CH642680A5 - DNA transfer vector, process for its preparation and its use - Google Patents

Description

The present invention relates to a DNA transfer vector which contains in its nucleotide sequence a sub-sequence of the structure of a gene of a higher organism. These new DNA transfer vectors can have, for example, a nucleotide sequence which contains the genetic code for a specific protein, for example insulin or growth hormone.

Furthermore, the present invention relates to a method for producing the DNA transfer vector according to the invention and its use for producing genetically modified microorganisms.

When a DNA transfer vector is produced which contains in its nucleotide sequence a sub-sequence which has the genetic code for a specific protein, this specific nucleotide sequence is first isolated and then DNA is synthesized with this specific nucleotide sequence. If the DNA transfer vector according to the invention is transferred into a microorganism as the host, the DNA is replicated in this.

In the following, in particular working procedures for the isolation of insulin genes and growth hormone genes, their purification, transfer and replication in a microbial host and their subsequent characterization are dealt with.

A preferred DNA transfer vector according to the invention is a plasmid in which a specific nucleotide sequence, which originates from a higher organism, is inserted. These plasmids can be used to produce genetically modified microorganisms that contain the specific nucleotide sequence from the higher organism as part of their genetic makeup. Bacteria are preferred microorganisms for the modification of which the DNA transfer vectors according to the invention are used.

The following symbols and abbreviations are used in the present description.

DNA = deoxyribonucleic acid RNS = ribonucleic acid cDNS = complementary DNA (enzymatically synthesized from an mRNA sequence)

mRNA = messenger RNA

tRNS = transfer RNS

dATP = deoxyadenosine triphosphate dGTP = deoxyguanosine triphosphate dCTP = deoxycytidine triphosphate

A = adenine

T = thymine

G = guanine

C = cytosine

Tris = 2-amino-2-hydroxy-ethyl-1,3-propanediol EDTA = ethylenediamine tetraacetic acid ATP = adenosine triphosphate TTP = thymidine triphosphate

The biological meaning of the base sequence of DNA is that of storing genetic information. It is known that the base sequence of the DNA is used as a code that determines the amino acid sequence of all proteins produced by the cell. In addition, parts of the sequence are used for regulatory purposes, to control the time of manufacture and the amount of each protein. The way these controlling elements work is only partially recognized. Finally, the base sequence in each strand serves as a template for the replication of the DNA that accompanies cell division.

The way in which the information from the base sequence of DNA is transferred to the amino acid sequence of proteins is a fundamental process that is universal for all living organisms.

It could be proven that every amino acid usually present in proteins is determined by the sequence of one or more trinucleotides or triplets. Each protein thus corresponds to a segment of the DNA with a trilet sequence that corresponds to the amino acid sequence of the protein. The following table illustrates the genetic code.

Genetic code

Phenylalanine (Phe) «Leucine (Leu) Isoleucine (Ile) Methionine (Met) Valine (Val)

Serine (Ser) and Proline (Pro) Threonine (Thr) Alanine (Ala) Tyrosine (Tyr) termination signal 55 termination signal

TTK

XTY

ATM

ATG

GTL

ORS

CCL

ACL

GCL

TAK

TAJ

TGA

Histidine (His) CAK

Glutamine (Gin) CAJ Asparagine (Asn) AAK Lysine (Lys) AAJ

Aspartic Acid (Asp) GAK Glutamic Acid (Glu) CAJ Cysteine (Cys) TGK

Tryptophan (Try) TGG Arginine (Arg) WGZ

Glyin (Gly) GGL

Key: Each triplet of three letters represents a tri-nucleotide of the DNA, with a 5 'end on the left and a 3' end on the right. The letters stand for the purine or 60 pyrimidine bases that make up the nucleotide sequence:

A = Adenine G = Guanine C = Cytosine 65 T = Thymine X = T or C if Y = A or G X = C if Y = CoderT Y = A, G, C or T if X = C

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Y = A or G, if X = TW = C or A, if Z = A or GW = C, if Z = C or TZ = A, G, C or T, if W = CZ = A or G, if W = A QR = TC, if S = A, G, C or T QR = AG, if S = T or CS = A, G, C or T, if QR = TC S = T or C, if QR = AG J = A or GK = T or CL = A, T, C or GM = A, C or T

The first step in the biological process of translating the nucleotide sequence into an amino acid sequence is the so-called transcription. In this stage, a segment of the DNA with the sequence responsible for the protein to be produced is transferred to an RNA. RNA is a polynucleotide similar to DNA, but with ribose instead of deoxyribose and uracil instead of thymine. The bases of the RNA are capable of the same type of base pairing as the bases of the DNA. The RNA resulting from the transcription of a DNA nucleotide sequence is therefore complementary to this copied sequence. This RNA is called mes-senger RNA (mRNA) because of its intermediate position between the genetic apparatus and the apparatus for protein synthesis of the cell.

In the cell, the mRNA serves as a template in a complex process in which numerous enzymes and organelles are involved within the cell and which leads to the synthesis of specific amino acid sequences. This process is called translation.

There are often further stages which serve to generate a functional protein from the amino acid sequence which is produced during translation. One example is the production of insulin.

The direct precursor of insulin is a polypeptide that contains proinsulin, which contains the two insulin chains A and B via another peptide C, see Steiner, D.F. Cunningham, D., Spigelman, L. and Aten, B., Science 157, 697 (1967). It has recently been reported that the first translation product of insulin mRNA is not proinsulin itself, but a pre-proinsulin that contains more than 20 additional amino acids at the amino end of the proinsulin, see Cahn, S.J. Keim, P. and Steiner, D.F., Proc. Nati. Acad. Be. USA 73, 1964 (1976), and Lomedico, P.T. and Saunders, G.F. Nucl. Acids Res. 3,381 (1976). The structure of the pre-proinsulin molecule can be represented schematically as follows: NH2- (pre-peptide) -B-chain- (C-peptide) -A-chain-COOH.

Numerous proteins of importance for medicine or research are found or produced in the cells of higher organisms such as vertebrates. These include, for example, the hormone insulin, other peptide hormones such as growth hormone, proteins involved in regulating blood pressure and numerous enzymes that are important for technical, medical or research purposes. It is often difficult to extract these proteins from the organism in usable amounts by extraction, especially in the case of proteins of human origin. There is therefore a need for techniques by which such proteins are produced in reasonable amounts by cells outside the organism. In some cases it is possible to obtain suitable cell strains using tissue culture techniques. However, the growth of cells in tissue cultures is slow, the medium is expensive, the conditions have to be carefully controlled and the yields are low. It is also often difficult to obtain a grown cell strain with the desired differentiated properties.

In contrast, microorganisms such as bacteria can be grown relatively easily in chemically defined media. The fermentation technology is already highly developed and easy to control. The organisms grow rapidly and high yields are possible. In addition, certain microorganisms have already been genetically characterized and are indeed among the best characterized and recognized organisms.

It would therefore be very desirable to be able to transfer a gene code for a protein of medical importance from an organism that normally produces the protein to a suitable microorganism. In this way, the protein could be produced by the microorganism under controlled growth conditions and obtained in desired amounts. It is also possible that the total cost of producing the desired protein could be significantly reduced by such a process. The ability to isolate and transfer the gene sequence that determines the production of a particular protein into a microorganism of a precisely known genetic structure also opens up the valuable opportunity for research to investigate how the synthesis of such a protein is controlled and how the protein is processed after synthesis becomes. Isolated genes can also be modified so that they encode protein variants with other therapeutic or functional properties.

Surprisingly, it was found that the desired goals can be achieved by a DNs transfer vector which contains a sub-sequence with the structure of a gene of a higher organism in its nucleotide sequence. Such DNA transfer vectors can be used to modify a microorganism to contain this sequence.

An object of the present invention is therefore a DNA transfer vector which contains in its nucleotide sequence a sub-sequence with the structure of a gene of a higher organism, which was created by transcription on a gene of a higher organism.

Preferred DNA transfer vectors according to the invention are those which have a nucleotide sequence which encodes the A chain or the B chain of human insulin.

Other preferred DNA transfer vectors according to the invention are those which, in their nucleotide sequence, have a sub-sequence with the structure of a gene of a higher organism which codes for growth hormone, in particular such an amino acid sequence which codes for human growth hormone.

Another object of the present invention is a method for producing the DNA transfer vector according to the invention which contains in its nucleotide sequence a sub-sequence with the structure of a gene of a higher organism, which is characterized in that a) isolating cells which contain mRNA encoding the desired protein b) extracting the mRNA from the cells, wherein an RNase inhibitor is present which prevents RNase-induced decomposition of the mRNA, or in the absence of such an RNase inhibitor, c) isolating an mRNA which is in the is essentially free of proteins, DNA and other RNA,

d) a double-stranded cDNA is synthesized in which one strand has a nucleotide sequence which is complementary to that of the mRNA and a cDNA is produced which has a nucleotide sequence which encodes the desired protein

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e) provides a DNS transfer vector which has suitable reactive ends which are capable of being connected to one another or connected to the double-stranded DNA and f) connecting the DNS transfer vector to the double-stranded cDNA which has a nucleotide sequence encoding the desired protein in a DNA ligase catalyzed reaction to give a DNA transfer vector which has in its nucleotide sequence a sub-sequence encoding the desired protein.

Furthermore, the present invention relates to the use of the DNA transfer vector according to the invention which contains in its nucleotide sequence a sub-sequence with the structure of a gene of a higher organism, which was created by transcription to a gene of the higher organism in order to modify a microorganism that it contains this sequence.

The previous knowledge of enzyme-catalyzed reactions is explained below:

Reverse transcriptase catalyzes the synthesis of the DNA complementary to an RNA template in the presence of the RNA template, an oligo-deoxynucleotide primer and the four deoxynucleoside triphosphates dATP, dGTP, dCTP and TTP. The reaction is initiated by the non-covalent binding of the oligodeoxynucleotide primer to the 3 'end of the mRNA, followed by the stepwise addition of the appropriate deoxynucleotides, according to the base pairing rule, to the 3' end of the growing chain. The molecule obtained as a product can be described as hairpin-shaped, it contains the starting RNA and a complementary single strand of DNA. Reverse transcriptase can also catalyze a similar reaction in which a single strand of DNA serves as a template, in which case the product is a hairpin-shaped double strand of DNA with a single strand of DNA connecting a pair of ends; compare Aviv, H. and Leder, P., Proc. Nati. Acad. Be. 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 are capable of hydrolysing phosphodiester bonds in double-stranded DNA, whereby the sequence of the DNA strand is cleaved. If the DNA is in the form of a closed loop, it is converted into a linear form. The main feature of an enzyme of this type is that the hydrolytic effect only occurs where there is a specific nucleotide sequence. This sequence is called the recognition site (cleavage site) of the restriction endonuclease. Restriction endonucleases were isolated from various sources and their nucleotide sequence and recognition sites were determined. Some restriction endonucleases hydrolyze the phosphodiester bonds in both strands in the same place, so that blunt ends are formed. Others catalyze the hydrolysis of bonds a few nucleotides apart, creating single-stranded regions at each end of the cleaved molecule. These single-stranded ends are self-complementary, therefore cohesive, and can be used to re-bind the hydrolyzed DNA. Since each DNA susceptible to cleavage by such an enzyme must contain the same recognition site, the same cohesive ends are produced, so that it is possible to link heterologous DNA sequences with other, similarly obtained sequences; see Roberts, R.J., Crit. Rev. Biochem. 4,123 (1976). The recognition sites are relatively rare, but the general usability of the restriction endonucleases has been greatly expanded by the chemical synthesis of double-stranded oligonucleotides with the sequences of the recognition sites. Virtually any DNA segment can thus be coupled to a different segment simply by appending the corresponding restriction oligonucleotide to the ends of the molecules and subjecting the product to the hydrolytic action of the corresponding restriction endonuclease, whereby the required cohesive ends are obtained; see Heynecker, H.L. Shine, J. Goodman, H.M. Boyer, H.W. Rosenberg, J., Dickerson, R.E. Narang, S.A., Itakura, K., Lin, S. and Riggs, A.D., Nature 263, 748 (1976), and Scheller. R.H. Dickerson, R.E. Boyer, H.W. Riggs, A.D. and Itakura, K., Science 196, 177 (1977).

SI endonuclease is an enzyme which is capable of hydrolysis of the phosphodiester bonds of single-stranded DNA or single-stranded intermediates in otherwise double-stranded DNA; see Vogt, V.M., Eur. J. Biochem. 33, 192 (1973).

DNA ligase is an enzyme that causes the formation of a phosphodiester bond between two DNA segments with a 5'-phosphate or a 3'-hydroxyl, which are formed, for example, by two DNA fragments that are held together by cohesive ends. The normal function of the enzyme is presumably to connect single-stranded intermediates in an otherwise double-stranded DNA molecule. However, under appropriate conditions, DNA ligase can catalyze the blunt end connection where two blunt end molecules are covalently bound; see Sgaramella, V., Van de Sande, J.H., and Khorana, H.G., Proc. Nati. Acad. Sci., USA 67, 1468 (1970).

Alkaline phosphatase is an enzyme that hydrolyzes phosphate ester bonds including the 5'-terminal phosphate groups in DNA.

Another step in the overall process to be described is the insertion of a specific DNA fragment into a DNA vector, for example a plasmid. A plasmid is any autonomous DNA-replicating unit that can be present in a microbial cell next to the genome of the host cell. A plasmid is not genetically linked to the host cell chromosomes. Plasmid deoxyribonu-small acids are double-stranded ring molecules, generally with molecular weights on the order of a few million, although in some the molecular weight is greater than 108. They usually represent only a small percentage of the total DNA of the cell. The plasmid DNA can generally be separated from the DNA of the host cell due to the large size difference. The plasmids can replicate regardless of the rate of host cell division, and in some cases their rate of replication can be affected by the experimenter by changing the growth conditions. Although the plasmid is in the form of a closed ring, a DNA segment can be introduced into the plasmid artificially, a recombined plasmid having an enlarged molecular size being formed without its ability to replicate or express any genes of the plasmid being significantly impaired. The plasmid therefore serves as a useful vector for transferring a DNA segment into a new host cell. Plasmids suitable for recombining DNA typically contain genes that may be useful for reasons of selection, for example genes of drug resistance.

To illustrate the practice of the invention, the isolation and transfer of rat insulin gene are described in detail. In this case, insulin was chosen because of its central importance from the perspective of clinical medicine and from the perspective of basic research. The disclosed method can be readily applied by one of ordinary skill in the art to isolating the insulin gene from other organisms, including humans.

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Insulin was first isolated in 1922. This hormone is known to be used today to treat diabetes. Slaughterhouses supply cattle and pigs with pancreas as a source of insulin, but hormone depletion is developing as the number of diabetics worldwide increases. In addition, some diabetics develop an allergic reaction to bovine and pig insulin. The possibility of producing human insulin in quantities that meet global demand would therefore be extremely desirable. The production of human insulin in bacteria is a technique that could be used to achieve this goal. Progress in this direction has so far been thwarted by the fact that there was no technique for introducing the insulin gene into bacteria. The present invention provides such a technique.

The ability to produce a DNA with a specific sequence that provides the genetic code for a specific protein allows the nucleotide sequence to be modified by chemical or biological means in such a way that the protein ultimately produced is also modified. In this way, for example, one could produce a modified insulin that is tailored to special medical needs. The genetic ability to produce an insulin-like amino acid sequence with the essential functional properties of the insulin can therefore be transferred to a microorganism. Similar considerations apply to the case of growth hormones.

The ability to transfer the genetic code for a certain protein, which is required in the normal metabolism of a higher organism, into a microorganism, such as a bacterium, opens up new possibilities for the production of such proteins. This also creates the possibility of multiplying or replacing such proteins by proteins which have been produced by microorganisms which have been modified by the use of DNA transfer vectors according to the invention. The proteins produced in this way can be used where the ability of the higher organism to produce these proteins has been damaged.

It is also possible to establish a symbiosis between microorganisms which have been modified by the DNA transfer vector according to the invention and people with chronic or acute deficiency diseases. For this purpose, these genetically modified microorganisms can be implanted or otherwise incorporated to compensate for the pathological metabolic error.

In the method according to the invention for producing the DNA vector according to the invention, a specific nucleotide sequence containing genetic information is isolated and DNA is synthesized using this specific nucleotide sequence. When the DNA transfer vector according to the invention is used, it is then transferred into a host microorganism and this microorganism is modified in the desired manner.

The method according to the invention and the use according to the invention are now explained in more detail using the example of specific DNA sequences which are transferred in a microorganism, specifically into a bacterium.

These specific examples are used to explain the production of DNA transfer vectors which have a sub-sequence of the gene for rats pre-proinsulin, or the gene for rat growth hormone or the gene for human growth hormone in their nucleotide sequence.

According to the procedures described here, it can be assumed that this method is well applicable to the transfer of any DNA sequence of a higher organism, such as a vertebrate, to any microorganism. A higher organism is defined here as any eucaryotic organism with differentiated tissues, including, for example, insects, mollusks, plants, vertebrates, including mammals, the latter being cattle, pigs, primates and humans. A microorganism is understood to mean any microscopic living organism that falls under the name Protist, whereby it can be procaryotic or eucaryotic and can consist, for example, of a bacterium, protozone, algae and fungi, including yeasts. In the present method, a selected cell population is first isolated using a new method. Intact mRNA is extracted from the cells by a method in which practically all RNase activity is suppressed. The intact messenger RNA from the extract is purified by column chromatography and then subjected to the action of reverse transcriptase, this action taking place in the presence of the four deoxynucleoside triphosphates required for the synthesis of a complementary (cDNA) strand. The product of this first reverse transcriptase reaction is further processed in a process that selectively removes the ribonucleotide sequence. The remaining deoxynucleotide sequence, which is complementary to the starting mRNA, is incubated in a second reaction with reverse transcriptase or DNA polymerase in the presence of the four deoxynucleoside tri-phosphates. The resulting product is a double cDNA structure, the complementary strands of which are connected at one end by a single-stranded loop. This product is then treated with single stranded nuclease that cleaves the single stranded loop. The resulting double-stranded cDNA is then extended by adding a specific DNA at both ends, which has the sequence of a recognition or cleavage site of a restriction enzyme. The addition is catalyzed by a DNA ligase. The extended cDNA is then treated with a restriction endonuclease, self-complementary single-stranded ends being formed at the 5 'ends of each strand of the double helix.

A plasmid DNA with a recognition site for the same restriction endonuclease is treated with this enzyme to cleave the polynucleotide strand and generate self-complementary single-stranded nucleotide sequences at the 5 'ends. The 5'-terminal phosphate groups on the single-stranded ends are removed to prevent the plasmid from forming a ring structure capable of transforming a host cell. The cDNA prepared above and the plasmid DNA are then incubated together in the counter-type of DNA ligase. Under the reaction conditions described, a viable, closed ring can only be formed from plasmid DNA if a segment of the cDNA is inserted. The plasmid containing the cDNA sequence is then placed in a suitable host cell. Cells that have taken up a viable plasmid can be recognized by the appearance of colonies with a feature contributed by the plasmid, such as drug resistance. Pure bacterial strains containing the recombined plasmid with the built-in cDNA sequence are then grown and the newly combined plasmid is then reisolated. In this way, large amounts of recombined plasmid DNA can be produced, from which the specific cDNA sequence can be isolated again by endonucleolytic cleavage with the corresponding restriction enzyme.

The inventive method and the inventive use are based on the isolation of any nucleotide sequence from a higher organism, including humans, and their transfer into one

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Microorganism applicable as a host. The method proves useful for transferring a genetic code for a specific protein of medical or technical value and for the microbiological synthesis of such proteins. To illustrate the invention, the nucleotide sequence coding for insulin was isolated from rats, transferred into bacteria and replicated there. In the same way, the nucleotide sequence coding for rat growth hormone was isolated, transferred and replicated in bacteria. The method is applicable to the transfer of a nucleotide sequence isolated from human cell material, for example for human insulin and growth hormone or other polypeptide hormones.

The present invention comprises a method for isolating a DNA molecule with a specific nucleotide sequence which can be used for transfer into a microorganism, the original nucleotide sequence of the DNA being found in the microorganism after the multiplication.

The stages that make up the process according to the invention can be divided into three general categories:

1. The isolation of a desired cell population from a higher organism

There are two sources for a genetic sequence encoding a specific protein: the DNs of the underlying organism itself, and an RNA translation of the DNA. According to the current safety regulations of the National Institutes of Health, it is stipulated for the USA that human genes of all kinds can only be introduced into recombined DNA and then into bacteria after they have been cleaned very carefully, or if they are in specially equipped (P4) facilities works, see U.S. Federal Register, Vol. 41, No. 131, July 7, 1967, pp. 27902-27943. Any method of actual utility for the production of human protein, such as the present method, therefore preferably works by isolating the specific mRNA that has a nucleotide sequence encoding the desired protein. In this way, one has the further advantage that the mRNA can be purified more easily than DNA extracted from cells. In particular, one can also take advantage of the fact that in highly differentiated organisms such as vertebrates, it is possible to identify a specific cell population of special localization in the organism, the function of which mainly consists in the production of the protein in question. Such a population can also be present during a temporary stage of development of the organism. In such cell populations, a large proportion of the mRNA isolated from the cells has the desired nucleotide sequence. The choice of the cell population to be isolated and the method used for the isolation can be of essential importance with regard to the initial purity of the mRNA.

In most tissues, glands and organs, the cells are held together by a generally fibrous network of connective tissue, which consists mainly of collagen, but which may also contain other structural proteins, polysaccharides and mineral deposits. The isolation of cells from a particular tissue inevitably requires methods for releasing the cells from the connective tissue matrix. The isolation and purification of a specific differentiated cell type therefore comprises two main stages: the separation of the cells from the connective tissue, and the separation of the desired cells from all other cell types present in the tissue. The procedures provided according to the invention are applicable to the isolation of numerous cell types from different tissues. For example, the isolation of Langer-Hans islands from pancreas, which are suitable for the isolation of insulin-encoding mRNA, is described.

Insulin-producing cells can also come from other sources, for example from the pancreas of calf fetuses and from cultured islet tumor cells. The isolation of pure islet cells is much easier in these cases, especially when using pure cell cultures. In these cases, the method for isolating the islet cells described above is not necessary, but the method mentioned remains advantageous because of its general applicability.

Often the proportion of desired mRNA can be increased if the cell responses to stimuli from the environment are used correctly. For example, treatment with a hormone can lead to increased production of the desired mRNA. Other methods include growing at a certain temperature or in the presence of a specific nutrient medium or other chemical substance. When rat growth hormone mRNA is isolated, treatment of cultured pituitary cells with thyroid hormone and glucocorticoids significantly increases the level of growth hormone mRNA in a synergistic manner.

2. Extraction of the mRNA

An essential feature of the present invention is the virtually complete removal of the RNase activity in the cell extract. The mRNA to be extracted is a single-stranded polynucleotide, unpaired with a complementary strand. The hydrolytic cleavage of a single phosphodyne bond in the sequence would therefore render the entire molecule unusable for the purpose of transferring an intact genetic sequence into a microorganism. As already mentioned, the RNase enzyme is widespread, active and exceptionally stable. It is found on the skin, survives the usual rinsing processes for glass equipment and occasionally contaminates the stocks of organic chemicals. The difficulties are particularly acute when working with extracts from pancreatic cells, since the thyroid is a source of digestive enzymes and is therefore rich in RNase. However, the problem of RNase contamination arises in all tissues and the method disclosed herein for removing RNase activity is applicable to all tissues. The surprising effectiveness of the method is described using the example of successfully isolating intact mRNA from isolated islet cells of the pancreas.

When the cells are torn open and in all the processes which are used to isolate the RNA which is practically free of protein, a combination of a chaotropic anion, a chaotropic cation and a disulfide bond-releasing agent is used according to the invention. The common effect of these agents is described using the example of their use in the isolation of practically uninjured mRNA from isolated Langerhans islets from rat pancreas.

The choice of suitable chaotropic ions is based on their solubility in aqueous media and their availability. Suitable chaotropic cations are, for example, guanidinium, carbamoylguanidinium, guanylguanidium, lithium ion and the like. Suitable chaotropic anions are, for example, iodide, perchlorate, thio-cyanate, diiodosalicylation and the like. The effectiveness of salts formed by combining such cations and anions depends in part on their solubility. For example, lithium diiodosalicylate is a stronger one

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Denaturing agent as guanidinium thiocyanate, but it has a solubility of only about 0.1 M, and it is also relatively expensive. The preferred combination of cation and anion is in guanidinium thiocyanate, since this salt is easily accessible and readily soluble in aqueous media, up to about 5 molar solutions.

As is known, thiol compounds such as ß-mercaptoethanol cleave intramolecular disulfide bonds in proteins by means of a thiol-disulfide exchange reaction. Numerous thiol compounds are active in this direction, for example β-mercaptoethanol, dithiothreitol, cysteine, propanol-dimerkaptan and the like. An essential property is the solubility in water, since the thiol compound must be present in a large excess over the intramolecular disulfides in order to complete the exchange reaction. ß-Merkaptoethanol is preferred because of its availability and low price.

When inhibiting RNase during RNA extraction from cells or tissues, the effect of a chaotropic salt is directly dependent on its concentration. The preferred concentration is therefore the highest possible concentration. The success of the method according to the invention with regard to the preservation of intact mRNA during the extraction is presumably based on the rapid denaturation of the RNase and the extent of the denaturation. This may explain the superiority of guanidinium thione cyanate over hydrochloride, despite the fact

that the hydrochloride as a denaturing agent is only slightly weaker. The effectiveness of a denaturant is defined as the threshold concentration required to completely denature a protein. On the other hand, the rate of denaturation of numerous proteins depends on the concentration of the denaturing agent in relation to the threshold concentration, increased from the fifth to the tenth power; see Tanford, C.A. Adv. Prot. Chem. 23, 121 (1968). This relationship shows qualitatively that a weakly stronger denaturing agent than guanidinium hydrochloride can denature a protein many times faster at the same concentration. The kinetic relationship between RNase denaturation and mRNA preservation during their extraction from the cells was apparently not yet recognized at the time of the invention. The above considerations suggest that the preferred denaturant would be one that has a low threshold concentration combined with high water solubility. For this reason, guanidinium thiocyanate is preferred over lithium diiodosalylate, although the latter compound is a stronger denaturing agent because the solubility of guanidinium thiocyanate is much higher and it can therefore be used in a concentration which allows a faster RNase inactivation. The above considerations also explain the preference for guanidinium thiocyanate over the comparatively soluble hydrochloride, since the former is a somewhat stronger denaturant.

The use of a disulfide bond cleaving agent in combination with a denaturing agent enhances and increases the action of the latter by fully unfolding the RNase molecule. The thiol compound accelerates the progress of the denaturation process by preventing rapid renaturation that can occur if the intramolecular disulfide bonds remain intact. Furthermore, any RNase remaining in the mRNA preparation and contaminating it becomes practically inactive, even in the absence of denaturing agent and thiol. Disulfide bond cleaving agents with thiol groups are effective to some extent at any concentration, but a large excess of thiol groups is generally preferred over the intramolecular disulfide bonds so that the equilibrium reaction proceeds towards disulfide cleavage. On the other hand, numerous thiol compounds are malodorous and unpleasant to process at high concentrations, so that an upper concentration limit arises in practice. Concentrations in the range of 0.05 M to 1.0 M were found to be effective for β-mercaptoethanol, and concentrations of 0.2 M are considered optimal when isolating undegraded RNA from rat pancreas.

The pH of the medium during the mRNA extraction from the cells can range from pH 5.0 to 8.0.

After the cells have been broken open, the RNA is secreted from the mass of cell protein and DNA. Several methods are known for this purpose, all of which are suitable. A common practice is an ethanol precipitation procedure, in which the RNA fails selectively. According to the invention, the precipitation stage is preferably bypassed and the homogenate is layered directly onto a 5.7 molar cesium chloride solution which is located in a centrifuge tube, after which centrifugation is carried out; compare the description of Gli-sin, V., Crkvenjakov, R., and Byus, C., Biochemistry 13, 2633 (1974). This method is preferred because it constantly maintains an environment that is unfavorable for RNase, so that RNA can be obtained in high yield and free of DNA and protein.

The above procedures lead to the purification of the entire RNA from the cell homogenate. However, only part of this RNA is the desired mRNA. In order to further purify this mRNA, the fact is used that in the cells of higher organisms mRNA is further processed in the cell after transcription, with binding to polyadenylic acid. Such mRNA with poly-A sequences attached to it can be selectively isolated by chromatography on columns with cellulose to which oligothymidylate is bound, see Aviv, H. and Leder, P. loc. cit. The above methods provide essentially pure, intact mRNA from starting materials that are rich in RNase. The purification of the mRNA and the subsequent steps carried out in vitro can be carried out in practically the same way with any mRNA, regardless of its organism of origin.

Under certain conditions, for example when using cells from tissue cultures as the source of the mRNA, the RNase contamination can be so low that the above step of RNase inhibition is not necessary. In these cases, the prior art methods for reducing RNase activity may be sufficient.

3. Formation of the cDNA

Figure 1 is a schematic representation of the remaining stages of the process. The first step is to create a DNA sequence that is complementary to the purified mRNA. Reverse transcriptase is chosen as the enzyme for this reaction, although in principle any enzyme could be used which is capable of forming a complementary DNA strand on the basis of the mRNA used as a template. The reaction can be carried out under the known conditions, using the mRNA as a template and a mixture of the four deoxynucleoside triphosphates as a precursor of the DNA strand. One of the deo-xynucleoside triphosphates in a-stelhing is expediently labeled with 32P in order to follow the course of the reaction and to control the work-up and separation, for example by chromatography and electrophoresis, and to be able to provide quantitative yield information; see Efstratiadis, A. et al., loc. cit. As can be seen from the drawing, the product of the reaction with the reverse transcriptase is a double-stranded hairpin shape with non-covalent Bin5

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between the RNA strand and the DNA strand.

The product of the reaction with the reverse transcriptase is obtained from the reaction mixture using standard methods. A combination of phenol extraction, chromatography on “Sephadex G-100” (manufacturer Pharmacia Inc. Uppsala, Sweden) and precipitation with ethanol are expediently used.

Once the cDNA is enzymatically synthesized, the RNA template can be removed. Numerous methods for the selective degradation of RNA in the presence of DNA are known. The preferred method is alkaline hydrolysis, which is highly selective and can be easily controlled by pH adjustment.

After alkaline hydrolysis and subsequent neutralization, the 32D-labeled cDNA can be concentrated by precipitation with ethanol if desired.

A double-stranded hairpin-shaped cDNA is synthesized using the appropriate enzyme, for example with DNA polymerase or reverse transcriptase. The reaction conditions are similar to those described above, including the use of a 32P-labeled nucleoside triphosphate. Reverse transcriptase is available from a variety of sources, including avian myeloblastosis virus (the virus is available from Life Sciences Incorporated, St. Petersburg, Florida, which it produces under a contract with the National Institutes of Health).

After the formation of the cDNA hairpin, it may be advisable to recover the DNA from the reaction mixture. Here too, it is useful to use phenol extraction, chromatography on "Sephatex G-100" and precipitation with ethanol to purify the DNA product and to remove contaminating protein.

The hairpin structure can be converted to a conventional double-stranded DNA by removing the single-stranded loop that connects the ends of complementary strands. Various enzymes are available for this purpose, which bring about a specific hydrolytic cleavage of single-stranded DNA regions. An enzyme suitable for this purpose is the Sl nuclease from Aspergillus oryzae (the enzyme can be obtained from Miles Research Products, Elkhart, Indiana). Treatment of the hairpin-shaped DNA with S1 nuclease leads to cDNA molecules with base-paired ends in high yields. Extraction, chromatography and precipitation with ethanol are carried out as described above. The use of reverse transcriptase and S1 nuclease in the synthesis of double-stranded cDNA transmissions from mRNA was described by Efstratiadis, A. et al., Loc. cit. described.

If necessary, the proportion of cDNA molecules with blunt ends can be increased by treatment with E. coli DNA polymerase I in the presence of the four deoxynu-cleoside triphosphates. The combination of exonuclease and polymerase activity of the enzyme results in the removal of all protruding 3 'ends and the filling in of all protruding 5' ends. In this way, the participation of the majority of the cDNA molecules in the subsequent linking reactions is ensured.

The next step in the process is to treat the ends of the cDNA product so that there are appropriate sequences at each end that have a recognition site for a restriction endonuclease. The choice of the DNA fragment to be added to the ends is determined by reasons of expediency in handling. The sequence to be added to the ends is selected according to the restriction endonuclease chosen, and this choice is in turn based on the choice of the DNA vector with which the cDNA is to be recombined. The plasmid chosen should have at least one site that responds to restriction endonuclease cleavage. The plasmid pMB-9 has, for example, a recognition site for the Hind III enzyme. Hind III is isolated from Hemophilus influenza and made according to the Smith, H.O. and Wilcox, K.W., J. Mol. Biol. 51, 379 (1970). The Haoph III enzyme from Hemophilus aegyptious is synthesized by the method of Middleton, J.H., Edgell, M.H. and Hutchinson III, C.A. J. Virol. 10.42 (1972). An enzyme from Hemophilus suis, called Hsu I, catalyzes the hydrolysis at the same recognition sites as Hind III. These two enzymes are therefore considered to be functionally interchangeable.

It is expedient to use a chemically synthesized double-stranded decanucleotide with the recognition sequence for Hind III for binding to the ends of the cDNA double strand. The double-stranded decanucleotide has the sequence shown in Fig. 1, see Heyneker, H.L., et al. and Scheller, R.H., et al., loc. cit. Various such synthetic sequences with recognition sites are available today so that the ends of the double DNA can be prepared in such a way that they respond to the action of one of the numerous restriction endonucleases.

The recognition site sequence can be bound to the ends of the cDNA in a known manner. The method of choice is a reaction called "blunt end linkage" and catalyzed by a DNA ligase purified by the method of Panet, A., et al., Biochemistry str. 12, 5045 (1973) was. The blunt end linkage reaction was described by Sgaramella, V., et al., Loc. cit. described. The product of linking a blunt-ended cDNA with a large molar excess of a double-stranded decanucleotide to a Hind III endonuclease recognition site is a cDNA with this Hind III recognition sequence at each end. Treatment of the reaction product with Hind III endonuclease leads to cleavage at the recognition site and formation of single-stranded self-complementary 5 'ends, see FIG. 1.

Use of DNA transfer vectors according to the invention for modifying microorganisms

In principle, a large number of virus and plasmid DNA can be used for recombining with the DNA transfer vectors according to the invention, which were produced by the method explained above. The basic prerequisites for the modification of microorganism cells are that the DNA transfer vector must be capable of entering a cell of the microorganism, its replication takes place in this host cell, and that it also has a genetic determinant that enables those host cells, that have picked up the vector. For public security reasons, the selection range should be limited to those transfer vector types that appear suitable for the respective experiments, in accordance with the guidelines of the National Institutes of Health mentioned above. The list of approved DNA transfer vectors is constantly expanding as new vectors are developed and recognized by the relevant committee of the National Institutes of Health. According to the invention, of course, the use of any virus and plasmid DNA is provided which possess the abilities described, including those which are still pending approval. Suitable transfer vectors, the use of which is approved today, are various derivatives of bacteriophage X (see, for example, Blattner, FR, Williams, BG, Biechi, AE, Denniston-Thompson, K, Faber, HE, Furlong, LA, Grunwald, DJ., Kiefer, DO, Moore, DD,

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Schümm, J.W., Sheldon, E.L. and Smithies, O., Science 196, 161 [1977]) and derivatives of the plasmid col El (see, for example, Rodriguez, RL, Bolivar, S., Goodman, HM, Boyer, HW and Betlach, MN, ICN-UCLA Symposium on Molecular Mechanisms In The Control of Gene Expression, DP 5 Nierlich, WJ, Rutter, CF, Fox. Eds. [Academic Press, NY, 1976] pp. 471-477). Plasmids from col El are relatively small, they have molecular weights of the order of a few million and have the property that the number of copies of plasmid DNA per host cell can be increased from 20 to 40 under normal conditions to 1000 or more if one treating the host cell with chloramphenicol The ability to increase the amount of genes in the host cell, under certain circumstances, allows the host cell, under the control of the experimenter, to primarily produce proteins that are encoded by the genes on the plasmid of col El are therefore preferred transfer vectors according to the invention The suitable derivatives of col El include the plasmids pMB-9, which carry the gene for tetracycline resistance, 20 and pBR-313, pBR-315, pBR-316, pBR- 317 and pBR-322, which have a gene for ampicillin resistance in addition to the tetracycline resistance gene It is a reasonable possibility to select the cells which have been successfully infected by the plasmid, since 25 colonies of these cells grow in the presence of the active substances, while cells without the plasmid do not grow or form no colonies. In the examples in this description, a plasmid from colE1 was always used which, in addition to the drug resistance gene, had a Hind III recognition site.

As with the plasmid, the choice of a suitable host can also be made from a very broad spectrum, although the range has been narrowly limited for reasons of public safety. An E. coli strain, designated X-35 1776, was developed and approved by the National Institutes of Health for procedures of the type described, requiring P2 facilities; compare Curtiss, III, R., Ann. Rev. Microbiol., 30, 507 (1976). E. coli RR-1 can be used if P3 facilities are present 40. As in the case of the plasmids, the invention of course provides for the use of strains of any host cells which can be used to house the chosen vector, including protists and other bacteria, for example yeast.

Recombined plasmids are obtained by mixing plasmid DNA treated with restriction endonuclease with cDNA, both of which contain end groups treated analogously. In order to keep the possibility that cDNA segments enter into combinations with one another to a minimum, the plasmid DNA is used in molar excess over the cDNA. This procedure has so far resulted in the majority of the plasmids circulating without an inserted cDNA fragment. The subsequently transformed cells then mainly contain plasmid and no plasmids recombined with cDNA. The result is a very tedious and time-consuming selection process. In the prior art, this problem has been solved by attempting to produce DNA vectors with a restriction endonuclease recognition site in the center of a suitable labeling gene such that the insertion of the cDNA divides the gene, causing loss of the function encoded by the gene .

A method of reducing the number of colonies to be examined for recombined plasmids is preferably used. Here, plasmid DNA, which was cut off with the restriction endonuclease, is treated with alkaline phosphatase (commercially available enzyme). Treatment with alkaline phosphatase removes the 5'-terminal phosphate groups from the ends of the plasmid generated with the endonuclease, and the self-assembly of the plasmid DNA is prevented. Ring formation and transformation thus depend on the insertion of a DNA fragment which has 5'-phosphorylated ends. This method reduces the relative frequency of transformations without recombination to less than 1 to 10 + 4.

The method according to the invention is based on the fact that the reaction catalyzed by DNA ligase takes place between a 5'-phosphate-DNA end group and a 3'-hydroxyl-DNA end group. If the terminal 5'-phosphate group is removed, no binding reaction occurs. If double-stranded DNS is to be connected, two situations are possible, as shown in Table 1:

case

Table 1 Respondents

Ligase product

II

III

-OH

H "0 P0-

5 *

O? O3H2 + 2 3HO-

3 '

-OH OH

H, 0-P-0-0H-

31

■ OH -OH

HOHO-

3 »5»

O-P-O

-O-P-0 ± 2H2 °

3 '5f

O-P-O

-OHH O

- + H2 °

no reaction

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12

In Table 1, the double-stranded DNA is shown schematically by two parallel lines, the 5 'and 3' end groups depending on the hydroxyl or phosphate groups. In case I there are 5'-phosphate groups on both reactive end groups, with the result that both strands are covalently bound. In case II, only one of the strands to be connected has a terminal 5'-phosphate group, with the result that a single-stranded covalent bond takes place, while there is discontinuity in the other strand. The non-covalently linked strand remains connected to the molecule in a known manner due to hydrogen bonds between complementary base pairs on the opposite strands. In case III, none of the ends of the reactants has a 5'-phosphate group, so that no binding reaction can occur.

Undesired binding reactions can therefore be prevented by treating corresponding ends of the reactants whose connection is to be prevented by removing the 5'-phosphate groups. Any method of removing 5 'phosphorus groups that do not otherwise damage the DNA can be used. Hydrolysis catalyzed by the enzyme alkaline phosphatase is preferred.

The method described above is also useful when a linear DNA molecule has to be cleaved into two sub-fragments, typically with a restriction endonuclease, and then reconstituted in the original sequence. The sub-fragments can be cleaned separately and the desired sequence can be reconstituted by reconnecting the sub-fragments. The enzyme DNA ligase, which catalyzes the end-to-end linkage of DNA fragments, can be used for this purpose, see Sgaramella, V., Van de Sande, J.H. and Khorana, H.G., Proc. Nati., Sci USA 67, 1468 (1970).

If the sequences to be joined are not blunt, the ligase obtained from E. coli can be used, see Modrich, P. and Lehman, I.R., J. Biol. Chem. 245, 3626 (1970).

The reconstitution of the original sequence from sub-fragments obtained by restriction endonuclease is greatly improved if a method is used which prevents reconstitution in the wrong sequence. This is done by treating the cDNA fragment of homogeneous length of the desired sequence with an agent which removes the 5'-terminal phosphate groups of the cDNA before cleavage of the homogeneous cDNA with a restriction endonuclease. Alkaline phosphatase is preferably used again. The 5'-terminal phosphate groups are a structural precondition for the later binding action of the DNA ligase, which is used to reconstitute the cleaved subfragments. Ends without a 5'-terminal phosphor group cannot therefore be bound covalently. The DNA subfragments can only be bound at those ends that have a 5'-phosphate group.

This method prevents the most important undesired binding reaction, namely the connection of two fragments with the reverse sequence, that is to say back-to-front instead of front-to-back. Other possible side reactions such as dimerization and cyclization are not prevented, since these take place in a type II reaction according to Table 1. These side reactions are less disadvantageous because they lead to physically separable and identifiable products, which is not the case when recombining in the wrong direction.

To illustrate the method described above, cDNA encoding rat insulin was isolated and recombined with a plasmid. The DNA molecules became

Transformation of E. coli X-1776 used. The cells to be transformed were selected by growth on a medium containing tetracycline. A recombined plasmid DNA obtained from transformed cells contained an inserted DNA fragment of approximately 410 nucleotides in length. Other new combinations isolated in the same way were also analyzed. The inserted fragments were released from the plasmid with Hind III or HSU I endonu clease and the DNA sequence io was determined according to the method of Maxam, A.M. and Gilbert, W., Proc. Nati. Acad. Be USA, 74, 560 (1977). The nucleotide sequences of the inserted DNA fragments were overlapped and contained the entire coding reaction for rat proinsulin I and 13 of 23 amino acids of the prepep-15 tid sequence. A summary of the nucleotide sequence of this region is shown below.

In a similar manner, cDNA encoding rat growth hormone was isolated, recombined with a plasmid and transferred into E. coli. After extensive replication in E. 20 coli, a nucleotide sequence of approximately 800 nucleotides in length was isolated, which contained the entire coding sequence for rat growth hormone and parts of the precursor peptide and part of the 5'-untranslated region.

The described method is generally applicable to the isolation and purification of a gene from a higher organism, including human genes, its transfer into a microorganism and replication in the microorganism. New recombined plasmids containing all or part of the isolated gene 30 are also described. Furthermore, previously unknown new microorganisms are described which contain a gene from a higher organism as part of their genetic makeup. The following examples describe in detail the method used for the isolation, purification and transfer of rat insulin in E. coli. In the examples, recombinant plasmids are also characterized which contain parts of rat insulin, rat growth hormone, human insulin and human growth hormone, as well as the genes mentioned contain the new microorganisms.

example 1

To produce purified rat islet cells, the pancreas of an anesthetized rat is infused with 45 Hank's saline solution by retrograde infusion into the pancreatic duct. Hank's salt solution is a well-known and commercially available standard solution. Then the pancreas is removed, crushed in Hank's solution at 0 ° C and digested with collagenase and trypsin inhibitor. All procedures are carried out at 0 to 4 ° C unless otherwise stated. The digestion conditions are extremely critical. Two shredded pancreases in a total volume of 8 ml of Hank's solution are placed in a 30 ml glass tube. All glass parts had been treated with silicone (Handelsbe-55 Drawing Siliclad, manufacturer Clay-Adams Division, Becton-Dickinson Inc., Parsippany, New Jersey). The incubation mixture contained 12 mg collagenase, an enzyme obtained from Clostridium histolyticum (produced essentially according to the method of Mandl, I, Mackeman, eJD and Howes, EL, J. Clin. Invest. 32, 1323 (1943) - type CLS IV, manufactured by Worthington Biochemical Corporation, Freehold, New Jersey) and 1 mg soybean trypsin inhibitor, manufactured by Sigma Chemical Company, St. Louis, Missouri. Incubation was carried out at 37 ° C. for 25 minutes with 65 shakes at 90 movements per minute. Constant monitoring was required to ensure that collagen nose digestion was at an optimal level. If the incubation was too short, the islet cells were released incompletely,

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if the incubation is too long, their decomposition begins. After the incubation, the glass tube was centrifuged at 200 x G for 1 min. The supernatant liquid was decanted off and the cake was washed with Hank's solution, this process was repeated five times. After the last centrifugation, the cake was suspended in 15 ml of the commercial product Ficoll (manufacturer Pharmacia Chemical Company, Uppsala, Sweden) with a density of 1.085. A layer of 8 ml Ficoll density 1.080 was added and a layer of 5 ml Ficoll density 1.060 then the glass tube was centrifuged in a vibrating beaker rotor for 5 min at 500 x G and then 5 min at 2000 x G. The acinar cells remained on the ground, the islet cells rose in a gradient and formed a layer between the two upper layers. The islet cell layer contained contaminating ganglion cells, lymph nodes and connective tissue. Large fragments of impurities were removed from the layer material. The rest of the preparation was freed from visible contamination by hand using a micropipette under the microscope. The cell preparation was then diluted in Hank's solution and centrifuged. The supernatant liquid was decanted and the cell cake was stored frozen in liquid nitrogen.

The islets collected from 200 rats were homogenized in 4-molar guanidinium thiocyanate solution (trade name Tridom, manufacturer: Fluka AG, Chemische Fabrik, Buchs), which was 1-molar buffered with β-mercaptoethanol and pH 5.0, at 4 ° C. The homogenate was overlaid over 1.2 ml of 5.7 molar cesium chloride solution which contained 100 mmol of ethylenediaminetetraacetic acid and centrifuged for 18 hours at 37 revolutions / minute in a SW 50.1 rotor of a Beckman ultracentrifuge at 15 ° C. The RNA travels to the bottom of the centrifuge tube.

Polyadenylated RNA was prepared by chromatographing the entire RNA preparation on oligo (dT) cellulose according to the method of Aviv, H., and Leder, P. loc. cit. isolated.

Reverse transcriptase from avian myeloblastosis virus (obtained from Life Science Inc., St. Petersburg, Florida) was used to transcribe the entire polyadenylated RNA from the Langerhans Islands of rats into cDNA. The reactions were carried out in 50 mM Tris-HCl, pH 8.3, with 9 mM MgCk, 30 mM NaCl, 20 mM β-mercaptoethanol, 1 mM each of 3 non-radioactive deoxyribonucleoside tri-phosphates, 250 (iM of the fourth, in the a-position with 32P-labeled deoxynucleoside triphosphate, specific activity 50-200 Curie / mol, 20 μg / ml 01igo-dT12_i8 (manufacturer Collaborative Research, Waltham, Massachusetts), 100 μg / ml polyadenylated RNA and 200 units / ml reverser The mixture was incubated for 15 min at 45 ° C. After the addition of EDTA-NA2 to 25 mM, the solution was extracted with an equal volume of phenol saturated with water, and the aqueous phase was then filled on a Sephadex G-100 Column 0.3 cm in diameter and 10 cm high in 10 mM Tris-HCl, pH 9.0, 100 mM NaCl, 2 mM EDTA, and the eluted nucleic acid was mixed with ammonium acetate (pH 6.0 to 0.25 M) Ethanol precipitated, the precipitate was centrifuged off and the cake was freshly added in 50 ml prepared 0.1 molar sodium hydroxide solution and incubated at 70 ° C. for 20 min in order to hydrolyze the RNA. The mixture was neutralized with 1 molar sodium acetate solution, pH 4.5, and the 32P cDNA product was precipitated with ethanol and redissolved in water. Samples of single-stranded cDNA were analyzed on native polyacrylamide gel according to the method of Ding-man, C.W. and Peacock, A.C., Biochemistry 7,659 (1968). The gels were dried and the 32P-DNA was analyzed by autoradiography using a Kodak No-Screen NS-2T film (manufacturer: Eastman Kodak

Corp., Rochester, New York). The cDNA was heterodisperse, as can be seen from the electrophoresis. It contained at least one major cDNA of approximately 450 nucleotides (compared to known standards).

Example 2

The single-stranded cDNA obtained according to Example 1 was treated with reverse transcriptase in order to produce the complementary strand. The reaction mixture contained 50 mM Tris-HCl, pH 8.3.9 mM MgCLî, 10 mM dithiothreitol, 50 mM each of three unmarked deoxyribonucleoside triphosphates, 1 mM of a nucleoside triphosphate labeled with 32P in the a position with the specific effect 1 -10 Curie / millimole, 50 fxg / ml cDNA and 220 units / ml reverse transcriptase. The reaction mixture was incubated at 45 ° C. for 120 min. The reaction was stopped by adding EDTA-Na2 to 25 mM, then it was extracted with phenol, chromatographed on Sephadex G-100 and precipitated with ethanol. A sample of the reaction product from 500 to 1000 cpm was analyzed by gel electrophoresis as described in Example 1. A heterodisperse band centered around 450 nucleotides in length was observed when compared to standard samples. Samples of the DNA reaction products of Examples 1 and 2 were treated separately with excess restriction endonuclease Hae III and analyzed by gel electrophoresis. Both products were cleaved by the endonuclease so that two bands of radioactivity were observed on gel electrophoresis. The bands resulting from the cleavage of the double-stranded cDNA represented cleavage products of essentially the same chain length as in the cleavage of the single-stranded cDNA.

Example 3

The double-stranded reaction product of Example 2 was at a concentration of 2-5 (ig / ml with 30 units of S1 nuclease with an activity of 1200 units / ml (manufacturer: Miles Laboratories, Elkhart, Indiana) in 0.03 M sodium acetate , pH 4.6.0.3 M sodium chloride, 4.5 mM ZnCh incubated for 30 min at 22 ° C. and for a further 15 min at 10 ° C. With the addition of Tris-base to 0.1 M final concentration, EDTA to 25 mM and E. coli-tRNA (produced according to the method of Ehrenstein, G., Methods in Enzymology, ed. SP Colowick and NO Kaplan, vol. 12A, p. 588 [1967]) to 40 (ig / ml After extraction of the reaction mixture with phenol and chromatography on Sephadex G-100, the eluted 32P cDNA was precipitated with ethanol, which resulted in a high yield of cDNA molecules with base-paired ends, which link the blunt ends chemically synthesized decanucleotides were required. Hind III decamers were prepared by the method of Scheller, R.H., Dickerson, R.E., Boyer, H.W., Riggs, A.D. and Itakura, K. Science 196, 1977 (1977). The Hind III decamers were linked to the cDNA by incubation at 14 ° C. in 66 mM Tris-HCl, pH 7.6, 6.6 mM MgCh, 1 mM ATP, 10 mM dithiothreitol, with 3 mM Hind III decamers with 105 cpm / pmole and T4 DNA ligase, about 500 units / milliliter, for 1 hour. The reaction mixture was then heated to 65 ° C for 5 min to inactivate the ligase. Then KCl to 50 mM final concentration, β-mercaptoethanol to 1 mM final concentration and EDTA to 0.1 mM final concentration were added before digestion at 150 units / ml, which was carried out at 37 ° C. for 2 hours. Hind III and Hae III endonucleases are commercially available preparations available from New England Bio-Labs, Beverly, Massachusetts. The reaction product was analyzed by gel electrophoresis as described in Example 1. It became a peak

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which corresponded to a sequence of about 450 nucleotides, and fragments of the cleaved Hind III decamers were also observed.

Example 4

Plasmid pMB-9-DNA (preparation: S. Rodrigues, RL, Boliver, F., Goodman, HM, Boyer, HW and Betlach, M., ICN-UCLA Symposium on Molecular and Cellular Biology, DP Wierlich, WJ Rutter and CF Fox, Eds., (Academic Press, New York 1976), pp. 471-477) was digested at the Hind III site with Hsu I endonuclease, then with alkaline phosphatase (BAPF type, Worthington Bio-chemical Corp., Freehold , New Jersey). The enzyme was present in the reaction mixture in an amount of 0.1 unit / microgram DNA, the reaction mixture was incubated in 25 mM Tris-HCl for pH 8 for 30 min at 25 ° C., followed by phenol extraction to remove the phosphatase . After ethanol precipitation, the phosphatase-treated plasmid DNA was added to cDNA that had cohesive ends to Hind III in a molar ratio of 3 moles of plasmid to 1 mole of cDNA. The mixture was incubated in 66 mM Tris, pH 7.6.6.6 mM MgCh, 10 mM dithiothreitol and 1 mM ATP for 1 hour at 14 ° C. in the presence of 50 units / ml T4 DNA ligase.

The reaction mixture was then added directly to a suspension of E. coli X-1776 cells, which were prepared for transformation as follows: the cells were grown to a cell density of approximately 2 × 108 cells / ml in 50 ml medium, the 10th g / 1 tryptone, 5 g / 1 yeast extract, 10 g / 1 sodium chloride, 2 mM NaOH, 100 µg / ml diaminopi-melinic acid and 40 µg / ml thymine and showed 37 ° C. The cells were collected by centrifugation under 5000 x G for 5 minutes at 5 ° C, suspended in 20 ml of cold 10-millimolar sodium chloride solution, centrifuged again and resuspended in 20 ml of transformation buffer containing 75 mM CaCh, 140 mM NaCl and 10 mM Tris pH 7 , 5 contained, and left in ice for 5 min. The cells were then centrifuged and resuspended in 0.5 ml of transformation buffer. The transformation was carried out by mixing 100 ni of the cell suspension with 50 ul of recombined DNA (1 ^ ig / ml). The mixture was incubated at 0 ° C for 15 min, then treated at 25 ° C for 4 min and at 0 ° C for 30 min. The cells were then transferred to agar plates for growth under selected conditions.

The recombined plasmids were selected with

5 micrograms / ml tetracycline when transformed with the Hind III product. A new combination called pAU-1 was isolated. Crude plasmid preparations with 2-5 [ig DNA (isolated from pAU-1) were treated with excess Hsu I-5 endonuclease. Then 10 mM EDTA-Na2 and 10% (weight / volume) cane sugar were added and the mixture was broken down on 8% polyacrylamide gel. The DNA was found in a position approximately 410 base pairs in length. In a similar experiment, the plasmid pBR-322 was used as the u transfer vector. All conditions remained as described, only the last selection of the recombined clones was carried out on plates containing 20 μg / ml ampicillin.

15 Example 5

After elution from the gel, DNA was labeled by incubation with y-32P-ATP and the enzyme polynucleotide kinase under the conditions of Maxam and Gilbert loc. cit. described conditions. The enzyme catalyzes the transfer 20 of a radioactive phosphate group from y-32P-ATP to the 5 'ends of the DNA. The enzyme was obtained from E. coli by the method of Panet, A., et al, Biochemistry 12, 5045 (1973). The DNA labeled in this way was cleaved with Hae III endonuclease according to the procedure of Example 2, 25 and two labeled fragments of approximately 265 and 135 base pairs were separated from one another on a polyacrylamide gel under the conditions described in Example 1. The isolated fragments were subjected to specific cleavage reactions, then the sequence analysis was carried out according to the method of Maxam and Gilbert, loc. cit. The sequence in Table 2 below relates to a compilation of the results of these test series and similar test series with cDNA and plasmid vectors from colE1 such as pMB-9 and pBR-322. At the 5 'end, a sequence of an estimated 50 to 120 nucleotides in length is indefinite, and the poly-dA segment at the 3' end is of different lengths. This sequence corresponds to the current state of knowledge, although further examinations may of course open up additional details or may necessitate a minor revision of some areas. The corresponding amino acid sequence of rat proinsulin I begins with the triplet position labeled 1 and ends with the triplet position labeled 86. Some uncertainty remains regarding the sequence underlined in the dashed line:

Table 2

^ / indefinite? ccc CTC CTC GTC CÏC ÏGG GAC ccc MG CCT CCT CAG CCT TTT GTC MA CA0 CAC CTT TGT

10th

20th

30th

GGT CCT CAC CTG GTG GAG GCT CTG TAC CTG GTG TGT GGG GAA CGT GOT TTC TTC TAC ACA CCC AAC TCC CGT CGT

40

50

GAA GTG GAG GAC CCG CAA GTG CCA CAA CTG GAG CTC GCT GGA GGC CCG GAG GCC GGG GAT CTT CAG ACC TCG GCA

60

70

80

CTG GAG GTT GCC CCG CAG AAG CGT GGC ATT GTG GAT CAG TGC TGC ACC AGC ATC TGC TCC CTC TAC CAA CTG GAG 86

AAC TAC TGC AAC TGA GTTÇAATCAATTCCCGATCCACCCCTCTGCAATGAATAAAGCCTTTGAATGACC-poly A

underlined with dashed line = areas of existing uncertainty

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Example 6

A nucleotide sequence encoding human insulin is isolated, purified, and inserted into a plasmid according to the instructions of Examples 1 to 4, starting from human pancreatic tissue which comes from a suitable source, for example a donated pancreas or a fresh corpse or a human insulinoma. A microorganism is produced essentially according to the instructions of Example 4, with a nucleotide sequence coding for the A and B chains of human insulin. The known amino acid sequence of the A chain is:

1 10

Gly-Ile-Val-Glu-Gln-Cys-Cys-Thr-Ser-Ile-Cys-Ser-Leu-Tyr-Glu-Leu-Glu-Asn-20

Tyr-Cys-Asn

The amino acid sequence of chain B is:

1 10 ■

Phe-Val-Asn-Glu-f3is-Leu-Cys-Gly-Ser-His-Leu-Val-Glu-Ala — Leu-Tyr-Leu-Val-

20 30

Cys-Gly-Glu-Arg-Gly-Phe-Phe-Tyr-Thr-Pro-Bys-Thr

The amino acid sequences are numbered from the end with a free amino group, see Smith, L.F., Diabetes 21, (Erg. 2), 458 (1972).

Example 7 25

For genes of non-human origin, US safety regulations do not require isolation of a cDNA as high as that of human cDNA. It is therefore possible to isolate the cDNA containing the entire rat growth hormone structural gene by isolating electrophoretically separated DNA of the expected length of about 800 base pairs (corresponding to the known amino acid length of the growth hormone). Raised pituitary cells from rats (sub-clone of cell strain GH-1, to be obtained from the American Type Culture Collection) were used as the source of rat growth hormone mRNA, see Tashjian, A.H., et. al., Endochrinology 82, 342 (1968). If these cells are grown under normal conditions, the growth hormone mRNA makes up only 1 to 3% of the total poly-A-containing RNA. However, the amount of 40 growth hormone mRNA was increased over the amount of other mRNA in the cell by the synergistic action of thyroid hormones and glucocorticoids. The RNA was obtained from 5 x 108 cells, which were grown in a suspension culture and disposed for the production of growth hormone by adding 1 mM dexamethasone and 10 mM L-triiodothyronine to the medium 4 days before the cells were collected. The polyadenylated RNA was isolated from the cytoplasmic membrane fraction of the cultured cells, see Martial, J.A., Baxter, J.D., so Goodman, H.M. and Seeburg, P.H., Proc. Nat. Acad. Be. USA 74, 1816 (1977), and Bancroft, F.C., Wu "G. and Zubay, G., Proc. Nat. Acad. Be. USA 70,3646 (1973). The mRNA was then further purified essentially according to the instructions in Examples 1, 2 and 3 and transcribed into double-stranded 55 cDNA. After gel electrophoresis fractionation, a weak but distinct band corresponding to a DNA approximately 800 base pairs in length was observed.

Treatment of the entire cDNA obtained by transcription from 60 of the mRNA of the cultured pituitary cells with Hhal endonuclease gave two main DNA fragments (after electrophoretic separation) corresponding to approximately 320 nucleotides (fragment A) and 240 nucleotides (fragment B). Nucleotide sequence analysis of fragments A 65 and B according to the procedure of Example 5 showed that these fragments were in fact portions of the coding region for rat growth hormone, based on the known amino acid sequence for rat growth hormone and by comparison with other known growth hormone sequences; see Wallis, M. and Davies, RVN, Growt Hormone 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, Erg. 2, pp. 120-121 (National Biomedicai Research Foundation, Washington, D.C., 1976). If the double-stranded cDNA with 800 base pairs is treated analogously with Hhal-endonuclease after the electrophoretic isolation described above, two fragments are obtained under the main cleavage products which correspond in length to fragments A and B.

Since the approximately 800 base pair rat growth hormone cDNA was not purified using restriction endonuclease treatment, the DNA had to be treated to remove unpaired single-stranded ends. In practice, these unpaired ends are removed from each of the nucleotides dATP in 25 p.160 mM Tris-HCl, pH 7.5, 8 mM MgCh, 10 mM β-mercaptoethanol, 1 mM ATP and 200 | iM,

dTTP, dGTP and dCTP. The mixture was incubated with a unit of E. coli DNA polymerase I at 10 ° C for 10 min to remove all protruding 3 'ends and fill in all protruding 5' ends. DNA polymerase I is a commercial product (Boehringer-Mannheim Biochemicals, Indianapolis, Indiana).

Chemically synthesized Hind III binders were added to the rat growth hormone cDNA with about 800 base pairs according to the procedure of Example 3. The plasmid pBR-322 with an ampicillin resistance gene and a single Hind III site in the tetracycline resistance gene was pretreated with Hind III endonuclease and alkaline phosphatase according to the procedure of Example 4. The plasmid treated in this way was combined with the rat growth hormone cDNA with the 800 base pairs in a DNA ligase reaction according to Example 3. The mixture for the ligase reaction was used to transform a suspension of E. coli X-1776 cells which had been treated according to Example 3. The recombined colonies were discarded due to their growth on nutrient plates containing ampicillin and their inability to grow to 20 µg / ml tetracycline. 10 such colonies were obtained which contained the plasmid with an insertion of about 800 base pairs. The release was by Hind III. Cleavage.

The rat growth hormone DNA (RGH-DNA) with 800 base pairs was isolated in preparative amounts from the recombined clone pRGH-1 and the nucleotide se-

642 680

16

quenz was determined as described in Example 5. The nucleotide sequence included portions of the 5 'untranslated region of the rat growth hormone and a sequence of 26 amino acids found in the growth hormone protein precursor prior to secretion. The mRNA sequence deduced from the gene sequence is shown in Table 3 below. The predicted amino acid sequence agrees well (with the exception of positions 1 and 8) with the partial sequence of the rat growth hormone according to Wallis and Davies, loc. cit., which has residues 1-43, 65-69, 108-113, 133-143 and 150-190.

Table 3

DNA nucleotide sequence of a strand containing the entire sequence encoding rat growth hormone. The corresponding amino acids are listed along with their position number in relation to the amino end. Amino acids with negative numbers correspond to the sequence of pro-growth hormone. The corresponding mRNA sequence is the same, only T is replaced by U in the mRNA:

10th

Table 3

51 GTCGACAGATCACTGAGTGGCG

Met Ala Ala ATG CCT CCA

Trp Pro Gill Glu Ala Gly Ala leu Pro Ala Met Pro Leu Ser TGG CCT CAA GAC GCT GGT CCT TTA CCT GCC ATG CCC TTC TCC

Ala Ala Asp Thr Tyr Lys Glu Phe Glu Arg Ala Tyr Ile Pro CCT CCT GAC ACC TAC AAA CAG TTC CAG CCT CCC TAC ATT CCC

60

Ser Glu Thr Ile Pro Ala Pro Thr Gly Lys Glu Glu Ala Gin TCA CAG ACC ATC CCA GCC CCC ACC GGC AAG GAG GAG GCC CAG

Ser Trp Leu Gly Pro Val Gin Phe Leu Ser Arg Ile Phe Thr TCA TGG CTG CCG CCC CTG CAG TTT CTC AGC AGG ATC TTT ACC

120 '

Asp Leu Clu Clu Gly Ile Gin Ala Leu Met Gin Glu Leu Clu CAC CTG CAA CAC GCC ATC CAG GCT CTG ATG CAC GAG CTG GAA

Phe Asp Ala Asn Met Arg Ser Asp Asp Ala Leu Leu Lys Asn TTT CAC CCC AAC ATG CGC AGC GAT GAC GCT CTG CTC AAA AAC

180

Tyr Leu Are Val Met Lys Cys Arg Arg Phe Al.a.Glu Ser Ser TAC CTG CCC GTC ATG AAC TGT CGC CGC TTT CCG GAA AGC ACC

Asp Ser Cln CAC TCT CAU

Scr Leu Phe ACT CUC TTT

1.0

Glu Gly Gin CAG 'GGA CAG

Cln Arg Thr GAG AGA ACT

100

Asn Ser Leu AAC AGC CTC

Asp Gly Ser GAC GGC AGC

160

Tyr Gly Leu TAT GGG CTC

Thr Pro Trp Leu Leu Thr Phe Ser Leu Leu Cys Leu Leu ACT CCC TCCi CTC CTC ACC TTC AGC CTG CTC TCC CTG CTG

20th

Ala Asn Ala Val Leu Arg Ala Gin Iiis Lt-u Iiis Gin Leu CCC AAT GCT CTG CTC CCA CCC CAG CAC CTG CAC CAC CTG

Arg Tyr Ser Ile Gin Asn Ala Gin Ala Ala Phe Cys Phe CGC TAT TL'C ATT CAG AAT CCC CAG CCT CCT ITC TCC TTC

80

Asp Met Glu Leu Leu Arg Phe Ser Leu Leu Leu Ile Gin GAC ATC GAA TTC CTf CGC TTC TCG CTC CTG CTC ATC CAG

Met Phe Gly Thr Ser Asp Arg Val Tyr Glu Lys Leu Lys ATC TTT GGT ACC TCG GAC CGC GTC TAT CAG AAA CTC AAG

UiO.

Pro Arg Ile Gly Cln Ile Leu Lys Gin Thr Tyr Asp Lys CCC CGT ATT CCG CAG ATC CTC AAG CAA ACC TAT CAC AAC

Leu Ser Cys Phe Lys Lys Asp Leu His Lys Ala Glu Thr CTC TCC TGC TIC AAG AAG CAC CTC CAC AAG GCA GAC ACC

Cys Ala Phe TGT GCT TTC

TAC GCACACACTGGTGTCTCTGCCGCACTCCCCCGTTACCCCCCTGTACT CTGGCAACTGCCACCCCTACACTTTGTCCTAATAAAATTAATGATGCATCATATC poly (A) 31

Example 8

Human growth hormone mRNA is isolated essentially according to the instructions of Example 7, only the biological starting material is human pituitary tumor tissue. Five benign human pituitary tumors, which had been frozen in liquid nitrogen after surgical removal and each weighed 0.4 to 1.4 g, were dissolved in 4-molar gunidinium thiocyanate solution, which was 1-molar in mercaptoethanol and at pH 5 , 0 had been buffered, thawed at 4 ° C and homogenized. The homogenate was layered over 1.2 ml of 5.7 molar cesium chloride solution, which contained 100 mmol of ethylenediaminetetraacetic acid, and centrifuged at 37,000 revolutions / minute in a SW 50.1 rotor of a Beckmann ultracentrifuge at 15 ° C. for 18 hours. The RNS came to the bottom of the centrifuge tube. Further purification with a column with oligo-dT and sucrose grain sedimentation was carried out as in Examples 1, 2 and 3. About 10% of the growth hormone coded in this way was estimated from the incorporation of a radioactive amino acid precursor into precipitation material from anti-growth hormone in a cell-free translation system from wheat germ; compare Roberts, B.E. and 45 Patterson, B.M., Proc. Nat. Acad. Be. USA 70, 2330 (1973). The cDNA of human growth hormone is produced essentially as described in Example 7. The cDNA is fractionated by gel electrophoresis and the material thus migrating to an approximately 800 nucleotide length is selected for cloning. This fraction is treated with DNA polymerase I as described in Example 6, then the Hind III links are added. The cDNA is then recombined with the alkaline phosphatase-treated plasmid pBR-322 and 55 DNA ligase. E. coli X-1776 is transformed with the recombinant DNA and a strain containing the growth hormone DNA is selected. This strain is grown in preparative amounts, the DNA is isolated therefrom and its nucleotide sequence is determined. The cloned-60 growth hormone DNA contains the nucleotides encoding the entire amino acid sequence of human growth hormone. The first 23 amino acids of human growth hormone are

ELN-Phe-Pro-Thr-Ile-Pro-10 20

Leu-Ser-Arg-Leu-Phe-Asp-Asn-Ala-Met-Leu-Arg-Ala-His-Arg-Leu-His-Gln-Leu-.

17th

642 680

The rest of the sequence is shown in Table 4:

Table 4

Nucleotide sequence of a strand of human growth hormone DNA. The numbers refer to the

Amino acid sequence of human growth hormone starting at the amino end. The DNA sequence corresponds to the mRNA sequence for human growth hormone, only replaces the T in the mRNA U.

Table 4

24 34 40 43

Ala Plie Asp Tlir Tyr Gin Glu Plie GÌu Glu Ala Tyr Ile Pro tys «lu Cln Lys Tyr Ser Plm I.cu Cln Asn Pro Cln.

5 'C CCC TTT CAC ACC TAC CAC GAG TTT CAA CAA CCC TAT ATC CCA AAC CAA CAC AAG TAT TCA ITC CTC CAC AAC CCC CAC

60

Thr Ser Leu Cys Phe Ser Glu Ser Ile Pro Tlir Pro Ser Asn Asg Glu Glu Tlir Gin Gin Lys Ser Asn Leu Clu Leu Leu ACC TCC CTC TGT TTC TCA GAG TCT ATT CCG ACA CCC TCC AAC ACC CAG CAA ACA CAA CAO AAA TCC AAC CTA CAC CTC CTC

80 100

Arg Ile Ser Leu Leu Leu Ile Gin Ser Trp Lau Glu Pro Val Gin Phe Leu Arg Ser Val Phe Ala Asn Aan Leu Vai'Tyr CCC ATC TCC CTG CTG CTC ATC CAG TCG TCG CTC RAG CCC CTC CAG TTC CTC ACC ACT CTC TTC CCC AAC AAC CTC CTG TAC

120

Cly Ala Ser 'Asp Ser Asn Val Tyr Asp Leu Leu Lys Asp Leu Clu Clu Cly Ile Gin Thr Leu Met Cly Arg Leu Glu Asp GCC CCC TCT CAC AGC AAC GTC TAT CAC CTC CTA AAG GAC CTA CAG CAA GCC ATC CAA ACC CTC ATG CCC ACG CTC CAA CAC

140

Gly Ser Pro Arg Thr Gly Gin Ile Phe Lys Gin Thr Tyr Ser Lys Phe Asp Thr Aan Ser Iiis Asn Hls Asp Ala Leu Leu CCC ACC CCC CCC ACT GGG CAC ATC TTC AAG CAG ACC TAC AGC AAG ITC GAC ACA AAC TCA CAC AAC CAT CAC GCA CTA CTC

160 180

Lys Asn Tyr Gly Leu AAC AAC TAC CCG CTG

180

Leu Tyr Cys Phe Arg Lys Asp Met Asp Lys Val Glu Thr Phe Leu Arg Ile V.ìl Gin Cys Arg Ser CTC TAC TCC ITC AGG AAC CAC ATG CAC AAC CTC GAG ACA TTC CTC CGC ATC CTG CAI! TCC CCC TCT

191

Val Clu Gly Ser Cys Gly Phe

CTC CAG CGC ACC TCT CGC TTC TAC CTCCCCGCCTGCCATCCCTCTCACCCCTCCCCAGTCCCTCTCCTCCCC

The method according to the invention makes it possible for the first time to isolate a nucleotide sequence coding for a specific regulatory protein of a higher organism, such as a vertebrate, furthermore, the transfer of the genetic information contained therein can be carried out in a microorganism in which the unlimited replication is possible. The disclosed method can be applied to the isolation and purification of human insulin gene and its transfer into a microorganism. Human growth hormone gene and other protein genes can also be isolated, transferred and replicated in a microorganism. Some new recombined plasmids are disclosed which have a structure in their nucleotide sequence which has been obtained by transcription from a gene of a higher organism. New microorganisms are revealed,

which are modified so that they contain a nucleotide sequence which has arisen by transcription from a gene of a higher organism. The examples illustrate the * 5 invention on the transfer of rat gene for proinsulin I into a strain of Escherichia coli and the transfer of rat gene for growth hormone on a strain of E. coli. The sequence of the major part of the transferred gene was determined in each case and was found to encode the 50% total amino acid sequence of rat proinsulin I or rat growth hormone.

The microorganism described here was deposited with the ATCC under the accession number 31392. The plasmid-containing rat insulin gene, which can be transferred independently to other strains of microorganisms, was deposited with the ATCC under the accession number 40003.

1 sheet of drawings

Claims (13)

642 680 2nd 2. DNS transfer vector according to claim 1, marked 1. DNA transfer vector, which contains in its nucleotide sequence net by a nucleotide sequence which encodes the A chain of a sub-sequence with the structure of a gene of a higher human insulin and consists of organism by transcription on a gene of the higher organism arose. 5 5 'GGL.ATM_GTL GAJ, CAJ TGK TGK_ACL OR S ATM, TGK QR,? S17 1 2 3 4 5 6 7 8 9 9 10 11 X13TYi3TAK14GAJ15X16TY16GAJ17AAK18TAK19TGK20AAK21 3 ' where A = deoxyadenyl, G = deoxyguanyl, C = deoxycy- A, G, C or T if QR "= TC, and T or C if QRn = tosyl, T = thymidyl, J = A or G, K = T or C, L = A, AG and the subscripts n the amino acid position TC or G, M = A, C or T, Xn = T or C if Yn = A 15 in human insulin followed by the nucleotide sequence or G and C if Yn = C or T, Yn = A, G, C or T, if it corresponds to the genetic code, where the amino Xn = C and A or G, if X „= T, Wn = C or A, if Zn are numbered amino acid positions from the amino end. = G or A, and C if Zn = C or T, Zn = A, G, C or 3. DNA transfer vector according to claim 2, characterized T, if Wn = C, and A or G, if Wn = A, QRn = TC, net by another nucleotide sequence that links the B chain of if Sn = A, G, C or T, and AG, if S " = T or C, Sn = 2 "encodes human insulin and consists of 5 '—- TTK1GTL2AAK3GAJ4CAK5X6TY6TGK7GGLgQRgSgCAlCioX11TY11GTL12 GAJ13GCLUX15TY15TAK16XL7TY17GTL18TGK19GGL2QGAJ21W22GZ22GGL23 IIK24TIK25TAK26ACI-27CCL28AAJ29ACL30 -3 ' 4. DNA transfer vector according to claim 3, thereby 30 5. DNA transfer vector according to claim 1, characterized in that for the A and B chains characterized in that the gene of the higher organism coding for human insulin nucleotide sequences Coded growth hormone. by a nucleotide sequence (NaNbNc) j within the part 6. DNA transfer vector according to claim 5, sequence 5'-ACL30 (NaNbNc) jGCLi 3 'are linked by a human amino acid sequence where Na, Nb and Nc can mean A, T, G or C and j 35 nucleotide sequence encoding growth hormone which is any integer of Represents 0 to 100, provided that NaNbNc means neither TAJ nor TGA. 5 * -TTK1CCL2ACL3A, m4CCL5X6TY6QR7S7W8GZ8X9TY9TTK10GAK11AAX12GCL13ATGX15TY15 ^ 16 ^ Zi6GC ^ il7C ^ i8 ^ 19GZi9 ^ 20 ^ 20 ^ '^ 21 ^^ 22 ^ 23' ^ 23 CCL24TTK25CAK26ACL27TAK23CAJ7gCAJ30TTK3ICAJ32CAJ33ACL34TAK35ATN36 CCL37AAJ33CAJ3gCAJ40AAJA1TAK42QR43S43TTK44XA5TY45CAJ46AAKA7CCL4aCAJ49 ACL50 ^ R5lS51X52TY52TCK53TTK5A ^ R55S55GAJ56 ^ R57S57A ™ 58CCL59ACL60CCL61 ^ R62S62 AAK63W64GZ64GAJ65CAJ66ACL67CAJ63CAJ69AAJ70C * R7lS71AAK72X73TY73GAJ74X75TY75X76TY7 W77CZ77A ™ 78QR79S79X80TYS0X81TY31X82TY82A ™ 83CAJ84QR85T35T87C83 ™ CCL89CTL9OCAJ91TTK92X93TY93W94GZ94 ^ R95S95GTL96rTIC97GCL9SAAK99AAKl00XioiTY101 CTL102TAK103GGL104GCLI05QR106S 106GAKio7QR108S 108AäK109GTL1 10TAKi 1 lCAKi 12 X113TY113X114TY114AAJ115GAK'116X117TY117CAJ118GAJ119GGL120A ™ 121CAJ122ACL123 X124TYi24ATGGGL126WI27GZl27X123TY12SGAJ129GAK130GGL13lQR132SL32CCL133 W134GZ134ACL135GGL136CAJ137A ™ 138TTK139AAJ140CAJ1A1ACL142T'UC143QR144S1A4 AAJ145TTK146GAK1A7ACL148'UK149QR150S150CAK151AAK152CAK153GAK154GCL155X156TY156 3rd 642 680 x157Tïl57AJU15SAA1C159TAKl60CGL16lX162Tï162X163Tï163'rAK164TCK165rrK166H167GZ167 AAJ168GAK169AIGCAK171AAJ172CTI'173GAJi74ACL175ITK176X177Tï177W178GZl78A ™ 179 GTL1S0CAJI81TGK182H183GZ183I3R184SX84GTI-ÌS5GAJ186KTGII18T187GGII18 where A = deoxyadenyl, G = deoxyguanyl, C = deoxycy-> »toyl, T = thymidyl, J = A or G, K = T or C, L = A, TC or G, M = A, C or T, Xn = T or C if Yn = A or G, and C if Yn = C or T, Yn = A, G, C or T, if Xn = C, and A or G, if Xn = T, Wn = C or A, if Z "= G or A, and C, if Zn = C or T, Zn = A, G, 15 C or T, if Wn = C, and A or G, if Wn = A, QR" = TC, if Sn = A, G, C or T, and AG if Sn = T or C, Sn = A, G, C or T if QRn = TC, and T or C if QRn = AG and the subscripts Denote amino acid position in human growth hormone, to which the nucleotide sequence corresponds according to the genetic code, where the amino acid positions are numbered from the amino end. 7. A method for producing the DNA transfer vector according to claim 1, which contains in its nucleotide sequence a 25 sub-sequence with the structure of a gene of a higher organism, characterized in that a) cells are isolated which contain mRNA, which encode desired protein b) extract the mRNA from the cells, wherein an RNase-30 inhibitor is present which prevents RNase-induced decomposition of the -mRNA, or, in the absence of such an RNase inhibitor, extract c) isolate an mRNA which essentially is free of proteins, DNA and other RNAs, 35 d) synthesizing a double-stranded cDNA in which one strand has a nucleotide sequence which is complementary to that of the -mRNA and a cDNA is thereby produced which has a nucleotide sequence which encodes the desired protein 40 e) provides a DNA transfer vector which has suitable reactive ends which are capable of being connected to one another or connected to the double-stranded DNA and f) the DNS transfer vector with the double-stranded 45 cDNA that has a nucleotide sequence that encodes the desired protein in a DNA ligase-catalyzed reaction to give a DNA transfer vector that has a sub-sequence in its nucleotide sequence that encodes the desired protein. 50 8. The method according to claim 7, characterized in that one produces a DNA transfer vector, in which the nucleotide sequence has a sub-sequence that encodes insulin by a) producing from the pancreas of an insulin, the organism is isolated, which Contain insulin-encoding mRNA, b) the mRNA is extracted from the islet cells in the presence of the RNase inhibitor, so that the RNase degradation of the mRNA is prevented, 60 c) isolates the mRNA essentially free of protein, DNA and other RNA, d) synthesizing a double-stranded cDNA, one strand of which has a nucleotide sequence which is complementary to the mRNA, wherein a cDNA with 65 nucleotide sequence coding for insulin is obtained, e) provides a DNA transfer vector with reactive ends that can be joined together or with double-stranded cDNA, and f) connects the DNA transfer vector and the double-stranded cDNA with the nucleotide sequence coding for insulin. 9. The method according to claim 8, characterized in that in step (b) an RNase inhibitor is used which contains a chaotropic cation, a chaotropic anion and an agent which cleaves disulfide bonds. 10. The method according to claim 8, characterized in that the reactive ends possessing DNA molecules are linked in step (f) according to a method in which a certain part of the reactive ends is prevented from linking to one another, the method consisting in pretreating a certain portion of the reactive ends with a reagent capable of removing the 5'-terminal phosphate groups, and incubating DNA molecules with pretreated and untreated reactive ends together with a DNA ligase that connects between the reactive ends manufacturing reaction catalyzes except for the pretreated reactive ends, which are not linked together in the ligase-catalyzed reaction. 11. The method according to claim 7, characterized in that a DNA transfer vector is produced which has a sub-sequence in its nucleotide sequence which encodes growth hormone by a) isolating pituitary cells which contain growth hormone-encoding mRNA, b) the mRNA is extracted from the pituitary cells in the presence of an RNase inhibitor, thereby preventing the RNase degradation of the mRNA, c) isolates the mRNA which is essentially free of protein, DNA and other RNA, d) synthesizing a double-stranded cDNA, one strand of which has a nucleotide sequence which is complementary to the mRNA, wherein a cDNA with a nucleotide sequence coding for growth hormone is obtained, e) provides a DNA transfer vector which has reactive ends capable of binding to one another or to double-stranded cDNA, and f) connects the DNA transfer vector to the double-stranded cDNA with a nucleotide sequence coding for growth hormone. 12. The method according to claim 11, characterized in that one uses an RNase inhibitor which contains a chaotropic cation, a chaotropic anion and a disulfide bond cleaving agent. 13. The method according to claim 11, characterized in that the reactive ends possessing DNA molecules are linked in step (f) by a method in which a certain part of the reactive ends is prevented from linking to one another, the method consisting in that a certain part of the reactive ends is treated with a reagent which is capable of removing the 5'-terminal phosphate groups, and the DNA molecules with pretreated and untreated reactive ends are incubated together with a DNA ligase which connects between the ends manufacturing 642 680 4th The reaction catalyzes except for the pretreated ends, which are not joined together in the ligase-catalyzed reaction. 14. Use of the DNA transfer vector according to claim 1, which contains in its nucleotide sequence a sub-sequence with the structure of a gene of a higher organism, which was created by transcription to a gene of the higher organism in order to modify a microorganism so that it contains this sequence. 15. Use according to claim 14, characterized in that a DNA transfer vector is used which has in its nucleotide sequence a sub-sequence with the structure of a gene which codes for insulin in order to produce a microorganism which contains a nucleotide sequence which codes for insulin by mixing a microorganism with this DNA transfer vector linked to the cDNA. 16. Use according to claim 14, characterized in that a DNA transfer vector is used which has in its nucleotide sequence a sub-sequence with the structure of a gene encoding growth hormone in order to produce a microorganism which has a nucleotide sequence which encodes growth hormone by mixing a microorganism with the DNA transfer vector associated with the cDNA. 17. Use according to any one of claims 14-16, characterized in that the DNA transfer vector used is a plasmid and that a bacterium is used as the microorganism to be modified.