CS227002B2 - Method of preparing vectors for transferring deoxyribonuclec acid - Google Patents

Description

(54) A method for producing a DNA transfer vector

The invention relates to a method for producing a DNA transfer vector, in particular to isolate a specific nucleotide sequence comprising a genetic information code for a specific protein, to synthesize DNA comprising the specific nucleotide sequence, and to transfer the DNA to a microorganism in which the DNA can be propagated. The invention further relates to the isolation of the insulin 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 mRNA sequence) mRNA - RNA involved in messenger RNA tRNA - transfer RNA dATP - desoxyadenosine triphosphate dGTP - desoxyguanosine triphosphate dCTP - desoxycytidirttriphosphate

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 into functional proteins. Insulin will be exemplified. Genetic code phenylalanine (Phe) TTK leucine (Leu) XTY isoleucine (Ile) ATM methionine (Met) ATG valine (Valj GTL serine (Ser) ORS proline (Pro) CCL threonine (Thrj ACL alanine (Ala) GCL tyrosine (Tyrj TAK.

termination signal TAJ termination signal TGA histidine (His) CAK glutamine (Gln) CAJ asparagine (Asn) AAK lysine (Lys) AAJ aspartic acid (Asp) GAK glutamic acid (Glu) CAJ 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 or G X = C when Y, 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, -11 ZA or GW = C, if ZC or TZ - A, G, C or T, if WCZ = A or G, if WA QØ = TC, if SA, G, C or T QR = AG if ST or GS = A, G, C or T if QR TC S = T or C if QR AG

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

Bgl I treatment site - a sequence of GCCNNNN and 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 - CCAGG or CCTGG sequence specifically cleaved at the arrow site by Eco RII endonuclease.

Hae II endonuclease treatment site - sequence of both AGCGC and T or AGCGC; C or GGCGC and T or GGCGC * - 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 - GTGCA β sequence specifically cleaved at the arrow site by Pst I endonuclease

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

Sst I endonuclease site - GAGCT β 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 B linked together by another peptide C as described by Steiner, D. F., Cunningham D., Spigelman L. and Eten B., Science.

WITH

157, 697 (1967). Recently, it has been reported that the initial translation product of insulin mRNA is not inherent proinsulin but preproinsulin which contains more than 20 additional amino acids at the amino terminus of proinsulin as described by Cahn SJ, Keitm P. and Steiner F. D “Why. Nati. Acad. Sci. USA 73, 1964 (1976) and in Lomedico Ρ. T. and Saunders G.F., Nucl. Acids Res. 3, 381 (1976). The structure of the preproinsulin molecule can be schematically represented as

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

In proteins of higher organisms, such as vertebrates, many proteins can be found that are of medical or research interest. These include, for example, insulin hormone, other peptide-like hormones, such as growth hormone, blood pressure-controlling proteins, and various enzymes of industrial, medical or research interest. It is often very difficult to obtain these proteins in a usable amount by extraction from organisms, this problem being particularly troublesome 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 a cell line with the 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 said protein to be produced by microorganism-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 protein to a microorganism with a well-defined genetic structure would be of great value to investigate the way the protein is synthesized and to investigate the protein's post-synthesis behavior. 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 triphosphates, dATP, dGTP, dCTP and TTP. The reaction begins with the non-covalent binding of the olidodesoxynucleotide, which binds to the 3 ‘mRNA position, followed by sequential binding of the respective 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. 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 loop. with the DNA strand. These procedures have been described in Aviv H. and Leder P., Proc. Nati. Acad. Sci. USA 69, 1408 (1972) and Efstratiadis A., Kafotos, F. C., Maxam A. F. and Maniatis T., Cell 7, 279 (1976).

Restriction endonucleases are enzymes that are capable of hydrolyzing phosphodiester bonds in the double strand of DNA, thereby disrupting DNA continuity. 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 bydrolysis of linkages that are separated by only a few nucleotides, leaving free portions of individual chains that are cleaved from the parent molecule. The terminal portions of these strands may be complementary and may be used 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 in which the enzymes may 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 HL, Shine J., Goodman. 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).

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. M., 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 part of a strand in a double stranded DNA molecule. 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 JH, and Khorana H.

G., Why. Nati. Acad. Sci. USA 67, 1468 (1970).

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

The next step to be described is to incorporate a specific DNA fragment into a 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 linked to the host cell chromosome. A DNA plasmid exists as a double-stranded circular molecule with a molecular weight of several millions, although although the weight of some molecules is 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 difference in the size 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. A 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 in the world 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 makes it possible 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 proteins 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 the microorganisms modified by the method of 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 the sick person.

The invention therefore 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 producing a DNA encoding a nucleotide sequence coding for insulin, so that cells containing the mRNA coding for insulin are isolated, mRNA is extracted from the cells, and purified and cDNA is synthesized from the mRNA. to form a cDNA having a nucleotide sequence coding for insulin, and the cDNA thus obtained is reacted with a DNA transfer vector to produce a DNA transfer vector with a nucleotide sequence coding for insulin, characterized in that from cells with a containing the mRNA coding for insulin extracts the mRNA by homogenizing the cells in the presence of a ribonuclease inhibitor containing guanidinium thiocyanate and β-mercaptoethanol at pH 5.0 to 8.0, thereby preventing the degradation of the mRNA by ribonuclease, whereupon the cDNA is reacted with a DNA transfer vector in two steps, wherein in the first step the DNA transfer vector is enzymatically hydrolyzed by donulases of the H'nd III or Hsu I family to produce a DNA transfer vector with reactive ends capable of interconnecting or linking to a cDNA with a nucleotide sequence coding for insulin and, in a second step, enzymatically linking the DNA transfer vector, and cDNA with a nucleotide sequence coding for insulin using a DNA ligase in the presence of adenosine triphosphate 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 will be 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, either prokariotic or eukariotic, 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 desoxynucleotides, 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 the sites to n ^; where the 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 strand and produce similar free ends capable of binding to the 5 'terminal end. The 5‘-phosphating groups are removed so that the plasmid cannot form an annular structure that changes the host cell. Then the cDNA prepared above and the DNA plasmid are incubated together 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 that has received the altered viable plasmid, which is to be determined by the formation of colonies with altered properties, such as resistance to certain chemicals. Pure bacterial strains containing the recombinant plasmid are then grown and the recombinant plasmid isolated again.

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 carrying 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 process 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 an O 'DNA of the organism and, moreover, a modified RNA of the organism. According to the common 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 been thoroughly purified previously as specified. equipment. (Federal Register, vol. 41, no. 131, July 7, 1967, pg. 27 902 to 27 943. * This means that any process which could be used to produce ^one human protein, it is preferable to isolate specific mRNA This procedure has the additional advantage that mRNA is easier to purify than DNA extracted directly from cells, and in particular, it is beneficial to take advantage of the fact that, in the above differentiated organisms, such as vertebrates, it is possible to find a specific population of cells at a specific site in the body, the essential function of which is to produce the desired protein, and the population of cells may only exist for a transient developmental period of the organism. a large portion of the mRNA isolated from the cell, the desired nucleotide sequence, it is therefore possible to select a preferred cell population and good isolation method in terms of 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. Way! According to the invention, different cell types can be isolated from different tissue types. An example is the isolation of the 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 the 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, by the action! hormone may produce increased production of the desired mRNA. Other methods are, for example, growing at a certain temperature, supplying a certain nutrient or other chemical. By isolating rat growth hormone mRNA, the desired proportion of growth hormone mRNA could be substantially increased by treating rat pituitary cells grown in tissue culture simultaneously with thyroid hormone * and glucocorticoids.

2. Extraction of mRNA

Important! The object of the method according to the invention is, as far as possible, to completely remove the activity of the ribonuclease 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 linkage in the chain would render the entire molecule unusable for transferring intact genetic sequences to the microorganism. As noted, 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 the 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.

The method of the invention uses a protein-free combination of chaotropic anion, chaotropic cation, and an agent that disrupts disulfide bonds during cell disruption and all procedures required to isolate RNA. The efficacy of the simultaneous action of all of the above agents has been demonstrated when used to isolate intact mRNA in good yield from isolated islets of rat 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, carbamoylguanidine, guanylguanidino, lithium, and the like. Suitable chaotropic anions are iodide, perchlorate, thiocyanate, diodosalicylate, and the like. For example, lithium diodsalicylate is more effective than guanidinium thiocyanate, 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 (3-mercaptoethanol) are known to disrupt intramolecular disulfide bonds in proteins by a double exchange, and a number of thiol compounds, such as (3-mercaptoethanol, dithiotreitol, cysteine, propanoldimercaptan and the like) are known to be effective. they must be soluble in water, since they have to be used in large excess with respect to the number of intramolecular disulphide bonds in order to complete the double substitution, and the most preferred substance in this sense is (? -mercaptoethanol, since 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 completely denature the protein. On the other hand, the degree of denaturation of many proteins depends on the concentration of denaturing agent, which sometimes needs to be increased 5x to 10x. These relationships have been described in Tamtord C.A., Adv. Prot. Chem. 23, 121 (1968). Qualitatively, this relationship states that a denaturing agent that is only slightly more effective than guanidime hydrochloride can denature a 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. When the above relationships are correct, the preferred denaturing agent is a low threshold concentration and high water solubility composition. For this reason, guanidium thiocyanate is preferable to lithium diodsalicylate, although the latter is a much 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 each agent also provides an answer to why guanldinium thiocyanate is preferable to the relatively equally soluble hydrochloride; the reason is that the first of these is a slightly stronger denaturant.

The disulfide-disrupting agent is used in admixture with a denaturing agent * because it has a potentiating effect on ribonuclease denaturation. The thiol compound prevents rapid denaturation, which can occur if intramolecular disulfide bonds remain intact. In addition, 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 the intramolecular disulfide bonds in order 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, effective concentrations are in the range of 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 extraction of -mRNA from the cells is performed should have a pH in the range of 5.0 to 8.0.

After cell disruption, 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 carrying out the process of the invention is to omit the precipitation step and superimpose the honiogeneate directly onto a 5.7M precision 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 obtains high-yield, RNA-free and protein-free RNA.

The above method can be used to obtain purified RNA from cellular hornogenate. 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 polyamide acid binding upon its formation. The mRNA portion containing the polyadenylic acid sequence can be selectively isolated by column chromatography of the cellulose to which the oligochymidylate is bound as described in Aviv H. and Leder P., supra. The above procedures are sufficient to obtain pure, intact mRNA 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

FIG. 1 schematically illustrates a process relating to the remaining steps of the process of the invention. The first step is to generate a sequence of complementary DNA to the purified mRNA. The enzyme used for this reaction is usually reverse transcryptase, 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 conditions described in the known literature using mRNA 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 a ³² P-labeled form in order to monitor the progress of the reaction and isolate the product in a timely manner 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 product of the reverse transcryptase reaction is a double strand hairpin structure with a non-covalent bond between the RNA strand and the DNA strand.

The product of the reaction using a reverse transcryptase structure has a double strand hairpin shape, with a non-covalent bond between the RNA strand and the DNA strand.

The reaction product using reverse transcryptase is removed from the reaction mixture by conventional means. It has been found advantageous to use a combination of phenol extraction, Sephadex G-100 chromatography (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, ⁵² 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.

After formation of the hairpin cDNA, it may be desirable to recover purified DNA from the reaction mixture. As mentioned above, it is advantageous 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 connecting the 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 SI nuclease isolated from Aspergillus oryzae. This enzyme is available from Miles Research Products, Elkhart, Indiana. Treatment of the hairpin-shaped DNA structure with SI nuclease in a high yield yields a cDNA molecule with the appropriate end. After purification, chromatography and ethanol precipitation as described above, the purified product is obtained. The use of reverse transcriptase and SI nuclease in the synthesis of double-stranded cDNA as a structure corresponding to mRNA has been described in Efstraitiadise et al., Supra.

If desired, the proportion of cDNA molecule with corresponding ends can be increased by using E. coli DNA polymerase I in the presence of four desoxynucleoside triphosphates.

The combination of the exonuclease and polymerase activity of this enzyme results in the 3 3 free ends being removed and binding at all 5 zakon ends. This ensures maximum participation of the cDNA molecule in subsequent binding reactions.

A further step of the method according to the invention is to treat the end of the cDNA thus obtained so as to obtain sequences which contain sites of suitable restriction endonuclease action. 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 used for the recombination of 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 can act

III. Hind III is isolated from Hemophilus influenzae and purified according to the method of Smith H. O. and Wilcox K. W., J. Mol. Biol. 51, 379 (IS70). A similar Hae III enzyme from Hemophilus aegypticus is purified as described by Middleton, J. H., Edgell. H. and Hutchison III, C.A., J. Virot. 10, 42 (1972). The enzyme from Hemophilus suis, designated Hsu I, catalyzes specifically by hydrolysis at the same site as HindIII. Thus, the two enzymes may be interchanged as far as function is concerned.

It is preferred to use a chemically synthesized double stranded decanucleotide that contains a site where Hind III can act and to 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. And 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 filled so that the resulting substance is sensitive 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 according to the method described by 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 sites suitable for Hind III restriction endonuclease is cDNA with the indicated 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. Generation of recombinant transfer vector

DNA

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 to be multiplied in the cell, and that the vector contains a genetic determinant to separate those host cells that the vector contain. From a safety point of view, it is necessary to limit the choice of these vectors so that the vectors meet the requirements of the National Institutes ot Health (NIH). The list of permitted DNA transfer vectors is continually expanding with the discovery of new vectors and subject to approval by the NIH Recombinant DNA Safety Committee, the invention contemplating the possible use of any DNA of viral or plasmid origin with the necessary capabilities, including those that will only be allowed by the NIH. Suitable vectors that are already allowed include a variety of lambda bacteriophage derivatives (Blattner, F. R., Williams B. G., Blechl A.

E., Denniston-Thompson, K., Faber Η. E., Furlong L, A., Gruinwald DJ, Riefer DO, Moore DD, Schumm JW, Sheldon EL and Smithies O., Science 196, 161 [1977], and col E1 plasmid derivatives, as described, for example, in Rodriguez RL, Bolivar S ., Goodman Η. M., Boyer HW and Betlach Μ. N. ICN-UCLA Symposium on Molecular Mechanisms In The Control ot Gene Expression, D.P. Nierlich, W.J. Rutter, C.F. Fox, Eds. (Academic Press, NY, 1976), pp. 471-477. Col E1-derived plasmids are relatively small, have a molecular weight of several million, and have the property that the number of plasmid DNA copies in a single host cell can be increased from 20 to 40 per 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 the 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 gene for ampicillin resistance. The presence of resistance genes is an advantageous feature for recognizing cells that have been successfully infected with the plasmid, since colonies of such227002 cells will grow in the presence of the substance, whereas cells that do not contain the plasmid will die. In the above experiments 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 enzyme site.

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

Plasmid pBR-322 was similarly described in Bolivar F. et al., Gene, 2, 95 (1977), which describes the synthesis and properties of this plasmid. Plasmid pBR-322 has a molecular weight of 2.7 χ 10 ⁶ daltons. (controlled by Bolivar F., Gene, 4, 121 [1978]) and carries the ampicillin resistance (Aβ ^R ) gene and the tetracycline resistance (Tc ^R ) gene. 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 pBR-322 carries single Eco RI sites, two Hinc II sites, five Eco RII sites, three Bgl I sites, twelve Alu I sites, twelve Hae sites II and seventeen seats for Hae III. A detailed description of these sites in plasmid pBR-322 is given on page 103 of the first reference. When the pBR-322 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 pBR-322 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 introduced into the Pst I site, recombinant molecules can be found in selection for ampicillin (Ap ^s ) sensitive colonies having Tc ^R. Similarly, if:. by incorporating DNA 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.

Also plasmid pBR-323 has been described in detail. The synthesis and properties of this plasmid have been described by Bolivar F. et al., Gene 2, 75 (1977). Plasmid pBR-313 has a molecular weight of 5.8 χ 10 ⁶ daltons and contains the ampicillin resistance gene (Ap ^R ) and the tetracycline resistance gene (Tc ^R ). Will sense -plasmid contains only ^one site for restriction endonuclease recognition site Hind III, Sal I -a Bam- HI in the Tc ^R gene. A full analysis of plasmid pBR-313 was performed and a map of restriction endonuclease sites was constructed as shown on page 84 of this publication. Incorporation of foreign DNA into any Hind III, Sal I or Bam HI treatment site results in the loss of Tc ^R. Thus, reconstituting molecules can be found in the selection of strains with Aβ ^R and Tc ^s . The vector can be characterized by antibiotic resistance, DNA sequence, and molecular weight.

The third plasmid described in detail is pMB 9. This plasmid can be prepared according to Rodriguez RL et al., Supra, and Bolivar F. et al., Gene, 2775 (1977). The plasmic pMB 9 has a molecular weight of 3.5 χ 10 ⁶ daltons and contains a gene for resistance to tetracycline Tc ^R. It has a single site of action for each of the endonucleases Eco RI, Hind III, Sal I or Bam-HI. The last three endonucleases are located in the Tc ^R gene. When foreign DNA is inserted into the Hind III, Sal I, or BamHI site, Tc ^R is lost. A vector containing foreign DNA at one of these sites can be characterized by this property. This vector can further be characterized by analyzing the DNA sequence of the incorporated portion, by comparing the molecular weight by analyzing the endonuclease sites.

Plasmid pS-101 has also been described in detail. Cohen S. H. et al., Pro-c. Nat. Aca-d. Sci. USA, 70, 1293 (1973) describes the synthesis and properties of this plasmid. Further plasmid properties can be found in Co-he S. N. et al., P-roc. Nat. Aca-d. Sci. USA, 70, 3240 (1973), Boerer H. W. et al., Recomhinant Molecules, Beers R.

F. and Basset, EG, Eds, Raven Press, New York on page 13 (1977) and Cohen SN et al., Recombinant Molecules, on page 91. Plasmld pS-101 has a molecular weight of 5.8 x χ 10 ⁶ contains dalt-one and contains a gene for resistance to tetracycline Tc ^R. In the region of this gene it contains a single site for the action of each of the endonucleases Hind III, Sal I and Bam HI. In addition, it contains a single Eco RI site, one Hpa I site, one Sr I site, and four Hinc II sites. When this plasmid is digested with Hind III, the Tc ^R is lost. The same is true for the cleavage at the Sal I or Bam HI site, when the DNA is inserted into one of these sites can be found in the selection of the recombinant molecule. A vector containing foreign DNA at any of these sites can be characterized by a loss of antibiotic resistance. The vector can be further characterized (including by removing the inserted DNA and determining and comparing the molecular weight), (b) constructing a map of sites for endonuclease treatment, and (c) determining the sequence of the inserted DNA.

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 et al., Supra, describe the synthesis and properties of these phages. These phages have also been 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, incorporating foreign DNA into the EcoRI site of Charon 16A will result in the loss of the lac5 gene. Growth of recombinant phage on bacteria Lac ^- results in the formation of colorless colonies. This means that the vector containing the foreign DNA at the appropriate site can be characterized by genetic properties, such as 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.

AgtWES has been described as well. ΛΒ, a bacteriophage lambda derivative. 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 Rl and Sst I restriction endonuclease sites. This phage contains four Bam HI sites to yield 5.4 χ 10 ³ fragments.

19.3 χ 10 ³ , 3.8 χ 10 ^3, and 11.4 χ 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 R1 and digested with Sst I. This prevents recombination with lambda B. It is also possible to incorporate foreign DNA with Eco · R1 cleavage sites into the phage at the Eco R1 cleavage site. Phages can be cloned by in situ hybridization as described by Benton WD and Davis RW, Science, 196, 180 (1977). The vector may be characterized by (a) removing the inserted DNA and determining and comparing molecular weights, (b) analyzing restriction endonuclease sites, and (c) determining the sequence of the inserted DNA.

An E. coli strain, designated X-1776, has been developed and NIH approval has been obtained for the use of this strain and using the P2 apparatus as described in 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 generated by mixing restriction endonuclease-treated DNA plasmids and cDNA with appropriately processed 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 plasmids in large excess usually resulted in the binding of the plasmid chains to the ring without simultaneously 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 selected gene, 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 foisphatase, which is available from several sources, for example, Worthington Biochemical Corporation, Freehodl, New Jersey. Alkaline phosphatase removes the 5'-terminal phosphate groups from the end of the plasmid after endonuclease treatment, thereby preventing the end portions of the DNA plasmids from binding together. As a result, the formation of the ring depends on the incorporation of a DNA fragment containing 5'-terminal phosphate groups. As described above, can be decreased relative frequency transformation without rekomblnace to less than 1-10 ^+4.

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

Case

TABLE I Reagents

Product using ligase

I 3 ‘

----- OH

4 "

----- OPO3H2

5 ‘

5 ‘3‘ 5 ‘

H2O3PO ----- Q— P — O - HO ----- O — P — O-3 * 5 ‘3‘ + 2 H2O

II 3 ‘

------ OH

------ OH

5 ‘

5 * 3 * 5 *

II / O3P — O ----- OI — P — O- +

HO ----- OH HO-3 ‘5‘ 3 * + H 2 O

III 3 ‘

--------- OH +

5 ‘b

HO ---- O reactions

HO ----- 3 ‘

In Table I, the 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 I, 5‘-phosphate is present at both ends, resulting in covalent bonding of both chains. In case II, only one chain has a terminal 5‘-phosphate group, so that after covalent bonding the discontinuity remains on the other 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 f-phosphate groups from the non-bonded termini. Any method for removing 5 způsobu-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, the rat insulin insulin cDNA was isolated and recombined with the plasmid, the DNA molecules 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.

Nati. Acad. Sci. USA 74,560 (1977). The nucleotide sequence in the DNA fragment overlaps and contained the complete coding sequence for rat prolnsulin I as well as 13 of the 23 amino acids of the prepeptide 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 growth in E. coli cells, which sequence contained all of the code for rat growth hormone including portions of the peptide precursor and a portion of the 5 'region that was not transferred.

The method described above can generally be used to isolate and purify any gene from a higher organism, including human genes, and to transmit and multiply these genes in cells of microorganisms. The novel recomibinant plasmids contain all or part of the isolated gene as described above. New microorganisms, as yet unknown, have also been described whose genetic system has been altered 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 plaismides that contain a portion of the rat insulin gene, the gene for ^; rat growth hormone, the human insulin gene and the human growth hormone gene.

The invention will be illustrated by the following examples.

Example 1

The described procedures illustrate the extraction and isolation of rat insulin insulin mRNA, the synthesis of complementary DNA and the description of the properties of complementary DNA. Purified rat islets of Langerhans is obtained by infusing the pancreas of anesthetized rats with Hanks solution with a retrograde infusion of the gland. Haniks' solution is a standard mixture of salts, is known and can be obtained from a number of manufacturers, for example, the Grand Island Biological Supply Company, 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 the gland is not digested. Two rat pancreas in 8 ml Hanks medium are placed in a 30 ml glass tube. All glass tubes were pretreated with silicone. Siliclad (Ciay-Adaims Division, Becton-Dickinson, Inc., Persippany, New Jersey) was used for this purpose, and the incubation mixture contained 12 mg of collagenase, an enzyme prepared from Clostridium histolyticum as described in Mandl I., Mackennan. JD and Howes EL, J. Clin Invest., 32, 1323 (1943), used type CLS IV (Worthington Biochemical Corporation, Freehold, New Jersey) and 1 mg soybean trypsin inhibitor, (Sigma Chemical Company, St. Louis). Incubation should be carried out at 37 ° C for 25 minutes with constant shaking at 90 oscillations per minute, and the mixture should be observed at all times to ensure that collagenase digestion is to the optimum extent. If the incubation is too short, Langerhans islet cells are not completely released, but if the incubation is too long, the cells will be disrupted. and centrifuged at 200 g for 1 minute. 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 (Pharmacia Chemical Company, Uppsala, Sweden) at a density of 1.085. A 1.080 Ficoll layer and a 1.060 Ficoll layer of 5 ml are then added, after which the tube is centrifuged for 5 minutes at 500 g and then for 5 minutes at 2000 g. As a result, the acine cells remain at the bottom of the tube and a layer between the two upper layers of the formulation. The islet cell band also contains ganglion 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 chapped nitrogen.

Islet cells from 200 rats are co-homogenized in 4 M guanidinium thiocyanate (Tridom, Fluka AG Chemische Fabrik, Buchs, Switzerland) containing 1 M β-mercaptoethanol and a 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 Comipany, 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 Aviv H. and Leder P, supra.

Reverse transcriptase from avian myeloblastosis virus (DJ Beard, Life Science Inc., St. Petersburg, Florida) is then used to transfer the entire islet polyadenylate RNA structure from rat islets to cDNA. The reaction is carried out in 50 mM trts-buffered hydrochloric acid at pH 8.3 containing 9 mM MgCl 2, 30 mM NaCl, 20 mM (S-mercaptoethanol, 1 mM each of 3 nonradioactive deoxyribonucleoeide triphosphates, 250 µM fourth labeled desoxynucleoside triphosphate). · in position aa radioactive phosphorus' specific activity 50-200 curies of 1 mol, 20 ug / ml oligo-dTi ₂ _i8 (Collaborative Research, Waltham, Massachusetts), 100 ug / ml polyadenylated RNA and 200 units / ml reverse transcriptase The mixture is incubated for 15 minutes at 45 DEG C. Sodium ethylenediaminetetraacetic acid is then added to a concentration of 25 mM and the solution is extracted with an equal volume of water-saturated phenol, after which the aqueous phase of the column is 0.3 x 10 cm. containing Sephadex G-100 in 10 mM hydrochloric acid tris buffer, pH 9.0 containing 100 mM NaCl and 2 mM EDTA, and the nucleic acid is precipitated with ethanol after addition of ammonium acetate at pH 6.0 to a concentration of The precipitate is separated by centrifugation, the sediment is dissolved in 50 μl of freshly attached 0.1 M sodium hydroxide solution, and incubated at 70 ° C for 20 minutes for RNA hydrolysis. Then. The mixture was neutralized by addition of 1 M sodium acetate at pH 4.5 and ³² P-cDNA was ethanol precipitated and redissolved in water. Single-stranded cDNA fractions were analyzed on a polyacrylamide gel as described. 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-2T film (Eastman Kodat Gorporalion, 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 with the 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 contains 50 mM hydrochloric acid tris buffer, pH 8.3, 9 mM MgCl 2, 10 mM dithiothreitol, 50 mM each of the three unlabeled desoCyrihonucleoside triphosphates, 1 mM labeled nucleoside triphosphate and radioactive phosphorus at specific activity 1 to 10. curie per mM, 50 µg / ml cDNA and 220 units / ml reverse transcriptase. The reaction mixture was incubated at 45 ° C 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 could be detected by comparison with standard samples. Aliquots of the DNA reaction products of Example 1 and Example 2 were then independently coated with excess restriction endonuclease Hae III and analyzed by gel electrophoresis. Both products were cleaved by the 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 same length as the band produced by the cleavage of the single strand cDNA.

Example 3

In this example, the binding of the Hind III-treated decanucleotide chains to the rat double-stranded cDNA of Langenhans islets as described in Example 2. The reaction product of Example 2, with a double-strand at a concentration of 2-5 µg / ml, was described. reacted with 30 units of SI nuclease with an activity of 1200 units / ml (Miles Laboratories, Elkhar, Indiana) in 0.03 M sodium acetate at pH 4.6 with 0.3 M sodium chloride and 4.5 mM magnesium chloride at a temperature of 22 ° C for 30 minutes, then the mixture is further incubated for 15 minutes at 10 ° C. The reaction was constructed by adding tri-s-buffer to a concentration of 0.1 M, EDTA to a concentration of 25 mM and tRNA from E. coli, prepared according to the method of Ehrenstein G., Methods in Enzymology, SP Co-lowick and NO Kaplan, Eds., Vol. 12A, p. 588 (1967) to an amount of 40 µg / ml. The reaction mixture was extracted with phenol, chromatographed on Sephadex G-100, and ³² P-cDNA labeled with ethanol. 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 Schelle-RH, Dickerson RE, Boyer-HW, Riggs AD, and Itakura K., Science 196, 177 (1977). 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, 10 mM dithiothreitol, 3 mM Hind III decamers ⁵⁰⁰ cpm / pmol and T4 DNA ligase at about 500 units / ml for 1 hour. The reaction mixture is then heated at 65 ^° C for 5 minutes to inactivate the ligase. Potassium chloride is added to a concentration of 50 mmol, (3-mercaptoethanol to a concentration of 1 mM and EDTA to a concentration of 0.1 mM, and the mixture is 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 Wngland Biol-Labs, Beverly, Massachusetts), and the reaction product is analyzed by gel electrophoresis in the same manner as in Example 1, showing a peak of approximately 450 nucleotides in sequence. and, in addition, fragments of the cleaved decamers of Hind III.

Example 4

In this example, the formation of the plasmid recombinant and the properties of the rsk-o-mbinant after propagation will be described. The plasmid pMB-9 DNA was prepared according to the method of Rodriguez R. L., Boliver F., Goodman. M., Boyer HW and Betlach M., in ICN-UCLA Symposim on Molecular and Cellular Biology, DP Wierlich, WJ Rutter and CF Fox, Eds., (Academic Pres, New York 1976) pp. 471-477 in place of Hind III, using Hsu I endonuclease, and then treated with BAPF-type alkaline phosphatase (Worththington Viochemical Corporation, Freehand, 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 hydrochloric acid pH 8 for 30 min at 65 ° C and then is extracted with phenol to remove phosphatase. After ethanol precipitation, the thus obtained plasmid DNA is added to a cDNA containing Hind III ends in a molar ratio of 3 moles of plasmid per mole of cDNA. The mixture was incubated in 66 mM Tris buffer at -pH 7.6, the mixture further containing 6.6 mM magnesium chloride, 10 µM 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 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 a 50 ml medium containing 10 g / liter of Trypton, 5 g / ml. 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 g and at 5 ° C, then resuspended in 20 ml cold 10 mM sodium chloride, centrifuged again, and resuspended in 20 ml buffer containing 75 mM calcium chloride. , 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 μΐ of said cell suspension with 50 μ DNA 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 μξ / / ml, the selected recomibinant, designated pAU-1, is isolated and the crude plasmid with 2-5 μξ DNA isolated from pAU-1 is digested with excess Hsu I endonuclease. EDTA to a concentration of 10 mM and 10% sucrose (w / v) and the mixture is separated into an 8% polyacrylamide gel. The DNA has approximately 410 base pairs. In a similar experiment, plasmid pBR-322 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 μ% 1 / ml.

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 P-ATP and with the enzyme polynucleotide kinase under the conditions described in Maxama and Gilbert, supra. The enzyme catalyses the activity of the above-mentioned group of radioactive phosphate on the 5'-DNA closure. 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 265 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. Maxam 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 pMB-9 and pBR-322. With respect to the 5'-end, an unspecified sequence of approximately 50 to 120 nucleotides remains, and the poly-dA segment at the 3'-end 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 small 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.

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Example 1a

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

Example 1b

The method of Examples 1 and 1a is repeated, but the pH of guanidinium thiocyanate and β-mercaptoethanol are varied by homogenizing islet cells. PH 6.0, 7.0 and 8.0 were used. At all of these values, the same results were obtained, i.e. the degradation of the ribonuclease-mRNA was prevented.

Example 3a

Eco-R1 site decanucleotides were ligated to the cDNA of Example 2 as described in Example 3. Eco R1 site decamers were prepared as described in Scheller et al., Supra, and have the sequence 5'-CCGAATTCGG- 3 '. After coupling, the product is treated with Eco-R1 under the same reaction conditions as described for Hsu I or Hind III. The Eco R1 enzyme was commercially obtainable (New England Biolabs). 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 steps was observed. - fragments of cleaved decamers.

Example 4a

i) The procedure of Example 4 was repeated using plasmid pBR-322 prepared according to Bolivar et al., Gene 2, 95, (1977) instead of plasmid pMB-9 DNA. All conditions were maintained except for the final selection of recombinant clones, which was carried out on plates containing 20 µg / ml tetracycline. The incorporated portion was removed as described above to yield 410 base pair DNA of the sequence shown in Table 1.

ii) The procedure of Example 4 was repeated using plasmid pBR-313 DNA prepared according to Bolivar et al., Gene 2, 75 (1977) instead of plasmid pMB-9 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.

iii) The procedure of Example 4 was repeated using the plasmid pSC-101 DNA prepared by the method of Co-hen et al., Proc. Nat. Sci., USA, 70, 1293 (1973) instead of plasmid pMB-9 DNA. All conditions are maintained including selection of recombinant clones. The incorporated portion was 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 HB-101 was used instead of E. coli X-1776. All conditions were otherwise maintained and the same results were obtained.

Example 4c

i) The transfer vector used was an Ohava 16A DNA prepared according to the method of Blattner, supra. 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-R1 endonuclease at its site of treatment and then treated with alkaline phosphatase as in Example 4. Ethanol precipitation. The phosphatase-treated cDNA was added to the cDNA containing the Eco Rl-terminated cDNA at a molar ratio of 2 moles of vector per mole of cDNA. The mixture is bound by treatment with: T4 DNA ligase as in Example 4. The mixture is directly added to the E. coli X-1776 cell suspension prepared according to Example 4 and the transformation is also carried out according to Example 4. Recombinant phages are isolated and grown. was on lac bacteria on plates containing 80 µg / ml

5-chloro-4-bromo-3-indolyl-β-D-galactoside (X6), recombinant phages containing the cDNAs incorporated into the Eco R1 Ch-Aaron 16A treatment site are isolated and give rise to colorless plaques. The recombinant after selection is isolated and treated with an excess of Eco R1 endonuclease, and the mixture is analyzed as in Examples 4 and 5. The 410 bp DNA is obtained with the sequence shown in Table 1. The mixture can also be used to generate recombinant phages in vitro. as described in Sternberg N. et al., Gene 1, 255 (1977). Recombinant phage properties may also be demonstrated by in situ hybridization as described by Benton W. D. and Davis R. W., Science 136, 180 (1977).

ii) Charon-3A DNA, prepared as described in Blattner &apos; s publication, is used as a vector instead of Charon 16A DNA. Otherwise, the conditions described for Charon 16A DNA are retained. Selection of recombinant phages is performed by culturing phages 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 227002 portion was removed as described above to obtain 410 base pair DNA with the sequence shown in Table 1.

III) AgtWES.AB DNA, prepared as described by Tiemier et al., is used as a vector instead of Charon 16A. All conditions are otherwise the same as Charon 16A. The selection of recombinant phages is carried out by hybridization according to the above-mentioned Benton and Davis puncturing. The insert is removed to give 410 base pairs DNA with the sequence shown in Table 1.

Example 4d

The method of Example 4c is repeated, but using E. coli RRB, E. coli HB-101, E. coli DP-50 or E. coli DP-50 SupF instead of E. coli X-1776. All conditions are otherwise the same and the same results are obtained.

Example 1 a d 6

Disclosed is the isolation and purification of DNA with a nucleotide sequence coding for human insulin and the synthesis of a vector containing both DNA and the formation 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 (S-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 the method of 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 double-stranded cDNAs were added. The product is treated with Hind III or Hsu I and analyzed as described in Example 3. A peak corresponding to a sequence of 450 nucleotides can be observed except for fragments cleaved at the site of treatment. Hind III The human cDNA was then inserted into plasmid pMB-9 This procedure is carried out according to Example 4 as well as the transformation of E. coli X-1776 and the selection of recombinant plasmids. The insert is removed with Hsu I and analyzed according to Example 4. DNA of 450 nucleotides is obtained. It is possible to demonstrate cloned human insulin which contains nucleotides that code for the entire amino acid sequence of human insulin. The amino acid sequence of chain A is as follows:

Gly — Ile — Val — Glu — Gin — Cys — Cys—

Thr-Ser-Ile-Cys-Ser-Leu-Tyr-Flu-20

Leu — Glu — Asn — Tyr — Cys — Asm

The amino acids in chain B have the following sequence:

Phe — Val — Asn — Glu — His — Leu — Cys—

Gly — Ser — His — Leu — Val — Glu — Ala—

Leu — Tyr — Leu — Val — Cys — Gly — 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 ij Example 6 was repeated except that plasmid pBR-322 was used instead of plasmid pMB-9 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.

The procedure of Example 6 was repeated except that plasmid pBR-313 DNA was used instead of plasmid pMB-9 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.

iii} The procedure of Example 6 was repeated using plasmid pSC-101 DNA instead of plasmid pMB-9 DNA. All conditions are otherwise the same as in Example 4a (iiij. The insertion portion is removed to give 450 nucleotide DNA with the sequence described in Example 6.

Example 6b

The procedure of Examples 6 and 6a was repeated except that E. coli RRI or E. coli HB-101 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 R1 site of Example 3a.

i) Incorporate human insulin Eco cDNA

Recombinant phages were isolated and the insert was removed according to Example 4c (i) to obtain DNA of 450 nu227002 cleotides with the sequence described in Example 6.

ii) Incorporating human-insulin cDNAs with Eco Rl-binding sites into the Charon 3A vector of Example 4c (ii). The recombinant phages were isolated and analyzed according to Example 4c (ii) to obtain 450 nucleotide DNA with the sequence described in Example 6.

iii) The plasmid AgtWES.AB was used as a vector for transfer of human insulin cDNA after treatment with Ec-o R1 as described in Example 4c (iii). Recombinant phages were isolated and analyzed. The insert is removed to give DNA of 450450 nucleotides with the sequence described in Example 6.

Example 6d

Example 6c is repeated, using E. coli RRI, E. coli HB-101, E. coli DP-50 or E. coli DP-50 SupF instead of E. coli X-1776. The same conditions are used and the same results are obtained.

Claims (5)

Subject CLAIMS 1. A method for producing a DNA transfer vector with a nucleotide sequence coding for insulin by isolating cells containing the mRNA coding for insulin, extracting the mRNA from the cells, and then purifying and synthesizing the cDNA from the mRNA. to form a cDNA having a nucleotide sequence coding for insulin, and the cDNA thus obtained is reacted with a DNA transfer vector to produce a DNA transfer vector with a nucleotide sequence coding for insulin, by extracting the mRNA from cells containing insulin-coded mRNA by homogenizing the cells in the presence of a ribonuclease inhibitor containing guanidinium thiocyanate and (3-mercaptoethanol at pH 5.0 to 8.0, thereby preventing mRNA degradation by ribonucleases, whereby the cDNA is reacted with the DNA transfer vector in two steps, wherein in the first step the DNA transfer vector is enzymatically hydrolyzed by a restriction endonuclease from a group of Hidn III or Hsu I to form a DNA-transfer vector with reactive ends capable of joining or linking to a cDNA with a nucleotide sequence coding for 'insulin' and, in a second step, the DNA-transfer vector and cDNA are zymatically coupled with a nucleotide sequence coding for insulin using a DNA ligase in the presence of adenosine triphosphate and producing a vector for transferring the nucleotide sequence coding for insulin. 2. Method of claim 1 for preparing a DNA transfer vector with sledem- nucleotides coding for insulin ^1, characterized in that after the first stage still enzymatically hydrolyzes any řosfáťová 5'-end group of the vector transferring DNA with reactive endings to form a preprocessed DNA transfer vector with reactive endings that are not capable of reconnecting but which are capable of being coupled to cDNA having a nucleotide sequence that encodes insulin. 3. A method according to claim 1 or 2, characterized in that a ribonuclease inhibitor containing 4 M guanidinium thiocyanate with 0.05 to 1.0 M / 3-m-ercaptoethanol is used. 4. The method of claim 3, wherein the ribonuclease inhibitor is 0.2 M / 3-mercaptoethanol. 5. The process according to claim 2, wherein the enzymatic hydrolysis is carried out by treatment with an alkaline phosphatase.