WO2001049839A2 - Method for expressing gene and use thereof - Google Patents

Abstract

The present invention relates to a method of converting one of DNA, RNA and protein to any one of DNA, RNA and protein by the 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 rotues, and a medicament for expressing an objective protein in vivo in the body of human or animal.

Description

DESCRIPTION

Method for Expressing gene and Use Thereof

Techncal Field

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 .

Background Art

Since 1990 's, various results of researches on so- called gene vaccination have been reported. First of all, Wolff et al . published a paper entitled "Direct Gene Transfer into Mouse Muscle in vitro" in 1990, and it was reported therein for the first time that DNA expression vector was administered into a mouse by intramuscular injection, and thereby a protein encoded by the gene was expressed in muscular cells thereof, which continued for at least two months (1). This provided a clue to today's researches on gene vaccination.

As a basic principle of the gene vaccine, for example, 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.

That is, it is thought that marker molecules are expressed in the cells by employing a gene vaccine, thereby making it possible to selectively attack infected cells. As a result, since 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.

Following Wolff et al . , Johwston et al. published in 1992 that as a result of lodging DNA plasmid into an ear of a mouse by a gene gun, an antigen was expressed several weeks later at a probability of 30/34 (2). In addition, Robinson et al . published in 1993 that as a result of evaluating the vaccine effects of a DNA plasmid encoding a protein specific to an influenza virus from the viewpoint of a method of administration thereof, the evaluation order was intramuscular and intraperitoneal injection < topical administration to muscous membrane < lodging by a gene gun (3). Thereafter, Tang et al . published in 1997 that an antibody to a protein encoded by a DNA was expressed in mouse serum only by topical administration of the DNA vector onto the epidermis at a high ratio of 96 % (4). In connection with the mechanism of .this topical administration onto the epidermis, Scott et al. made it clear in 1997 that by tipical administration of a virus vector onto the epidermis of a depilated mouse, the gene is introduced into the epidermis tissue at a high efficiency ( 5 ) .

Moreover, ius et al . published general review about DNA vaccination in 1997 (6).

Recently, Debs et al . made it clear in 1999 that when an aqueous DNA plasmid solution was topically administered onto the epidermis of a mouse, a protein encoded by the DNA was expressed in a localized state in epidermis keratin cells and hair f.ollicles, and that the level was comparative to the case of the hypodermic injection of the same gene (7).

Subsequently, Khavari et al . made it clear for the first time in 1999 that when an aqueous DNA plasmid solution was topically administered onto the intact epidermis of a mouse, gene transfer occurs through hair follicles . ( 8 ) .

Moreover, Silva et al. made it clear for the first time in 1999 that as a result of the intramuscular injection of a plasmid DNA encoding a tuberculous bacilli protein to a mouse which was infected with tuberculous bacilli for 8 weeks four times at a two- week interval, the number of tuberculous bacilli in the lungs of the mouse began to decrease two months later, and decreased sharply five months later, and that thus, the DNA vaccine was effective for a remedy of tuberculosis of a mouse (9). Thus, results of researches on gene vaccines have been reported successively since 1990. However, though each of them employs a method of administering DNA or RNA to a body of human or animal through a gene expression vector as a basic principle, the influence of the expression vector upon the body has not fully been elucidated yet. Besides, it is pointed out that the influence of the expression vector upon the body becomes a problem; though it is thought that a method not employing the expression vector is preferred, no effective solution has^* been found yet.

On the other hand, for a gene expression method, since it was elucidated that the semi-conservation and semi-replication of a genome DNA is the body of heredity with a Watson-Click DNA double helix model (1953) as a leading theory, the irreversible route of a DNA-RNA ( RNA) -protein has been the central dogma of gene expression. And during this, the existence of a reverse transcriptase in a retrovirus was made clear, and at instant, the RNA-DNA reverse transcription seemed to destroy the central dogma. However, 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. Moreover, 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.

Subjects for the Invention to be Solved

Under these circumstances, 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.

The above, gene expression route means that not only irreversible gene expression based on the central dogma [DNA— RNA (mRNA) — protein] , but also reversible gene expression [protein = RNA (mRNA) =DNA] is present in vivo. As a more important matter, it is indicated that 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). It is suggested from the fact that 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. These facts signify that gene information can be stored in vivo in both directions in the gene expression reactions of DNA— RNA — protein, and DNA— protein reversibly, and that the optional gene expression can be caused therein.

It is suggested that 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. In view of the above, the present inventors have 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".

It is an objective of the present invention to provide a method of converting one of DNA, RNA and protein to any one of corresponding DNA, RNA or protein, for example, in vitro, by this new gene expression route.

Further, it is another objective of the present invention to provide a method of producing DNA, RNA or protein by the above gene expression route.

It is still another objective of the present invention to provide a reagent having an action of promoting a reaction of the above gene expression route. It is still another objective of the present invention to provide a medicament for expressing an objective protein in vivo by an administration thereof to a human or animal body.

It is still another objective of the present invention to provide a method of expressing an objective protein in vivo by administering the above medicament to a human or animal body.

Means for Solving the Subjects

The present invention for solving the above subjects comprises the following technical means.

(1) 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 ) ,

3) conversion from a protein to a DNA (reverse direct expression ) , or

4) direct conversion from DNA to a protein (direct expression ) .

(2) A method of converting one of DNA, RNA ^"and protein to any one of corresponding DNA, RNA or protein according to the following reactions containing calmodulin or a substance having a calmodulin-like function (referred to as CaM):

1) conversion from an RNA to a DNA (reverse transcription) ,

2) conversion from a protein to an RNA (reverse translation) , 3) conversion from a protein to a DNA (reverse direct expression ) ,

4) direct conversion from a DNA to a protein (direct expression ) , 5) replication of a. DNA

6) transcription from a DNA to an RNA, or

7) translation from an RNA to a protein.

(3) A method according to above (1) or (2), wherein an mRNA or an RNA is converted to a DNA by reverse transcription.

(4) A- method according to above (1) or (2), wherein a protein is converted to an RNA by reverse translation.

(5) A method according to above (1) or (2), wherein a protein is directly converted to a DNA by reverse expression.

(6) A method according to above (1) or (2), wherein a DNA is directly converted to a protein by direct expression .

(7) A method of producing a DNA, an RNA or a protein, wherein a DNA, an RNA or a protein is produced by a' method according to any one of above (1) to (6)

(8) 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).

(9) 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.. (10) 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.

(11) A reagent according to above (8), which has an action of promoting a reaction of directly reverse- expressing a protein to a DNA.

(12) A reagent according to above (8), which has an action of promoting a reaction of directly expressing a DNA to a protein. (13) A reagent according to above (8), which has an action of promoting a reaction of replicating a DNA.

(14) 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.

(15) A medicament according to above (14), which is a medicament for expressing an objective antibody in vivo.

(16) 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.^"

(17) 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.

(18) 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. (19) 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.

(20) 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.

(21) A method of producing RNA from DNA by transcription, which comprises incubating the DNA in the solution containing calmodulin, NTP to prepare the corresponding RNA, and then isolating the RNA from the solution.

(22) A method of producing RNA from protein by reverse translation, which comprises incubating the protein in the solution containing calmodulin, tRNA and NTP to prepare the RNA, and then isolating the RNA from the solution.

(23) 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

3) isolating the DNA from the solution.

(24) 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., and

1) extracting the protein to characterize profiles thereof, and/or

2) overlaying the product on a gel containing substrate or a membrane containing chemical reagent, and incubating thereof to characterize biological and chemical activity of the protein.

(25) 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 . (26) 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.

Embodiments for Performing the Invention

Next, the present invention will be described in more detail.

A new gene expression route found by the present inventors is shown in Fig. 1 in contrast with a conventionally known gene expression route. In the drawing, the new gene expression route is shown in the upper part, and the conventional gene expression route is shown in the lower part.

As shown in Fig. 1, the new gene expression route comprises reversible expression systems of non- chromosomal genes.

In the drawing, DNA (genomic, genome type) shows a genomic DNA, DNA (genetic, gene type) shows a non- genomic DNA, and CaM shows calmodulin or substances having the properties as same as that of calmodulin or calmodulin-like function. In the present invention,- CaM is defined as to mean substances containing calmodulin and substances having the above calmodulin-like function.

As shown in Fig. 1, 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.. In addition, the present inventors have confirmed that each reaction of the gene expression route is promoted by the presence of CaM.

Hence, the use of calmodulin or substances having a calmodulin-like function is preferable and important in the present invention. In a reaction system containing CaM, for example, a degree of dissociation, 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. In 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+. S g ^• of calmodulin and/or 0.1 M of AMP, ADP and ATP were subjected to a chromatograph according to a high-pressure liquid chromatography (HPLC, 6 kg/cm2 and 0.5 ml/min) to detect at A280 nm. The calmodulin was dissociated to a tetramer (65 Da), a dimer (30 KDa) and a monomer (15 KDa).

In the present invention, CaM (calmodulin and substances having the calmodulin-like function) is defined that 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. Moreover, such substances having the calmodulin-like function and an action of promoting the reactions of the above gene expression systems can be employed similarly.

Here, 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. In the present invention, such substances are generally referred to as CaM.

In 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. In this case, 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. Hence, 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. Thus, according to the gene expression route 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.

Next, with a view to more simply explaining the present invention, the case of a specific gene and a specific protein (M2 gene, human p53 gene, and proteins coded by these genes) will be described as an example.

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). As a result of reacting an mRNA sample prepared from the M2 gene in a reaction system in vitro containing CaM, it was revealed that a double-stranded M2 DNA was formed from the mRNA by reverse transcription. Moreover, as a result of reacting the human p53 gene (human tumor suppressor gene) as a sample in a reaction system in vitro containing CaM, it was revealed that the p53 DNA was replicated. In addition, as a result of reacting the human p53 gene as an RNA sample in a reaction system in vitro containing CaM, it was revealed that the p53 DNA was formed from the p53 RNA by reverse transcription. Similarly, the transcription process from the p53 DNA to the p53 RNA was confirmed.

Further, it was revealed that the p53 DNA was directly converted to a p53 protein (direct expression). Moreover, as a result of reacting the p53 protein (or M2 protein) in a reaction system in vitro containing CaM, it was revealed that the p53 protein (or M2 protein) was directly converted to a p53 DNA (or M2 DNA) (reverse direct expression).

As a result of comparing the base sequence of the 53 DNA (or M2 DNA) reversely expressed from the p53 protein (or M2 protein) with the base sequence of the human p53 DNA (or M2 DNA), it. was confirmed that both were almost identical, though not completely identical. This shows that there is little mutation in the DNAs synthesized by reverse expression. Conventionally, a promoter has been needed for gene expression; however, no promoter is needed in the above method and reverse expression can be correctly realized, and hence, this method is useful as a new method of synthesizing DNA.

Since the gene expression route of the present invention allows the reactions to be promoted in a reaction system containing CaM, the CaM is useful as a reaction-promoting reagent having an action of promoting the reactions. As preferable 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. In this case, proper subsidiary components can be incorporated in addition to these components. Moreover, 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. . *

Since 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.

As shown in examples to be described later, as a result of topical administering a human p53 DNA (human tumor^" suppressor gene) to a mouse to examine its carcinogenesis inhibitory effect, it was revealed that a high tumor suppressor effect can_.be obtained. Thus, the fact that, by the topical or oral administration of a true naked DNA, RNA or protein, a corresponding gene is expressed in vivo and a corresponding protein is formed is very important as a proof supporting the existence of a new gene expression route. Next, the usefulness of the present invention will further be described.

This method can be utilized for the expression of a useful gene in vivo. By the topical, oral administration or injection of DNA (cDNA) and RNA (mRNA) of genes which are thought to be useful, proteins encoded by the genes can be formed in vivo and their effect can be imparted to human or animal body. . Similarly in culture cells, proteins and genes can be formed. In these cases, the effect can further be improved by adding CaM together with the genes. In the method of the present invention, 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.

Moreover, this method can be utilized for synthesizing a gene from a specific protein and obtaining thereof.

At present, in order to determine the sequence from a protein whose gene DNA sequence is unknown, alternative methods are adopted of detecting an mRNA to make cDNA by a reverse transcription or synthesizing a cDNA from an amino acid sequence of a protein. By employing a reverse expression system according to the present invention, 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.

There exists on the upper and the lower of a gene ORF a part controlling and adjusting the expression of the gene. By administering this control sequence DNA to human or animal body by topical administration to the skin or oral administration can be controlled disadvantageous gene expression and excessively expressed genes in vivo. In this case, the expression of the control DNA (effect) can be controlled by administering CaM additionally. This method is useful, for example, for the control of aging genes, the control of mast genes and the control of neurotransmitters .

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. In this case, calmodulin or a substance having a calmodulin-like function is used as CaM. They are used as basic compounds, and other subsidiary components can properly be combined therewith. The dNTP, NTP, and amino acid mixture may properly be designed according to an objective DNA, RNA or protein.

As 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. Moreover, by the addition of CaM can its effect further be heightened. In this case, 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.

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.

In Fig. 40, preferably, 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. Preferably, 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. In this case, 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.

In Fig. 40, preferably, 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.

As a preferable example, 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.

In the reverse transcription route of this invention, DNA is produced from RNA by incubating the

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. Next, in the reverse direct expression route of this invention, 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.

Next, in the reverse translation route of this invention, RNA is produced from protein by incubating the protein in the solution containing calmodulin, tRNA and NTP.

In these route, 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.

In the routes,, reaction conditions such as temperature, time, solution and the like are determined properly in accordance with the conditions as described in the subsequent Examples.

In this invention, since 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.

Brief Description of the Drawings

Fig . 1

Fig. 1 shows a diagram showing general concepts of new gene expression routes (above) and conventional gene expression routes (below). ' Fig., 2

Fig. 2 shows a diagram of the reverse transcription of M2 mRNA with CaM. Fig. 3 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

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

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

Fig. 7 shows a diagram of the reverse direct expression of human p53 protein and M2 recombinant protein with CaM. Fig. 8

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

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

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 ^'

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

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

Fig. 14 shows the relationship between the human p53 DNA and RNA topically administrered and its antitumor effect. Fig. 15

Fig. 15 hows an electrophoresis photograph of M2 DNA formed by the reverse direct expression of the M2 protein/CaM topically administered. Fig. 16

Fig. 16 shows an electrophoresis photograph of M2 DNA formed by the replication of the M2 DNA administered (intraperitoneal injection/topical administration) . Fig. 17

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

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

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

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

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

Fig. 23 shows a diagram of the sequence determination of M2 DNAs obtained by the DNA replication, reverse transcription, reverse direct expression and translation. ^Fig- 24

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

Fig. 25 shows the relationship between the molecular weight of calmodulin and retention time. Fig. 26

Fig. 26 shows a diagram showing the concept of the active mechanism of CaM. Fig. 27

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

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

Fig. 29 shows schematic of the method of reverse transcription and translation in vitro of GST-60 b RNA. Fig. 30

Fig. 30 shows isolation of GST-60 bp DNA from pGEX- 6p-l/60 bp by restriction enzymes EcoRV and Xho I. Fig. 31

Fig. 31 shows preparation and confirmation of GST- 60 bp DNA by digestion with DNAase I. Fig. 32

Fig. 32 shows preparation and confirmation of GST- 60 bp fusion protein from E. coli transformant lysates. Fig. 33 Fig. 33 shows reverse transcription from GST-60 b RNA to GST-60 bp DNA in vitro by Calumodulin. Fig. 34

Fig. 34 shows translation from GST-60 bp DNA to GST-60 b RNA by CaM with NTP. Fig. 35

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 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

Fig. 37 shows reverse translation from GST-60 bp protein to GST-60 b RNA by CaM. Fig. 38

Fig. 38 shows original 60 bp DNA used to produce recombinant GST fusion protein and clones, sequenced. Fig. 39

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 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 ^•

Fig. 42 shows M2 DNA produced from M2 protein by the method of Eastern blot II. Fig. 43

Fig. 43 shows comparison of reverse transcription activity between bovine brain CaM and human brain CaM using GST-60 bp RNA.

Examples

Next, the present invention will be described specifically according to examples. These examples are for describing the present invention in accordance with specific examples so that those skilled in the art could carry out the present invention and not for restricting the scope of the present invention. Example 1 In this example, double-stranded DNA was synthesized from mRNA by reverse transcription in vitro (see Fig. ^•2).

(1) Preparation of M2 mRNA

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).

(2) Reverse Transcription of M2 mRNA

According to the following reaction system, 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 μ 1 PCR buffer (10-fold concentration) , 2

25 mM MgCl₂ 2 β 1

Poly T primer (20 //M) 2 1

0.1 M DTT 2 1

10 mM dNTT mixture 4 /; 1 Bovine CaM (1 mg/ml) 1 /; 1

5 mM Ca(H₂P0₄ )₂ 1 β 1

Anti-CaM rabbit IgG 1 1

DEPC-water -4-5 β 1 Total 20// 1

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.).

(3) PCR

Next, 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.

DNA sample (M2 DNA) ^• 1 1

25 mM MgCl₂ 2 1

Upper and lower primers 1 β 1 and 1 1 PCR buffer (10-fold concentration) 5 1

2 mM dNTP mixture . 5 β 1

AmpliTaq Gold ® (Perkin-Elmer Inc.) 0.5 1 (DNA polymerase, 5ϋ/ β 1)

Water 34.5 1 Total 50 β 1

According to the above was performed 45-cycles of the PCR. (4) Detection of DNA

Subsequently, the reacted fluid was subjected to ethidium bromide-agarose gel electrophoresis, and gel plate thereof was photographed under UV irradiation to detect M2 DNA. The results are shown in Fig. 3. In Fig. 3 are shown 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.

(5) Determination of the Sequence

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) .

Example 2 (1)

In the following examples 2 (1) to (3), replication of DNA, transcription of DNA, and synthesis of DNA from RN by reverse transcription were performed in vitro, respectively (see Figs. 4-5). (1) Preparation of DNAs

Employing M2 DNA and human p53 DNA, these DNAs were replicated 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.

DNA sample (M2 DNA or human p53 DNA) 1 /_/ 1 Upper and lower primers (20 β 1 ) 1/.1 and 1 1 PCR buffer (10-fold concentration) 2 β 1

25 mM MgCl₂ 2 μ 1

5 mM NTP mixture 4 β 1

Bovine CaM (1 mg/ml) 1 β 1 5 mM Ca(H₂P0₄ )₂ 1 β 1

Anti-CaM rabbit igG 1 β 1

Water 6-7 β 1

Total 20 /.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. ) .

(2) PCR

Next, 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.

DNA sample (M2 DNA or p53 DNA) 1 β I 25 M MgC12 2 # 1

Upper and lower primers (20 M) l β 1 and 1 / 1

PCR buffer (10-fold concentration) 5 β 1

2 M dNTP mixture 5 μ 1

AmpliTaq Gold ® (Perkin-Elmer Inc.) 0.5 1 ( DNA polymerase , 5U/ 1 )

Water 34 . 5 β 1

Total 50 1

According to the above was performed 45-cycles of the PCR.

(3) Detection of DNAs

In the same manner as in Example 1, DNAs were detected . The results are shown in Fig. 6. In Fig. 6 are shown results of the cases of 1) p53 DNA, 2) p53 DNA + CaM + anti-CaM antibody, 3) CaM, and 4) p53 DNA + CaM.

As shown in Fig. 6, the replication of the p53 DNA from the p53 DNA and the p53 DNA/CaM was confirmed. (5) Determination of the Sequence

In the same manner as in Example 1, sequence thereof was determined (see Example 17).

Example 2 (2) (1) Transcription of DNAs

Employing M2 DNA and p53 DNA, 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.

DNA sample (M2 DNA or p53 DNA) l β 1

Upper and lower primers (20 β M) l β 1 and 1 /z 1 PCR buffer (10-fold concentration) 2 μ 1

25 mM MgCl₂ 2 β 1 5 mM NTP mixture 4 β 1

Bovine CaM (1 mg/ l) 1 ; 1

5 mM Ca(H₂P0₄ )₂ 1 β 1

Anti-CaM rabbit IgG 1 1 Water 6-7 β 1

Total 20 β 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.).

(2) Reverse Transcription (RT)-PCR

Next, the formed M2 DNA or p53 DNA was reverse- transcribed (RT), and then subjected to PCR. The reaction system of the PCR is shown below.

Sample (M2 RNA or p53 DNA) 1 ju 1

25 mM MgCl₂ 2 1

Upper and lower primers (20 M) 1 1 and 1 /ι 1 PCR buffer (10-fold concentration). 5 1 2 mM dNTP mixture 5 1

Superscript ® (Lifetec Oriental) 1 // 1

(RNA-dependent DNA polymerase)

DEPC-water 33.5 β 1

Total 50 1 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.

(3) Detection of DNAs The formed RNAs were detected as DNAs- by RT-PCR. In the same manner as in Example 1, DNAs were detected.

The results are shown in Fig. 6. In Fig. 6 are shown 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

In the same manner as in Example 1, sequence thereof was determined (see Example 17).

Example 2 (3)

(1) Reverse Transcription of RNAs

Employing M2 RNA and p53 RNA, DNAs 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 ι 1 Upper and lower primers (20 M) 1 j 1 and 1 i 1 PCR buffer (10-fold concentration) 2 / 1

25 mM MgCl₂ 2 1

0.1 M DTT 2 # 1

10 mM dNTP mixture 4 β 1 Bovine CaM (1 mg/ml) 1 1

5 mM Ca(H₂P0₄ )₂ 1 β 1

Anti-CaM rabbit IgG 1 1

DEPC-water 6-7 1

Total 20 β 1 The reaction solution was put into a microtube, and incubated at 37 °C for 60 minutes, and then DNAs were extracted by SepaGene ® Kit (manufactured by Sanko Co., Ltd. ) .

(2) PCR

Next, the DNAs formed from the RNAs by reverse transcription were amplified by a PCR.

The reaction system of the PCR is shown below. Sample (p53 DNA) 1 β 1

25 mM MgCl₂ 2 μ 1

Upper and lower primers (20 β M) l β 1 and 1 1

PCR buffer (10-fold concentration) 5 μ 1

2 mM dNTP mixture 5 1 A pliTaq Gold ® ( Perkin-Elmer Inc.) 0.5 1 (DNA polymerase, 5U/ β 1)

Water 34.5 β 1 ^■

Total 50 β 1 According to the above was performed 45-cycles of the PCR.

(3) Detection of DNAs

In the same manner as in Example 1, DNAs were detected . The results are shown in Fig. 6. In Fig. 6 are shown results of the cases of 1) p53 RNA + CaM + anti- CaM antibody, 2) p53 RNA, and 3) p53 RNA + CaM. As shown in Fig. 6, it was confirmed that DNAs were formed from the p53 RNA/CaM by reverse transcription. (4) Determination of the Sequence

In the same manner as in Example 1, sequence thereof was determined (see Example 17).

Example 3

In this example, DNAs were synthesized from proteins by reverse direct expression in vitro (see Fig. 7). (1) Reverse Direct Expression of Proteins

^"Employing M2 protein and p53 protein, DNAs were synthesized from these proteins by reverse direct expression according to the following reaction system. Volume of nuclease-free water to be added was properly adjusted according to a case of adding CaM, an anti-CaM antibody and a case of adding neither. Protein sample 1 1

(M2 protein or p53 protein (1 mg/ml)) Bovine CaM (1 mg/ml) 1 β 1

100 mM Ca(H₂ P0₄ )₂ 1 1

Anti-CaM rabbit IgG 1 β 1 Mixture (in vitro translation kit (manufactured by Ambion, Inc.)) 20 β 1

( tRNA-containing erythrocyte lysates: 16.7 # 1 master mixture (amino acid mixture): 1.3 β l Nuclease-free water: 2-3 β 1) Total 25 u 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.). ( 2 ) PCR

Next, 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.

Sample DNA (M2 DNA or p53 DNA) 1 β 1

25 mM MgCl₂ 2 1

Upper and lower primers (20 M) l β 1 and 1 1 each 1 β 1

PCR buffer (10-fold concentration) 5 β 1

2 M dNTP mixture 5 ^ 1

AmpliTaq Gold ® ( Perkin-Elmer Inc.) 0.5 β 1 (DNA poly erase, 5U/ β 1) Water 34.5 1

Total 50 β 1

According to the above was performed 45-cycles of the PCR.

(3) Detection of DNAs

In the same manner as in Example 1, DNAs were detected ..

The results are shown in Fig. 8. In 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. As shown in Fig. 8, it was confirmed ^"that M2' DNAs were formed from the M2 protein by reverse direct expression, ( 4 ) Determination of the Sequence in tne same manner as in Example 1, sequence thereof was determined (see Example 17).

Example 4 Direct Expression of DNAs to Proteins in vitro (1) Method

According to the following reaction system, 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.

DNA sample (M2 DNA or p53 DNA (20 β g/ml) 2 β 1 Bovine CaM (1 mg/ml) 1 / 1 100 mM Ca(H₂P0₄ )₂ 1 1

Anti-CaM rabbit IgG 1 μ 1

Mixture (in vitro translation kit) ( RNA-containing erythrocyte lysates: 16.7 1 master mixture: 1.3μ 1 Nuclease-free water: 1-2 1)

Total 25 β 1

The 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) .

That is, employing 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.

(2) Results

The results are shown in Table 1. As is apparent from the table, it was revealed that M2 protein was formed from the M2 DNA, and human p53 protein was formed from the human p53 DNA, and that the formation thereof was inhibited by the DNA + anti-CaM antibody.

to ISO

CD on o n

Table Direct expression from DNA to protein by CaM

M2 protein produced Human p53 protein produced

Gene DNA used (ng/ml) (ng/ml)

None ND ND

M2 DNA 11 +3.5^* ND

M2 DNA + anti-CaM 0.7 ±0.6 ND

Human p53 DNA ND 2.4+1.1

Human p53 DNA + anti-CaM ND ND

^*Mean i standard deviation of triplicates.

Example 5

Reverse Translation of Protein to RNA in vitro

(1) Method

According to the following reaction system, RNA was synthesized from M2 protein by reverse translation.

M2 protein (1 mg/ml) 1 β 1

Bovine CaM (1 mg/ml) 1 β 1

100 mM Ca(H₂ P0₄ )₂ 1 β 1

10 mM NTP mixture 1 β 1 Anti-CaM rabbit IgG 1 β 1

Mixture (in vitro translation kit) (tRNA-containing erythrocyte lysates: 16.7 1 master mixture: 1.3 β 1 Nuclease-free water: 2 β 1 ) Total 25 / 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.).

(2) Reverse Transciption (RT)-PCR

Next, the formed RNA was reverse-transcribed to DNA by reverse transcription (RT)-PCR according to the following reaction system, and the DNA was amplified, and then detected.

Sample RNA (M2 RNA) 1 β 1

25 mM MgCl₂ 2 β 1

Upper and lower primers (20 β M ) l β 1 and 1 β 1 PCR buffer (10-fold concentration) 5 β 1 2 mM dNTP mixture 5 β 1

Superscript ® 1 β 1 (RNA-denpendent DNA poly erase)

AmpliTaq Gold ® ( Perkin-Elmer Inc.) 0.5 1 (DNA polymerase, 5U/ β 1)

DEPC-water 34.5 β 1

Total 50 β 1

(3) Detection of DNA In the same manner as in Example 1, DNA was detected by ethidium bromide-2 % agarose gel electrophoresis. The results are shown in Fig. 9. In Fig. 9 are shown results of the cases of 1) M2 protein, and 2) M2 protein + anti-CaM antibody. As shown in Fig. 9, it was confirmed that M2 DNA was formed from the M2 protein.

Example 6

Reverse Translation from Protein to RNA in vitro

In the same manner as in Example 5, 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.

It was revealed that the p53 DNA was detected in the case of the p53 protein + CaM, but not ditected in the case of the p53 protein + CaM + anti-CaM antibody.

Example 7

Reverse Direct Expression from Protein to DNA in vitro In the same manner as in Example 3, p53 DNA was synthesized from p53 protein by reverse direct expression in vitro. The results are shown in Fig. 11.

The p53 DNA was detected in the case of the p53 protein + CaM ( l β g or 0.2 g).

Example 8

In this example, p53 DNA (human tumor suppressor gene) was administered to mice and its carcinogenesis suppressor effect (antitumor activity) was tested. One week before. i.m. injection of living Balb/c 3T/12-3 cells (tumor cells) to Balb/c mice including three males and three females at a rate of 10⁵ cells/mouse, human p53 DNA (5 g/mouse) and bovine CaM ( 5 β g/mouse). were topically administered to them. Fig. 12 shows the relationship between the topical administration of the human p53 DNA/CaM and the antitumor activity.

The cumulative mortality (p53 DNA:Q , p53 DNA + CaM: Δ ) 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.

As a result, for the cumulative mortality, Δ (p53 DNA + CaM) was 1/6 of that of the controls, and for the diameter of the formed solid tumor tissue, A (p53 DNA + CaM) was 1/6 of that of the controls; thus the rate of formation thereof was small as compared with those of the controls..

Example 9 In this example, p53 DNA and an anti-CaM antibody were topically administered to mice and their antitumor activity was tested.

One week before i.m. injection of living Balb/c 3T/12-3 cells (tumor cells) to Balb/c mice including three males and three females at a rate of 10⁵ 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 :Δ ) 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 (φ ) of solid tumor tissue formed were measured.

As a result, 8 weeks after the injection of tumor cells, for the cumulative mortality, Δ (DNA + anti- CaM) was 4/6 of that of the controls, □ (DNA) was 2/6 of that of the controls, and for the diameter of the formed solid tumor tissue, A (DNA + anti-CaM) was 4/6 of that of the controls and ■ (DNA) w.as 2/6 of that of the controls; thus the rates of formation thereof for both A and ■ were rather small as compared with those of the controls.

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.

Example 10

Antitumor Activity by the topical Administration of DNA or RNA

The human p53 DNA and RNA were topically administered to mice, and their antitumor activity was examined.

One week before i.m. injection of living Balb/c 3T/12-3 cells (tumor cells) to Balb/c mice including three males and three females at a rate of 10⁵ 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.

The cumulative mortality (D AiQ , RNA: Δ ) 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 (φ ) of solid tumor tissue formed were measured. As a result, for the cumulative mortality, □ (DNA) was 2/6 of that of the controls andΔ (RNA) was 0/6 of that of the controls, and for the diameter of the formed solid tumor tissue, ■ (DNA) was 2/6 of that of the controls andA (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 .

Example 11

Reverse Direct Expression from Protein to DNA in vivo

The reverse direct expression from M2 protein to M2 DNA in Balb/c mice topically administered with the M2 protein was examined.

The M2 protein (10/ g/mouse) and the M2 protein (10 β 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

Manheim). The results of determining the number of DNA copies under laser irradiation while amplifying the M2 DNAs in mouse tissues (brain, liver, lymphocyte) by cycle PCR according to cyber green are shown in Table 2. Moreover, a photograph of the electrophoresis showing the results of the detection of M2 DNAs according to an ordinary PCR is shown in Fig. 15.

It has been found from' these results that DNAs were formed from the protein + CaM by reverse direct expression .

(SJ

I able bπect ot <ϋaM on reverse expression trom Mi. protein to M2 DNA in topically administered balb/c mice. M2 DNA was detected by thermal cycler PCR amplification with cyber-green.

M2 DNA detected from mice by quantative PCR after 2 weeks

Topical administration Brain Liver Lymphocytes

(copies/mg) (copies/mg) (copies/10⁵ cells)

M2 protein (10 μg/mouse) 180 267 19.2

M2 protein (10 μg/mouse) 3185 1288 451

+ bovine CaM (5 μg/mouse)

Exampl e 12

Replication of DNA in vivo Compared by the

Administration Routes

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.

It has been found from these results that topical and oral administrations are more effective as administration routes.

to

CJ1 O cn cπ

t

Table Comparison of administration routes on in vivo replication of M2 oo DNA in ballb/c mice. M2 DNA detection was carried out by thermal cycler PCR with cyber-green.

M2 DNA detection from mice by quantitative PCR after 2 weeks

Route Dose/mouse Brain Liver Lymphocytes (copies/mg) (copies/mg) (copies/10⁵ cells)

Intraperitoneal 5 μg 105 8.3 4.4 injection Oral administration 5 g 854 .333 6.7

Example 13

Effect of CaM in Topical Administration

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.

It has been found that the DNA + CaM is effective for the formation of DNAs. On the other hand, the IHN virus M2 mRNA (1 // g/mouse) and bovine CaM ( 5 β g/ ouse) 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. 18.

It has been found that the 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.

Example 14

Reverse Transcription from RNA to DNA in vivo

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.

Example 15

Replication of DNA in vivo

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.

Example 16

Reverse Direct Expression from Protein to DNA in vivo

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.

Example 17

Determination of the Sequences of the Obtained Gene DNAs

In the present example, the sequences of the IHN virus M2 DNA and the human p53 DNA were determined. (1) Determination of the Sequence of IHN Virus M2 Gene ^■ CDNA

As shown in 22, 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.

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, and 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.

In the drawing, for example, ( ) shows the mutation of amino acid. As is apparent from Fig. 23, little mutation is found as compared with the original M2 gene DNA. 0

(2) Determination of the Sequence of Human p53 Gene DNA

As shown in Fig. 24, 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.

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, and Clone 6 shows p53 DNA obtained from a Balb/c mouse one month after topical administration of the p53 RNA in vivo, respectively.

In the drawing-, for example, ( ) shows the mutation of amino acid. As is apparent from Fig, 24, little mutation is found as compared with the original human p53 gene DNA.

(3) It has been found from the above (1) and ( 2 ) that reverse transcription and reverse direct expression are correctly performed by the DNA expression systems employing CaM according to the present invention, and that DNAs can be synthesized by these DNA expression systems.

Since these DNA expression systems need no promoter, and therefore, these are very useful as new ^■ DNA synthesizing systems.

Example 18

Reagent kits for promoting the following reactions which contain the following components were prepared. (D CaM, Ca(H₂P0₄)₂, dNTP (for reverse transcription)

(2) CaM, Ca(H₂P0₄)₂, tRNA, NTP (for reverse translation)

(3) CaM, Ca(H₂P0₄)₂, tRNA, dNTP (for reverse direct expression) (4) CaM, Ca(H₂P0₄)₂, tRNA, amino acid mixture (for direct expression)

(5) CaM, Ca(H₂P0₄)₂, dNTP (for replication of DNA)

(6) CaM, Ca(H₂P0₄)₂, NTP (for transcription) (7) CaM, Ca(H₂P0₄)₂, tRNA, amino acid mixture (for translation)

Example 19

A medicinal composition containing a protein and CaM was prepared from the following arrangement.

Protein (1 mg/ml) 1 // 1

CaM (1 mg/ml) 1 // 1

100 mM Ca(H₂ P0₄ )₂ 1 β 1

Example 20

A medicinal composition containing RNA and CaM was prepared from the following arrangement.

RNA 1 β 1

CaM (1 mg/ml) 1 / 1 100 mM Ca(H₂P0₄ )₂ 1 ^ 1

Example 21

A medicinal composition containing DNA and CaM was prepared from the following arrangement. DNA 1 β 1

CaM (1 mg/ml) 1 1

100 mM Ca(H₂P0₄ )₂ 1 // 1

Example 22 (Preparation of. GST-60 bp DNA)

(-1) isolation of PUC 118/60 bp plasmid

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.).

The sequence of the 60 bp oligo DNA chemically synthesized by the present inventors is shown in Fig. 28

(2) Digestion of PUC 118/60 bp plasmid with EcoRI

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 β 1

EcoRI (12 U/ 1 } Takara Shuzo Co., Ltd.) 2 / 1 Buffer solution H (10-fold; Takara Shuzo Co., Ltd.)

1 β 1 (which consists of 500 mM Tris-HCl (pH 7.5), 100 M MgCl₂ , 10 mM Dithiothreitol and 1000 mM NaCl)

Total 10 1

(3) Subcloning into pGEX-6p-l plasmid

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:

60 bp DNA (10 ng// 1) 7// 1 pGEX-6p-l (20 ng/ 1; Amersham Pharmacia Biotech Co. ) ' 1^' β 1 Ligation enzyme solution (DNA Ligation Kit Ver. 1; Takara Shuzo Co., Ltd.) 8 / 1

Total 16 β 1

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.).

(4) Double digestion with EcoRV and Xho I

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/ β 1) ^■ 50 β 1 Xho I (8 13/ β 1 ; Takara Shuzo Co., Ltd.) 3 / 1 EcoRV (14 \3/ β 1.; Takara Shuzo Co., Ltd.) 3/ 1 Buffer solution H (10-fold; Takara Shuzo Co., Ltd.)

7 β 1 (which consists of 500. mM Tris-HCl (pH 7.5), 100 mM MgCl₂ , 10 mM Dithiothreitol and 1000 mM NaCl)

Pure water 7 / 1

Total 70 1

The digested sample was subjected to agarose gel electrophoresis to prepare linear DNA including GST-60 bp DNA (Fig. 30 shows agarose gel electrophoresis of GST-60 bp DNA from pGEX-6p-l/60 bp by restriction enzymes EcoRV and Xho I), and then the DNA was isolated from the gel by Centrifuga filter Microcori YM-3® (Millipore Co. ) .

( 5 ) Treatment with RNase A

The isolated DNA was treated with RNase A at 37°C for 30 min in the following solution:

Extracted GST-60 bp DNA (2 ng// 1) 10 / 1

RNase A (10 g/ β 1 ; Nippon Gene Co., Ltd.) 2 β 1

100 mM Tris-HCl (pH 8.0) 2 β 1

Nuclease-free water 6 // 1 Total 20 β 1

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.

Example 23 (Preparation of GST-60 b RNA)

(1) PCR amplification of GST-60 bp DNA

The subcloned 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

Primers 3 and 4 at 20 β M 1 + 1 / 1

2 mM dNTP 5 1

25 mM MgCl₂ 3 // 1

PCR buffer solution (10-fold) 5 1 (which consists of 500 mM KCl and 100 mM Tris-HCl

(PH 8.3)) A pli Taq Gold® at 5 ύ/ β 1 (PE Biosystems Inc.)

0.5 β 1 Pure Water 33.5 β 1

Total 50 β 1

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.

Primer 3 ( sense) :

5 ' -AACAGTATTCATGCTCCCTAT-3 ' Primer 4 (antisense):

5 ' -AGAGGTTTTCACCGTCATCAC-3 '

(2) Subcloning into pGEM-T easy plasmid

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 :

GST-60 bp DNA (10 ng/ /_/ 1) 7 β 1 pGEM-T easy (50 ng/ // 1; Promega Co.) 1 // 1 T4 DNA ligase (3 Weiss U/ / 1; Promega Co.) 1// 1 T4 DNA ligase buffer solution (10-fold) 1 1 (which consists of 300 mM Tris-HCl (pH 7.8), 100 mM MgCl₂ , 10 mM Dithiothreitol and 1000 mM NaCl)

Total 10 β 1 The subcloned pGEM-T easy/GST-60 bp DNA was inserted into E. coli MV 1184 to transform thereof, and the clone including GST-60 bp DNA was selected by DNA sequencing with SP6 and T7 primers, and then the plasmid DNA was isolated by FlexPrep® Kit (Amersham Pharmacia Biotech Co. )

(3) Digestion of the selected clone with Sph I The selected clone DNA was digested with Sph I at 37 °C overnight to prepare liner plasmid DNA in the following solution:

Selected clone plasmid DNA (10 ng/// 1) 25 / 1 Sph I (12 13/ 1; Takara Shuzo Co., Ltd.) 2 /_/ 1 Buffer solution H (10-fold; Takara Shuzo Co., Ltd.)

3 β 1 (which consists of 500 mM Tris-HCl (pH 7.5), 100 mM MgCl₂ , 10 mM Dithiothreitol and 1000 mM, NaCl)

Total 30 β 1

The linear plasmid DNA was confirmed by 2 % agarose gel electrophoresis.

(4) Transcription by SP6 RNA polymerase (MEGA script SP6 Kit (Ambion Inc.))

The linear plasmid DNA was transcribed into RNA including GST-60 b at 37 °C for 3 hr in the following solution :

Linear plasmid DNA (2 ng/// 1) 5 /_/ 1 SP6 RNA polymerase 2 β 1

50 mM NTP (ATP, CTP, GTP, UTP ) mixture 4X 2 / 1

Buffer solution (10-fold) 3 // 1

Nuclease-free water 3 / 1 Total 20 β 1

(5) Digestion with DNase I

The transcribed sample was digested with DNase I at 37 °C for 30 min in the following solution: Transcribed sample 20 β 1

DNase I (2ϋ/ 1; Ambion Inc.) 2 1

Total 22 β 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.

Example 24

(Preparation of GST-60 bp fusion protein) (1) Isolation of GST-60 bp fusion protein

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

(Amersham Pharmacia Biotech Co. ) and then to Glutatione Sepharose 4 B column (Amersham Pharmacia Biotech Co.) to prepare GST-60 bp fusion protein. (2) Treatment with RNase A and DNase I

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

100 mM Tris-HCl (pH7.8) 2 β 1

Nuclease-free water 6 1

Total 20 β 1

RNase in the solution was inhibited by adding

RNasin (2 U/ / 1 ; Promega Co.), and then the reacted fluid was treated DNase I at 37 °C for 30 min in the following solution:

Reacted fluid _ 20 1 DNase I (70 U/ 1 ; Takara Shuzo Co., Ltd.) 4 / 1

25 mM MgCl₂ 6 // 1

Total 30 β 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).

Example 25

(Reverse transcription; GST-60 b RNA → GST-60 bp DNA) (1) Reverse transcription

GST-60 b RNA was reverse transcribed into DNA at 30 °C for 30 min in the following solution:

GST-60 b RNA at 10 ng/ / 1 1 // 1 Bovine calmoduline (Cam; Wako Pure Chemical Co.,

Ltd. ) at l β g/ml 1 1

2.5 M dATP 1 1

2.5 mM dCTP 1 _/ 1

2.5 mM dGTP 1 / 1 2.5 mM dTTP 1 // 1

0.1 M Ca(H₂HP0₄ )₂ 1 _/ 1

25 mM MgCl₂ 2 / 1

PCR buffer (10-fold; Perkin Elmer Inc.) 2. / 1

0.1 M DTT (dithiothreitol) 2 // 1 Nuclease-free water 6-8 // 1

* Total 20 β 1

The reacted fluid was subjected to SepaGene (Sanko Co., Ltd.) to extract nucleic acid, and DNA sample was dissolved in 20// 1 water.

(2) PCR amplification with primers 1 and 2

The DNA sample was subjected to PCR amplification by AmpliTaq ® PCR system (Perkin-Elmer Inc.) with primers 1 and 2 in the following solution:

DNA sample 1 β 1

Primers 1 and 2 (20 /_/ M) 1 + 1 β 1

2 mM dNTP mixture 5 1

AmpliTaq Gold ® (5 O/ β 1; Perkin-Elmer Inc.) 0.5 β 1

PCR buffer (10-fold; Perkin Elmer Inc.) 5 β 1

25 mM MgCl₂ 3 // 1

Pure water 33.5 / 1 Total 50 β 1

Primer 1 ( sense) :

5 ' -TCGGATCTGGAAGCCCTGTT-3 ' Primer 2 (antisense): 5'-ACGTTCAGGACCCATAAGTTT-3'

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 β g/ ml, and as the result, 96 bp, 162 bp and 225 bp DNA as PCR products were prepared.

Fig. 33 shows reverse transcription from GST-60 b RN to GST-60 bp DNA in vitro.

Example 26

(Transcription; GST-60 bp DNA -> GST-60 b RNA) (1) Transcription of DNA to RNA The GST-60 bp DNA was transcribed into RNA at 30 °C for 60 min in the following solution:

GST-60 bp DNA at 10 ng/ // 1 1 / 1

Bovine cal oduline (CaM; Wako Pure Chemical Co., Ltd. ) at l β g/ml 1 / 1 1.25- M ATP 1 β 1

1.25 mM CTP 1 β 1

1.25 mM GTP 1 β 1

1.25 mM UTP 1 1 0.1 M Ca(H₂P0₄ )₂ 1 1

Nuclease-free water 13-14 / 1

Total 20 1

The reacted fluid was treated with DNase at 37 °C for 30 min and then subjected to SepaGene (Sanko Co., Ltd.) to extract nucleic acid, and RNA sample was dissolved in 20/ 1 water.

(2) RT-PCR amplification with primers 1 and 2 The 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:

RNA sample 1 β 1

Primers 1 and 2 1 + 1 β 1 10 mM dNTP mixture 4 / 1

0.1 DTT (dithiothreitol) 2.5 1 RNasin (RNase inhibitor; Promega Inc.) at 40 U/ β 1 0.25 β 1

Nuclease-free water 14.25 1 +

Enzyme mixture 1 1 (reverse transcriptase + DNA polymerase)

RT-PCR buffer (5-foϊd; Roche Inc.) 10 β 1

Nuclease-free water 14 / 1 Total 50 β 1

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

- 7 min → 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 RT-PCR products were prepared.

Fig. 34 shows transcription of GST-60 bp DNA to GST-60 b RNA with NTP.

Example 27 (Reverse direct expression; GST-60 bp protein → GST-60 b DNA) (1) Reverse direct expression

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:

GST-60 bp fusion protein at l g/ / l 1 t l Bovine calmoduline at 1 // g/ / 1 1 1

Retic Lysate ITV® Kit (Ambion Inc.) 20 β 1 (which contains reticulocyte lysate, calf liver tRNA, riboso e and amynoacyl-tRNA synthetase)

1.25 mM dATP 1 β 1

1.25 mM dCTP 1 // 1

1.25 mM dGTP 1 β 1 1.25 M dTTP 1 β 1

0.1 M Ca(H₂P0₄ )₂ 1 /_/ 1

Nuclease-free water 3-4 / 1

Total 30 β 1

The reacted fluid was subjected to SepaGene (Sanko Co., Ltd.) to extract nucleic acid, and DNA sample was dissolved in 20 1 water.

(2) PCR amplification with primers 1 and 2 The DNA sample was subjected to PCR amplification by AmpliTaq® PCR System (Perkin-Elmer Inc.) with primers 1 and 2 in the following solution:

DNA sample 1 1

Primers 1 and 2 (20 M) 1 + 1 // 1 2 mM dNTP mixture 5 // 1

AmpliTaq Gold ® ( 5 U/ / 1 ; Perkin-Elmer Inc.)

0.5 1

PCR buffer (10-fold; Perkin-Elmer Inc.) 5 / 1

25 mM MgCl₂ 3 β 1 Pure water 33.5 1

Total 50 β 1

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.

' Example 28

(Direct expression; GST-60 bp DNA → GST-60 bp protein) (1) Direct expression

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:

GST-60 b ssDNA at 10 ng/// 1 1^' /_/ 1

Retic Lysate ITV® Kit (A bion Inc.) 20 β 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.,

Ltd. ) at 1// g/ 1 1 // 1 and/or Anti-CaM sheep antiserum (polyclone; Chemicon International Inc.) 1 // 1 Nuclease-free water 8-9 / 1

Total 50 β 1

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.

Example 29

(Reverse translation; GST-60 bp protein → GST-60 b RNA) (1) Reverse translation The GST-60 bp fusion protein prepared was treated with DNase I, and heated at 100 °C for 5 min 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 RNA by reverse translation at 30 °C for 60 min in the following solution:

GST-60 bp fusion protein at 10 ng/ / 1 1 / 1 Bovine calmoduline (CaM; Wako Pure Chemical Co., Ltd. ) at l β g/ β 1 1 1

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 β 1

1.25 M GTP 1 β 1

1.25 mM UTP 1 β 1

0.1 M Ca(H₂P0₄ )₂ 1 // 1 Nuclease-free water 3-4 / 1

Total 30 β 1

The reacted fluid was subjected to SepaGene (Sanko Co., Ltd.) to extract nucleic acid, and RNA sample was dissolved in 20// 1 water.

(2) RT-PCR amplification with primers 1 and 2

The 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:

RNA sample 1 β 1

Primers 1 and 2 1 + 1 β 1

10 mM dNTP mixture 4 // 1

0.1 DTT (dithiothreitol, 2.5 1 RNasin (RNase inhibitor; Promega Inc.) at 40 U/ β 1 0.25 1

Nuclease-free water 14.25 // 1

+

Enzyme mixture 1 β 1 (reverse transcriptase + DNA polymerase)

RT-PCR buffer (5-fold; Roche Inc.) 10 β 1

Nuclease-free water 14 β 1

Total 50 β 1 The RT-PCR amplification was performed in the conditions of 50 °C - 30 min -> 94 °C - 2 min→ 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 + X sec (25 cycles) (X: Prolonged 5 sec per cycle) → 68 °C - 7 miri →- 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 RNA as RT-PCR products were prepared. Fig. 37 shows reverse translation of GST-60 bp protein to GST-60 b RNA.

Example 30

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. (including 20 amino acids) to prepare M2 protein on the membrane, and membrane strips thereof were overlaid on 3 % agarose gel plates containing salmon tests DNA (100 β 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 .

In the Fig. 41, 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, and 4 shows strip of M2 DNA membrane after Eastern blot reaction put on RNA- agarose gel plate.

Next, 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.

Effects of the Invention

As described above in detail, 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.

Referential Documents (1) Wolff et al., "Direct Gene Transfer into Mouse Muscle in vivo", Science 247, 1465-1468 (1990) (2) Johwston et al . , "Genetic immunization is a simple method for eliciting an immunene response", Nature 356, 152-154 (1992) , ^{' •} (3) Robinson et al., Proc. Natl. Acad. Sci. USA., 90, 11478-11482 (1993)

(4) Tang et al . , "Vaccination onto bare skin", Nature 388, 729-730 (1997) (5) Scott et al., "Topical Application of Viral Vectors for Epidermal Gene Transfer", J. Invest. Dermatol . , 10-8, 803-808 (1997)

(6) Liu et al., Ann. Rev. Immunol. 15, 617-648 (1997)

(7) Debs et al., J. Invest. Dermatol., 112 (3), 370-375 (1999)

(8) Khavari et al . , "Immunization via hair follicles by topical application of naked DNA to normal skin", Nature Biotech., 17, 870-872 (1999)

(9) Silva et al., "Therapy of tuberculosis in mice by DNA vaccination", Nature 400, 269-270 (1999)

Claims

1. 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) ,

(3) conversion from a protein to a DNA (reverse direct expression ) , or

(4) direct conversion from DNA to a protein (direct expression) .

2. A method of converting one of DNA, RNA and protein to any one of corresponding DNA, RNA or protein according to the following reactions containing calmodulin or a substance having a calmodulin-like function (referred to as CaM): (1) conversion from an RNA to a DNA (reverse, transcription) ,

(2) conversion from a protein to an RNA (reverse translation) ,

(3) conversion from a protein to a DNA (reverse direct expression) ,

(4) direct conversion from a DNA to a protein (direct expression) ,

(5) replication of a DNA

(6) transcription from a DNA to an RNA, or (7) translation from an RNA to a protein.

3. A method according to Claim 1 o 2, wherein an mRNA or an RNA is converted to a DNA by reverse transcription. ,

4. A method according to Claim 1 or 2, wherein a protein is converted to an RNA by reverse translation.

5. A method according to Claim 1 or 2 , wherein a ■ protein is directly converted to a DNA by reverse expression.

6. A method according to Claim 1 or 2 , wherein a DNA is directly converted to a protein by direct expression .

7. A method of producing a DNA, an RNA or a protein, wherein a DNA, an RNA or a protein is produced by a method according to any one of Claims 1 to 6.

8. 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 Claim 1 or 2.

9. . A reagent according to Claim 8, which has an action of promoting a reaction of reverse-transcribing an mRNA or an RNA to a DNA.

10. A reagent according to Claim 8, which has an action of promoting a reaction of reverse-translating a protein to an mRNA or an RNA.

11. A reagent according to Claim 8, which has an action of promoting a reaction of directly reverse- expressing a protein to a DNA.

12. A reagent according to Claim 8, which has an action of promoting a reaction of directly expressing a DNA to a protein.

13. A reagent according to Claim 8, which has an action of promoting a reaction of replicating a DNA.

14. 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.

15. A medicament according to Claim 14, which is a medicament for expressing an objective antibody in vivo.

16. 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 .

17. A method of expressing an objective protein in vivo by administering a medicament according to any one of Claims 14 to 16 to a human or an animal by topical, oral administration or injection.

18. 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.

19. 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.

20. 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.

21. A method of producing RNA^' from DNA by transcription, which comprises incubating the DNA in the solution containing calmodulin, NTP to prepare the corresponding RNA, and then isolating the RNA from the solution.

22. A method of producing RNA from protein by reverse translation, which comprises incubating the protein in the solution containing calmodulin, tRNA and NTP to prepare the RNA, and then isolating the RNA from the solution.

23. 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 3) isolating the DNA from the solution.

24. 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, and

1) extracting the protein to characterize profiles thereof, and/or .

2) overlaying the product on a gel containing substrate or a membrane containing chemical reagent, and incubating thereof to characterize biological and chemical activity of the protein.

25. 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.

26. 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.