CS227032B2 - Microorganism cultivating method - Google Patents

Description

The invention relates to a method of growing a microorganism, in particular to isolating a specific nucleotide sequence comprising a genetic information code for a specific protein, synthesizing a DNA comprising the specific sequence of nucleotides and transferring the DNA to a microorganism in which the DNA can be propagated. The invention further relates to the isolation of the growth hormone gene, the purification of these substances, their transfer and their multiplication in a microbial host, and the detection of their properties. A number of new products can be produced by the process of the invention. These products also include a recombinant plasmid containing a specific nucleotide sequence derived from a higher organism and a novel microorganism containing, as part of its genetic grouping, a specific nucleotide sequence derived from a higher organism.

The following abbreviations are used during the application:

DNA - desoxyribonucleic acid

RNA - ribonucleic acid cDNA - complementary DNA (enzymatically synthesized from the mRNA sequence) mRNA - RNA involved in messenger RNA tRNA - transferred RNA dATP - desoxyadenosine triphosphate dGTP - desoxyguanosine triphosphate dCTP - desoxycytidine triphosphate

A - adenine

T-thymine

G - guanine

C - cytosine

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

EDTA - ethylenediaminetetraacetic acid ATP - adenosine triphosphate TTP - thymidine triphosphate

The biological importance of the basic DNA sequence is that it stores genetic information. It is known that the base sequence in DNA is used as a code that determines the amino acid sequence in all proteins that a cell produces. In addition, part of this sequence is used for regulatory purposes, in particular to control the time and amount of production of individual proteins. The nature of these proceedings has so far been only partially clarified. Finally, the base sequence in each strand is used as a basis for DNA renewal as well as for DNA multiplication that accompanies each cell division.

The way that basic DNA sequence information is used to determine the amino acid sequence of proteins is a fundamental process that is broadly universal for all living organisms. It has been shown that each amino acid found in proteins is determined by one or more sequences of trinucleotides. It follows that for each protein it is possible to detect a corresponding DNA segment that contains a triplet sequence that corresponds to the amino acid sequence of the protein. The genetic code is shown in the following table.

The biological way of converting the nucleotide sequence as information into the amino acid sequence in the first step is that the local DNA segment with the sequence that determines the structure of the protein to be produced is first transformed into its copy by RNA. RNA is a polynucleotide that is similar to DNA except that ribose is replaced by desoxyribose and uracil is used instead of thymine. RNA bases can get into the same relationships as in DNA. As a result, it is possible to create a complement in the RNA to the DNA nucleotide sequence. This modified RNA is called mRNA because it is an intermediate link between the genetic system and the protein synthesis system in the cell.

The cell uses mRNA in complex processes, including enzymes and organelles, to synthesize a specific sequence of amino acids. This method is called mRNA translation.

In many cases, additional steps are required to convert the synthesized amino acid sequence and functional protein. Insulin will be exemplified. Genetic code phenylalanine (Phe) TTK leucine (Leu) XTY isoleucine (Ile) ATM methionine (Met) ATG valine (Val) CTL serine (Ser) OPS proline (Pro) CCL threonine (Thr) ACL alanine (Ala) GCL tyrosine (Tyr) ) TAK termination signal TAJ termination signal TGA histidine (His) CAK glutamine (Gln) CA) asparagine (Asn) AAK lysine (Lys) AAJ aspartic acid (Asp) CAK glutamic acid (Glu) CA) cysteine (Cys) TGK tryptophan (Try TGG arginine (Arg) WGZ glycine (Gly) GGL

Key:

Each group of three letters represents a DNA trinucleotide with 5 ‘on the left and 3‘ on the right. Single letters mean purine or pyrimidine bases that form a nucleotide sequence.

A = adenine

G = guanine

C = cytosine

T = thymine

X = T or C when Y and G

X ’== C if Y is C or T

Y = A, G, C or T when X is C

Y = A or G when X is T

W - C or A, if ZA or GWC, if ZC or TZ - A, G, C or T, if WCZ = A or C, if WA QR = TC, if SA, G, C or T QR = AG if ST or CS = A, G, C or T if QR TC S = T or C if QR AG) - A or G

Κ = T or C L = A, T, C or G M = A, C or T

Alu I treatment site - A sequence of AG and CT, specifically cleaved at the arrow site by Alu I endonuclease.

Bam HI treatment site - sequence G and ATCC, specifically cleaved at the arrow site by Bam HI endonuclease.

Bgl I treatment site - sequence GCCNNNN 1 NGGC, specifically cleaved at the arrow site by Bgl I endonuclease (N stands for any nucleotide).

Eco RI site - G and AATTC sequence specifically cleaved at the arrow site by Eco RI endonuclease.

Eco RII endonuclease site - a sequence of both CCAGG or CCTGG, specifically cleaved at the arrow site by Eco RII.

Hae II endonuclease treatment site - sequence of AGCGC 1 T or AGCGC i C or GGCGC i T or GGCGC 1 C, specifically cleaved at the arrow site by Hae II endonuclease.

Hinc II Endonuclease Treatment Site - GTTAAC or GTCAAG or GTCGAC sequence, specifically cleaved by Hinc II endonuclease.

Pst I endonuclease site - CTGCA and G sequence, specifically cleaved at the arrow site by Pst I endonuclease

Sal I endonuclease site - G and TCGAC sequence specifically cleaved at the arrow site by Sal I endonuclease

Sst I endonuclease site - GAGCT and C sequence, specifically cleaved at the arrow site by Sst I endonuclease

The immediate precursor of insulin is a polypeptide called proinsulin, which contains two insulin chains A and II linked together by another peptide C as described in Steiner, DR, Ounningham D., Spigelman L. and Eten B., Science 157, 697 (1967). Recently, it has been reported that the initial translation product of insulin mRNA is not proinsulin itself but preproinsulin, which contains more than 20 additional amino acids at the amino terminus of proinsulin, as described in Cahn SJ, Keim F. and Steiner FD, Why. Nati. Acad. Sci. USA 73, 1964 (1976) and in Lomedico P.T. and Saunders C.F., Nud. Acids Res. 3, 381 (1976).

The structure of the propreinsulin molecule can be schematically represented as

NH2 - (prepeptide j) - chain B- (C-peptide) - chain A-COOH.

In proteins of higher organisms, such as vertebrates, many proteins of medical or research interest can be found. These include, for example, the hormone insulin, other hormones of a peptide nature, such as growth hormone, proteins that act to control blood pressure, and various types of enzymes of industrial, medical or research interest. It is often very difficult to obtain these proteins in a usable amount of extractions and organisms, this problem is particularly burdensome in the case of human proteins. It is therefore necessary to find a way in which these proteins can be obtained in sufficient quantities outside the original organism.

In some cases, appropriate cell lines can be obtained and maintained as tissue cultures. However, cell growth in tissue cultures is slow, the growth environment is very expensive, the growing conditions must be accurately maintained and yields are relatively low. Often it is very difficult to maintain cell l ^! nii with desired properties.

In contrast, microorganisms such as bacteria can be easily grown in chemically defined nutrient media. The fermentation processes are highly sophisticated and can be well controlled. The growth of organisms is rapid and high yields can be obtained. Moreover, some microorganisms are precisely genetically defined and are actually one of the best defined organisms ever.

For these reasons, it is highly desirable to have a genetic code transfer for proteins of medical importance from the organism in which the protein is normally produced to the microorganism in question. In this way, it would be possible for the protein to be produced by the microorganism under controlled growth conditions so that it could be obtained in the desired amount. It is also possible in this way to substantially reduce the cost of producing this protein. In addition, the possibility of isolating and transferring a genetic sequence that determines the production of a certain protein to a microorganism and a well-defined genetic structure would be of great value for research into the way the protein is synthesized and for the post-synthesis behavior of that protein. In addition, the isolated genetic sequence could be converted to code for different proteins with altered therapeutic or functional properties.

The method according to the invention achieves this objective. The process of the invention involves a variety of reactions, including enzymatically catalyzed reactions. The nature of these enzymatic reactions, if known, is always described at the same time.

Reverse transcriptase catalyzes DNA synthesis in the presence of modified RNA, oligodesoxynucleotide and four desoxynucleoside triphosphate, dATP, dGTP, dCTP, and TTP. The reaction begins with the non-covalent binding of an oligodesoxynucleotide that binds to the 3 ‘mRNA position, then sequentially binds the corresponding desoxynucleotides, as determined by the nucleotide sequence of the mRNA, to the 3‘ position of the growing chain. The resulting molecule contains the original RNA together with complementary DNA that is bound by a single DNA loop. The reverse transcriptase can also catalyze a similar reaction using modified DNA, in which case the resulting product is a compound with two strands of DNA joined together by a single DNA strand loop. These procedures have been described by Aviv H. and Leder R., Proc. Nati. Acad. Sci. USA 69, 1408 (1972) and Efstratiadis A., Kafotos, F. C., Maxam A. F., and Manistis T., Cell 7, 279 (1976).

Restriction endonucleases are enzymes that are capable of hydrolyzing phosphodiester bonds in the double strand of DNA, thereby breaking the continuity of the DNA strand. When the DNA is in the form of a closed loop, it turns into a linear structure. An essential characteristic of this type of enzyme is that the hydrolytic action occurs only at the site where the specific nucleotide sequence is located. This site is essential for the action of a restriction endonuclease. Enzymes of this type have been isolated from various sources and have been characterized by their site of action in the nucleotide chain.

Some restriction endonucleases hydrolyze phosphodiester linkages in both chains at the same site, other enzymes of this type catalyze the hydrolysis of bonds that are separated by only a few nucleotides, leaving free portions of the individual chains that are cleaved from the parent molecule. The terminal portions of these strands can be subjected to digestion with this enzyme, mum, to rebuild the hydrolyzed DNA. Since each DNA that can be subjected to digestion with this enzyme must contain a site of action, the same terminal portions are formed so that heterologous DNA strands that have been released by the above enzyme can be joined in this way. These procedures have been described in Roberts, R.J., Crit. Roar. Biochem. 4, 123 (1976). The sites where these enzymes can act naturally occur relatively rarely, but the usefulness of these enzymes has been increased by chemical synthesis of the double-stranded oligonucleotides carrying the enzyme-appropriate sites. In this way, almost any DNA segment can be joined to another segment by simply linking another nucleotide with a restriction endonuclease site at either end of the molecule and then hydrolyzing the resulting product with the appropriate restriction endonuclease to produce the corresponding end as described by Heyneker H. L, Shine J, Goodman II. R., Bayer, H., Rosenberg, J, Dickerson, R. E, Narang, S.A, Itakura, K, Lin, S., and Riggs, A., Nature 263, 748 (1976), and Scheller, R., Dickerson. E, Boyer H. W, Riggs A. D. and Itakura K, Science 196, 177 (1977).

S1-endonuclease is an enzyme that highly specifically hydrolyzes phosphodiester bonds in single-stranded DNA or single-stranded DNA that otherwise contains two strands, as described in Vogt, V. H, Eur. J. Biochem. 33, 192 (1973).

DNA ligase is an enzyme that is able to catalyze the formation of phosphodiester linkage between two DNA segments with 5 ‘phosphate and 3‘ hydroxyl groups, as they can be formed from two DNA fragments. The normal function of this enzyme is to attach a single strand or a portion of a strand in a double-stranded DNA. However, under appropriate conditions, the DNA ligase can catalyze the joining of the free ends in a covalent manner as described by Sgaramella V, Van de Sande, J. H, and Khorana, H. G, Proc. Hetl. Acad. Sci. USA 67, 1468 (1970).

Alkaline phosphatase is an enzyme whose general ability is to hydrolyze phosphate esters including 5 ^! -terminal phosphate DNA.

The next step to be described is to incorporate a specific DNA fragment into a DNA vector, such as a plasmid. A plasmid is a term called an autonomous DNA replication unit, as found in a microbial cell, distinct from the genome of the host cell itself. The plasmid is not genetically bound to the host cell chromosome. A DNA plasmid exists as a double-stranded circular molecule with a molecular weight of several million, although some molecules are greater than 10 ⁸ , molecules of this type usually make up only a small proportion of the total DNA of a cell. The DNA plasmid can usually be separated from the DNA of the host cell, due to the large size difference of the molecule. Plasmids may proliferate independently of the cell division rate of the host cells, and in some cases their proliferation may be controlled by changes in growth conditions. Although the plasmid exists in the form of a closed ring, it is possible to artificially introduce a DNA segment into the plasmid so that a larger molecule recombinant plasmid is formed without affecting the reproductive capacity of the plasmid or its other ability. The plasmid therefore serves as a suitable vector for transferring a DNA segment into a host cell. The plasmids used to produce recombinant DNA typically contain genes that may be suitable for selection purposes, for example, genes for resistance to certain substances.

In order to describe the method of the invention in more detail, the method will be illustrated for the isolation and transfer of the rat insulin gene. Insulin was chosen for its great importance in clinical medicine and also because its metabolism and structure are well developed in basic research. The procedure described is also applicable to the isolation of the insulin gene of other organisms, including man, by any person skilled in the art.

Insulin was first isolated in 1922. At present, the use of this hormone for the treatment of diabetes is well known. The common source of insulin is the pancreas of cattle and pigs, but there is a lack of this hormone as the number of people with diabetes worldwide is rising; In addition, many of these patients develop allergic reactions against bovine and porcine insulin over time, with the corresponding adverse consequences. It would therefore be desirable to obtain human insulin in sufficient quantities. Production of human insulin in bacterial cultures would be a good solution to this problem. So far, however, all attempts at this cultivation have been hampered by the fact that until now it has not been possible to incorporate the insulin gene into a bacterial cell. The method according to the invention enables this incorporation.

The ability to obtain DNA with a specific sequence that is also the genetic code for a specific protein allows to modify the nucleotide sequence chemically or biologically so that the specific protein produced is also modified. In this way, for example, it is possible to produce modified insulin for certain medical purposes. The genetic ability to produce any amino acid chain suitable for incorporation into the insulin chain or having the essential functional properties of insulin can be transferred to the microorganism. Similar conditions can be provided for growth hormone.

The ability to transfer the genetic code for a specific protein necessary for the normal metabolism of a particular higher organism to a microorganism, such as a bacterium, opens up the possibility of culturing that protein in the form of a conventional culture. This also opens up the possibility of obtaining more of these proteinaceous substances and gradually replacing them with the proteins produced by the microorganisms in any case where the ability of a higher organism to produce these proteins normally fails. In this way, for example, it would also be possible to establish symbiotic relationships between microorganisms modified according to the invention and humans with acute or chronic disease, whereby the genetically determined microorganism could be implanted or otherwise associated with the human organism to compensate for the pathological metabolic state of a diseased human.

Accordingly, the invention relates to a method of isolating a specific nucleotide sequence comprising genetic information, synthesizing DNA with a specific nucleotide sequence, and transferring the DNA to a host microorganism.

Accordingly, the present invention provides a method for culturing a microorganism containing and replicating a DNA transfer vector with a nucleotide sequence coding for insulin by extracting the mRNA coding for insulin from the cells, purifying the mRNA, and synthesizing the cDNA from the mRNA. with a nucleotide sequence coding for insulin, whereupon the cDNA is reacted with a DNA transfer vector to form a DNA transfer vector with a nucleotide sequence coding for insulin, and the DNA transfer vector transforms a microorganism characterized by: that the mRNA is extracted from cells containing mRNA that encodes insulin by homogenizing these cells in the presence of a ribonuclease inhibitor containing guanidinium thiocyanate and β-mercaptoethanol at pH 5.0 to 8.0 to prevent degradation of the mRNA by ribonuclease and cDNA reported in reaction with a tsk DNA transfer vector by enzymatically hydrolyzing a restriction endonucleate DNA transfer vector from a group of Hind III or Hsu I to form a DNA transfer vector having reactive ends capable of being linked to or linked to a cDNA having a nucleotide sequence coding for insulin and, in a second step, enzymatically linked to a DNA and cDNA transfer vector having s ^; ed nucleotide coding for insulin using DNA ligase in the presence of ATP to produce a DNA transfer vector with a nucleotide sequence coding for insulin.

Examples of transfer of DNA with a specific nucleotide sequence to bacteria are the rat preproinsulin gene, the rat growth hormone gene, and the human growth hormone gene. It is understood that the method of the invention could be adapted to transfer any desired DNA sequence from a higher organism, for example a vertebrate, to any microorganism. A higher organism in this case is defined as a eukaryotic organism with differentiated tissues, that is, for example, insects, molluscs, plants, vertebrates, including mammals, such as cattle, pigs, primates and humans. Micro-organism means any micro-organism, whether prokaryotic or eukaryotic, including bacteria, protozoa, algae and fungi, including yeast. In carrying out the method of the invention, a selected cell population is first isolated in an improved manner. Intact mRNA is extracted from these cells in a new manner, which simultaneously suppresses all ribonuclease activity. Intact mRNA is purified from the extract by column chromatography and then treated with the enzyme reverse transcriptase, which acts in the presence of the four desoxynucleoside triphosphates required to synthesize the cDNA complementary strand. The product of the first reaction using reverse transcriptase is then processed by selectively removing the ribonucleotide sequence. The remaining sequence of deoxynucleotides, which is complementary to the original mRNA, is incubated during the second reaction using reverse transcriptase or DNA polymerase in the presence of four desoxynucleoside triphosphates. The resulting product is a double cDNA structure whose complementary strands are linked together at one end by a single strand loop. This product is then reacted with a specific nuclease that cleaves the loop. The resulting double-stranded cDNA is then set up at both ends by binding specific DNA that contains sites where a restriction enzyme can act. This addition is catalyzed by the enzyme DNA ligase. The resulting product is then treated with a restriction endonuclease to produce free-ended chains that can complement each other.

The same enzyme is treated with a DNA plasmid that contains sites suitable for the same restriction endonuclease to cleave the polynucleotide chain and produce similar free ends capable of binding to the terminal ends of the 5 ', 5 ' -phosphate group. the plasmid could not create an annular structure that would change the host cell. Then the cDNA and DNA plasmid prepared above. together they incubate in the presence of DNA ligase. Under the reaction conditions described above, viable closed circles of the DNA plasmid will only be formed if cDNA with the appropriate free ends is present. The altered plasmid is then inserted into a suitable host cell which has received the altered viable plasmid, which is to be determined by the formation of colonies with altered properties, for example, resistance to certain chemicals. Pure bacterial strains containing the recombinant plasmid are then grown and the recombinant plasmid is reisolated. In this way, large amounts of the recombinant plasmid can be prepared and thus the specific sequence of cDNA can be isolated again by digestion with the appropriate restriction enzyme.

Any desired sequence of nucleotides derived from a higher organism, including humans, can be isolated and transferred to the host microorganism by the basic method of the invention. The method is suitable for transferring the genetic code for a specific protein of medical or industrial importance and for the microbiological synthesis of such a protein. In illustrating the method of the invention, isolation of the genetic code for rat insulin was described, which was then transferred to bacteria and propagated. In a similar manner, the nucleotide sequence for rat growth hormone was isolated and this sequence was also transferred and expanded in bacteria. The method of the invention may also be applied to the transfer of a nucleotide sequence isolated from a human, including human insulin, growth hormone and other hormones of a polypeptide nature.

The method of the invention envisages a method of isolating a DNA molecule having a specific nucleotide sequence, transferring this material to a microorganism, and consequently multiplying the nucleotide sequence in said microorganism.

The sequence of steps encompassed by the method of the invention can be described in four basic groups.

1. Isolation of the desired cell coupling from a higher organism

There are two potential sources of genetic code for a specific protein. It is a DNA organism and, moreover, a modified RNA of that organism. According to conventional safety regulations adopted by the National Institutes of Health, human genes of any kind can be processed into recombinant DNA and then transferred to bacteria only if the genes have previously been thoroughly purified on a designated device. (Federal Register, Vol. 41, No. 131, Jul. 7, 1967, pp. 27 902 to 27 943.) This means that for any method that could be used to produce human proteins, it is preferable to isolate specific mRNA with a sequence of nucleotide, which is the genetic code for the protein of interest. This procedure has the additional advantage that mRNA is easier to purify than DNA extracted directly from cells. In particular, it is advantageous to utilize the fact that in the above-differentiated organisms, such as vertebrates, a specific population of cells can be found at a specific location in the organism, the essential function of all these cells being the production of the desired protein. It may also occur that said cell population exists only for a transient developmental period of the organism. In these cell populations, even a large portion of the mRNA isolated from the cell will have the desired nucleotide sequence, so it is possible to choose a preferred cell population and a preferred method of isolation in terms of the initial purity of the isolated mRNA.

In most tissues, glands and other organs, cells are usually associated with a fibrous connective tissue network, which essentially consists of collagen, but may contain other substances, such as proteins, polysaccharides or inorganic substances, depending on the type of tissue. Isolation of cells from a given tissue requires a technique that releases cells from the connective tissue matrix. Thus, the isolation and purification of a specifically differentiated cell type consists of two main steps, namely the separation of cells from connective tissue and the separation of cells of the desired type from other cells of the tissue. By the method of the invention, different cell types can be isolated from different tissue types. An example is the isolation of islets of Langerhans from the pancreas, which is necessary for isolation of insulin mRNA.

Insulin-producing cells can be obtained from other sources, such as the immature pancreas of the calf or from a tissue culture of tumors of these cells. Isolation of pure islet cells is much easier in these cases, especially in the case of tissue culture. Thus, it will not always be necessary to isolate cells directly from the pancreas, but in some cases this is advantageous.

It can often be shown that the proportion of the desired mRNA can be increased by external influences. For example, hormone treatment may result in increased production of the desired mRNA. Other methods are, for example, growing at a certain temperature, supplying a certain nutrient or other chemical. In the isolation of rat growth hormone mRNA, it was possible to substantially increase the desired proportion of growth hormone mRNA by treating rat pituitary cells grown in tissue culture simultaneously with thyroid hormone and glucocorticoids.

2. Extraction of mRNA

An important object of the method according to the invention is, as far as possible, to completely eliminate ribonuclease activity in the cell extract. Indeed, it is necessary to extract mRNA with a single polynucleotide strand without the additional complementary strand. This implies that hydrolytic cleavage of a single phosphodiester bond in the chain would render the entire molecule unusable for transferring the intact genetic sequence to the microorganism. As already mentioned, the ribonuclease enzyme is widespread, potent and exceptionally stable. It can be applied to the skin, it can withstand the usual washing-up procedures without damage and is often a pollutant for stored organic chemical compounds. The difficulties are particularly evident when it comes to extracting cells from the pancreas, since this gland is a source of digestive enzymes and is therefore rich in ribonuclease. However, the problem of this enzyme is associated with all tissues, and therefore a method of removing the ribonuclease activity of the invention is also applicable to all tissues. The exceptional efficacy of this method will be illustrated in the successful isolation of intact Langerhans islet cell mRNA.

In the method of the invention, a protein-free combination of chaotropic anion, chaotropic cation and disulfide bonding agent is used during cell disruption and during all procedures required for RNA isolation. The efficacy of the simultaneous action of all of the above agents has been demonstrated by their use to isolate intact mRNA in good yield from isolated islets of rat Langerhans islets.

The choice of suitable chaotropic ions is based on their solubility in aqueous media and their availability. Suitable chaotropic cations are, for example, guanidine ions, carbamoylguanidine, guanylguanidine, lithium and the like. Suitable chaotropic anions are iodide, perchlorate, thiocyanate, diodosalicylate, and the like. For example, lithium diodsalicylate is more effective than guanidinium IV cyanate, but its solubility is only 0.1 N and, moreover, is relatively expensive. Guanidinium thiocyanate is the preferred combination of cation and anion because it is readily available and highly soluble in aqueous media up to a concentration of 5 M.

Thiol compounds, such as p-mercaptoethanol, are known to disrupt intramolecular disulfide bonds in proteins by double-swapping. A number of thiol compounds are known to be effective in this regard, for example β-mercaptoethanol, dithiotreltol, cysteine, propanoldimercaptan and the like. The substances must be water soluble because they need to be used in large excess relative to the number of intramolecular disulfide bonds to double substitution can be completed. The most preferred substance in this sense is β-mercaptoethanol because it is readily available and relatively very cheap.

In inhibiting ribonuclease during RNA extraction from cells or tissues, the efficacy of a given chaotropic salt is directly dependent on its concentration. Therefore, the most preferred concentration is at the same time the highest available concentration. The success of the method of the invention in maintaining intact mRNA during extraction depends on the rate at which the ribonuclease is denatured and, moreover, the degree of denaturation. This appears to give the advantage of using guanidinium thiocyanate over hydrochloride, although otherwise hydrochloride is only slightly less effective as a denaturing agent. The efficiency of a denaturing agent is defined as the threshold concentration to be achieved to fully denature the white plane. On the other hand, the degree of denaturation of many proteins depends on the concentration of the denaturant, which sometimes needs to be increased 5 to 10 times. These relationships have been described in Tanford C.A., Adv. Prot. Chem. 23, 121 (1968). Qualitatively, this relationship states that a denaturing agent that is only slightly more effective than guanidinium hydrochloride can denature the protein several times faster at the same concentration. Relationships between kinetics of ribonuclease denaturation and mRNA preservation during cell extraction have not been identified or exploited. If the above relationships are correct, this means that the preferred denaturant is a low threshold concentration and high water solubility. For this reason, guanidinium thiocyanate is preferable to lithium diodsalicylate, although the latter is a far more effective denaturant, because the solubility of guanidinium thiocyanate is much higher and therefore can be used at a concentration that allows more rapid inactivation of ribonuclease. The above analysis of the effect of the individual agents also provides an answer to why guanidinium thiocyanate is preferable to the relatively equally soluble hydrochloride, the reason being that the former is a slightly stronger denaturant.

An agent that disrupts disulfide bonds is used in admixture with a denaturing agent because it has a potentiating effect on the denaturation of the ribonuclease. The thiol compound prevents rapid denaturation, which can occur if intramolecular disulfide bonds remain intact. Furthermore, it is achieved that any additional ribonuclease remaining in the isolated RNA would remain ineffective even in the absence of the denaturant. Disulfide-disrupting thiol compounds are effective at any concentration, although it is preferable to use a large excess of thiol groups relative to intramolecular disulfide bonds to ensure a successful intramolecular cleavage reaction. On the other hand, it has to be considered that many thiol compounds have an unpleasant odor, especially at higher concentrations, so that in practice there is an upper limit of their concentration. When using β-mercaptoethanol as an effective concentration in the range 0.05 to 1.0 M, the optimal concentration for isolation of intact RNA from rat pancreas is 0.2 M.

The medium in which the mRNA is extracted from the cells should have a pH in the range of 5.0 to 8.0.

After cell disruption, the RNA is separated from

DNA and other cellular proteins. A number of methods have been developed for this purpose, all of which are useful and described in the literature. A common method is ethanol precipitation, where RNA is selectively precipitated. A preferred method for use in the process of the invention is to omit the precipitation step and superimpose the homogenate directly onto a 5.7 M cesium chloride solution in a centrifuge tube followed by centrifugation as described by Glisin, V., Crkvenjakov R., and Byus C. Biochemistry 13, 2633 (1974). This method is most advantageous because it maintains a ribonuclease unfavorable environment and thus yields high yield RNA free of DNA and protein.

The above method can be used to obtain purified RNA from a cell homogenate. Only part of this RNA is the desired mRNA. In order to further purify the desired fraction, it is advantageous to take advantage of the fact that in the cells of higher organisms, mRNA is further expanded by the cell by polyadenylic acid binding upon its formation. This fraction of mRNA that contains the polyadenylic acid sequence _; can be selectively isolated by chromatography on a cellulose column to which the oligothymidylate is bound as described in Aviv H. and Leder P, supra. The above procedures are sufficient to obtain pure, intact mRNAs from ribonuclease-rich sources. Further purification of this portion and other in vitro procedures can be performed essentially the same for any type of mRNA regardless of source.

In certain circumstances, for example, where the source of mRNA is a tissue culture, ribonuclease contamination may be so low that it is not necessary to use the above-described method of ribonuclease inhibition. In these cases, conventional methods used to reduce ribonuclease activity are sufficient.

3. Generation of cDNA

Figure 1 schematically illustrates the procedure for the remaining steps of the method of the invention. The first step is the generation of a complementary DNA sequence for mRNA purification. The enzyme used for this reaction is usually reverse transcriptase, although in principle any enzyme can be used to generate a complementary DNA strand using mRNA as a support. The reaction can be carried out under the conditions described in the known literature using mRŇA and a mixture of four desoxynucleoside triphosphates as a precursor of the DNA strand. It is preferred that one of the desoxynucleoside triphosphates be used in the ³² P-labeled form in order to monitor the progress of the reaction and isolate the product in time after separation of the ballasts, for example by chromatography or electrophoresis. Radioactive phosphorus labeling is also advantageous for quantitative determination of yield as described in Efstratiadis A., et al., Supra.

As further shown in FIG. 1, the production of the 1B reaction using a reverse transcriptase is a double strand hairpin structure, and a non-covalent bond between the RNA strand and the DNA strand.

The reaction product using reverse transcriptase is removed from the reaction mixture by conventional means. It has been found advantageous to use a combination of phenol extract, chroinatography on Sephadex G-100 (Pharmacia Inc, Uppsala, Sweden) and ethanol precipitation.

Once enzymatic cDNA synthesis has occurred, the base RNA can be removed. A variety of methods for selectively degrading RNA in the presence of DNA are known in the literature. The preferred method is alkaline hydrolysis, which is highly selective and can be easily controlled by adjusting the pH.

Optionally, after alkaline hydrolysis followed by neutralization, ³² P-labeled cDNA can be concentrated by ethanol precipitation.

Hairpin-shaped double-stranded cDNA synthesis is accomplished using an appropriate enzyme, such as DNA polymerase or reverse transcriptase. Reaction conditions similar to those described above are used, including the use of a position-labeled nucleoside triphosphate and radioactive phosphorus. Reverse transcriptase can be obtained from a variety of sources. A suitable and common source is avian myeloblastosis virus. This virus can be obtained from Dr. Beard, Life Sciences Incorporated; St. Petersburg, Florida, where the virus is produced in collaboration with the National Institutes of Health. .....

Generation of hairpin cDNAs may be desirable to obtain purified DNA from the reaction mixture. As mentioned above, it is preferable to use phenol extraction, Sephadex G-100 column chromatography and ethanol precipitation to obtain purified protein-free DNA.

The hairpin-like structure can be converted to a conventional double-stranded DNA structure by removing a single loop that connects the two ends of the complementary strands. A variety of enzymes capable of hydrolytically cleaving single DNA strands can be used for this purpose. A suitable enzyme of this use is S1 nuclease isolated from Aspergillus oryzae. This enzyme is available from Miles, Research Products, Elkhart, Indiana. By applying S1 nuclease to the hairpin-like DNA structure, a cDNA molecule with the appropriate ending is obtained in high yield. Extraction by chromatography and ethanol precipitation as described above gave the purified product. The use of reverse transcriptase and S1 nuclease in the synthesis of double-stranded cDNA as a structure corresponding to mRNA has been described in Efstratiadis et al., Supra.

If desired, the proportion of the corresponding end-cDNA molecule can be increased by using E. coli DNA polymerase I in the presence of four desoxypucoside triphosphates. Combination exonukleázového polymerase and the effect of this enzyme has the effect of removing the free ends ^3: .a bond formed at all 5 'end. This ensures maximum participation of the cDNA molecule in subsequent binding reactions.

A further step of the method of the invention is to treat the end of the cDNA so obtained in order to obtain sequences which contain a restriction endonuclease site in place of the appropriate effect. The choice of the DNA fragment that binds to these ends depends on the reaction possibilities. The sequence to be attached to these ends is selected according to the restriction endonuclease chosen, and the choice of this enzyme further depends on the choice of the DNA vector to be recombined for cDNA. The selected plasmid should contain at least one site at which a restriction endonuclease may act. For example, plasmid pMB9 contains one site in which the restriction enzyme Hind III can act. Hind III is isolated from Hemophilus influenzae and purified according to the method of Smith H. C. and Wilcox K. W., J. Mol. Biol. 51, 379 (1970). A similar Hae III enzyme from Hemophilus aegypticus is purified as described by Middleton, J. H., Edgell. H., and Hutchison III, C.A., J. Virol. 10, 42 (1972). The enzyme from Hemophilus suia, designated Hsu I, catalyzes specifically by hydrolysis at the same site as Hind III. The two enzymes can therefore be exchanged for function.

It is preferred to use a chemically synthesized double stranded decanucleotide that contains a site in which Hind III can act and bind the strand to the ends of the cDNA. The double stranded decanucleotide has the sequence shown in Figure 1 and has been described by Heyneker H. L. et al. With Scheller R. H. et al. A variety of similar sequences are available which contain a site where the restriction enzyme can act so that the double stranded DNA ends can be occupied so that the resulting substance is susceptible to any restriction endonuclease selected.

Coupling of the above sequences with restriction endonuclease sensitive sites to the ends of the cDNA can be accomplished by any known method. A highly preferred method is a DNA ligase-catalyzed method purified by the method of Panet A. et al. Biochemistry 12, 5045 (1973). The binding reaction was described in Sgarellell, supra. The product of this reaction between the cDNA and the large molar excess of the double-stranded decanucleotide containing the sites suitable for Hind III restriction endonuclease is cDNA with said strands at each end. When treated with Hind III, this reaction product cleaves at the above sites to form a single 5 řetězce chain that can complement each other, as shown in Figure 1.

4. Construction of vector for recombinant DNA transfer

In principle, large amounts of virus and plasmid DNA can be used to generate recombinant cDNAs prepared as described above. Essential requirements are that the selected DNA transfer vector can be incorporated into the host cell, be multiplied within the host cell, and that the vector contains a genetic determinant to separate those host cells that contain the vector. From a safety point of view, it is necessary to limit the choice of these vectors to meet the requirements of the National Institutes of Health (NIHJ). The list of permitted vectors for DNA transfer is constantly expanding with new vectors and subject to NIH Recombinant DNA Safety Committes approval. any DNA of viral or plasmid origin with the necessary capabilities, including those that will be allowed by the NIH only later, suitable vectors that are already allowed include a variety of lambda bacteriophage derivatives (Blattner, FR, Williams BG, Blechl AE, Denniston-Thompson, K., Faber E., Furlong LA, Grunwalc] DJ, Kiefer DO, Hoore DD, Schomm,

J. W., Sheldon E. L. and Smithies O., Science 196, 161 (1977)) and col E1 plasmid derivatives, as described, for example, in Rodriguez R. L., Bolivar S., Goodman et al. M., Boyer HW and Betlach Μ. N. INC-UCLA Symposium on Molecular Mechanisms In The Control of Gene Expression, DP Nierlich, WJ Rutter, CF Fox, Eds, (Academic Press, NY, 1976), pp. 471-477. Plasmids derived from col E1 are relatively their molecular weight is of the order of several million and has the property that the copy number of plasmid DNA in a single host cell can be increased from the conventional 20 to 40 to 1000 or more by treating the host cell with chloramphenicol. The ability to increase the amount of gene in a host cell allows, under certain conditions, to force the host cell to produce primary proteins, the codes of which are carried by the genes contained in the plasmids. These col E1 derivatives are therefore preferred vectors for use in carrying out the process of the invention. Suitable col E1 derivatives are, for example, plasmids pMB9 carrying a tetracycline resistance gene and plasmids pBR-313, pBR-315, pBR-316, pBR-317 and pBR-322, which contain, in addition to the tetracycline resistance gene, a resistance gene ampicillin. The presence of resistance genes is an advantageous feature for recognizing cells that have been successfully infected with the plasmid, since colonies of such cells will grow in the presence of

27032, whereas cells that do not contain the plasmid die. In the experiments described above and in the examples below, a plasmid derived from col E1 was used, which contained, in addition to the gene for resistance to a particular substance, a Hind III site.

As in the plasmid selection, the host cell can be selected from a relatively wide range of options, but this amount has to be narrowed for safety reasons.

Plasmid pBR322 was similarly described in Bolivar F. et al., Gene, 2, 95 (1977), which describes the synthesis and properties of this plasmid. Plasmid pBR322 has a molecular weight of 2.7 X 10 ⁶ daltons (controlled, Bolivar F., Gene, 4, 121 (1978)) and carries the ampicillin resistance gene (Ap ^R ) and the tetracycline resistance gene (Tc ^R ). It carries a single restriction endonuclease Pst I site in the Aβ ^R gene. It carries the only endonuclease site of Hind III, Sal I and Bam HI in the region of the Tc ^R gene. In addition, plasmid pBR322 carries a single Eco RI site, two Hinc II sites, five Eco RII sites, three Bgl I sites, twelve Alu I sites, twelve Hae II sites, and seventeen sites. for Hae III. A detailed description of these sites in plasmid pBR322 is given on page 103 of the first reference. When the pBR322 plasmid is digested, the Aβ ^R gene is lost, and DNA can be inserted into the Pst I restriction endonuclease site. Similarly, when the plasmid pBR322 is digested with the restriction endonuclease Hind III or Sal I or Bam HI with the insertion of DNA, the Tc ^R gene is lost. When DNA is incorporated into the Pst I site, recombinant molecules can be found in selection for ampicillin (Ap ^s ) sensitive colonies having Tc ^R. Similarly, when DNA is inserted into a Hind III, Sal I or Bam HI treatment site, recombinant molecules can be found in the selection of colonies that are Aβ ^R and Tc ^s . A vector containing foreign DNA inserted at any of the four sites can be characterized with respect to antibiotic resistance such as Aβ ^with Tc ^R or Aβ ^{R with} Tc ^s . The vector can be further characterized by removing the inserted DNA and determining the molecular weight of both products as compared to the molecular weight of the starting materials. Further characterization can be accomplished by constructing a complete restriction endonuclease site map in this vector. It is also possible to determine the sequence of the inserted DNA.

Plasmid pBR313 has also been described in detail. The synthesis and properties of this plasmid have been described in Bolivar F., et al., Gene 2, 75 (1977). Plasmid pBR313 has a molecular weight of 5.8 X 10 ⁶ daltons and contains an ampicillin resistance gene. (Ap ^{R is a} tetracycline resistance gene (Tc ^R j. This plasmid contains a single restriction endonuclease site Hind III, Sal I and Bam HI in the region of the Tc ^R gene. A full analysis of plasmid pBR313 was performed and a site map was generated restriction endonucleases, this map is shown on page 84. Incorporation of foreign DNA into any Hind III, Sal I or Bam HI site results in the loss of Tc ^R , thus recombinant molecules can be found in the selection of strains with Aβ ^R and Tc ^s . The vector can be characterized by antibiotic resistance, a DNA sequence of molecular weight.

The third plasmid that has been described in detail is pMB9. This plasmid can be prepared according to Rodriguez, RL et al., Supra, and Bolivar F. et al., Gene, 2, 75 (1977). Plasmid pMB9 has a molecular weight of 3.5 X 10 ⁶ daltons and contains a gene for resistance to tetracycline Tc ^R. It has a single site for the action of each of Eco RI, Hind III, Sal I or Bom HI endonucleases. The site of action of the last three endonucleases is found in the Tc ^R gene. When foreign DNA is inserted into the Hind III, Sal I or Bam HI treatment site, Tc ^R is lost.

A vector containing foreign DNA at one of these sites can be characterized by this property. This vector can also be characterized by DNA sequence analysis of the inserted portion, molecular weight comparison, and endonuclease sites.

Plasmid pSCIO1 has also been described in detail. Cohen S. H. et al., Proc. Nat. Acad. Sci. USA, 70, 1293 (1973) describes the synthesis and properties of this plasmid. Additional plasmid properties can be found in Cohen, S. N., et al., Proc. Nat. Acad. Sci. USA, 70, 3240 (1973); Boyer

HW et al., Recombinant Molecules, Beers, RF and Basset, EG, Eds, Raven Press, New York on page 13 (19771 and Cohen SN et al., Recombinant Molecules, on page 91. Plasmid pSCIO1 has a molecular weight of 5.8 X IO ³ daltons and contains the gene for tetracycline resistance Tc ^R. In the region of this gene contains a single site for the action of each of the endonucleases Hind III, Sal I and Bam HI. it also contains a single site for the Eco RI, one site for the Hpa I, one site for Sr I, and four sites for Hinc II, and when this plasmid is digested with H'nd III, the Tc ^R is lost, and the same is true for Sal I or Bam HI cleavage at incorporation. DNA at any one of these sites can be found in the selection of the recombinant molecule, and the vector containing the foreign DNA at any one of these sites can be characterized by a loss of antibiotic resistance. (b) constructing mg227032 py sites for endonuclease action; and (cj) determining the DNA sequence.

For example, the phage for vectors include derivatives of phage lambda, which is called Charon, particularly Charon 3A, Charon 4A, and Charon 16A. These phages have been described in detail. Blatner and others mentioned above. ci describe the synthesis and properties of these phages. These phages were also mapped, including endonuclease sites, as shown on page 161 of the above publication. The genotype of each phage is shown on page 164 together with the result of cleavage at different sites. For example, by incorporating foreign DNA into the Eco RI site of Charon 16A, it causes the loss of the lac5 gene. The growth of recombinant phage on lac * bacteria results in the formation of colorless colonies. That is, a vector containing foreign DNA at a particular site can be characterized by genetic properties, for example, colorless spots on lac * bacteria. The vector can be further characterized by removing the inserted DNA and determining the molecular weight of both products while comparing to the molecular weight of the starting products. Further characterization can be accomplished by constructing a map of endonuclease sites in the vector. It is also possible to detect the sequence of the inserted DNA.

GtWES has been described as well. B, a derivative of lambda bacteriophage. The synthesis and basic properties of this phage have been described in Tremeier D. et al., Nature 263, 526 (1976). Other properties of this phage have been described in Leder P. et al., Science, 196, 175 (1977). The genotype of this phage has been identified in both publications. The second publication also identifies the placement of both Eco RI and Sst I restriction endonuclease sites. This phage contains four Bam HI sites to yield 5.4 X 10 ³ , 19.3 X 10 ³ fragments,

3.8 X 10 ³ and 11.4 X 10 ³ base pairs. The analysis of restriction endonuclease sites is shown on page 527 of the above publication. The lambda B fragment is removed with Eco RI and digested with Sst I. This avoids recombination with the lambda B fragment. It is also possible to incorporate foreign DNA with Eco RI cleavage sites into the phage at the Eco RI cleavage site. The phages can be cloned by in situ hybridization as described by Benton WD and Davis RW, Science, 196, 180 (1977J). The vector can be characterized by (a) removal of the incorporated DNA and determination and comparison of molecular weights (bj by analysis of sites of action) restriction endonucleases; and (c) determining the sequence of the incorporated DNA.

An E. coli strain, designated X-1776, was developed, and NIH approval was obtained for the use of this strain and using the P2 apparatus as described by Curtiss, III, R., Ann. Roar. Microbiol., 30, 507 (1976). E. coli RR-1 is suitable when a P3 type device can be used. As in the case of plasmids, it is understood that any host cell strain is contemplated in the practice of the method of the invention if the strain is capable of transmitting the selected vector, including non-bacterial hosts such as yeast, if such strains NIH will be allowed in the future.

Recombinant plasmids are produced by mixing a plasmid DNA that has been treated with a restriction endonuclease and a cDNA with appropriately treated end groups. In order to minimize cDNA binding to each other, the plasmid is added in a large molar excess. In the previously described methods, the addition of the plasmid in large excess usually resulted in the binding of the plasmid chains to the ring, while at the same time incorporating the cDNA fragment. The cells so treated usually contained predominantly a plasmid without recombinant cDNA. As a result, the process was very time consuming. Attempts have therefore been made to obtain DNA vectors with a restriction endonuclease site in the middle of the gene of choice, so that incorporation of the recombinants divides the gene, thereby causing a loss of function encoded by said gene.

Preferably, a method is used to reduce the number of colonies to be monitored when a recombinant plasmid is used. The method consists in treating the plasmid first treated with a restriction endonuclease with alkaline phosphatase, which is available from several sources, for example, Worthington Biochemical Corporation, Freehold, New Jersey. Alkaline phosphatase removes the S? Terminal phosphate groups from the end of the plasmid after endonuclease treatment, thereby preventing the terminal portions of the plasmid DNA from binding together. As a result, ring formation depends on the incorporation of a DNA fragment containing 5'-terminal phosphate groups. In the above manner, the relative frequency of transformation without recombination can be reduced to less than 1 to 10 ⁺⁴ .

The invention is based on the fact that the DNA ligase catalyzed reaction is performed between the 5'-phosphate end group of the DNA and the 3'-hydroxyl end groups of the DNA. If the terminal 5'-phosphate groups are removed, the reaction does not occur. When double-stranded DNA binds, three types of situations can occur as shown in Table I.

Table I Case

Reactants

Product after use of ligase

3 ‘ ----- OH ----- OPO3H2 5 ‘ + 5 ‘ H2O3PO— HIM- 3 ‘ 3 ‘ 5 ‘ 5 ‘ O — P — O --- 0 — P — 0 —-- 3 ‘ + 2HzO 3 ‘ 5 ‘ 3 ‘ 5 * ----- OH H2O3P — O— - O — P — O --- - + + H2O ----- OH OH— OH HO --- - 5 * 3 ‘ 5 ‘ 3 ‘ 3 ‘ 5 ‘ ----- OH HIM + About reaction ----- OH HIM 5 ‘ 3 ‘

In Table I, double-stranded DNA is schematically represented by solid parallel lines, the corresponding 5‘- and 3‘-end groups being labeled with a hydroxyl or phosphate residue according to their nature. In case of I, 5 fosf-phosphate is present at both ends, resulting in covalent bonding of both chains. In case II, only one string has a terminal. 5‘-phosphate group, so that after covalent bonding the discontinuity remains on the next chain. A chain that is not covalently bonded remains linked to the bound molecule by hydrogen bonds that occur between the two chains, as is known in the literature. In case of III, there is no binding at any of the termini, since they do not contain a 5‘-phosphate group.

Undesirable binding reactions can be avoided by removing 5‘-phosphate groups from the non-bonded termini. Any method for removing 5'-phosphate groups can be used for this purpose as long as the DNA structure is not disrupted. A preferred procedure is an alkaline phosphatase catalyzed hydrolysis.

To illustrate the above procedures, rat insulin insulin cDNA was isolated and recombined with a plasmid. DNA molecules were used to convert E. coli X-1776. Transformed cells were selected by growth on a culture medium containing tetracycline. One recombinant DNA plasmid obtained from the transformed cells contained a DNA fragment containing approximately 410 nucleotides. In addition, other recombinants were obtained and isolated in a similar manner, which were also analyzed. The fragments were released from the plasmid using Hind III or Hsu I, and then subjected to DNA sequence analysis as described by Maxam A. M. and Gilbert W., Proc. Bati. Acad. Sci. USA 74,560 (1977). The nucleotide sequence in the DNA fragment overlaps and contained the complete coding sequence for rat proinsulin I as well as the 13 to 23 amino acids of the prepentide sequence. The complete nucleotide sequence for the above product was determined.

In a similar manner, the cDNA code for rat growth hormone was isolated, recombined with the plasmid, and transferred to E. coli cells. In this way it was possible to re-isolate a nucleotide sequence of approximately 800 nucleotides after successful propagation in E. coli cells, which sequence contained the entire code for the rat growth hormone including portions of the peptide precursor and a portion of the 5‘-region that was not transferred.

In general, the above method can be used to isolate and purify any gene from a higher organism, including human genes, and to transmit and multiply these genes in the cells of microorganisms. The novel recombinant plasmids contain all or part of the isolated gene as described above. New microorganisms, which are still unnamed, have also been described, whose genetic system has been modified to include genes from a higher organism. Specific examples will now be described in which each step of the method of the invention will be described in detail with respect to the isolation, purification and transfer of the rat insulin gene to E. coli cells, thereby explaining in more detail the applicability of the method of the invention. The following examples also characterize recombinant plasmids that comprise a portion of the rat insulin gene, the rat growth hormone gene, the human insulin gene, and the human growth hormone gene.

The invention will be illustrated by the following examples.

He did

The described procedures elucidate the extraction and isolation of rat insulin insulin mRNA, synthesis of complementary DNA, and description of the properties of complementary DNA. Purified cells of rat pancreatic islets can be obtained by the anesthetized rat pancreas subjected infusion Hanks solution retro _t grádní infusion outlet of this gland. Hanks' solution is a standard mixture of salts, is known and can be obtained from a number of manufacturers, such as Grand Island Biological Supply

Gompany, Grand Island, New York. The pancreas is then removed, crushed in Hanks' solution at 0 ° C and digested with collagenase and soybean trypsin. All procedures are performed at 0-4 ° C unless otherwise noted. In particular, it is necessary to maintain these conditions when digesting the gland. Two rat pancreas in 8 ml Hanks medium are placed in a 30 ml glass tube. All glass tubes were pretreated with silicone.

Siliclad, Clay-Edams Division, Becto-Dickinson Inc, Persippany, New Jersey was used for this purpose. The incubation mixture contained 12 mg of collagenase, an enzyme prepared from Clostridium histolyticum, as described by Mendl I., Maekennan, J. D. and Howes E. L., J. Clin. Invest. 32, 1323 (1943), the CLS type (Worthington Biochemical Corporation, Freehold, New Jersey) and 1 mg soybean trypsin inhibitor (Sigma Chemical Company, St. Louis, Missouri) were used. Incubation should be performed at 37 ° C for 25 minutes with shaking at 90 oscillations per minute. The mixture should always be observed to ensure that collagenase digestion is performed to the optimum extent. If the incubation is too short, Langerhans islet cells are not completely released, whereas if the incubation is too long, the cells are disrupted. After incubation, the tube is centrifuged for 1 minute at 200 g. The supernatant is decanted and the segment is washed with Hanks' solution and the procedure is repeated 5 times. After the last centrifugation, the sediment is suspended in 15 ml of Ficoll (Pharmacie Chemical Company, Uppsala, Sweden) at a density of 1.085. Then a 1.080 Ficoll layer and a 1.060 Ficoll Density layer were added, after which the tube was centrifuged at 500g for 5 minutes and 2000g for 5 minutes. As a result, the acine cells remained at the bottom of the tube and the islet cells formed a layer between the two upper layers of the formulation. The islet cell band also contains ganlial cells, lymphatic cells and connective tissue cells. However, large fragments were removed. The cells thus obtained are placed under a microscope under which visible contaminants can be removed manually using a micropipette. The cells are then diluted with Hanks solution and centrifuged again. The supernatant is decanted and the sediment is stored in liquid nitrogen.

Islet cells from 200 rats are co-homogenized in 4 N guanidinium thiocyanate (Tridom, Fluka AG Chemische Fabrik, Busch, Switzerland) containing 1 M / S-mercaptoethanol and buffer to maintain the pH of the solution at 5.0 at 4 ° C . The homogenate is layered on 1.2 ml of 5.7 M cesium chloride solution containing 100 mM EDTA and centrifuged at 37,000 rpm for 18 hours in a SW 50.1 ultra-centrifuge rotor at 15 ° C. (Beckman Ultracentrifuge Instrument Company, Fullerton, Calif.) When centrifuged, RNA reaches the bottom of the tube.

RNA with polyadenylate groups is isolated by chromatography of all RNA on oligo- (dT) -cellulose as described in Avlv H., and Leder, supra.

P.

Reverse transcriptase from avian mycloblatosis virus (DJ Board, Life Science Inc, St. Petersburg, Florida) is then used to transfer the structure of whole islet polyadenylate RNA from rat islets to cDNA. The reaction is carried out in 50 mM hydrochloric acid tris buffer pH 8.3 containing 9 mM MgCl 2, 30 mM NaCl, 20 mM β-mercaptoethanol, 1 mM each of 3 nonradioactive desoxyribonucleoside triphosphates, 250 μΜ of the fourth labeled desoxynucleoside triphosphate, and radioactive phosphorus with a specific activity of 50 to 200 curie per mole, 20 µg / ml oligo-dTi18.1 (Collaborative Research, Waltham, Mass.), 100 µg / ml polyadenylated RNA and 20C Units / ml reverse transcriptase. The mixture was incubated for 15 minutes at 45 ° C. Ethylenediaminetetraacetic acid sodium salt is then added to a concentration of 25 mM and the solution is extracted with an equal volume of water-saturated phenol, then the aqueous phase is chromatographed on a 0.3 X 10 cm column containing Sephadex G-100 in 10 mM tris buffer hydrochloric acid pH 9.0 containing 100 mM NaCl and 2 mM EDTA. The nucleic acid is precipitated with ethanol after addition of ammonium acetate at pH 6.0 to a concentration of 0.25 M. The precipitate is collected by centrifugation, the sediment is dissolved in 50 μϊ of freshly prepared 0.1 M sodium hydroxide solution and incubated at 70 ° C for 20 minutes to RNA hydrolysis. The mixture was then neutralized by addition of 1 M sodium acetate at pH 4.5 and ³² P-cDNA was precipitated with ethanol and redissolved in water. Single chain cDNA fractions were analyzed on polyacrylamide gel as described by Dingman CW and Peacock AC, Biochemistry 7, 659 (1968). The gel was dried and ³² P-cDNA was detected by autoradiography on a Kodak No-Screen NS-ST film (Hustman Kodak Corporation, Rochester, New York). The material was heterodisperse as judged by electrophoresis. It contained at least one major species of 450 nucleotides cDNA, as evidenced by comparison with a known standard.

Example 2

In this example, the synthesis and properties of the double-stranded cDNA containing the sequence of rat insulin as described above will be described. The single strand cDNA of Example 1 is treated with reverse transcriptase to synthesize the complementary strand. The reaction mixture contained 50 mM hydrochloric acid tris buffer pH 8.3, 9 mM MgCl, 10 mM dithiothreitol, 50 mM each of the three unlabeled desoxyribonuleoside triphosphates, 1 mM radio-phosphorus labeled nucleoside triphosphate at a specific activity of 1 to 10 curie per mM, 50 µg / ml cDNA and 220 units / ml reverse transcriptase. The reaction mixture was incubated at 45 degrees Celsius for 120 minutes. The reaction is stopped by adding 25 mM sodium EDTA, the mixture is extracted with phenol and chromatographed on Sephadex G-100 and then precipitated with ethanol. The reaction product fraction of 500-1000 cpm was analyzed by gel electrophoresis as described in Example 1. A heterodisperse band of 450 nucleotides in length was detected by comparison with standard samples. Aliquots of the DNA reaction products of Example 1 and Example 2 were then independently digested with excess Hae III restriction endonuclease and analyzed by gel electrophoresis. Both products were cleaved by said endonuclease so that two radioactive bands were observed during gel electrophoresis. The band produced by the digestion of the double stranded cDNA had approximately the products of this length as the band produced by the cleavage of the single stranded cDNA.

Example 3

In this example, the binding of the decanucleotide chains after Hind III treatment to the double-stranded islet cDNA of Langerhans islets as described in Example 2. The reaction product of Example 2 with a double strand at a concentration of 2-5 µg / ml is reported. in reaction with 30 units of SI nuclease with an activity of 1200 units / ml (Miles Laboratories, Elkhart, Indiana) in 0.03 M sodium acetate at pH 4.6 and 0.3 M sodium chloride with 4.5 M zinc chloride at 22 ° C. ° C for 30 minutes, then the mixture is further incubated for 15 minutes at 10 ° C. The reaction was constructed by adding tris-buffer to 0.1 M, BDTA to 25 mM, and E. coli tRNA prepared according to the method of Ehrenstein G., Methods in Enzymology, SP Colowick and NO Kaplan, Eds. , St. 12A, p. 588 (1967) to 40 μ% 1 / ml. The reaction mixture was extracted with phenol and chromatographed on Sephadex

G-100 and ³² P-cDNA labeled are ethanol precipitated. In this way, the paired-ended cDNA molecules required to bind chemically synthesized decanucleotides are obtained in high yield. Hind III decamers were prepared as described by Scheller RH, Dickerson, RE, Boyer HW, Riggs AD, and Itakura K., Science 196, 177 (1977). The binding of Hind III decamers to cDNA is performed by incubation at 14 ° C in 60 mM Tris-buffered saline pH 7.6 in the presence of

6.6 mM magnesium chloride, 1 mM ATP, mM dithiothreitol, 3 mM Hind III decamers 10 ⁵ cpm / pmol T4 DNA ligase in an amount of about 500 units / ml for 1 hour, the reaction mixture was then heated to 65 ° C for 5 minutes to inactivate the ligase. Potassium chloride was added to a concentration of 50 mmol, β-mercaptoethanol to a concentration of 1 mM and EDTA to a concentration of 0.1 mM, after which the mixture was digested with 150 units / ml of Hsu I or Hind III endonuclease for 2 hours at 37 ° C. . Hind III and Hae III endonucleases are commercially available (New England Biol-Labs, Beverly, Massachusetts). The reaction product was analyzed by gel electrophoresis in the same manner as in Example 1, and showed a peak that deduced a sequence of approximately 450 nucleotides and, moreover, Hind III cleavage fragment fragments.

Example 4

In this example, the formation of the plasmid recombinant and the properties of the recombinant after propagation will be described. The plasmid pMB-9 DNA was prepared according to the method of Rodrlguez R. L., Boliver F., Goodman. M., Boyer H. W. and Botlach M., in the INC-UCLA Symposium on Molecular and Cellular Biology, D. P. Wierlich,

KJ Rutter and CF Fox, Eds. (Academic Press, New York 1976) pp 471-477, at the Hind III site using Hsu I endonucleases, and then treated with BAPF-type alkaline phosphatase (Worthington Biochemical Corporation) , Freehold, New Jersey). The enzyme is present in the reaction mixture in an amount of 0.1 unit / microgram of DNA, the reaction mixture is incubated in 25 mM Tris buffer with pH 8 hydrochloric acid for 30 minutes at 65 ° C and then extracted with phenol to remove phosphatase. After ethanol precipitation, the DNA plasmid thus obtained is added to a cDNA containing Hind III ends in a molar ratio of 3 moles of plasmid per mole of cDNA. The mixture is incubated in 66 mM Tris buffer at pH 7.6, the mixture further comprising

6.6 mM magnesium chloride, 10 mM dithiothreitol and 1 mM ATP. Incubation is continued for one hour at 14 ° C in the presence of 50 units / ml T4 DNA ligase.

The resulting mixture was added directly to a 227032 suspension of E. coli X-1776 cells prepared as follows: The cells were grown to a density of 2 x 10 ⁸ cells / ml in 50 ml medium containing 10 g / liter Trypton, 5 g 1 liter of yeast extract, 10 g / liter of sodium chloride, 2 mM sodium hydroxide, 100 µg / ml diaminopimelic acid and 40 µg / ml thymine at 37 ° C. Cells were harvested by centrifugation for 5 minutes at 5000 grams at 5 ° C, then resuspended in 20 ml of cold 10 mM sodium chloride, centrifuged again, and resuspended in 20 ml of buffer containing 75 mM chloride calcium, 140 mM sodium chloride and 10 mM tris buffer, pH 7.5, after which the cells were allowed to stand on ice for 5 minutes and then centrifuged and resuspended in 0.5 ml of the same buffer. The transformation is then carried out by mixing 100 µl of said cell suspension with 50 µl of 1 µg / ml recombinant DNA. The mixture is incubated at 0 ° C for 15 minutes, then at 25 ° C for 4 minutes and at 0 ° C for 30 minutes. The cells are then transferred to agar plates for further growth.

Study of recombinant plasmids is performed at a concentration of tetracycline of 5 [mu] g / ml, the selected recombinant, designated pAU-1, is isolated and the crude plasmid of 2-5 [mu] g DNA isolated from pAU-1 is digested with excess Hau I endonuclease. sodium EDTA to a concentration of 10 mM and 10% sucrose (w / v) and the mixture is separated on an 8% polyacrylamide gel. The DNA has approximately 410 base pairs. In a similar experiment, plasmid pBR322 was used. All conditions corresponded to the above conditions except that the final selection of recombinant clones was performed on plates containing ampicillin at a concentration of 20 µg / ml.

T a b u 1 k a 1

Example 5

The DNA of pAU-1 of Example 4 was further purified by 6% polyacrylamide gel electrophoresis. After elution from the gel, the DNA is labeled by incubation with gamma- ³² U-ATP and polynucleotide kinase enzyme under the conditions described in Maxama and Gilbert, supra. The enzyme catalyzes the activity of the above-mentioned group of radioactive phosphate at the 5'-end of the DNA. The enzyme is obtained from E. coli as described in Panet A. et al. Biochemistry 12, 5045 (1973). The labeled DNA was digested with Hae III endonuclease as described in Example 2, and the two labeled fragments containing 165 and 135 bases were separated on a polyacrylamide gel under the conditions described in Example 1. The thus isolated fragments were subjected to specific digestion and base sequence analysis as described. in Maxama and Gilbert, supra. The sequence shown in Table 1 below is based on the results of the above experiments and the results of similar experiments performed using cDNA and col E1 derived vectors, for example pM8-9 and pBR322. With respect to the 5-terminus, an unspecified sequence of approximately 50 to 120 nucleotides remains and the poly-dA segment at the 3'-terminus is of variable length. This sequence is the most detailed information available so far, it goes without saying that in the course of future experiments some minor changes may be necessary or other parts of the chain will be clarified. The corresponding amino acid sequence of rat proinsulin I starts at the triplet indicated by 1 and ends at the triplet indicated by 86. The uncertainties remain in the region of the sequence that is underlined by the dashed line.

(not specified) —- GCC CTG CTC CTC TGTC 10 GAG CCC AAG CCT GCT CAG 1 GCT TTT GTC AAA 20 CAG CAC CTT TGT GGT CCT CAC CTG 30 GTG GAG GCT CTG TAC CTG TGT GGG GAA CGT 40 GGT TTC TTC TAC ACA CCC AAC TCC CGT CGT 50 GAA GTG GAG GAC CCG CAA GTG CCA CAA GTG GAG 60 CTG GCT CGA GGC CCG GAG GCC GCG GAT CTT CAG 70 ACC TGG GCA CGT GAG GTT CCC GGG CAG 80 GAA CTG AAG CGT CCC ATT GTG GAT CAG TGG TGC ACC AGC ATC TGC TCC CTC TAC GAG AAC TAC TCC AAC TGA GTTCAATCAAT-

TCCCGATCCACCCTCTCCAATGAATAAACCCTTTGAATGAGC-poly A

Example 6

The nucleotide sequence coding for human insulin is isolated, purified and inserted into the plasmid as described in Examples 1-4, starting from human pancreas tissue isolated from a suitable source, such as a dead human or pancreatic tumor. In this way, a modified microorganism according to Example 4 can be obtained whose nucleotide sequence corresponds to the code for the A chain and the B chain of human insulin. The known amino acid sequence for human insulin A chain is as follows:

10 Gly-Ile-Val-Glu-Cys-Cys-Thr-Ser-Ile-Cys

-Ser-Leu-Tyr-Glu-Leu-Glu-Asn-Tyr-Cys-Asn

Hereinafter, the amino acid sequence for human insulin B chain will be listed:

10 Phe-Val-Asn-Glu-His-Leu-Cys-Gly-Ser-His20

-Leu-Val-Glu-Ala-Leu-Tyr-Leu-Val-Cys-Gly-30

- Glu - Arg - Gly - Phe - Phe - Tyr - Thr - Pro - Lys - Thr

The amino acid sequence is numbered from the free amino acid end as described in Smith L. F., Diabetes 21 Supplement 2, 458 (1972).

Example 1a

The method of Example 1 is repeated, but the concentration of β-mercaptoethanol varies with the islet cell homogenization. The concentrations of (3-mercaptoethanol used were 0.05 M, 0.2 M, 0.6 M and 0.8 M. At all the concentrations of β-mercaptoethanol used, the same results were achieved, i.e. degradation of the mRNA by ribonuclease was excluded.

Example 1b

The procedure of Examples 1 and 1a is repeated, but the pH of guanidinium thiocyanate and (3-mercaptoethanol is changed in islet cell homogenization) using pHs of 6.0, 7.0 and 8.0, all of which produce the same results, i.e. that it was possible to prevent mRNA degradation by the ribonuclease.

Example 3a

Eco RI site decanucleotides bind to the cDNA of Example 2 as described in Example 3. Eco RI site decamers were prepared as described in Scheller et al., Supra, and have the sequence 5 sled-CCGAATTCGG-3 ‘. After coupling, the product is treated with Eco RI under the same reaction conditions as described for Hsu I or Hind III. Eco RI is commercially obtainable (New England Biolabsj. The reaction product was analyzed by gel electrophoresis in the same manner as in Example 1, and a peak corresponding to a sequence of approximately 450 nucleotides was observed, except for fragments of cleaved decamers. 4 using plasmids pBR322 prepared according to Bolivar et al., Gene 2, 95, (1977) instead of plasmids pMB9 DNA All conditions were retained except for the final selection of recombinant clones on plates containing 20 µg / ml The incorporated portion was removed as described above to yield 410 base pair DNA of the sequence shown in Table 1.

ii) The method of Example 4 was repeated using plasmids pBR313 DNA prepared according to Bolivar et al., Gene 2, 75 (1977) instead of plasmids pMB9 DNA. All conditions were retained except for the final selection of recombinant clones, as described above, to obtain 410 base pair DNA with the sequence shown in Table 1.

iiij The procedure of Example 4 was repeated using plasmids pSCIO1 DNA prepared according to the method of Cohen et al., Proc. Nat. Acad. Sci, USA, 70, 1293 (1973) instead of pMB9 DNA plasmids. All conditions are maintained including selection of recombinant clones. The insert is removed as described above to obtain 410 bp DNA with the sequence shown in Table 1.

Example 4b

The procedure of Examples 4 and 4a was repeated except that E. coli RRI or E. coli HB101 was used instead of E. coli X-1776. All conditions are otherwise maintained and the same results have been achieved.

Example 4c

i) Charon ISA DNA prepared as described in the above-mentioned Blattner publication is used as the transfer vector. The terminations were combined by incubation at 42 ° C for 60 minutes in 0.1 M tris-HCl, pH 8.0 and 10 mM magnesium chloride. The vector was digested with Eco RI endonuclease at its site and then treated with alkaline phosphatase as in Example 4. After ethanol precipitation, the phosphatase treated cDNA vector was added to the cDNA containing the Eco RI digest at a 2 molar ratio of 2 moles of vector per 1 mol cDNA. The mixture was ligated with T4 DNA ligase as in Example 4. The mixture was directly added to the E. coli X-1776 cell suspension prepared as described in Example 4 and transformed also as described in Example 4. Recombinant phages were isolated and grown to lac Bacteria on plates containing 80 µg / ml

5-chloro-4-bromo-3-indolyl- [beta] -D-galactoside (X6j), recombinant phages containing cDNA inserted into the Eco RI site of Charon 16A are isolated and give rise to colorless plaques. isolate and treat with excess Eco RI endonuclease, and the mixture is lysed as described in Examples 4 and 5. The 410 bp DNA sequence of the sequence shown in Table 1 is obtained. The mixture can also be used to generate recombinant phages in vitro as described in the publication Sternherg N. et al, Gene, 1, 255 (1977). Properties of _t recombinant phages can also be demonstrated in situ hybridization, which was described by WD Benton and RW Davis,

Science 196: 180 (1977).

ii) Charon 3A DNA, prepared as described in the above-mentioned Blattner publication, is used as a vector instead of Charon IBA DNA. Otherwise, the conditions described for Caron 16A DNA are retained. Recombinant phage selection is performed by culturing phage on lac ⁺ bacteria on X6-containing plates with colorless plaque isolation and then hybridizing or restriction endonuclease treatment as also described in the above-mentioned Blattner publication. The incorporated portion was removed as described above to obtain 410 bp DNA with the sequence shown in Table 1.

lil) gtWES. B DNA, prepared as described in Tiemier et al., Is used as a vector instead of Charon 16A. All conditions are otherwise the same as Charon 16A. Selection of recombinant phages is performed by hybridization according to Benton and Davis, supra. The insert is removed to give 410 bp DNA with the sequence shown in Table 1.

Example 1d 4d

The method of Example 4c is repeated, but using E. coli RRI, E. coli HB101, E. coli DP50 or E. coli DP50 SupF instead of E: coli X-1776. All conditions are otherwise the same and the same results are obtained.

Example 6

Disclosed is the isolation and purification of DNA with a nucleotide sequence coding for human insulin and the synthesis of a vector containing DNA as well as the generation of a microorganism strain that contains this DNA as part of its genetic information.

Islet cells are isolated from human pancreas by the method of Example 1. This tissue is obtained from deceased humans or from insulinomas. Islet cells mixed from several pancreas are homogenized in 4 M guanidinium thiocyanate containing 0.2 M / 3-mercaptoethanol at pH 5.0 as described in Example 1. Polyadenylated RNA is isolated by chromatography as described above by Aviva and Leder. Single-stranded cDNA was prepared using reverse transcriptase and hydrolyzed cDNA according to Example 1. Double-stranded cDNA according to Example 2 was prepared and digested with the nuclease SI according to Example 3. According to the same example, human insulin insulins were added to the double-stranded cDNAs. allowing binding to non-Hind III digestion. The product was treated with Hind III or Hsu I and analyzed as described in Example 3. A peak corresponding to the 450 nucleotide sequence was observed except for the fragments cleaved at the Hind III site. Then, the human insulin cDNA containing the Hind III-binding sites was inserted into plasmid pBM9. This procedure was carried out according to Example 4 as well as transformation of E. coli X-1776 and selection of recombinant plasmids. The insert is removed with ITsu I and analyzed according to Example 4. DNA of 450 nucleotides is obtained. it is possible to detect cloned human insulin which contains nucleotides which code for the entire amino acid sequence of human insulin. The amino acid sequence in chain A is as follows:

10

Gly-Ile-Val-Glu-Cys-Cys-Thr-Ser-Ile-Cys

-Ser-Leu-Tyr-Glu-Leu-Glu-Asn-Tyr-Cys-Asn.

The amino acids in chain B have the following sequence:

10

Phe-Val-Asn-Glu-His-Leu-Cys-Gly-Ser-His20

-Leu-Val-Glu-Ala-Leu-Tyr-Leu-Val-Cys-Gly-30

- Glu - Arg - Gly - Phe - Tyr - Thr - Pro - Lys - Thr.

The amino acid sequence is numbered from the end at which the free amino group is located.

Example 6a

i) Example 6 was repeated except that plasmid pBR322 was used instead of plasmid pMB9 DNA. All conditions are otherwise identical to those in Example 4a (i). The insert is removed to obtain 450 nucleotide DNA with the sequence described in Example 6.

ii) The procedure of Example 6 was repeated except that plasmid pBR313 DNA was used instead of plasmid pMB9 DNA. All conditions are the same as in Example 4a (ii). The insert is removed to give 450 nucleotide DNA with the sequence described in Example 6.

iiij The procedure of Example 6 was repeated using plasmid pSCIO1 DNA instead of plasmid pMB9 DNA. All conditions are otherwise the same as in Example 4a (iii). The insert is removed to give 450 nucleotide DNA with the sequence described in Example 6.

3β

Example 6b

The procedure of Examples 6 and 6a was repeated except that E. coli RRI or E. coli HB101 was used instead of E. coli X-1776. All conditions are identical and the results obtained are identical.

Example 6c

Using the method of Example 6, human insulin cDNA was prepared and treated with chemically synthesized chains bound at the Eco RI site of Example 3a.

i) Incorporation of Eco RI treated human insulin cDNAs into the Eco RI treatment site of Charon 16A according to Example 4c (i). Recombinant tags were isolated and the incorporated portion removed as in Example 4c (i). DNA of 450 nucleotides was obtained with the sequence described in Example 6.

ii) Incorporating the human insulin cDNA terminated for Eco RI site binding into the Charon 3A vector of Example 4c (ii). Recombinant phages were isolated and analyzed according to Example 4c (iij) to obtain 450 nucleotide DNA with the sequence described in Example 6.

iiij Plasmid gtWES. B is used as a vector for the transfer of human insulin cDNA after Eco RI treatment according to Example 4c (iiij). Recombinant phages are isolated and analyzed. The insert is removed to give 450 nucleotide DNA with the sequence described in Example 6.

Example 6d

Example 6c is repeated, using E. coli RRI, E. coli HB101, E. coli DP50 or E. coli DP50SupF instead of E. coli X-1776. The same conditions are used and the same results are obtained.

The method of the invention makes it possible, for the first time, to isolate a nucleotide sequence that is a specific regulatory protein code for a higher organism, such as a vertebrate, and at the same time allows the transfer of genetic information contained therein to a microorganism. The method of the invention can be used to isolate, purify and transfer the human insulin gene to a microorganism. Similarly, it is possible to isolate the human growth hormone gene and other proteins of a protein nature, and to transfer and multiply these genes to a microorganism.

In carrying out the method of the invention, several new recombinant plasmids have been discovered, each containing a nucleotide sequence or part of a sequence of nucleotides corresponding in structure to a higher organism gene. It has also been possible to modify several new microorganisms to contain a nucleotide sequence carrying a structure transferred from a higher organism gene. By the method of the invention, for example, it was possible to successfully transfer the rat preinsulin I gene into an E. coli strain and to transfer the rat growth hormone gene also to an E. coli strain. The sequence of the major part of the transferred gene was also determined in each of these cases and it was found that the code for the complete amino acid sequence of rat proinsulin I or rat growth hormone could be transferred, compared with known genetic codes common to all life forms.

The invention has been described in connection with specific embodiments, but it will be understood that it can be used for a variety of purposes which essentially concern the transmission of information structures. Therefore, the invention cannot be limited to the contents of the present examples.

Claims (5)

SUBJECT 1. A method for growing a microorganism containing and replicating a DNA-transfer vector with a nucleotide sequence coding for insulin by extracting from the cells an mRNA coding for insulin, purifying the mRNA, synthesizing a cDNA with the sequence coding for this mRNA. of nucleotides coding for Insulin, whereupon the cDNA is reacted with a DNA transfer vector to produce a DNA transfer vector with a nucleotide sequence coding for insulin, and the DNA transfer vector transforms a microorganism characterized in that mRNA is extracted from cells containing mRNA that encodes insulin by homogenizing these cells in the presence of a ribonuclease inhibitor containing guanidinium thiocyanate and β-mercaptoethanol at pH 5.0 to 8.0 to prevent degradation of the mRNA by ribonuclease and the cDNA is reacted with a DNA transfer vector by enzymatically hydrolyzing a DNA transfer vector by a restriction endonuclease of the Hi group in a first step nd III or Hsu I to produce a DNA transfer vector having reactive ends capable of being linked to or linked to a nucleotide sequence coding for insulin and, in a second step, enzymatically linked to a DNA sequence and a nucleotide sequence cDNA, which encodes insulin by using a DNA ligase in the presence of ATP to produce a DNA transfer vector with a nucleotide sequence encoding insulin. 2. The method of claim 1 for culturing a microorganism containing and replicating a DNA transfer vector with a nucleotide sequence coding for insulin, characterized in that, after the first step, it is subjected to enzymatic hydrolysis of any 5'-phosphate end capping in the vector. for transferring DNA with reactive ends to form a DNA vector with ends that are incapable of binding to one another but capable of binding to cDNA having a nucleotide sequence that encodes insulin. * 3. The method of claim 1 wherein the ribonuclease inhibitor comprises 4 M guanidinium thiocyanate and 0.05 to 1.0 M (3-mercaptoethanol). 4. The method of claim 3, wherein the inhibitor comprises 0.2 M (3-mercaptoethanol). 5. The method of claim 2, wherein the enzymatic hydrolysis is an alkaline phosphatase.