene ORF from the protein and determining its sequence.
- This method can further be utilized for the control of gene expression in vivo.
- the reagent of the present invention can be prepared by properly combining CaM, a calcium ion/a phosphoric acid ion, tRNA, dNTP, NTP, and an amino acid mixture.
- CaM calcium ion/a phosphoric acid ion
- tRNA a calcium ion/a phosphoric acid ion
- dNTP calcium ion/a phosphoric acid ion
- tRNA a calcium ion/a phosphoric acid ion
- dNTP calcium ion/a phosphoric acid ion
- tRNA a calcium ion/a phosphoric acid ion
- dNTP calcium ion/a phosphoric acid ion
- NTP a substance having a calmodulin-like function
- amino acid mixture may properly be designed according to an objective DNA, RNA or protein.
- a preferable medicament according to the present invention can be exemplified a medicinal composition containing a DNA, an RNA or a protein, CaM and a calcium ion/a phosphoric acid ion.
- the medicament is characterized in that a true naked gene ORF or protein not combined in a vector is employed, and is essentially different from those employing a DNA connected to a conventional vector.
- CaM can its effect further be heightened.
- other proper subsidiary components and carriers can be incorporated.
- the subsidiary components and carriers for the medicament and the configurations thereof may be proper and are not particularly restricted so far as they can be injected, orally administered or topically administered.
- Fig. 27 The summarized flows of genetic information among chemically synthesized oligo DNA, RNA and oligopeptide corresponding to the DNA sequence in vitro is shown in Fig. 27 together with preferable materials to be used in their reaction systems. These flows describe the following reaction systems in the six directions which are composed of reverse transcription from RNA to DNA, transcription from DNA to RNA, direct expression from DNA to protein, reverse direct expression from protein to DNA, reverse translation from protein to RNA and translation from RNA to protein, and their detection methods (italic type) in the reaction systems are represented in brief.
- This invention provides a method of characterizing profiles such as molecules and the like, biological and chemical activity of protein produced from DNA by direct expression route on a membrane to be used for electrophoresis , and a method of characterizing profiles of gene DNA produced from protein by reverse direct expression on the membrane.
- the former method has been named as “Eastern blot I” and the latter method has been named as “Eastern blot II” by the present inventors.
- Fig. 40 shows preferable schematic of new method of the Eastern blot I and preferable schematic of new method of the Eastern blot II devised from the principles of direct expression and reverse direct expression of this invention.
- a sample of known or unknown gene DNA is subjected to alkali denaturation to prepare ssDNA, and the DNA is transfer to a membrane and cross-linked to the membrane by UV-cross linker.
- membrane of cellulose, vinyl compounds or the like to be used for electrophoresis are used in this method.
- the DNA cross-linked to the membrane is incubated in the reaction solution containing tRNA, ribosome, aminoacyl-tRNA synthetase and amino acids to prepare the corresponding protein.
- the reaction solution containing reticulocyte lysate, tRNA and amino acids is preferably used.
- Produced sequence of amino acids on the membrane is washed by water, and then extracted to characterize profiles such as molecules and the like of the protein and/or overlaid on a gel containing substrate or membrane containing chemical reagent and incubated to characterizing biological and chemical activity of the protein.
- This new method of Eastern blot I is a convenient and useful method for testing the characteristics of the protein which is produced by direct expression of the corresponding gene DNA on a membrane.
- a sample of known or unknown protein is transfered to positively charged membrane, and incubated in the reaction solution containing tRNA, dNTPs and calumodulin to prepare dsDNA by reverse direct expression, and washed by water, and then the produced DNA is extracted to characterize profiles of the DNA.
- Gene DNA of which the sequence of sense chain corresponds with RNA sequence spliced is prepared.
- This new method of Eastern blot II is preferably used for a preliminary step of PCR amplification of DNA, DNA sequencing, and characterizing gene DNA ho ology, as same as a method for characterizing profiles of gene DNA produced on a membrane from protein by reverse direct expression.
- a sample of known or unknown protein is converted to the corresponding DNA by reverse direct expression route of this invention, and the produced DNA is subjected to a DNA amplification such as PCR amplification and the like to obtain the amplified DNA corresponding to the protein sample, and a method containing these steps of preparation of DNA is encompassed in the scope of this invention as far as it contains the reverse direct expression route of this invention.
- DNA is produced from RNA by incubating the reverse transcription route of this invention.
- RNA in the solution containing calmodulin and dNTP, and in the transcription route of this invention RNA in the solution containing calmodulin and dNTP, and in the transcription route of this invention.
- RNA is produced from DNA by incubating the DNA in the solution containing calmodulin and NTP.
- DNA is produced from protein by incubating the protein in the solution containing calmodulin, tRNA and dNTP, and in the direct expression of this invention, protein is produced from DNA by incubating the DNA in the solution containing tRNA, ribosome, amynoacyl-tRNA synthetase and amino acids, or in the solution containing reticulocyte lysate and amino acid mixture.
- RNA is produced from protein by incubating the protein in the solution containing calmodulin, tRNA and NTP.
- the solution to be used are not limited to the above described solutions, and in each route of. this invention, the solution containing materials having similar or equivalent function thereto can be used properly.
- reaction conditions such as temperature, time, solution and the like are determined properly in accordance with the conditions as described in the subsequent Examples.
- gene DNA is amplified by incubating the DNA with calmodulin
- DNA in a sample containing known or unknown gene DNA subjected to a DNA chip is amplified by incubating the gene DNA with calmodulin, and thereby the gene DNA hybridized with the DNA chip can' be detected preferably.
- Fig. 1 shows a diagram showing general concepts of new gene expression routes (above) and conventional gene expression routes (below).
- Fig. 2 shows a diagram showing general concepts of new gene expression routes (above) and conventional gene expression routes (below).
- Fig. 2 shows a diagram of the reverse transcription of M2 mRNA with CaM.
- Fig. 3 shows an electrophoresis photograph of M2 DNA formed by the reverse transcription of M2 mRNA in vitro with CaM.
- Fig . 4 Fig.4 shows a diagram of the preparation of human p53 RNA and DNA.
- Fig. 5 shows a diagram of the preparation of human p53 RNA and DNA.
- Fig. 5 shows a diagram of the replication and transcription of human p53 DNA and the reverse transcription of human p53 RNA with CaM.
- Fig. 6 shows a diagram of the replication and transcription of human p53 DNA and the reverse transcription of human p53 RNA with CaM.
- Fig. 6 shows an electrophoresis photograph of p53 DNA/RNA (RNA being detected as DNA) formed by the replication and transcription of human p53 DNA in vitro and the reverse transcription of human p53 RNA with CaM.
- Fig. 7 shows an electrophoresis photograph of p53 DNA/RNA (RNA being detected as DNA) formed by the replication and transcription of human p53 DNA in vitro and the reverse transcription of human p53 RNA with CaM.
- Fig. 7 shows a diagram of the reverse direct expression of human p53 protein and M2 recombinant protein with CaM.
- Fig. 8 shows a diagram of the reverse direct expression of human p53 protein and M2 recombinant protein with CaM.
- Fig. 8 shows an electrophoresis photograph of M2 DNA formed by the reverse direct expression of M2 recombinant protein in vitro with CaM.
- Fig. 9 shows an electrophoresis photograph of M2 DNA formed by the reverse direct expression of M2 recombinant protein in vitro with CaM.
- Fig. 9 shows an electrophoresis photograph of M2 RNA (being detected as M2 DNA by a reverse transcription (RT)-PCR) formed by the reverse translation of M2 protein in vitro with CaM.
- Fig. 10 shows an electrophoresis photograph of M2 RNA (being detected as M2 DNA by a reverse transcription (RT)-PCR) formed by the reverse translation of M2 protein in vitro with CaM.
- Fig. 10 shows an electrophoresis photograph of M2 RNA (being detected as M2 DNA by a reverse transcription (RT)-PCR) formed by the reverse translation of M2 protein in vitro with CaM.
- Fig. 10 shows an electrophoresis . hotograph of p53 RNA (being detected as p53 DNA by a reverse transcription (RT)-PCR) formed by the reverse translation of human p53 protein in vitro with CaM.
- Fig. 11 shows an electrophoresis . hotograph of p53 RNA (being detected as p53 DNA by a reverse transcription (RT)-PCR) formed by the reverse translation of human p53 protein in vitro with CaM.
- Fig. 11 shows an electrophoresis . hotograph of p53 RNA (being detected as p53 DNA by a reverse transcription (RT)-PCR) formed by the reverse translation of human p53 protein in vitro with CaM.
- Fig. 11 shows an electrophoresis photograph of p53 DNA formed by the reverse direct expression of human p53 protein in vitro with CaM.
- Fig. 12 shows an electrophoresis photograph of p53 DNA formed by the reverse direct expression of human p53 protein in vitro with CaM.
- Fig. 12 shows the relationship between the human p53 DNA/CaM topically administered and its antitumor effect.
- Fig. 13 Fig. 13 shows the relationship between the human p53 DNA/anti-CaM antibody topically administered and its antitumor effect.
- Fig. 14 shows the relationship between the human p53 DNA/anti-CaM antibody topically administered and its antitumor effect.
- Fig. 14 shows the relationship between the human p53 DNA and RNA topically administrered and its antitumor effect.
- Fig. 15 shows the relationship between the human p53 DNA and RNA topically administrered and its antitumor effect.
- Fig. 15 hows an electrophoresis photograph of M2 DNA formed by the reverse direct expression of the M2 protein/CaM topically administered.
- Fig. 16 hows an electrophoresis photograph of M2 DNA formed by the reverse direct expression of the M2 protein/CaM topically administered.
- Fig. 16 shows an electrophoresis photograph of M2 DNA formed by the replication of the M2 DNA administered (intraperitoneal injection/topical administration) .
- Fig. 17 shows an electrophoresis photograph of M2 DNA formed by the replication of the M2 DNA administered (intraperitoneal injection/topical administration) .
- Fig. 17 shows an electrophoresis photograph of M2 DNA (one week and two weeks later) in tissues (brain, liver, lymphocyte) formed by the replication of the M2 cDNA topically administered.
- Fig. 18 shows an electrophoresis photograph of M2 DNA (one week and two weeks later) in tissues (brain, liver, lymphocyte) formed by the replication of the M2 cDNA topically administered.
- Fig. 18 shows an electrophoresis photograph of M2 DNA (one week and two weeks later) in tissues (brain, liver, lymphocyte) formed by the replication of the M2 cDNA topically administered.
- Fig. 18 shows an electrophoresis photograph of M2. DNA (one week and two weeks later) in tissues (brain, liver, lymphocyte) formed by the translation of the M2 mRNA topically administered.
- Fig. 19 shows an electrophoresis photograph of M2. DNA (one week and two weeks later) in tissues (brain, liver, lymphocyte) formed by the translation of the M2 mRNA topically administered.
- Fig. 19 shows an electrophoresis photograph of M2 DNA (one month later) in tissues (brain, liver, lymphocyte) formed by the translation of the M2 mRNA administered (intraperitoneal injection, topical and oral administrations).
- Fig. 20 shows an electrophoresis photograph of M2 DNA (one month later) in tissues (brain, liver, lymphocyte) formed by the translation of the M2 mRNA administered (intraperitoneal injection, topical and oral administrations).
- Fig. 20 shows an electrophoresis photograph of p53 DNA (one month later) in tissues (brain, liver, lymphocyte ) • formed by the replication of the human p53 DNA administered (topical and oral administrations).
- Fig. 21 shows an electrophoresis photograph of p53 DNA (one month later) in tissues (brain, liver, lymphocyte ) • formed by the replication of the human p53 DNA administered (topical and oral administrations).
- Fig. 21 shows . an electrophoresis photograph of M2 DNA (one month later) in tissues (brain, liver, lymphocyte) formed by the reverse direct expression of the M2 protein • administered (topical and oral administrations ) .
- Fig. 22 Fig. 22 is a diagram of the sequence determination of M2 DNAs and human p53 DNAs obtained by the DNA replication, reverse transcription, reverse direct expression and translation.
- Fig. 23 is a diagram of the sequence determination of M2 DNAs and human p53 DNAs obtained by the DNA replication, reverse transcription, reverse direct expression and translation.
- Fig. 23 shows a diagram of the sequence determination of M2 DNAs obtained by the DNA replication, reverse transcription, reverse direct expression and translation.
- Fi g- 24 shows a diagram of the sequence determination of M2 DNAs obtained by the DNA replication, reverse transcription, reverse direct expression and translation.
- Fig. 24 shows a diagram of the sequence determination of human p53 DNAs obtained by the DNA replication, reverse transcription, reverse direct expression and translation of.
- Fig. 25 shows a diagram of the sequence determination of human p53 DNAs obtained by the DNA replication, reverse transcription, reverse direct expression and translation of.
- Fig. 25 shows the relationship between the molecular weight of calmodulin and retention time.
- Fig. 26 shows the relationship between the molecular weight of calmodulin and retention time.
- Fig. 26 shows a diagram showing the concept of the active mechanism of CaM.
- Fig. 27 shows a diagram showing the concept of the active mechanism of CaM.
- Fig. 27 shows the summarized flow of genetic information among chemically synthesized oligo DNA, RNA and oligopeptide corresponding to the DNA sequence in vitro.
- Fig. 28 shows the summarized flow of genetic information among chemically synthesized oligo DNA, RNA and oligopeptide corresponding to the DNA sequence in vitro.
- Fig. 28 shows schematic of the method of direct expression, reverse direct expression and reverse translation in vitro from synthesized GST-60 bp oligo DNA and GST-60 bp protein.
- Fig. 29 shows schematic of the method of direct expression, reverse direct expression and reverse translation in vitro from synthesized GST-60 bp oligo DNA and GST-60 bp protein.
- Fig. 29 shows schematic of the method of reverse transcription and translation in vitro of GST-60 b RNA.
- Fig. 30 shows schematic of the method of reverse transcription and translation in vitro of GST-60 b RNA.
- Fig. 30 shows isolation of GST-60 bp DNA from pGEX- 6p-l/60 bp by restriction enzymes EcoRV and Xho I.
- Fig. 31 shows isolation of GST-60 bp DNA from pGEX- 6p-l/60 bp by restriction enzymes EcoRV and Xho I.
- Fig. 31 shows isolation of GST-60 bp DNA from pGEX- 6p-l/60 bp by restriction enzymes EcoRV and Xho I.
- Fig. 31 shows isolation of GST-60 bp DNA from pGEX- 6p-l/60 bp by restriction enzymes EcoRV and Xho I.
- Fig. 31 shows preparation and confirmation of GST- 60 bp DNA by digestion with DNAase I.
- Fig. 32 shows preparation and confirmation of GST- 60 bp DNA by digestion with DNAase I.
- Fig. 32 shows preparation and confirmation of GST- 60 bp fusion protein from E. coli transformant lysates.
- Fig. 33 shows reverse transcription from GST-60 b RNA to GST-60 bp DNA in vitro by Calumodulin.
- Fig. 34 shows reverse transcription from GST-60 b RNA to GST-60 bp DNA in vitro by Calumodulin.
- Fig. 34 shows translation from GST-60 bp DNA to GST-60 b RNA by CaM with NTP.
- Fig. 35 shows translation from GST-60 bp DNA to GST-60 b RNA by CaM with NTP.
- Fig. 35 shows reverse direct'.expression from GST-60 bp fusion protein to GST-60 bp DNA in vitro with CaM, NTP and reticulocyte lysate.
- Fig. 36 shows direct expression from GST-60 bp DNA to GST-60.
- bp fusion protein in vitro with reticulocyte lysate. and amino acids.
- Fig. 37 shows reverse direct'.expression from GST-60 bp fusion protein to GST-60 bp DNA in vitro with CaM, NTP and reticulocyte lysate.
- Fig. 36 shows direct expression from GST-60 bp DNA to GST-60.
- bp fusion protein in vitro with reticulocyte lysate. and amino acids.
- Fig. 37 shows reverse direct'.expression from GST-60 bp fusion protein to GST-60 bp DNA in vitro with CaM, NTP and reticulocyte lysate.
- Fig. 36 shows direct expression from GST-60 bp DNA to GST-60.
- Fig. 37 shows reverse translation from GST-60 bp protein to GST-60 b RNA by CaM.
- Fig. 38 shows reverse translation from GST-60 bp protein to GST-60 b RNA by CaM.
- Fig. 38 shows original 60 bp DNA used to produce recombinant GST fusion protein and clones, sequenced.
- Fig. 39 shows original 60 bp DNA used to produce recombinant GST fusion protein and clones, sequenced.
- Fig. 39 shows DNA clones produced from GST-60 b RNA and GST-60 bp fusion protein, and cDNA derived from RNA clones produced from GST-60 bp fusion protein.
- Fig. 40 shows schematic of new methods "Eastern blot I" and "Eastern bolt II” devised from the principles of direct expression and reverse direct expression.
- Fig. 41 Fig. 41 shows DNase and RNase activities of M2 protein produced from M2 DNA by the method of Eastern blot I.
- Fig. 42 shows M2 DNA produced from M2 protein by the method of Eastern blot II.
- Fig. 43 shows M2 DNA produced from M2 protein by the method of Eastern blot II.
- Fig. 43 shows comparison of reverse transcription activity between bovine brain CaM and human brain CaM using GST-60 bp RNA.
- Example 1 In this example, double-stranded DNA was synthesized from mRNA by reverse transcription in vitro (see Fig. • 2).
- M2 gene of pathogenic RNA (single-stranded) rahbdo virus IHN (infectious hematopoietic necrosis) was infected to cultured cells of rainbow trout gonad (RTG- 2). Subsequently, nucleic acid was extracted and treated with .DNase I to prepare mRNA. The obtained mRNA was employed as M2 mRNA sample (IHN virus mRNA).
- M2 DNA was synthesized from the M2 mRNA by reverse transcription in vitro.
- DEPC shows diethyl pyrocarbonate .
- Volume of DEPC-water to be added was properly adjusted according to a case of adding calmodulin (CaM), an anti-CaM antibody and a case of adding neither.
- mRNA sample (IHN virus mRNA) 1 ⁇ 1 PCR buffer (10-fold concentration) , 2
- the reaction solution was put into a microtube, incubated at 37 °C for 60 minutes, and treated with RNase A, and then DNA was extracted by SepaGene® Kit (manufactured by Sanko Co., Ltd.).
- the M2 DNA formed from the IHN virus mRNA by reverse transcription was amplified by PCR employing M2 primers.
- the reaction system of the PCR is shown below.
- AmpliTaq Gold ® (Perkin-Elmer Inc.) 0.5 1 (DNA polymerase, 5 ⁇ / ⁇ 1)
- Fig. 3 results of the cases of 1) CaM, 2) M2 mRNA treated with RNase + CaM, 3) M2 mRNA, 4) M2 mRNA + CaM, and 5) M2 mRNA + CaM + anti-CaM antibody. It is apparent from Fig. 3 that double-stranded DNA was formed from the mRNA by reverse transcription.
- An objective M2 DNA was scraped from a predetermined band of the above plate, extracted, ligated to a pCR 2.1 vector of Escherichia coli, and the vector was introduced into Escherichia coli and incubated to proliferate the cells; then plasmid was extracted from the cells, and subjected to a sequencer to read a sequence of the prasmid by employing primers (see Example 17) .
- Bovine CaM (1 mg/ml) 1 ⁇ 1 5 mM Ca(H 2 P0 4 ) 2 1 ⁇ 1
- the reaction solution was put into a icrotube, and incubated at 37 °C for 60 minutes, and then DNAs were extracted by SepaGene ® Kit (manufactured by Sanko Co., Ltd. ) .
- the M2 DNA or the p53 DNA formed by the DNA replication was amplified by PCR.
- the reaction system of the PCR is shown below.
- AmpliTaq Gold ® (Perkin-Elmer Inc.) 0.5 1 ( DNA polymerase , 5U/ 1 )
- Fig. 6 results of the cases of 1) p53 DNA, 2) p53 DNA + CaM + anti-CaM antibody, 3) CaM, and 4) p53 DNA + CaM.
- RNAs were synthesized from these DNAs by transcription according to the following reaction system. Volume of water to be added was properly adjusted according to a case of adding CaM, an anti-CaM antibody and a case of adding neither.
- Bovine CaM (1 mg/ l) 1 ; 1
- the reaction solution was put into a microtube, incubated at 37 °C for 60 minutes, and then DNAs were extracted by SepaGene ® Kit (manufactured by Sanko Co., Ltd.).
- the formed M2 DNA or p53 DNA was reverse- transcribed (RT), and then subjected to PCR.
- RT reverse- transcribed
- the reaction system of the PCR is shown below.
- the reaction solution was incubated at 50 °C for 30 minutes to perform RT reaction, and then the DNAs were amplified by 45-cycles of the PCR.
- RNAs were detected as DNAs- by RT-PCR. In the same manner as in Example 1, DNAs were detected.
- Fig. 6 results of the cases of 1) p53 DNA + CaM, and 2) p53 DNA. As shown in Fig. 6, the transcription from the p53 DNA + CaM to RNA was confirmed. (4) Determination of the Sequence
- RNAs were synthesized from these RNAs by reverse transcription according to the following reaction system. Volume of water to be added was properly adjusted according to a case of adding CaM, an anti-CaM antibody and a case of adding neither.
- RNA sample (M2 RNA or p53 RNA) 1 ⁇ 1
- Upper and lower primers (20 M) 1 j 1 and 1 i 1 PCR buffer (10-fold concentration) 2 / 1
- DNAs were synthesized from proteins by reverse direct expression in vitro (see Fig. 7).
- Anti-CaM rabbit IgG 1 ⁇ 1 Mixture (in vitro translation kit (manufactured by Ambion, Inc.)) 20 ⁇ 1
- the M2 DNA and p53 DNA formed from the M2 protein and p53 protein by reverse translation were amplified by PCR.
- the reaction system of the PCR is shown below.
- Fig. 8 The results are shown in Fig. 8.
- Fig. 8 are shown results of the cases of 1) CaM, 2) protease- treated M2 protein + CaM, 3) M2 protein + CaM, anti-CAM antibody, 4) M2 protein - + CaM, and 5) M2 protein.
- proteins were synthesized from M2 DNA or p53 DNA by direct expression. It was confirmed that the DNAs included no RNA. Volume of nuclease-ffee water to be added was properly adjusted according to a .case of adding CaM, an anti-CaM antibody and a case of adding neither.
- RNA-containing erythrocyte lysates 16.7 1 master mixture: 1.3 ⁇ 1 Nuclease-free water: 1-2 1)
- reaction solution was put into a microtube and incubated at 37 °C for 90 minutes, and then proteins were detected by ELISA (enzyme-linked immunpsorbent assay) .
- an anti-M2 protein rabbit IgG an anti-p53 protein rabbit IgG (primary antibody) and an anti-rabbit IgG goat serum combined with horseradish peroxidase (secondary antibody), M2 protein and p53 protein were detected by ELISA.
- RNA was synthesized from M2 protein by reverse translation.
- Bovine CaM (1 mg/ml) 1 ⁇ 1
- the reaction solution was put into a microtube, incubated at 37 °C for 90 minutes, and treated with DNase I, and then RNA was extracted b SepaGene ® Kit (manufactured by Sanko Co., Ltd.).
- RNA reverse-transcribed to DNA by reverse transcription (RT)-PCR according to the following reaction system, and the DNA was amplified, and then detected.
- RT reverse transcription
- AmpliTaq Gold ® ( Perkin-Elmer Inc.) 0.5 1 (DNA polymerase, 5U/ ⁇ 1)
- p53 RNA was synthesized from p53 protein by reverse translation in vitro.
- the p53 RNA was detected as p53 DNA by RT-PCR. The results are shown- in Fig. 10.
- the p53 DNA was detected in the case of the p53 protein + CaM ( l ⁇ g or 0.2 g).
- p53 DNA human tumor suppressor gene
- carcinogenesis suppressor effect antiitumor activity
- the cumulative mortality (p53 DNA:Q , p53 DNA + CaM: ⁇ ) and the mean diameter of solid tumor tissue formed at the administration site (p53 DNA: ⁇ , p53 DNA + CaM:A ) were recorded.
- Tumor cells were administered by injection to mice including three males and three females to employ them as controls, and the cumulative mortality ( O ) and the diameter (# ) of solid tumor tissue formed were measured.
- Example 9 In this example, p53 DNA and an anti-CaM antibody were topically administered to mice and their antitumor activity was tested.
- Balb/c 3T/12-3 cells tumor cells
- Balb/c mice including three males and three females at a rate of 10 5 cells/mouse, human p53 DNA and an anti-CaM rabbit IgG were topically administered to them.
- Fig. 13 shows an inhibitory effect by the anit-CaM rabbit antibody to the antitumor activity of the mice topically administered with the human p53 DNA.
- the cumulative mortality (D A: ⁇ , DNA + anti-CaM : ⁇ ) and the diameter of solid tumor tissue formed at the injection site (DNA: ⁇ , DNA + anti-CaM: A. ) were recorded.
- Tumor cells were administered by injection to mice including three males and three females to employ them as controls, and the cumulative mortality ( O ) and the diameter ( ⁇ ) of solid tumor tissue formed were measured.
- the antitumor activity was reduced in the case of the p53 DNA + anti-CaM, which suggested that the endogeneous CaM of the mouse was effective.
- the human p53 DNA and RNA were topically administered to mice, and their antitumor activity was examined.
- Balb/c 3T/12-3 cells tumor cells
- Balb/c mice including three males and three females at a rate of 10 5 cells/mouse, human p53 DNA or RNA was topically administered to them.
- Fig. 14 shows the relationship between the topical administration of the human p53 DNA and RNA to the mice and their antitumor activity.
- RNA: ⁇ The cumulative mortality (D AiQ , RNA: ⁇ ) and the mean diameter of solid, tumor tissue formed at the administration site (DNA: ⁇ , RNA:A ) were recorded. Tumor cells were administered by injection to mice including three males and three females to employ them as controls, and the cumulative mortality ( O) and the diameter ( ⁇ ) of solid tumor tissue formed were measured.
- O the cumulative mortality
- ⁇ (DNA) was 2/6 of that of the controls
- RNA was 0/6 of that of the controls
- ⁇ (DNA) was 2/6 of that of the controls
- RNA was 1/6 of that of the controls; thus the rate of formation . thereof for ⁇ (DNA) was rather small and that forA (RNA) was remarkably small as compared with those of the controls .
- the M2 protein (10/ g/mouse) and the M2 protein (10 ⁇ g/mouse) + bovine CaM (5/ g/mouse) were topically administered to the Balb/c mice, and two weeks after the administration,, the number of M2 DNA copies formed in vivo was examined by a light cycler PCR (Behringer
- the M-2 DNA (5 / g/mouse) was administered to Balb/c mice by intraperitoneal injection and topical administration, and two weeks after the administration, the M2 DNA in mouse tissues (lymphocyte, liver, brain) replicated in vivo were examined according to electrophoresis, are shown in Fig. 16. Moreover, " similarly, the M2 DNA was administered to the mice by intraperitoneal injection and oral administration, and two weeks after the administration, the number of the M2 DNA copies were detected and determined by a light cycler PCR according to cyber green, and the results are shown in Table 3.
- the IHN virus M2 cDNA ( l g/mouse) and bovine CaM (5 g/mouse) were topically administered to the skin, of Balb/c mice, and one week later, four weeks later, the replication of the M2 DNA in vivo was examined by electrophoresis. The results are shown in Fig. 17.
- RNA + CaM has a high activity for the formation of DNAs and has a high effect of maintaining the formation of DNAs over a long period of time.
- the M2 mRNA (50 ng/mouse) was administered to Balb/c mice by intraperitoneal injection, topical administration and oral administration, and one month later, M2 DNAs in mouse tissues (lymphocyte, liver, brain) were amplified by PCR, and detected by electrophoresis. The results are shown in Fig. 19.
- the M2 DNAs were detected at a high activity in the brain for the intraperitoneal injection, in the liver and the brain for the tipical administration, and in the lymphocyte for the oral administration.
- the p53 DNA (50 ng/mouse) was administered to Balb/c mice by topical administration to the skin and oral administration, and one month later, the p53 DNAs in mouse tissues (lymphocyte, liver, brain) were amplified by PCR, and detected by electrophoresis. The results are shown in Fig. 20. The p53 DNAs were detected in a high activity in all cases.
- the M2 protein (1 g/mouse) was administered to Balb/c mice by topical administration to the skin and oral administration, and one month later, M2 DNAs in mouse tissues (lymphocyte, liver, brain) were amplified by PCR, and detected by electrophoresis. The results are shown in Fig. 21'. The . M2 DNAs were detected in a particularly high activity for the topical administration.
- the M2 DNA ligated to pCR 2.1 vector was digested by EcoRI, and by employing upper and lower primers, the sequence thereof was determined by reading in both directions of sense chain and anti-sense chain . of each clone. The results are shown in Fig. 23.
- Clone 1 shows M2 DNA obtained by the reverse transcription of the IHN virus mRNA in vitro in Q . the presence of CaM
- Clone 2 shows M2 DNA obtained by the reverse direct expression of the recombinant M2 protein in vitro in the presence of CaM
- Clone 3 shows M2 DNA obtained from a Balb/c mouse one month after topical administration of the IHN virus mRNA in 5 vivo, respectively.
- the human p53 DNA ligated to pCR 2.1 vector was digested by EcoRI, and by employing 5 upper and lower primers, the sequence thereof was determined by reading in both directions of sense chain and anti-sense chain of each clone. The results are shown in Fig. 24.
- Clone 4 shows p53 DNA obtained by the reverse transcription of the p53 RNA in vitro in the presence of. CaM
- Clone 5 shows p53 DNA obtained by the reverse direct expression of the .p53 protein in vitro in the presence of CaM
- Clone 6 shows p53 DNA obtained from a Balb/c mouse one month after topical administration of the p53 RNA in vivo, respectively.
- CaM Ca(H 2 P0 4 ) 2 , NTP (for transcription)
- CaM Ca(H 2 P0 4 ) 2 , tRNA, amino acid mixture (for translation)
- a medicinal composition containing a protein and CaM was prepared from the following arrangement.
- PUC 118 (Takara Shuzo Co., Ltd.)/60 bp DNA plasmid was inserted into E. coli JM 109 to transform thereof and then the transformed clone was selected, and the plasmid DNA was isolated from the clone by FlexPrep® Kit (Amersham Pharmacia Biotech Co.).
- PUC 118/60 bp plasmid was digested with EcoRI at 30 °C overnight to prepare 60 bp DNA in the following solution: PUC 118/60 bp plasmid (5ng//z 1) , 7 ⁇ 1
- the 60 bp DNA was ligated into EcoRI site of pGEX- 6p-l plasmid at 15 °C overnight to prepare subcloned pGEX-6p-l/60 bp plasmid in the following solution:
- the ' subcloned pGEX-6p-l/60 bp plasmid was inserted into E. coli MV 1184 to transform thereof, and the plasmid DNA was isolated by FlexPrep® Kit (Amersham Pharmacia Biotech Co.).
- the pGEX-6p-l/60 bp plasmid was digested with EcoRV and Xho I at 37°C for 4 hr to prepare DNA including GST-60 bp DNA in the following solution: pGEX-6p-l/60 bp plasmid ( 2ng/ ⁇ 1) ⁇ 50 ⁇ 1 Xho I (8 13/ ⁇ 1 ; Takara Shuzo Co., Ltd.) 3 / 1 EcoRV (14 ⁇ 3/ ⁇ 1.; Takara Shuzo Co., Ltd.) 3/ 1 Buffer solution H (10-fold; Takara Shuzo Co., Ltd.)
- the isolated DNA was treated with RNase A at 37°C for 30 min in the following solution:
- the solution was subjected to SepaGene (Sanko Co., Ltd.) to extract DNA, and thereby GST-60 bp DNA (2038 bp) was prepared.
- the GST-60 bp DNA and GST-60 bp DNA digested with DNase I were subjected to agarose gel electrophoresis to confirm the preparation thereof as shown in Fig. 31.
- pGEX-6p-l/60 bp plasmid was subjected to PCR amplification by using Primer 3 (sense) and Primer 4 (antisense) in the following solution: pGEX-6p-l/60 bp (2ng//z 1) 1/ 1
- PCR buffer solution (10-fold) 5 1 (which consists of 500 mM KCl and 100 mM Tris-HCl
- the PCR was performed in the conditions of hot start 95°C - 12 min ⁇ 94 °C - 30 sec ⁇ 50 °C - 30 sec ⁇ 72 °C - 30 sec (45 cycles) ⁇ 4°C , to prepare GST-60 bp DNA as the PCR product.
- the GST-60 bp DNA (PCR product) was ligated into pGEM-T easy vector at 4 °C overnight to prepare subcloned pGEM-T easy/GST-60 bp in the following solution :
- the linear plasmid DNA was confirmed by 2 % agarose gel electrophoresis.
- the linear plasmid DNA was transcribed into RNA including GST-60 b at 37 °C for 3 hr in the following solution :
- transcribed sample was digested with DNase I at 37 °C for 30 min in the following solution: Transcribed sample 20 ⁇ 1
- the solution was subjected to SepaGene (Sanko Co., Ltd.) to extract RNA, and thereby GST-60 b RNA (970b) was prepared. No contamination with DNA thereof was confirmed by PCR amplification as same as the foregoing PCR amplification.
- the pGEX-6p-l/60 bp vector prepared in Example 22 was inserted into E. coli MV 1184 to transform thereof, and the transformant was subjected to sonification
- the GST-60 bp fusion protein was treated with RNase A at 37°C for 30 min in the following solution:
- GST-60 bp protein (1 / g/ / 1) 10 1 RNase A (10 / g// / l; Nippon Gene Co., Ltd.) 2 / 1
- the solution was heated at 80 °C for 5 min to inhibit DNase I in the solution, and thereby GST-60 bp protein was prepared. No contamination with DNA or RNA thereof was confirmed by PCR amplification or RT-PCR amplification.
- Fig. 32 shows preparation and confirmation of GST- 60 bp fusion protein from E. coli transformant lysates by SDS-PAGE. stained ' with coomassie brilliant blue (CBB).
- CBB coomassie brilliant blue
- GST-60 b RNA was reverse transcribed into DNA at 30 °C for 30 min in the following solution:
- the reacted fluid was subjected to SepaGene (Sanko Co., Ltd.) to extract nucleic acid, and DNA sample was dissolved in 20// 1 water.
- the DNA sample was subjected to PCR amplification by AmpliTaq ® PCR system (Perkin-Elmer Inc.) with primers 1 and 2 in the following solution:
- the PCR amplification was performed in the conditions of hot start 95°C - 12 min ⁇ 68 °C - 30 sec
- Fig. 33 shows reverse transcription from GST-60 b RN to GST-60 bp DNA in vitro.
- Bovine cal oduline (CaM; Wako Pure Chemical Co., Ltd. ) at l ⁇ g/ml 1 / 1 1.25- M ATP 1 ⁇ 1
- RNA sample was dissolved in 20/ 1 water.
- Enzyme mixture 1 1 (reverse transcriptase + DNA polymerase)
- the RT-PCR was performed in the conditions of 50 °C - 30 min ⁇ 94.2 °C - 2 in ⁇ 94 °C - 30 sec ⁇ 50 °C - 30 sec ⁇ 68 °C - 45 sec (10 cycles) ⁇ 94 °C - 30 sec ⁇ 50 °C - 30 sec ⁇ 68 °C - 45 sec (25 cycles) ⁇ 68 °C
- Fig. 34 shows transcription of GST-60 bp DNA to GST-60 b RNA with NTP.
- the GST-60 bp fusion protein prepared in Example 24 was treated with DNase I, and heated at 100°C and cooled, and then treated with RNase A and the RNase A was inhibited with RNasin (Promega Inc.) to prepare protein sample.
- the protein sample was converted to DNA by reverse direct expression at 30 °C for 30 min in the following solution:
- Retic Lysate ITV® Kit (Ambion Inc.) 20 ⁇ 1 (which contains reticulocyte lysate, calf liver tRNA, riboso e and amynoacyl-tRNA synthetase)
- the reacted fluid was subjected to SepaGene (Sanko Co., Ltd.) to extract nucleic acid, and DNA sample was dissolved in 20 1 water.
- AmpliTaq Gold ® 5 U/ / 1 ; Perkin-Elmer Inc.
- the PCR amplification was performed in the conditions of hot start 95°C - 12 min ⁇ 68 °C - 30 sec ⁇ 50 °C - 30 sec ⁇ 94 °C - 30 sec (45 cycles) ⁇ 4°C , and then the solution was subjected to 2 % agarose gel electrophoresis containing ethydium bromide at 0.5 // g/ ml, and as the result, 96 bp, 162 bp .and 225 bp DNA as PCR products were prepared.
- Fig. 35 shows reverse direct expression of GST-60 bp fusion protein to GST-60.
- bp DNA in vitro with NTP and reticulocyte lysate.
- the GST-60 bp dsDNA prepared was heated at 100°C for 5 min and cooled to prepare GST-60 b ssDNA.
- the DNA sample was converted to protein by direct expression at 37 °C for 120 min in the following solution:
- Retic Lysate ITV® Kit (A bion Inc.) 20 ⁇ 1 (which contains reticulocyte lysate, calf liver tRNA, riboso e and amynoacyl-tRNA synthetase) 0.13 M amino acid mixture (including 20 amino acids ) 20 // 1 and/or Bovin calmodulin (CaM; Wako pure Chemical Co.,
- GST-60 bp fusion protein was detected by western blot using anti-GST rabbit IgG (primary antibody) and enzyme-linked anti-rabbit IgG sheep serum (secondary antibody) .
- Fig. 36 shows direct expression of GST-60 bp DNA to GST-60 bp fusion protein in vitro with reticulocyte lysate and amino acids.
- Retic Lysate ITV® Kit (Ambion Inc.) 20 / 1 (which contains reticulocyte lysate, calf liver tRNA, ribosome and amynoacyl-tRNA synthetase) 1.25 mM ATP , 1 // I 1.25 mM CTP 1 ⁇ 1
- RNA sample was dissolved in 20// 1 water.
- RNA sample was subjected to RT-PCR amplification by Titan® One Tube RT-PCR System (Roche Inc.) with primers 1 and 2 in the following solution:
- Enzyme mixture 1 ⁇ 1 (reverse transcriptase + DNA polymerase)
- M2 DNA was subjected alkali denaturation to prepare ssDNA, transfered to membrane, and cross-linked to the membrane by UV-cross linker.
- the DNA was incubated with Retic Lysate ITV ® Kit (Ambion Inc.; containing reticulocyte lysate, calf liver tRNA, riboso e and aminoacyl-tRNA synthetase) and amino acid mixture.
- M2 protein on the membrane was prepared by preparing M2 protein on the membrane, and membrane strips thereof were overlaid on 3 % agarose gel plates containing salmon tests DNA (100 ⁇ g/ml) or calf liver RNA (100// g/ml),- and then incubated at 30 °C for 30 hr to characterize DNase and RNase activities of M2 protein produced from M2 DNA. The results of the test are shown in Fig. 41. Plaque on the gel represents positive for DNase and RNase activities .
- 1 shows strip of M2 DNA membrane without reaction put on DNA-agarose gel plate as a control
- 2 shows strip of M2 DNA membrane af er Eastern blot reaction put on DNA-agarose gel plate
- 3 shows strip of M2 DNA membrane without reaction put on RNA- agarose gel plate as a control
- 4 shows strip of M2 DNA membrane after Eastern blot reaction put on RNA- agarose gel plate.
- M2 protein is transfered to positively charged membrane, incubated in the reaction solution containing tRNA and 4 dNTP with bovine calmodulin to prepare dsDNA by reverse direct expression, and then subjected to PCR amplification to detect the product.
- Fig. 42 shows M2 DNA produced from M2 protein by the method of Eastern blot II and products as comparative examples.
- the present invention relates to a method of converting one of DNA, RNA and protein to any one of DNA, RNA and protein, and the following particular effects can be exhibited by the present invention: 1) any one of DNA, RNA and protein can be synthesized from one of DNA, RNA and protein by the above gene expression routes; 2) DNA, RNA or protein can be produced in vitro by the above conversion methods; 3) a reagent having effects of promoting the actions of the above gene expression routes including the following processes can be provided: reverse transcription from RNA to DNA, reverse translation from protein to RNA, reverse direct expression from protein to DNA, direct conversion (direct expression) from DNA to protein and replication of DNA; 4) a medicament for expressing an objective protein in vivo by administering one of DNA, RNA and. protein to human being or animal can be provided; and 5) a method of administering the medicament to human being or animal can be provided.
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Cited By (2)
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WO2002036150A1 (en) * | 2000-11-06 | 2002-05-10 | Umeda Jimusho Ltd. | Gene-digesting gene and enzyme having vaccine-like effect |
WO2003027286A1 (en) * | 2001-09-19 | 2003-04-03 | Japan Science And Technology Agency | Method of synthesizing polynucleotide via reverse translation from protein and oligonucleotide to be used in this method |
Citations (4)
Publication number | Priority date | Publication date | Assignee | Title |
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WO1986003780A1 (en) * | 1984-12-21 | 1986-07-03 | Techniclone Research Partners I | Method for electrically immortalizing lymphoid cells |
WO1990011092A1 (en) * | 1989-03-21 | 1990-10-04 | Vical, Inc. | Expression of exogenous polynucleotide sequences in a vertebrate |
US5437291A (en) * | 1993-08-26 | 1995-08-01 | Univ Johns Hopkins | Method for treating gastrointestinal muscle disorders and other smooth muscle dysfunction |
WO1998018489A1 (en) * | 1996-10-30 | 1998-05-07 | The Uab Research Foundation | Enhancement of tumor cell chemosensitivity and radiosensitivity using single chain intracellular antibodies |
-
2001
- 2001-01-09 WO PCT/JP2001/000048 patent/WO2001049839A2/en active Application Filing
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WO1986003780A1 (en) * | 1984-12-21 | 1986-07-03 | Techniclone Research Partners I | Method for electrically immortalizing lymphoid cells |
WO1990011092A1 (en) * | 1989-03-21 | 1990-10-04 | Vical, Inc. | Expression of exogenous polynucleotide sequences in a vertebrate |
US5437291A (en) * | 1993-08-26 | 1995-08-01 | Univ Johns Hopkins | Method for treating gastrointestinal muscle disorders and other smooth muscle dysfunction |
WO1998018489A1 (en) * | 1996-10-30 | 1998-05-07 | The Uab Research Foundation | Enhancement of tumor cell chemosensitivity and radiosensitivity using single chain intracellular antibodies |
Non-Patent Citations (2)
Title |
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CRAIG D ET AL: "Plasmid cDNA-directed protein synthesis in a coupled eukaryotic in vitro transcription-translation system" NUCLEIC ACIDS RESEARCH,GB,OXFORD UNIVERSITY PRESS, SURREY, vol. 20, no. 19, 1992, pages 4987-4995, XP002088195 ISSN: 0305-1048 * |
SONNEMANN JUERGEN ET AL: "Elongation in a Dictyostelium in vitro translation system is affected by calmodulin antagonists." FEBS (FEDERATION OF EUROPEAN BIOCHEMICAL SOCIETIES) LETTERS, vol. 329, no. 1-2, 1993, pages 183-188, XP001010426 ISSN: 0014-5793 * |
Cited By (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO2002036150A1 (en) * | 2000-11-06 | 2002-05-10 | Umeda Jimusho Ltd. | Gene-digesting gene and enzyme having vaccine-like effect |
WO2003027286A1 (en) * | 2001-09-19 | 2003-04-03 | Japan Science And Technology Agency | Method of synthesizing polynucleotide via reverse translation from protein and oligonucleotide to be used in this method |
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