KR840001441B1 - Recombinant dna transfer vector containing a gene from a higher organism - Google Patents

Abstract

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Description

Method for producing DNA delivery media containing nucleotide sequences for specific proteins of higher organisms

1 is a diagram illustrating the process of the present invention.

The present invention relates to a method of giving a microorganism the genetic ability to synthesize a protein according to a genetic code by separating and purifying a specific nucleotide sequence or gene from a higher organism and transferring the gene to the microorganism.

In particular, the present invention provides a method for isolating, purifying genes of a polypeptide such as insulin or growth hormone to microorganisms.

The present invention provides a method for preparing a DNA transfer vector comprising a nucleotide sequence that genetically encodes a specific protein of a higher organism, which consists of the following steps.

(1) isolates cells containing mRNA that drive-encode this particular protein from higher organisms capable of producing that particular protein,

(2) extracting mRNA from these isolated cells in the presence of a ribonuclease inhibitor to prevent degradation of the mRNA by ribonuclease,

(3) Isolate mRNAs on proteins, DNA and other RNAs,

(4) either strand synthesizes a double stranded cDNA having a nucleotide sequence complementary to that of the mRNA, thereby producing a cDNA having a nucleotide sequence that genetically encodes a particular protein,

(5) adding a DNA transfer mediator with reactant ends capable of binding double stranded cDNA,

(6) By combining DNA delivery media with a double stranded cDNA having a nucleotide sequence that encodes a specific protein, a DNA delivery mediator having a nucleotide sequence that encodes a specific protein is generated.

The present invention is directed to a method for separating specific nucleotide sequences containing genetic information for genetically encoding a specific protein, synthesizing DNA having the specific nucleotide sequence, and delivering the supporting DNA to a microorganism host capable of replicating the DNA. It is about. More specifically, the present invention relates to the isolation, purification, delivery of the insulin gene and growth hormone gene to the microorganism host, replication in the microorganism host and then their properties.

The new product is produced by the present invention. The sour product includes a non-binding plasmid containing specific nucleotide sequences derived from higher organisms and new microorganisms containing specific nucleotide sequences derived from higher organisms as part of the genetic factor structure.

The symbols and abbreviations used in the present specification are described as follows.

DNA-deoxyribonucleic acid

RNA-ribonucleic acid

cDNA-complementary DNA (enzymatically synthesized from mRNA sequences)

mRNA-IMR RNA

tRNA-transfer RNA

dATP-deoxyadenosine triphosphate

dGTP-deoxyguanosine triphosphate

dCTP-deoxycytidine triphosphate

A-Adenine

T-Thymine

G-guanine

C-cytosine

Tris-2-amino-2-hydroxyethyl-1,3-propanediol

EDTA-ethylenediamine tetraacetic acid

ATP-Adenosine Triphosphate

TTP-thymidine triphosphate

The base sequence of DNA is biologically important as it contains operational information. In DNA, the sequence of bases is known to be used as a code for specializing the amino acid sequence of all proteins produced by cells. Part of the base sequence is also used to control the time and amount of each protein produced.

Very little is known about the nature of these regulatory elements. Finally, the nucleotide sequence of each strand is used as a template for the replication of DNA involving cell division.

The use of information about the DNA sequence to determine the amino acid sequence of a protein is common and applies to all organisms. Each amino acid commonly found in proteins is known to be determined by one or more trinucleotides or tribasic sequences. Therefore, each protein has a corresponding segment of DNA that contains a series of triplets corresponding to the amino acid sequence of the protein. The genetic code is described in the accompanying table.

In a biological method of converting information about a nucleotide sequence into an amino acid sequence structure, so-called transcription is performed in the first step. In this step, a partial segment of DNA with an array that specifies the protein to be produced is first copied into RNA. RNA is a polynucleotide similar to DNA except that it contains ribose instead of deoxyribose and uracil instead of thymine. The bases of RNA can form homogeneous base pairs that relate to DNA. As a result, RNA transcription of the DNAnucleotide sequence is complementary to the copied sequence. These RNAs are called messenger RNAs (mRNAs) because of their position as mediators between the cell's genetic app-aratus and protein synthesis. In cells, mRNA is used as a template in complex processes involving a variety of enzymes and organs in the cell, synthesizing specific amino acid sequences. This process is called translation of mRNA. There is an additional step, sometimes called processing, which involves the conversion of amino acid sequences synthesized by translation into functional proteins. The case of insulin is given as an example.

Genetic code

Phenylalanine (Phe) TTK Histidine (His) CAK

Leucine (Leu) XTY Glutamine (Gln) CAJ

Isoleucine (Ile) ATM Asparagine (Asn) AAK

Methionine (Met) ATG Lysine (Lys) AAJ

Val GTL Aspartic Acid (Asp) GAK

Serine ORS Glutamic Acid (Glu) CAJ

Proline (C) CCL Cysteine (TGK)

Threonine (Thr) ACL Tryptophan (Try) TGG

Alanine (Ala) GCL Arginine (Arg) WGZ

Tyrosine (Tyr) TAK Glycine (Gly) GGL

End sign TAJ

End sign TGA

Key Points: A trinucleotide of DNA having a 5 'end on the left and a 3' end on the right of each three letter tribasic. Letters indicate getting putin or pyrimidine to form a nucleotide sequence.

A = Adenine

G = guanine

C = cytosine

T = Thymine

X = T or C (when Y is A or G)

X = C (when Y is C or T)

Y = A, G, C or T (X is C)

Y = A or G when X is T

W = C or A when Z is A or G

W = C (when Z is C or T)

Z = A, G, C or T (when W is C)

Z = A or G (when W is A)

QR = TC (When S is A, G, C or T)

QR = AG (when S is T or C)

S = A, C, C or T (when QR is TC)

S = T or C when OR is AG

J = A or G

K = T or C

L = A, T, C or G

M = A, C or T

A direct precursor of insulin is a single polypeptide called proinsulin, which contains two insulin chains A and B linked by different peptides C [Steiner, DF, Cunningham, D., Spigelman, L. and Aten, B. , Science 157,697 (1967).

Recently, it has been reported that the initial translation product of insulin mRNA is not proinsulin itself, but prepreinsulin containing more than 20 additional amino acids at the amino terminus of proinsulin [Cahn, SJ, Keim, P. and Steiner. , DF, Proc, Natl. Acad. Sci. USA 73, 1964 (1976) and Lomedico, P.T and Saunders, G, F., Nucl. Acids Res. 3,381 (1976).

The structure of the preproinsulin molecule can be represented by NH _2- (pre-peptide) -chain- (Cpeptide) -A chain-COOH.

Many proteins of value for medical or research purposes are found in or made by cells of higher organisms such as vertebrates. Examples include other peptide hormones such as hormone insulin, growth hormone, proteins for blood pressure control, and various enzymes of value for industrial, medical, or research use. It is quite difficult to extract these proteins from organisms and get enough, and this problem is particularly acute for human proteins. Therefore, a technique is needed to layer these proteins by cells outside the organism. In some cases, suitable cell lines can be obtained that can be incubated by tissue culture.

However, the growth of cells in tissue culture is slow, the medium is expensive and the ovulation conditions must be precisely controlled and the yield is low. Moreover, it is difficult to grow a cultured cell line having the desired characteristics.

On the other hand, microorganisms such as bacteria are relatively easy to grow in chemically defined media. Fermentation technology is so advanced that it is easy to control. The organism grows fast and high yields are possible. In addition, some microorganisms are fully genetically depicted and, in fact, are among the best described and understood organisms.

It is highly desirable to isolate genes that genetically encode medically important proteins from organisms that make proteins and deliver them to appropriate organisms. In this way, under controlled growth conditions, proteins can be made by the microorganisms and obtained in the desired amount. In addition, the total cost of producing the desired protein by this process can be substantially reduced. In addition, the ability to isolate and transfer gene sequences that determine the production of a particular protein to microorganisms with a clear genetic background is a study of how the synthesis of these proteins is regulated and what processes the proteins take after synthesis. Provides research methods of considerable value for Moreover, isolated genetic code sequences can be altered to genetically encode various proteins with altered therapeutic or functional properties.

The present invention provides a method for achieving the above-mentioned object. Methods have been published that consist of a series of complex steps, including enzyme-catalyzed reactions. The nature of this enzymatic reaction is described herein as is known in the art.

Reverse transcriptase is a DNA complementary to an RNA template strand in the presence of an RNA template, an oligo-deoxynucleotide primer and a 4-day deoxine nucleoside triphosphate, ie NATP, dGTP, dCTP, and TTP. It catalyzes the synthesis. The reaction begins by noncovalently binding the oligo-deoxinenucleotide primer to the 3 'end of the mRNA, and then to the 3' end of the grown chain with the appropriate deoxynucleotide determined by the base pairing relationship with the mRNA nucleotide sequence. Add egg meal. The product molecule is described as a hairpin structure containing the original RNA together with the complementary strand of DNA linked by a single strand of DNA loops.

Reverse transcriptase can also catalyze similar reactions using single-stranded DNA templates, in which case the resulting product is a hairpin structure of double-stranded DNA with loops of single-stranded DNA connecting a pair of ends [ Aviv, H. and Leder, P., Proc. Natl. Acad. Sci. USA 69,1408 (1972) and Efstratiadis, A., Kafatos, F.C., Maxam, A.F. And Maniatis, T., Cell 7,279 (1976).

Restriction endonucleases are enzymes that break down the linkage of DNA strands by hydrolyzing phosphodiester bonds in double-stranded DNA.

If the DNA is in the form of a closed loop, the loop is converted into a linear structure. An important feature of this type of appeal is that their hydrolysis can only occur where there are specific nucleotide sequences. This arrangement is called the recognition site of restriction endonucleases. Restriction endo nucleases obtained from various sources are isolated and clearly identified by their nucleotide sequence. Some restriction endonucleases hydrolyze phosphodiester bonds on both strands at the same position to produce blunt ends.

Other enzymes catalyze the hydrolysis of bonds separated separately by several nucleotides, creating a single stranded site free at each end of the cleaved molecule.

These single-stranded ends are self-complementary and have the ability to recombine hydrolyzed DNA. Since DNAs that are likely to be cleaved by these enzymes contain the same recognition site, the same binding ends are generated, allowing the heterologous sequence of the DNA treated with the restriction endonuclease to be combined with other sequences treated in a similar manner. [Roberts, RJ, Crit. Rev. Biochem. 4,123 (1976).

Restriction sites are relatively rare, but the general use of restriction endonucleases has been greatly extended by chemically synthesizing double stranded oligonucleotides with restriction site arrangements. Thus, in fact, the segment of DNA can bind tightly to any other segment by adding the appropriate restriction oligonucleotide to the molecular terminus, and by hydrolyzing this product with the appropriate restriction endonuclease, the necessary binding ends are produced [Heyneuker, HL]. , Shine, J., Goodman, HM, Boyer, HW, Rosenberg, J., Dickerson, RE, Narang, SA, Itakura, K., Lin, S. and Riggs, AD Nature. 263,748 (1976) and Scheller, R. H., Dickerson, R. E., Boyer, H. W., Riggs, A. D. and Itakurn, K., Science 196,177 (1977).

S1 endonucleases are enzymes with general specificity capable of hydrolyzing phosphodiester bonds in single-stranded gaps or loops within single-stranded DNA or double-stranded DNA [vogt, V.M., Eur. J. Bi-ochem. 33, 192 (1973).

DNA ligase is an enzyme capable of catalyzing the formation of phosphodiester bonds between two DNA segments, each having 5 'phosphate and 3' hydroxyl, which can be formed from two DNA fragments held together by binding ends. to be. The general function of the enzyme is believed to bind single strand nicks within other double stranded DNA molecules. However, under appropriate conditions, DNA ligase can blunt end ligation covalently linking two molecules with blunt ends [Sgaramella, V., Van de Sande, JH, and Khorana, HG, Proc]. . Nat]. Acad. Sci. USA 67. 1468 (1970).

Alkaline phosphatase is an enzyme with general specificity capable of hydrolyzing phosphate esters containing 5 'terminal phosphoric acid on DNA.

Another step, described in the whole process, is the insertion of specific DNA fragments into DNA media such as plasmids. Plasmid is a term used as a self-replicating DNA unit that can be found in microbial cells, unlike the genome of the host cell itself. The plasmid does not genetically bind to the chromosome of the host cell. Plasmid DNA, although some have a molecular weight of 10 ⁸ or more, generally exists in the form of double-stranded ring molecules of several million molecular weight, which usually comprise a very small percentage of the total DNA in the cell. Plasmid DNA can usually be separated from host cell DNA because there is a significant difference in size between the two. Plasmids can replicate regardless of the rate of host cell division, and in some cases, researchers can control their rate of replication depending on growth conditions. Even if the plasmid is in a closed ring state, it is possible to insert DNA segments into the plasmid by artificial methods to form a recombination plasmid with a larger molecular size, in which case the genetic factor to be transferred has a substantial effect on the replication or expression ability. Does not have Plasmids therefore serve as useful mediators for delivering DNA binding to new host cells. Plasmids useful in DNA recombination techniques typically contain genes suitable for selective purposes, such as drug resistance genes.

In order to illustrate the practice of the present invention, the isolation and transfer of the insulin gene of rats will be described in detail. Insulin was chosen here because it is very important in terms of clinical medicine and basic research. The published method can be used by people of ordinary skill in the art to isolate insulin genes from other organisms, including humans.

Insulin was first isolated in 1922. It is well known that this hormone is currently being used to treat diabetes. In the slaughterhouse, the pancreas of cows and pigs can be obtained from the insulin source, but as the number of diabetics increases worldwide, there is a deficiency of hormones. Moreover, some diabetics have allergic reactions to bovine and porcine insulin with toxic results. It is therefore highly desirable to produce enough human insulin to meet the needs of the world. Making human insulin from bacteria is one way to achieve this desirable purpose. However, prior to the present invention, since the technology for inserting the insulin gene into bacteria has not been developed, the process for achieving such a desirable object has not been carried out. The present invention provides such a technique.

By obtaining DNA with a specific sequence that can genetically encode a specific protein, we can modify the nucleotide sequence by chemical or biological methods, and finally the specific protein that is produced. This enabled the production of modified insulin, for example, made for particular medical applications. Thus, it is possible to give microorganisms the genetic ability to produce any amino acid sequence that has the important functional properties of insulin. Similar thoughts apply to growth hormone.

The ability to pass the genetic code for specific proteins necessary for the normal metabolism of certain higher organisms to microorganisms, such as bacteria, is important to enable the culture production of such proteins.

In addition, if the ability of higher organisms to function normally during the production of such proteins is impaired, it gives the important possibility to increase the yield of such proteins or to substitute them with proteins produced by the microorganisms modified according to the present invention. do. For example, by establishing a symbiotic relationship between the microorganism produced by the present invention and a person with chronic or acute necrosis, the genetically modified microorganisms as described herein may be implanted into or associated with a person and metabolized. To treat pathological deficiency.

A method of separating specific nucleotide sequences containing genetic information, synthesizing DNA having this specific nucleotide sequence, and delivering the DNA to a host microorganism has been published.

The present invention exemplifies a specific DNA sequence delivered to bacteria, including the structural gene of rat preproinsulin, the gene of mouse growth hormone, and the human growth hormone gene. Therefore, this method is thought to be very suitable for transferring desired DNA sequences from higher organisms such as vertebrates to any microorganism. Here, higher organisms are defined as eukaryotes with differentiated tissues, including insects, molluscs, plants and vertebrates to which mammalian mammals, such as cattle, pigs, primates, and humans belong. As known in the art, microorganisms are microscopic organisms belonging to protozoa and include proteases or eukaryotes, for example bacteria, protozoa, algae and yeast. In the present method, first, the selected cell population is isolated by an improved method.

In extracting complete mRNA from these cells by a new method, virtually all ribonuclease activity is inhibited. From this extract, complete messenger RNA is purified by column chromatography, followed by reverse transcriptase acting on the mRNA which acts in the presence of the four deoxynucleoside triphosphates required to synthesize complementary (cDNA) strands. The product of this first reaction using reverse transcriptase optionally removes the ribonucleotide sequence. The remaining deoxynucleotide sequence is complementary to the original mRNA and is incubated in the presence of four deoxynucleoside triphosphates in a second reaction with reverse transcriptase or DNA polymerase. The resulting material is a duplex cDNA structure with complementary strands joined together by a single strand of loops at one end. This product is then treated with a single strand specific nuclease that can cleave a single strand loop. The double-stranded cDNA thus produced is lengthened by adding specific DNA containing restriction enzyme recognition site arrays to both ends. In such additions, DNA ligase enzymes catalyze. The elongated cDNA is then treated with restriction endonucleases to produce a single strand of self-complementary at the 5 'end of each strand of the duplex.

Plasmid DNA having the recognition site for the same restriction endonuclease is treated with the enzyme to break down the polynucleotide strands and produce a single stranded nucleotide sequence that is self-complementary at the 5 'end. Removing the 5'-terminal phosphate group at the single-stranded end prevents the plasmid from forming a ring structure that can modify the host cell. The previously prepared cDNA and plasmid DNA are incubated together in the presence of DNA ligase. Only under the described reaction conditions can a viable closed ring of plasmid DNA be formed if it contains a segment of cDNA. The plasmid containing the cDNA array is then inserted into a suitable host cell. Cells that receive viable plasmids can be detected by the appearance of colonies with genetic properties derived from the plasmids, such as drug resistance. Once pure bacterial strains containing recombinant plasmids with coalesced cDNA arrays are grown, the recombinable plasmids can be isolated again.

In this way, a large amount of recombination plasmid DNA can be produced, and specific cDNA sequences can be re-isolated by endoaucleolytic cleavage with suitable restriction enzymes.

The basic process of the present invention is to isolate the desired nucleotide sequence obtained from high school students, including humans, and deliver it to the host microorganism. This method will be useful for the delivery of genes that genetically encode specific proteins of medical or industrial value and for the microbiological synthesis of such proteins. To describe the present invention, a nucleotide sequence that genetically encodes insulin is isolated from a mouse, delivered to bacteria, and reproduced therein. Similarly, nucleotide sequences that genetically encode rat growth hormone are isolated, transferred to bacteria, and then replicated in them.

The method can be applied to the delivery of nucleotide sequences isolated from humans, such as human insulin, growth hormone, as well as other polypeptide hormones.

The present invention includes a method for isolating DNA molecules of a particular nucleotide sequence and delivering it to a microorganism, where the original nucleotide sequence of DNA is found after replication has occurred in the delivery organism. A series of steps involving the process of the present invention can be generally classified into four categories.

1. Isolation of the desired population of lobster from higher organisms

There are two possible sources of genetic alignment for genetic coding of specific proteins: the DNA of the source organism itself and its RNA transcription. Currently, the National Institutes of Health safety regulations state that certain human genes can only be inserted into recombination DNA and subsequently into bacteria after very careful purification of the genes in a special high risk (P4) container. Federal Register, Vol. 41, No. 131, July 7, 1967, pp. 27902-27943].

Therefore, the preferred approach in any process available for the production of human proteins in this method is to isolate specific mRNAs with nucleotide sequences that genetically encode the desired protein. The advantage of this method is that it is much easier to purify mRNA than to extract DNA from cells. Especially in highly differentiated organisms, such as vertebrates, one can take advantage of the fact that it is possible to identify specific populations of cells with specific positions in organisms that contribute primarily to producing the protein in question. Optionally, these populations exist during the transitional phase of life. In this cell population, most of the mRNA separated from the cell has the desired nucleotide sequence. Therefore, the selection of the cell population to be separated and the separation method used therein are of substantial benefit in view of the initial purity of the mRNA to be separated therefrom.

Most tissues, gland, and organelles are usually bound by connective tissue fibrous networks, and in principle are composed of collagen, but depending on the tissue, they may contain other structural proteins, polysaccharides, and inorganic components. do. Separation of cells from selected tissues absolutely requires techniques to release the cells from the connective tissue matrix. Therefore, there are two important steps in the method of isolating and purifying specific differentiated cell types: cells from the connective tissue matrix. To separate the cells of the desired form from all other cell types found in the tissue. The principles of action described in the present invention can be applied to the separation of various cell types from various tissue sources. For example, we will describe a method for isolating Langerhans islands from the pancreas that is suitable for the isolation of mRNAs that genetically encode insulin.

Insulin-producing cells can be obtained from other sources, such as the fetal calf pancreas or cultured islet tumor cells. In such a case, especially in pure cell culture, it will be much simpler to isolate pure Langerhans islet cells. The Langerhans islet cell separation method set out above will not be necessary in this case but is advantageous because it is generally applicable.

It is frequently found that the response of cells to ambient stimuli can be used to increase the proportion of the desired mRNA. For example, treatment with hormones can increase the production of the desired mRNA. Other methods include growing at specific temperatures and exposing to certain nutrients or other chemicals. When the mRNA of mouse growth hormone was isolated, the pituitary cells of the cultured mice were treated with thyroid hormone and glucocorticoid to increase the mRNA percentage of growth hormone.

2. Extraction of mRNA

An important feature of the present invention is the complete elimination of ribonuclease activity in the cell extract. The mRNA to be extracted is a single strand of polynucleotide and is not paired with any complementary strand. Thus, hydrolyzing a single phosphodiester bond in the sequence may be because the entire molecule is obsolete to deliver the complete gene sequence to the microorganism. As mentioned above, enzyme ribonucleases are widely distributed and active but exceptionally stable. This enzyme is found on the skin and remains a common glass cleaning technique, sometimes contaminating organic chemicals.

There is a particular difficulty when dealing with extracts of pancreatic cells, because the pancreas is a source of digestive enzymes and is therefore rich in liponuclease. However, the problem of ribonuclease contamination is present in all tissues, and the method of eliminating ribonuclease activity disclosed herein can be applied to all tissues. The particular effect of this method in the present invention can be explained by the successful isolation of complete mRNA from Langerhans islet cells isolated from the pancreas.

In the present invention, chaotropic anions, chaotrope cations, and deansulphide lyases are used in combination during all the operations required to separate RNA from cell division and protein. The combined use of these materials was demonstrated by the high yield of separation of intact mRNA from the pancreatic islets of Langerhans.

When selecting suitable chaotrop ions, keep in mind the solubility and usefulness in the aqueous medium. Suitable chaotrope cations include guanidinium, carbamoylguanidinium, guanylguanidinium, and lithium.

Suitable chaotropes anions include iodine, perchlorate, thiocyanic acid, diiodosalicylic acid and the like. The relative effectiveness of salts formed by combining these cations and anions will be determined in part by the solubility of these salts. Lidioiosalicylate, for example, is a much more potent modifier than guanidinium thiocyanate, but is only about 0.1 M between solubility and relatively expensive. Guanidinium thiocyanate provides a preferred cation-anion bond, which is preferred because it is readily available and can dissolve up to about 5 M in an aqueous medium.

Thiol compounds, such as β mercaptoethanol, are known to break disulfide bonds in molecules of proteins by thiol disulfide interchange reactions. In addition to β-mercaptoethanol, various suitable thiol compounds are known to be effective, including dithiothreitol, cysteine, propanol dimercaptan and the like.

In order to complete the interchange reaction indispensably, the thiol compound must be present in an excessive amount on the intramolecular disulfide, so it is necessary to be water-soluble. Β-mercaptoethanol is suitable because it can be easily purchased at a suitable price.

The effect of a given chaotrope salt to inhibit ribonucleases during RNA extraction from tissues or cells is directly related to its concentration. The preferred concentration is therefore the highest concentration actually available. The success of the present invention to fully support mRNA during extraction is believed to depend on the rapid degeneration of the thibonuclease and the extent of denaturation. For example, although hydrochloride is only slightly less potent as a denaturant, it is for this reason that guanidinium thiocyanate is more prevalent than hydrochloride. The effect of denaturant is defined by the minimum concentration necessary to completely denature the protein. On the other hand, the rate of denaturation of various proteins increases from 5 to 10, depending on the denaturant concentration in proportion to the lowest threshold concentration of denaturant [Tanford, C.A., Adv. Prot, Chem. 23, 121 (1968).

This qualitative relationship suggests that only slightly stronger denaturants than guanidinium hydrochloride can denature proteins several times faster at the same concentration. The relationship between the rate of ribonuclease denaturation and the process of preserving mRNA when extracted from cells is not known or used before the present invention. If the above analysis is correct, it is believed that the preferred denaturant has a low minimum denaturation concentration combined with high water solubility. For this reason, although lithium diiodosalicylate is a more powerful denaturant, guanidinium thiocyanate is more preferred than lithium diiodosalicylate, and the solubility of guanidinium thiocyanate is much higher so that ribonucleacan is given at a given concentration. It can be used to deactivate the agent faster. The analysis also explains why guanidinium thiocyanate is more preferred than hydrochloride salts with nearly equivalent solubility, since guanidinium thiocyanate is a slightly stronger denaturant.

The use of disulfide hydrolysates with denaturants enhances the effect of denaturants by unfolding the ribonuclease molecule completely. Thiol compounds are thought to speed up the denaturation process by preventing rapid prototyping that can occur when intramolecular disulfide bonds remain intact. Furthermore, any contaminated ribonuclease remaining in mRNA production remains almost inactive even without denaturants and thiols. Generally speaking, disulfide bond degrading agents having thiol groups are somewhat effective at any concentration, although thiol groups that exceed significantly to intramolecular disulfide bonds are preferred to induce an interchange reaction in the direction of decomposing intramolecular disulfide. On the other hand, most thiol compounds have a bad smell and are difficult to handle at high concentrations, so there is an upper practical limit.

It was found that concentrations in the range of 0.05M to 1.OM were effective using β-mercaptoethanol, and 0.2M is considered to be the optimal concentration when undegraded RNA is isolated from mouse pancreas.

During the extraction of mRNA from the cells, the pH range of the medium should be in the range of pH 5.0-8.0.

After cell division, RNA is separated from cellular proteins and DNA. Various processes have been developed for this purpose, some of which are appropriate and all of them are well known in this field. A common method in the prior art is to use an ethanol precipitation process that selectively precipitates RNA. In the preferred method of the present invention, the homogeneous solution is poured directly into a 5.7 M cesium chloride solution in a sieving tube without passing through a precipitation step to form a layer. Centrifuge as described in C., Biochemistry 13, 2633 (1974). This method is desirable because it continuously maintains a bad environment for ribonucleases, allowing RNA to be recovered at high yield without DNA and protein.

Total RNA can be purified from the cell homogenate by the above-described procedure. However, only a fraction of the RNA recovered in this way is the desired mRNA. To further purify the desired mRNA, the fact that the mRNA in the cells of higher organisms is further purified by attachment with polyadenylic acid in the cell after transcription is transcribed. Such mRNAs containing attached poly A sequences can be selectively separated by column chromatography on a column of cellulose to which oligo-thymidylate is attached, as described above by Aviv, H. and Leder, p. . By the above-mentioned processes, pure, complete and translatable mRNA can be obtained from a rich source of lyonogenase. Purification of mRNA and subsequent in vitro procedures can be performed in the same way on any mRNA, regardless of the type of source organism.

In some circumstances, for example, when tissue culture cells are used as an mRNA source, there is very little ribonuclease contamination, and thus the ribonuclease inhibition method just described is not needed. In this case, conventional techniques of reducing ribonuclease activity are sufficient.

3. Formation of cDNA

The remaining steps of this process are illustrated in FIG. 1. The first step in this process is to form DNA sequences complementary to purified mRNA. In principle, any enzyme capable of forming complementary strands using mRNA as a template may be used, but the enzyme chosen for this reaction is a reverse transcriptase. This reaction can be set up under the conditions described in the prior art, using mRNA as a template and a mixture of four deoxynucleoside triphosphates as precursors to DNA strands. To track the progress of the reaction, label one of the deoxynucleoside triphosphates at 32 _p at the α position and quantitatively evaluate the recovery to recover the product after separation and separation such as chromatography and electrophoresis. In order to, it is convenient to tag.

[See above, Efstratiadis, A., et al.].

As shown in FIG. 1, the product of the reverse transcriptase reaction is a double-stranded hairpin structure having a noncovalent bond between the RNA strand and the DNA strand.

The product of reverse transcription enzyme reaction is removed from the reaction mixture by conventional methods known in the art. At this time, it is preferable to use a mixture of ethanol precipitation, chromatography using Separdex (trade name, Pharmacia Inc., Uppsala, Sweden) G-100, and ethanol precipitation. Once the cDNA has been enzymatically synthesized, the RNA template can be removed. Various methods of selectively degrading RNA in the presence of DNA are known in the art. One preferred method is alkaline hydrolysis, which has high selectivity and can be easily adjusted by pH adjustment.

Neutralize immediately after the alkaline hydrolysis reaction and if necessary concentrate the cDNA labeled 32 _p by ethanol precipitation.

Using a suitable enzyme such as DNA polymerase or reverse transcriptase to synthesize a double stranded hairpin cDNA. Reaction conditions use α-32 _p labeled nucleoside triphosphate, similar to that described above.

Reverse transcriptase can be obtained from a variety of sources. A suitable source is chicken myeloid leukemia (avain myeloblastosis) virus. This virus is found in Life Sciences Incorporated, St. D.J. in Petersburg, Florida. Obtained from Dr. Beard, Dr. Lee produces the virus under a contract with the National Institutes of Health.

It is convenient to purify DNA from the reaction mixture after forming the hairpin cDNA. As described above, it has been found to be convenient to purify DNA products free of contaminating proteins using the steps of phenol extraction, chromatography with Sephadex G-100 and ethanol precipitation. The hairpin structure can be converted to a conventional double stranded DNA structure by removing the single stranded loops that connect the ends of the complementary strands. For this purpose, various enzymes can be used which can specifically hydrolyze a single stranded region of DNA. Suitable enzymes for this purpose are Sl nucleases isolated from Aspergillus oryzae. This enzyme is available from Miles Research Products, Elkhart, Indiana. Treatment of DNA with hairpin-shaped structures with Sl nuclease can yield cDNA molecules with base pair ends with high yield. Extraction and kfmatograph the ethanol precipitation as described above. The use of reverse transcriptase and Sl nucleases to synthesize double stranded cDNA transcripts of mRNA has been described above by Efstratiadis et al.

In some cases, the ratio of cDNA molecules having blunt on can be maximized by treatment with E. coli DNA polymerase I in the presence of four deoxynucleoside triphosphates. This enzyme's activity as an exonuclease and polymerase acts to remove any 3'proximal end and fill any 5'proximal end. In this way, the subsequent binding reaction can maximize the proportion of cDNA molecules.

The next step in this process is to process the ends of the cDNA product to make a suitable arrangement containing restriction endonulease recognition sites at each end. Selection of the DNA fragment to be added to the terminal is convenient to handle.

The arrangement to be added at the end is selected based on the particular restriction endonuclease enzyme selected, which choice will depend on the choice of DNA media to recombine with the cDNA. The plasmid selected should have at least one site sensitive to restriction endonuclease degradation. For example, plasmid pMB9 contains one restriction site for enzyme hint III. Hind III is isolated from Hemophilus influenzae and is described by Smith, H.0., And Wilcox, K.W., J. Mol. Biol. 51, 379 (1970). HaeIII, an enzyme isolated from Hemophilus aegyptious, is described in Middleton, J.H., Edgell, M.H., and Hutchison III, C.A., J. Virol. 10, 42 (1972). HsuI, an enzyme isolated from Hemophilus suis, catalyzes isospecific hydrolysis at the same site as Hind III. Therefore, these two enzymes are thought to be functionally interchangeable.

To attach to the cDNA duplex end, it is preferable to use a double stranded decanucleotide that contains a cognitive sequence for Hind III and is chemically synthesized. Double-stranded decanucleotides have the arrangement shown in Figure 1. [Hyneker, H. L., et al., And Scheller, R. H., et al. Reference].

Such synthetic restriction site arrangements are useful for those skilled in the art because of their variety, thus providing the ends of the duplex DNA to be sensitive to the action of any of a wide variety of restriction endonucleases.

Attaching the restriction region to the ends of the cDNA can be accomplished using any method known to those skilled in the art. The method of choice is a reaction called blunt end ligation, catalyzed by DNA ligase purified by the method of Panet, A., et al., Biochemistry 12, 5045 (1973). The blunt deletion reaction was explained by Sgaramella, V, et al. The product of the blunt binding reaction that takes place between the cDNA having a blunt end and the excess mole double-stranded decanucleotide containing the hint III endo nuclease restriction site is a cDNA having a hint III restriction array at each end. Treatment of this reaction product with Hind III [endonuclease results in degradation at the restriction site and forms single stranded 5 ′ self-complementary ends as shown in FIG. 1.

4. Formation of Recombinant DNA Delivery Mediator

In principle, various viral and plasmid DNAs can be used to form recombinants with cDNA prepared by the methods described above. As important conditions, DNA delivery mediators must be able to enter host cells, replication can take place within host cells, and they must have genetic determinants to select host cells. However, for public safety, the range of choice is limited by the type of delivery media appropriate for the type of experiment to be performed, in accordance with the NIH baseline described above. The list of accredited DNA delivery media continues to grow as new mediators have been developed and approved by the NIH Recombinant DNA Safety Committee, and the present invention includes all viral DNA with the above-mentioned capabilities, including those that will later be NIH approved. The use of plasmid DNA is carefully considered. Suitable for use as currently approved delivery media are various derivatives of bacteriophage lambda [Blattner, FR, Williams, BG, Blechl, AE, Denniston Thompson, K., Faber, HE, Furlong, LA, Grunwald, DJ, Kiefer, DO , Moore, DD, Schumm, JW, Sheldon, EL, and Smithies, O., Science 196, 161 (1977)] and derivatives of plasmid Cole El [Rodriguez, RL, Bolivar, S., Goodman, HM, Boyer. H.W., and Betlach, M.N., ICN-UCLA Symposium on Mo1ecular Mechan-isms In The Control of Gene Expression, D.P. Nierlioh, W.J. Rutter, C. F. Pox, Eds, (Academic Press, NY, 1976), pp. 471-477.

Plasmids derived from Kohl El are relatively small, have molecular weights of several million, and the number of copies of plasmid DNA per host cell is usually 20-40, which can be increased to 1000 or more by treating chloramphenicol. There is a characteristic. If the genetic factor capacity can be increased in the host cell, under appropriate conditions, the host cell can produce a protein that is genetically encoded by the gene transferred on the plasmid. Therefore, derivatives of Kohl EI are preferred delivery vehicles for the method of the present invention. PBR-313, pBR-315, pBR-316, pBR- containing amplicilin resistance genes in addition to tetracycline resistance genes, including plasmid pMB-9 carrying tetracycline resistance genes as suitable derivatives of chol EI 317 and pBR-322. The presence of drug resistance genes is convenient for screening cells that have been successfully infected by the plasmid, since the clones of these cells grow even in the presence of the drug, while the cells that do not receive the plasmid do not grow or form clones. to be. In the experiments described herein as a specific embodiment of the present invention, plasmids derived from Kohl EI are used shearingly, which has a hint III site in addition to the drug resistance markers described above.

Along with the choice of plasmids, the choice of suitable hosts is usually very broad but narrowly restricted for public safety. E. coli strains designated X-1776 were developed and approved by NIH using a P2 containment system for the process described herein [Curtiss, III, R., Ann. Rev. Microbiol., 30, 507 (1976)].

E. coli RR-1 is suitable for use with P3 containment facilities. As in the case of plasmids, the present invention can be used with any host cell strain that can act as a transporter of selected mediators including protozoa other than bacteria, such as yeast, as long as the use of host cell strains is approved under the NIH baseline. It is understood that there is.

Recombinant plasmids are formed by mixing cDNAs containing residues treated in a similar manner to plasmid DNA treated with restriction endonucleases. To minimize the chance of cDNA fragments binding to each other, excess mole of plasmid DNA is added to cDNA. In the prior art, these methods resulted in the circularization of most plasmids without being inserted into cDNA fragments. The modified cells then contain mainly plasmids and no cDNA recombination plasmids. As a result, this selection process is very supportive and time consuming. A prior art solution to this problem was to design a DNA carrier with a restriction endonuclease site in the middle of a suitable marker gene, such that insertion of a recombinant would lead to loss of the ability to be genetically encoded by the gene to be separated between genes. .

Preferably, a method of reducing the number of clonies to be selected as recombination plasmids is used. The method involves treating alkaline phosphatase cleaved plasmid DNA with restriction endonucleases, which is commercially available from several sources, such as Worthington Biochemical Corporation in Freehold, New Jersey. Treatment with alkaline phosphatase removes the 5'-terminal phosphoric acid from the end of the plasmid generated by the endonuclease and prevents self-bonding of the plasmid DNA. As a result, it forms a circle, and the modification depends on the insertion of a DNA fragment containing a 5 'one phosphorylated terminus. The described method reduces the relative frequency of deformation to 1 to 10 ⁺⁴ or less in the absence of recombination.

The present invention is based on the fact that DNA ligase catalysis occurs between 5′-phosphate DNA residue and 3 ′ to hydroxyl DNA residue. If the terminal 5'-phosphate is removed, no binding reaction occurs. Three situations that can occur at which double-stranded DNA can be linked are shown in Table 1 below.

In Table 1, double stranded DNA is represented by straight parallel lines, each of which has hydroxyl (OH) or phosphoric acid (OPO ₃ H ₂ ). In the case of I, 5 'phosphate is at both reactant ends, resulting in both covalent bonds. In case II, only one of the strands to be bound has 5 'phosphate at the end, resulting in only one covalent single strand, leaving the other strand (strand not having 5' phosphate at the end) as a discontinuous single strand. . Each noncovalent strand is associated with a covalently bonded molecule by the interaction force between complementary base pairs and hydrogen bonds on opposite strands, as is well known in the art. In case III, none of the reactant ends have 5'-phosphate, so no binding reaction can occur.

Therefore, when no binding reaction is desired, the 5'-phosphate groups may be removed from the appropriate ends of the reactants to prevent the binding. Appropriate methods are used to remove only 5'-phosphate groups without damaging the DNA structure. Hydrolysis in which the alkaline phosphatase enzyme catalyzes is suitable.

The process described above is also useful when the linear DNA molecules are typically broken down into two subfragments using restriction endonuclease appeals and then reconstituted in their original arrangement. After refining these fragments separately, they can be recombined to reconstruct the desired arrangement. For this purpose, enzyme DNA ligase is used to catalyze the end-to-end reaction of DNA fragments [Sgarame-lla, V., Van de Sande, J.H., and Khorana, H.G., Proc. Natl. Acad. Sci USA 67, 1468 (1970).

If the sequences to be linked do not have blunt ends, ligase from E. coli is used [Modrich, P., and Lehman, I.R., J. Biol. Chem. 245,3626 (1970).

The efficiency of reconstructing the original sequence from the subdivision fragments produced by restriction endonuclease treatment is greatly increased by using a method that prevents reconstruction of an inappropriate sequence. To prevent this undesirable result, an array of uniformly lengthed cDNA fragments can be treated with a reagent that can remove the 5'-terminal phosphate group of the cDNA prior to disrupting homologous cDNAs with restriction endonucleases. As the enzyme, alkaline phosphatase is preferable. 5'-terminal phosphates are structural requirements for the subsequent ligation of DNA ligase used to reconstruct cleaved fragments. Therefore, residues without 5'-terminal phosphoric acid cannot be covalently linked. DNA segment fragments can only be bound at the terminal containing 5'-phosphate produced by restriction endonuclease digestion performed on isolated DNA fragments.

The most important of the above processes is the prevention of the formation of unwanted linking reactions, ie joining two fragments with opposite arrangements before and after instead of before and after. Other possible side reactions, such as dimerization and cyclization reactions, are caused by the reactions of Table 1, Case II above and cannot be prevented. These side reactions are physically separable, produce discernible products, and are almost nonsense because recombination in the opposite direction does not occur. To illustrate the above process, cDNAs genetically encoding murine insulin were isolated and recombined with the plasmid. These DNA molecules were used to modify E. coli X-1776. The variant was chosen to be grown on a medium containing tetracycline. One recombination plasmid DNA obtained from the modified cells was found to contain inserted DNA fragments about the length of about 410 nucleotides. Other recombinants isolated by similar processes were also obtained and analyzed. The inserted fragments were released from the plasmid by Hind III or HUS I endonuclease digestion and were treated with Maxam, A.M. And Gilbert, W., Proc. Natl. Acad. DNA sequences were analyzed by the method of Sci USA 74, 560 (1977). The nucleotide sequences of the inserted fragments were overlapping and found to contain regions of the entire genetic coding for I in murine proinsulin as well as 13 23 aminoacidosis of the prepeptide sequence. Composites with nucleotide sequences in this region are formed as follows.

In a similar manner, cDNA, which genetically encodes rat growth hormone, was isolated, recombined with plasmids and delivered to E. coli. After significant replication in E. coli, the nucleotide sequence of about 800 nucleotides in length was reisolated and found to contain a fully genetic sequence for RGH as well as the precursor peptide and the untranslated 5 'region.

The process just described is commonly used to isolate and purify genes from higher organisms, including humans, and to transfer these genes to or replicate in microorganisms. New recombination plasmids containing all or part of isolated genes have been described, and new microorganisms have been described so far that are not known at all and have genes from higher organisms as their genetic components. Examples that describe each step of the process of separating, purifying and delivering mouse insulin genes to E. coli are described in detail below to further clarify the features and utility of the present invention. The examples illustrate the recombination plasmid containing the mouse insulin gene, the mouse growth hormone gene, the human insulin gene and the genes of the human growth hormone, and the characteristics of the new microorganism containing the genes.

Example 1

Procedures described are extraction and isolation of rat insulin mRNA. The synthesis of DNA complementary to mRAN and the characteristics of this complementary DNA are described. To obtain purified islets of Langerhans cells, the anesthetized rat pancreas is filled with Hank's saline solution, which is possible by retrograde weeking to the pancreatic duct.

Hank salt solution is a standard salt solution mixture known in the art and can be purchased from a number of commercial sources such as Grand Island Biological Supply Company, Grand Island, New York. The pancreas was then cut and chopped in a 0 ° C. Hank solution and digested with clagenase and soybean trypsin inhibitor. All processes were conducted at 0-4 degrees Celsius unless otherwise noted. The conditions of the digestion were extremely important, and two 8 ml chopped rat pancreas in a total volume of Hank's solution were placed in a 30 ml glass test tube. All glass test tubes were pretreated with silicone. (Silicon: Silyclad, trade name, clay-Adams Division, Becton-Dickinson Inc., Parsippany. Using Mandl, I., Mackennan, JD and Howes, E. L., J. Clin. Invest. 32, 1323 (1943A), and 1 mg of soybean trypsin inhibitor from Signa Chemical Company, St. Louis, MO. The incubation was stirred for 25 minutes at a rate of 90 times per minute at 37 ° C. Continuous observation should be made to ensure that collagenase digestion proceeds to an optimal level. If the culture is too short, the Langerhans islet cells are incompletely released. If the culture is too long, the Langerhans islet cells begin to lyse. After incubation, the test tubes were centrifuged at 200 x G for 1 minute. The supernatant was discarded and the pellet was washed five times with Hank's solution. After the last centrifugation the pellet was suspended in 15 ml Picol (trade name, Pharmacia chelmical Company, Uppsala, Sweden) having a density of 1.85. Layered by adding 8 ml of picol with a density of 1.080 and 5 ml of picol with a density of 1.060, centrifuged in a wing rotor for 5 minutes at 500 × G of the test tube, followed by centrifugation at 2,000 × G for 5 minutes. Separated.

As a result of the process, the acinar cells remained at the bottom of the glass tube, and the Langerhans islet cells were inclined to form a layer between the two upper layers. The island of Langerhans contains contaminating gaglion cells, lymph nodes and connective tissue. Large contaminants were removed from the strip of materials in the strip. The remaining portions were placed under a dissecting microscope and, if contaminants were seen, removed using a micro pipette. The cell samples were diluted with Hank's solution and then centrifuged. The supernatant was drained and the cell pellet was stored frozen in liquid nitrogen.

Langerhans islet cells collected from 200 mice were homogenized in 4M thiocyanate guanidinium (Tridom, trade name, Fluka AG Chemische Fabrik, Buchs, Switzerland) containing 4 M of 1M β-metcaptoethanol buffered at pH 5.0. It was. 1.2 ml of 5.7 M CsCl containing 100 mM EDTA was poured into the homogenate, followed by centrifugation for 18 hours at 37,000 rpm in a SW 50.1 dipole at a Beckman ultracentrifuge at 15 degrees Celsius.

(Beckman Instrument Company, Fullerton, California) RNA was transferred to the bottom of the test tube.

The total RNA samples were isolated by chromatography on oligo (dT) -cellulose according to the methods of Aviv, H., and Leder, P. described above. DJ Beard, Life Science Inc., St. Chicken myeloid leukemia virus reverse transcriptase provided by St. Petersburg, Florida was used to transcribe total polyadenylate RNA isolated from the islets of rats to cDNA. These reactions were 50-200 per mole of 50 mM Tris-HCl, pH8.3, 9 mM MgCl ₂ , 30 mM NaCl, 20 mM beta-mercaptoethanol, 1 mM each of three non-radioactive deoxyribonucleoside triphosphates. 250 μM of the fourth deoxynucleoside triphosphate labeled Curiein alpha-32 _p , oligo-dT 12-18 20ug / ml, 100ug / ml polyadenylate RNA and reverse transcriptase from Collaborative Research, Waltham, Massachusetts It was fleshed at 200 units / ml. This mixture was incubated for 15 minutes at 45 degrees Celsius. After adding EDTA-NA _{2 up} to 25 mM, the solution was extracted with an equal volume of water saturated phenol and then 0.3 cm high by 10 cm in diameter containing 10 mM Tris-HCl, pH9.0, 100 mM NaCl, 2 mM EDTA. The liquid phase was chromatographed on Sephadex G-100 100 column. The nucleic acid eluted in the pore portion was precipitated with ethanol after 0.25M addition of ammonium acetate pH 6.0. The precipitates were collected by centrifugation, and the pellets were dissolved in 50 μl of freshly prepared 0.1M NaOH and incubated for 20 minutes at 70 degrees Celsius to hydrolyze the RNA. To this mixture was neutralized with the addition of 1 M sodium acetate pH 4.5, and the ³² P-cDNA product was precipitated with ethanol and dissolved again in water. Aliquots of single stranded cDNA were analyzed on natural polyacrylamide gels by the method of Dingman, CW, and Peacock, AC, Biochemistry 7,659 (1968).

The gel was dried and ³² P DNA was detected by radiograph using a Kodak No-Screen NS-2T film (trade name, Eastman Kodak Corporation, Rochester, New York). As determined by the electrophoresis mode, the cDNA was dispersed in various types, and compared with known standards, it contained at least one important cDNA, about 450 nucleotides in length.

Example 2

The synthesis and characterization of double-stranded cDNAs containing an array of murine insulin is described. The single stranded cDNA product of Example 1 was treated with reverse transcriptase to synthesize complementary strands. The reaction mixture is 50mM Tris _{-HCl, pH 8.3, 9mM MgCl 2} , 10mM dithiothreitol, by aryloxy having each interruption of the three kinds of labels ribonucleoside triphosphates 50mM, the specific activity per mmol of ^{32-alpha-1-10} Curie Nucleoside triphosphate labeled 1 P, 50 μg / ml cDNA and 220 units / ml reverse transcriptase were included. The reaction mixture was incubated for 120 minutes at 45 degrees Celsius. The reaction was stopped by adding 25 mM of EDTA-Na ₂ , extracted with phenol, chromatographed on Sephadex G-100, and ethanol precipitation was performed. An aliquot of the reaction product having 500 cpm to 1000 cpm was analyzed by gel electrophoresis as described in Example 1. Comparing the standard sample, a heterodisperse band with a center of about 450 nucleotides in length was observed. The DNA reactive aliquots of Examples 1 and 2 were each treated by digesting with excess restriction endonuclease Hae III and analyzed by gel electrophoresis in a similar manner. Both of these products were digested with endonucleases and two radio bands were observed on gel electrophoresis. The bands resulting from degrading the double stranded cDNA show degradation products of the same length as the bands resulting from digesting the single stranded cDNA.

Example 3

The blunt end binding of the Hind III decanucleotide conjugate and the murine Langerhans islet double stranded cDNA of Example 2 will be described. The double-stranded reaction product of Example 2 at concentrations of 2-5 μg / ml was treated with 30 units of Sl nuclease with 1200 / unit ml activity obtained from Miles Laboratories in Elkhart, Indiana, and 0.03 at 22 degrees Celsius. Incubated for 30 minutes in sodium M acetate, pH4.6, 0.3M sodium chloride, 4.5mM ZnCl ₂ and then further incubation at 10 degrees Celsius. Tris base is added until the final concentration is 0.1M, and 25 mM of EDTA is added. Von Ehrenstein, G., Methods in Enzymology, SP Colowick and NO Kaplan, Eds., Vol. Digestion was stopped by adding 40 μg / ml E. coli tRNA prepared by the method of 12A, p588 (1967). The reaction mixtures were extracted with phenol, chromatographed with Sephadex G-100, and ³² P-cDNA eluted at the pores was precipitated with ethanol. In this way, cDNA molecules having base-pair ends necessary for chemically synthesizing decanonide and blunt end bonds can be obtained with high yield. Hind III Decamers Scheller, RH, Dickerson. RE, Boyer, HW, Riggs, AD and Itakura, K., Science 196, 177 (1977). The binding of the Hind III decamer to cDNA was 66 mM Tris-HCl, pH 7.6, 6.6 mM MgCl ₂ , 1 mM ATP, 10 mM dithiothreitol, 3 mM Hind III decamer with 10 ⁵ cpm / pmol at about 14 degrees Celsius. Incubate for 1 hour in 500 units / ml of T ⁴ DNA ligase. The reaction mixture was then heated to 65 degrees Celsius for 5 minutes to inactivate ligase. KCl was added to a final concentration of 50 mM, beta-mergacethanethanol to a final concentration of 1 mM, and EDTA to a final concentration of 0.1 mM, followed by 150 units / ml of HSU I or Hind III endonuclease at 37 degrees Celsius for 2 hours. Digested.

Hind III and Hae III endonucleases are commercially available from the New England Institute of Biology, Beverly, Massachusetts. This reaction product was analyzed by gel electrophoresis as in Example 1, and a peak corresponding to about 450 nucleotide sequences with fragments of the degraded Hind III decamer was observed.

Example 4

The characteristics after formation and replication of the recombinable plasmid will be described. Rodriguez, RL Boliver, F., Goodm-an, HM, Boyer, HW, and Betlach, M., in ICN-UCLA Symoposium on Molecular and Cellular Bio-logy, DP Wierlich, WJ Rutter, and CF Fox, Eds., Plasmid pMB-9 DNA was prepared as described in (Academic Press, New York 1976) pp.471-477. This plasmid pMB-9 DNA was digested at the Hind III restriction site using Hsu I endonuclease, and then treated with alkaline phosphatase, BAPF type. This alkaline phosphatase is prepared from Worthington Biochemical Corporation. It was purchased from Freehold, New Jersey. This enzyme is present at about 0.1 units / μg DNA in the reaction mixture. This reaction mixture was 65 degrees Celsius in 25 mM Tris-HCl. After culturing for 30 minutes while maintaining pH8, phenol was extracted to remove phosphatase. After ethanol precipitation, the plasmid DNA treated with phosphatase was added to the cDNA containing the Hind III binding end in a molar ratio of 3 moles of plasmid to 1 mole of cDNA. This mixture was incubated for 1 hour in 66 mM Tris, pH 7.6, 6.6 mM MgCl ₂ , 10 mM dithiothreitol, and 1 mM APT in the presence of 50 units / ml T4 DNA ligase at 14 degrees Celsius.

The binding mixture was added directly to the suspension of E. coli X-1776 cells prepared for transformation as follows. Cells were about 2 × 10 ⁸ cells / ml in 50 ml of medium containing tryptone 10 g / 1, yeast extract 5 g / 1, NaCl 10 g / 1, NaOH 2 mM, diamino pimelic acid 100 ug / ml and thymine ⁴⁰ μg / ml Cultured at 37 degrees Celsius until grown to a cell density of. The cells were collected by centrifugation for 5 minutes at a rate of 5,000 × G at 5 degrees Celsius, then resuspended in 20 ml of chilled 10 mM NaCl and centrifuged as before to contain 75 mM CaCl 2, 140 mM NaCl and 10 mM Tris pH7.5. After resuspended in 20ml of the typical buffer solution and left for 5 minutes on ice. Cells were then centrifuged and resuspended in 0.5 ml typical buffer. Typical was performed by mixing 100 μl of cell suspension with 50 μl of recombinable DNA (1 μg / ml). The mixture was incubated for 15 minutes at 0 degrees Celsius and then incubated for 4 minutes at 25 degrees Celsius and then for 30 minutes at 0 degrees Celsius. Cells were then transplanted into agar plate medium to grow under selected conditions.

Recombinant plasmids were screened with 5 μg / ml tetracycline for typical Hind III sites.

A screened recombinant named PAU-1 was isolated. Zoflavin samples of 2 μg-5 μg DNA isolated from PAU-1 were digested with excess Hsu I endonuclease. EDTA-Na ₂ 10 mM and sugar 10% w / v (ie weight to volume) final concentration were added and the mixture was then dissolved on an 8% polyacrylamide gel.

DNA was found at a position corresponding to a length of about 410 base pairs. In a similar experiment, plasmid PBR-322 was used as the delivery medium. All conditions are as described above, except that only the final selection of the recombinant strain is carried out in a plate medium containing 20 μg / ml of ampicillin.

Example 5

DNA isolated from PAU-1 as described in Example 4 was further purified by electrophoresis on 6% polyacrylamide gel. After eluting from the gel, DAN was labeled by incubating with gamma-32P-ATP and an enzyme polynucleotide kinase under the conditions described by Maxam and Gilbert described above. The enzyme catalyzes the transfer of radioactive phosphate groups from gamma- ³² P-ATP to the 5'-end of DNA. This enzyme was obtained from colon colonies by the method of Panet, A., et al., Biochemistry 12, 5045 (1973). The labeled DNA was thus digested by Hae III endonucleases as described in Example 2, and two labeled fragments, polyacrylamide gels under the conditions described in Example 1, with about 265 and 135 base pairs, respectively Phase was separated. The separated fragments were subjected to specific degradation reactions according to the method of Maxam and Gilbert, and analyzed for alignment. The arrangement in Table 1 is based on the composite of findings from this series of experiments and finding a similar series of CDNAs using plasmid mediators derived from Cole El such as pMB-9 and pBR-322.

The arrangement between the 5 'end of the sequence and 50-120 nucleotides in length is not clear and the length of the poly dA segment at the 3'-end varies. This arrangement represents the best information currently available, and is understood to be more detailed as research continues, and in some ways requires some modification.

The corresponding amino acid sequence of rat proinsulin I begins at the tribase position indicated by 1 and ends at the tribasic position indicated by 86 and remains somewhat unclear as to the underlined arrangement.

TABLE 1

Example 6

As described in Examples 1-4, isolation and purification of the nucleotide sequence that genetically encodes human insulin and insertion into the plasmid may result in the human pancreas from a suitable source, such as a donated pancreas or a clean body, or human insulinoma. It begins by separating the tissues. As described in Example 4, microorganisms having nucleotide sequences that genetically encode human insulin A chains and B chains are produced.

Known amino acid sequences for the A chain of human insulin are as follows:

Known amino acid sequences for the B chain of human insulin are as follows.

The amino acid sequence is numbered from the terminal with free amino groups (see Smith, L.F., diabetes 21 (suppl. 2) 458 (1972)).

Example 7

Regarding the isolation and purification of DNA having a complete structural gene sequence for RGH, the synthesis of the delivery media containing the complete structural gene for RGH and the formation of a microbial strain containing the gene for RGH as part of the gene structure are described together. .

If it contains a non-human gene, U.S. Federal safety regulations do not require the separation of cDNA to a higher degree of purity as required by human cDNA. Thus, by separating electrophoretically separated DNA at about 800 base pairs in length, as determined from known amino acid lengths of RGH, cDNAs containing complete RGH structural genes can be isolated. Cultured rat pituitary cells, subtypes of the GH-1 cell line purchased from the American Type Culture Collection are used as a source of RGH mRNA (see Tashjan, A.H., et al., End-ochrinology 82, 342 (1968)).

For these cells, when grown under normal conditions, growth hormone mRNA is only 1-3% of the total poly-A containing RNA. However, thyroid hormones and glucocorticoids acted together to raise mRNA levels of growth hormones above the mRNA levels of other cells. Suspension cultures yielded RNA from cells grown to 5 × 10 ⁸ and incubated production of growth hormone by incorporating 1 mM dexamethasone and 10 mM-triiothytyronine in the medium for 4 days prior to cell collection. As described elsewhere, polyadenylation RNA was isolated from the cytoplasmic membrane of cultured cells [Martial, JA, Baxter, JD, Goodman, HM aud Seeburg, PH, Proc. Nat. Acad. Sci. USA 74, 1816 (1977), and Bancroft, FC, Wu, G, and Zubay, G., Proc. Nat. Acad. Sci. USA 70, 3646 (1973).

MRNA was further purified and transcribed into double-stranded cDNA as described in Examples 1,2 and 3 above. After sorting by gel electrophoresis, a clear band corresponding to DNA with about 800 base pairs in length was faintly observed.

Complete cDNA transcribed from the mRNA of cultured pituitary cells was treated with Hha I endonuclease and subjected to electrophoresis to obtain two important DNA fragments corresponding to about 320 nucleotides (fragment A) and 240 nucleotides (fragment B). did. Analysis of the nucleotide sequences of fragment A and fragment B as described in Example 5 showed that these fragments were compared to other known growth hormone sequences and based on the probable RGH amino acid sequence data and the coding region for RGH. Proved to be part of [Wallis, M. and Davies, RVN Growth Hor-mone And Related Peptides (Eds., Copecile, A. and Muller, EE), pp. 1-14 (Elsevier, New York, 1976), and Dayhoff, MO, Atlas of Protein Sequence and Structure, 5, suppl . 2, pp. 120-121 (Na-tional Biomedical Research Foundation, Washinton, D.C., 1976).

As described above, the treatment of 800 base pairs of double stranded cDNA isolated by electrophoresis with Hha I endonuclease revealed two fragments with lengths corresponding to A and B fragments between the major degradation products.

Since about 800 base pairs of RGH-cDNA are not purified by restriction endonuclease treatment, DNA must be processed to remove unpaired single stranded residues. To remove these residues, 25 μl liquid containing 60 mM Tris-HCl, 8 mM MgCl ₂ , 10 mM β-mercaptoethanol, 1 mM ATP and 200 μM each of dATP, dTTP, dGTP and dCTP before separation by electrophoresis. Treatment at The mixture was incubated for 10 minutes with 1 unit of E. coli DNA polymerase I at 10 degrees Celsius to remove the 3 'protruding end and fill the 5' protruding end by exonucleolysis. DNA polymerase I is commercially available from Boehringer-Mannheim Biochemicals, Indianapolis, Indi-ana.

As described in Example 3, about 800 base pairs of RGH-cDNA were treated with a chemically synthesized Hind III conjugate. Plasmid pBR-322 having a single Hind III site present in the ampicillin resistance gene and the tetracycline resistance gene was previously treated with Hind III endonuclease and alkaline phosphatase as described in Example 4. The treated plasmids were bound to 800 base pairs of RGH-cDNA in the DNA ligase reaction mixture as described in Example 3. The ligase reaction mixture was used to model a suspension of Escherichia coli X-1776 cells treated as described above in Example 3. Recombinant clones are distinguishable because they grow on nutrient plates containing ampicillin and cannot grow on 20 μg / ml tetracycline.

Ten such clones were obtained, all carrying a plasmid with an insert of about 800 base pairs liberated by Hind III digestion.

RGH-DNA of 800 base pairs was sufficiently separated from recombinant strain pRGH-1, and its nucleotide sequence was determined as described in Example 5. In this case the nucleotide sequence included not only the 26 amino acid sequence found in the precursor protein of the growth hormone before secretion but also the portions of the 5 ′ untranslated region of RGH. Messengers of mRNA sequences deduced from gene sequences are shown in Table 2. As described above by Wallis and Davides, the predicted amino acid sequences are partial amino acid sequences and 1 and 8 positions of the mouse growth hormone containing residues 1-43, 65-69, 108-113, 133-143 and 150-l9O. Except that it matches.

TABLE 2

A single stranded DNAnucleotide sequence containing a complete sequence that genetically encodes RGH. Corresponding amino acids are indicated with position numbers for the amino termini. A negative numbered amino acid indicates a pre-growth hormone sequence. The corresponding mRNA sequences are identical, but only T is replaced by U in the mRNA.

Example 8

The isolation and purification of the complete gene sequence genetically encoding HGH and the synthesis of recombination plasmids containing the complete structural gene for HGH and the generation of microorganisms with the complete structural gene for HGH will be described. .

Isolation of HGH mRNA is performed in the same manner as described in Example 7, except that the biological raw material is a human pituitary tumor tissue. Surgical removal of five good pituitary tumors, followed by rapid freezing in liquid nitrogen, cut and dissolved from 0.4g to 1.5g, respectively, and 4M thiocyanoic acid at 4 degrees Celsius containing 1M meractoethanol buffered at pH5.0 Homogenized in anidinium. The homogenate was poured into 1.2 ml of 5.7 M CsCl containing 100 mM EDTA, and at 15 degrees Celsius for 18 hours at a speed of 37,000 rpm in a SW 50.1 rotor of Beckman Instrument Company, Fullerton, California. Centrifugation was performed. RNA was collected at the bottom of the test tube. For further purification, oligo-dT columns and sugar gradient sedimentation methods were used as previously described in Examples 1,2 and 3. About 10% of the RNA thus isolated is used to genetically encode growth hormone, which has been found by mixing radioactive amino acid precursors with anti-growth hormone precipitates in a cell-free translation regimen derived from wheat embryos [Roberts, B.E. and Patterson, B. M., Proc. Nat. Acad. Sci. USA 70, 2330 (1973).

HGH-cDNA was prepared by carrying out as described in Example 7. HGH-cDNA is sorted by gel electrophoresis and selected for gastrointestinal strains to move the material to a position corresponding to about 800 nucleotides in length. The selected portion was treated with DNA polymerase I as described in Example 6 and then Himd III conjugates were added to the residue. DNA ligase was then used to recombine the cDNA with plasmid pBR-322 treated with alkaline phosphatase. E. coli X-1766 is typical of recombinant DNA and strains containing HGH DNA are selected. Strains containing HGH-DNA were grown by the prepared amount, and HGH-DNA was separated therefrom to determine their nucleotide sequence. Strained HGH-DNA was found to contain nucleotides that genetically encode the complete amino acid sequence of HGH. The first 23 amino acids of HGH

The rest of this arrangement is shown in Table 3.

TABLE 3

Nucleotide sequence of HGH-DNA single strand. The number is the amino acid sequence number of the HGH starting at the amino terminus. The DNA sequences shown in the table correspond to the mRNA sequences for HGH, but in the mRNA only T is replaced by U.

The method of the present invention for the first time branched a nucleotide sequence that genetically encodes a specific regulatory protein from higher organisms, such as vertebrates, so that the genetic information contained in the sequence can be transferred to a microorganism that can be infinitely replicated. The methods described apply to the isolation and purification of human insulin genes and transplanting them into microorganisms. Similar methods can be used to isolate human growth hormone genes and other protein genes, transfer them to microorganisms, and replicate there. Several new recombination plasmids have been identified, each containing sequences with the structure of higher organism genes in their eye-nucleotide sequences and transcribed from higher organism genes. Several new microorganisms have been known that are modified to contain nucleotide sequences, each of which has the structure of a higher organism gene, and are transcribed from the higher organism gene. The practice of the present invention has been demonstrated by grafting a mouse proinsulin I gene into an E. coli strain, and grafting a mouse growth hormone gene into an E. coli strain. The arrangement of the major parts of the transplanted gene was determined in each case and this arrangement was determined by reference to the genetic code generally known for all types of organisms and the complete amino acid sequence of each of the mouse proinsulin or mouse growth hormone. Has been found to genetically encode.

Although the present invention has been described with reference to specific embodiments, it can be used in various modifications. The application of the present invention is deemed to be generally within the scope of the present invention, and any modifications, applications or adaptations of the present invention which seek the principles of the present invention are not described herein but are known in the art related to the present invention or Or commonly practiced ones, which are apparent to those skilled in the art. The invention is not limited to the scope claimed by the claims.

Claims (1)

As shown and described in detail herein, in the method of preparing a DNA delivery medium containing a nucleotide sequence that genetically encodes a specific protein of rats and humans, the genetic coding of a specific protein from mice and humans capable of producing a specific protein To separate the cells containing the mRNA, extract mRNA from the isolated cells in the presence of a ribonuclease inhibitory composition to prevent ribonuclease degradation of the mRNA, with little protein, DNA and other RNA By separating only the mRNA, one strand synthesizes a double-stranded cDNA having a nucleotide sequence complementary to that of the mRNA, thereby producing a cDNA having a nucleotide sequence that genetically encodes a specific protein, and reacting the end of the reactant to bind the double-stranded cDNA. New gene that genetically encodes a specific protein by adding DNA transfer mediator The method of DNA transfer mediator is characterized by generating a DNA transfer mediator having a nucleotide sequence encoding a specific protein for genetic By combining double-stranded cDNA with a DNA transfer mediator having Leo suited arrangement.

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