FI64640C - FOERFARANDE FOER FRAMSTAELLNING AV EN KOMBINATIONS-DNA-MOLEKYLSOM INNEHAOLLER EN FOER INSULIN KODANDE NUCLEOTIDSEKVENS HOCSOM KAN ANVAENDAS VID FRAMSTAELLNING AV EN INSULIN PRODU RCENDE MICROORGANISM - Google Patents

Abstract

New DNA transfer vector contains in its nucleotide sequence a sub-sequence having the structure of and transcribes from, a gene of a higher organism. Specifically the sub-sequence is the code for human insulin A or B chain (or both), or human growth hormone (the nucleotide sequences of these codes are given). Also new are microorganisms including in their nucleotide structure such as a sub-structure. - The microorganisms are made by first isolating an appropriate messenger-RNA; purifying it, then using it to elaborate a double-strand complementary DNA (c-DNA) contg. the required code. This is then reacted with a DNA-transfer vector having reactive end-groups able to react with itself or with the c-DNA. - The modified microorganisms can be used to produce insulin or growth hormone by fermentation, which is easier and quicker than cultivation of cell lines and gives better yields.

Description

RSFl W (11) advertisement publication ¢ 4640 MA 1 1 '' UTLÄGGNI NGSStCRIFT U ^ C {45) Patent cyörnoity 12 1C 1933 (51) Kv.ik.'Jint.a.3 c /) 2 N 15/00 FINLAND — FINLAND (11) —ρ »· ηη« ιβΐΜΐη * 781675 (22) Hikemlipttvl - Ansdknlnpdig 26.05.78 (23) Alkupllvl — Gtltlghuttdig 26.05 * 78 (41) Become Public - Bllvlt offentllj 28.11.78

Patent · la Registry Board .... οη no o, 1 (44) NlhtSviksIpanon and kuLJulkalni date. - 31.UO.Oj

Patents and registers An »ök * n utlagd och utUkriften pubticsnd (32) (33) (31) Pyydetty« uolkeu »—B * jtrd prlorltat 27 · 05 · 77 21.0U.78, 26.0U.78, 26.0U.78 USA (US) 8013U3, 805023, 897709, 897710, 898887, 899002, 899003 (71) The Regents of the University of California, 2200 University Avenue,

Berkeley, California 9U720, USA (72) William J. Rutter, San Francisco, California, Howard Michael Goodman,

San Francisco, California, Axel Ullrich, San Francisco, California,

John Mitchell Chirgvin, San Francisco, California, Raymond Louis Pictet,

San Francisco, California, Peter Horst Seebure ^ San Francisco, California, USA (US), John Shine, Curtin, ACT, Australia-Australien (AU) (7U) Oy Kolster Ab (5U) Method of insulin-encoding nucleotide sequence-containing and insulin-producing micro The present invention relates to a method for the preparation of a recombinant DNA molecule for the production of a recombinant DNA molecule for use in the preparation of a recombinant DNA molecule for use in the manufacture of a recombinant DNA molecule. - for the production of a recombinant DNA molecule containing a sequence-containing and insulin-producing microorganism.

The following symbols and abbreviations are used in this specification: DNA - deoxyribonucleic acid RNA - ribonucleic acid cDNA - complementary DNA

(synthesized enzymatically from the mRNA sequence) mRNA - messenger RNA tRNA - transfer RNA

dATP - deoxyadenosine triphosphate 2 64640 dGTP - deoxyguanosine triphosphate dCTP - deoxycytidine triphosphate A - adenine T - thyrine G - guanine C - cytosine

Tris - 2-amino-2-hydroxyethyl-1,3-propanediol EDTA - ethylenediaminetetraacetic acid ATP - adenosine triphosphate TTP - thymidine triphosphate

Hind III recognition site - SEQ ID N AGCTT, specifically a broken arrow into the Hind ΙΙΙ endonucleases Hsu I recognition site - SEQ ID IaGCTT A, ti specifically cleaved by the arrow Hsu I endonuclease means.

HaeIII recognition site - SEQ ID GG ice, specifically a broken arrow mark Search ΙΙΙ endonucleases.

Col E1 - Colysinin E1-forming plasmid poly dA - polydeoxyadenylate oligo ^ 12-18 "° ligocieoxythymidylate (12-18 bases)

Alu I recognition site - SEQ ID AG ^ CT, specifically a broken arrow mark Alu I endonuclease means.

Bam HI recognition site - SEQ ID ^ G GATCC, specifically a broken arrow Bam HI - endonucleases.

BglII-recognition site - SEQ ID GCCNNNN ^ NGGC, specifically a broken arrow into the BglII endonucleases (Note: N stands for nucleotides).

Eco RI recognition site - SEQ ID ^ G AATTC, specifically a broken arrow into the Eco RI endonuclease means.

Eco RII recognition site - SEQ ID IcCAGG or icCTGG, cleaved specifically by means of the arrow into the Eco RLI endonuclease.

Search for the 11-recognition site - SEQ ID AGCGC iT, Jr. AGCGC C GGCGC 4'Γ or GGCGC I C, specifically cleaved by the arrow II endonuclease get through.

Hinc II recognition site - sequence GTTAAC, GTTGAC, GTCAAG or GTCGAC, specifically cleaved by Hinc II endonuclease.

3 6 4 64 0

Pst I recognition site - SEQ ID CTGCA G ^, specifically a broken arrow into the Pst I endonucleases.

Sal I recognition site - SEQ ID JrTCGAC G, specifically a broken arrow into the Sal I endonucleases. The Sst I - the recognition site - SEQ ID GAGCT ^, specifically a broken arrow into the Sst I-endonucleaasin means.

The biological significance of the base sequence of DNA lies in the fact that it is a repository of genetic information. It is known that the base sequence of DNA is the code by which the amino acid sequence of all proteins produced by a cell is determined. In addition, portions of the sequence may have the function of regulating the time and amount of protein production. The nature of the regulatory units is incompletely known. The base sequence of each strand is used as a template when DNA is replicated in cell division.

The way in which DNA base sequence information is used to determine the amino acid sequence of proteins is essentially the same for all living organisms.

It has been shown that each amino acid normally present in proteins is determined by one or more trinucleotides, i.e., the triplet sequence. Thus, for each protein, there is a corresponding DNA segment containing the triplet sequence corresponding to the amino acid sequence of the protein. The genetic code is shown in the following table.

Genetic code

Phenylalanine. (Phe) TTK Histidine (His) CAK

Leucine (Leu) XTY Glutamine (Gin) CAJ

Isoleucine (Ile) ATM Asparagine (Asn) AAK

Methionine (Met) ATG Lysine (Lys) AAJ

Välin (Vai) GTL Aspartic acid (Asp) GAK

Serine (Ser) QRS Glutamic acid (Glu) CAJ

Proline (Pro) CCL Cysteine (Cys) TGK

Threonine (Thr) ACL Tryptophan (Try) TGG

Alanine (Lower) GCL Arginine (Arg) WGZ

Tyrosine (Tyr) TAK Glycine (Gly) GGL

4,64640

Terminal signal TAJ

Terminal TGA

Key: Each three-letter triplet represents a DNA trinucleotide with a 5 'end on the left and a 3' end on the right. The letters represent the purine and pyrimidine bases from which the nucleotide sequence is formed.

A = adenine G = guanine C = cytosine T = thyrine

X = T or C if Y is A or G X = C if Y is C or T

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

Y = A or G if X is T

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

Transcription is the first step in the biological process by which nucleotide sequence information is converted to an amino acid sequence. In this step, the RNA is first copied with the DNA segment whose sequence defines the protein to be produced. RNA is a polynucleotide similar to DNA except that deoxyribose is replaced by ribose and thymine is replaced by uracil. The bases of RNA can settle in similar base pairs as the bases of DNA. Thus, RNA transcription of a DNA nucleotide sequence is complementary to the sequence to be copied. Such RNA is called messenger RNA (mRNA) because it acts as a mediator between the cellular genetic system and the protein-synthesizing system.

Within a cell, mRNA is used as a template in the complex process involving numerous enzymes and cellular systems that results in the generation of a particular amino acid sequence. This process is called mRNA translation.

Often, there are also additional steps in which the amino acid sequence synthesized in the translation process is converted to a functional protein. Insulin is an example of this.

The immediate precursor of insulin is a single polypeptide, called proinsulin, which contains two insulin chains, A and B, joined by peptide C, cf. Steiner, D. F., Cunningham, D., Spigelman, L., and Aten, B. Science 157, 697 (1967). According to recent information, the original translation product of insulin mRNA is not proinsulin but preproinsulin containing 20 additional amino acids at the amino terminus of proinsulin, cf. Cahn., S.J.,

Keim, P. and Steiner D.F., Proc. Natl. Acad. Sci. USA, 73, 1964 (1976) and Lomedico, P.T. and Saunders, G.F., Nucl.

Acids. Res. 3, 381 (1976). The structure of preproinsulin can be represented as follows: NH0 (pre-peptide) chain B- (chain O-chain A-C00H.

Many proteins of medical or research importance are located in or produced by cells of higher organisms, such as vertebrates. These include e.g. insulin hormone, other peptide hormones such as growth hormone, blood pressure regulating hormones and many hormones of industrial, medical or research importance. These proteins are often difficult to extract from the body in useful amounts, and this problem is quite severe for proteins of human origin. Thus, there is a need for methods by which such proteins can be produced in sufficient amounts in cells outside the organism. In certain cases, it is possible to provide suitable cell lines that can be maintained by tissue culture techniques. However, tissue culture is slow, the medium is expensive, the conditions are precisely controlled, and the yield is low. In addition, it is often difficult to maintain a cell line constant while maintaining the desired specific properties.

In contrast, microorganisms, such as bacteria, are relatively easy to grow in chemically defined media. Fermentation technology is advanced. It is possible to grow organisms quickly and in high yields. In addition, some microorganisms are thoroughly studied and known for their genes and properties.

Thus, it is highly desirable to be able to transfer the genetic code of a medically significant protein from an organism that normally makes the protein to a suitable microorganism. In this way, the microorganism can produce the protein under controlled growth conditions and the desired amount of protein can be obtained. Manufacturing costs may also be substantially reduced if the protein is prepared by such a method. In addition, the ability to isolate and transfer the genetic sequence that defines the production of a particular protein to a microorganism whose genetic background is well known provides a valuable means of research to know how the synthesis of this protein is regulated and how the protein is modified after synthesis..Genetic sequences possible? · also transformed to obtain proteins with altered therapeutic or functional properties.

The present invention includes a method for achieving the objects described above. This is a multi - step process involving enzyme - catalyzed reactions. The nature of these enzyme reactions will be described on the basis of facts known to date.

Reverse transcriptase (DNA polymerase) catalyzes the synthesis of DNA complementary to the RNA template strand in the presence of an RNA template, a DNA template, and four deoxynucleoside triphosphates, dATP, dGTP, dCTP, and dTTP. The reaction is initiated by oi-covalent binding of the DNA template to the 3 'end of the mRNA, followed by gradual insertion of the required deoxynucleotides to the 3' end of the growing chain as base pairs defined by the mRNA nucleotide sequence 7,64640. The resulting molecule can be considered as a hairpin structure containing the original RNA linked by a single strand of DNA to a complementary strand of DNA. Reverse transcriptase is also capable of catalyzing a similar reaction using a single-stranded DNA template, the resulting product being a double-stranded DNA hairpin with single-stranded DNA joining the strands at one end, cf. Aviv, H. and Leder, P., Proc. Natl.

Acad. Sci. USA 69, 1408 (1972) and Efstratiadis, A., Kafatos, F.C., Maxam, A.F. and Maniatis, T., Cell 7, 279 (1976).

Restriction endonucleases are enzymes capable of hydrolyzing phosphodiester bonds in double-stranded DNA to form a breakpoint in the DNA strand.

If the DNA is in the form of a closed loop, the structure of the loop becomes linear. The main property of this type of enzyme is that its hydrolytic effect is limited to a site with a specific nucleotide sequence. This sequence is called the restriction endonuclease recognition site. Restriction endonucleases have been isolated from a variety of sources and characterized by the nucleotide sequence of their recognition sites. ;

Some restriction endonucleases hydrolyze phosphodiester bonds at the same site in each strand such that. . . 1 a blunt head is created. Some catalyze the hydrolysis of bonds separated by a few nucleotides, creating a free single-stranded region at each end of the molecule. Such single-stranded ends are self-complementary and thus cohesive and can be used to reattach hydrolyzed DNA. Because a particular enzyme can be predicted to cleave DMA molecules with the same recognition site, the same cohesive ends are created, and thus it is possible to combine restriction,

endonuclease-treated heterologous DNA sequences I

to other sequences treated in the same manner, see

Roberts, R.J. Crit. Rev. Biochem. 4, 123 (1976). However, esterification sites are quite rare, but the utility of restriction endonucleases has been enhanced by synthesizing double-stranded oligonucleotides with a recognition site sequence. oligonucleotide, the product is exposed to the required restriction endonuclease to create the necessary cohesive ends, see Heyneker, HL, Shine, J., Goodman, HM, Boyer, HW, Rosenberg, J., Dickerson, RE, Narang, SA, Itakura, K ., Lin, S. and Riggs, AD, Nature 263, 748 (1976) and Scheller, RH, Dickerson, RE, Boyer, HW, Riggs, AD and Itakura, K., Science 196, 177 (1977).

S1 endonuclease is a general enzyme capable of hydrolyzing phosphodiester bonds in single-stranded DNA or single-stranded junctions in double-stranded DNA, cf. Vogt, V.M., Eur. J. Biochem. 33, 192 (1973).

DNA ligase is an enzyme capable of catalyzing the formation of a phosphodiester bond between two DNA segments having 5'-phosphate and 3'-hydroxyl, respectively, and which may consist of two DNA fragments held together by cohesive ends. The normal mode of action of an enzyme is considered to be that it combines several daughter DNA fragments generated in parallel with another strand. However, DNA ligase can, under suitable conditions, catalyze the fusion of blunt ends in which two blunt-ended molecules covalently join, cf. Sgaramella, V., Van de Sande, J.H. and Khorana, H.G., Proc. Natl. Acad. Sci. USA 67, 1468 (1970).

Alkaline phosphatase is a general enzyme capable of hydrolyzing phosphate esters, including the 5 'terminal phosphates of DNA.

In a sub-step of the method of this invention, a particular DNA fragment is inserted into a DNA vector, such as a plasmid. Plasmid is the name used for any independently replicating unit of DNA that is present in 9,64640 microbial cells and does not belong to the genome of the ram of the host cell.

The plasmid is not genetically bound to the chromosome of the host cell. Plasmid DNA exists as circular double-stranded molecules, usually having a molecular weight of a few million, some even more than 10, and usually representing only a small percentage of the total DNA of the cell. Plasmid DNA can usually be separated from host cell DNA due to its large size difference. Plasmids can replicate regardless of the rate of division of the host cell, and sometimes their rate of division can be regulated by changing the growth conditions. Although the plasmid exists as a closed ring, a DNA segment can be artificially inserted to form a recombinant plasmid with a higher molecular weight that has not substantially altered its ability to replicate or express its genes. Thus, the plasmid serves as a useful vector to transfer a DNA segment to a new host cell. Plasmids suitable for recombinant DNA technology are particularly those that contain genes of choice, such as genes that confer drug resistance.

To illustrate the practice of this invention, the isolation and transfer of the rat insulin gene will be described in detail. Insulin was chosen as the target because of its central role in clinical medicine and basic research. Those skilled in the art will be able to isolate the insulin gene from an organism, including a human, by the method described.

Insulin was first isolated in 1922. Today, the use of this hormone in the treatment of diabetes is well known. Although bovine and porcine pancreases from slaughterhouses are sources of insulin, there will be a shortage of this hormone as the number of diabetics in the world increases. In addition, some diabetics develop a harmful allergy to bovine and porcine insulin. Therefore, it would be highly desirable to be able to produce sufficient human insulin to meet the global need. If human insulin could be produced by bacteria, this goal would be achievable. Until the present invention, however, the goal has been hampered by the fact that there has been no method by which the insulin gene can be transferred to bacteria.

The method according to the invention is characterized in that a) the mRNA is isolated from the islet cells of the pancreatic tissue by homogenization in the presence of a ribonuclease inhibitor, for example a mixture of 4-m guanidinium thiocyanate and 0.05 to 1.0-m / 3-mercaptoethanol at pH 5. , 0 to 8.0, in which case no ribo-nuclease degradation of the mRNA occurs, b) a cDNA having an insulin-encoding nucleotide sequence is prepared from the obtained mRNA in a manner known per se by means of a reverse transcriptase enzyme, and said cDNA is double-stranded, c ) the restriction endonuclease recognition site sequences are ligated to the ends of the resulting double-stranded cDNA in a manner known per se, wherein the restriction endonuclease has the same enzyme as in d) below, after which the cDNA is cleaved by said restriction endonuclease III, Hsu I or Eco By RI, in a manner known per se, e.g. by incubation at pH 7.6 at 37 ° C for 2 hours to obtain an open plasmid vector with cohesive ends, e) hydrolysing the 5'-phosph of the plasmid vector obtained in d) -phosphate end groups, for example by treatment with alkaline phosphatase at pH 8.0 at 65 ° C for 30 minutes, whereby the cohesive ends mentioned in d) cannot be joined, f) the plasmid vector treated according to e) and the cDNA obtained in c) are joined by incubation in a manner known per se in the presence of DNA ligase and ATP, e.g. at pH 7.6 at 14 ° C for one hour, using a molar excess of a plasmid vector to obtain a recombinant DNA molecule containing a nucleotide sequence encoding insulin.

The practical aspect of the method of the present invention is illustrated by describing the isolation, transfer into bacteria, and replication of the nucleotide sequence of rat insulin. Similarly, the nucleotide sequence of rat growth hormone has been isolated, transferred, and replicated in bacteria. The method is also suitable for transferring a nucleotide sequence isolated from a human, such as human insulin and growth hormone, as well as other polypeptide hormones.

This invention includes a method by which a DNA molecule having a particular nucleotide sequence can be isolated and transferred to a microorganism, and the original nucleotide sequence of the DNA can be found in the organism after replication.

The various steps of the method contained in this invention can be divided into four groups.

1. Isolation of the desired cell from a higher organism

There are two possible sources for the genetic code of a particular protein, namely the DNA of the source organism or the RNA transcription of the DNA. According to the current safety requirements of the US National Institutes of Health, human genes, whatever they may be, can be inserted into recombinant DNA and then into bacteria only when the genes have been thoroughly purified or when there are facilities for particularly high-risk work ( P4), see Federal Register, Vol. 41, no. 131, 1967-07-07, pp. 27902-27943. Preferably, the starting point is to isolate a specific mRNA that contains the code of the desired protein. This approach also has the advantage that mRNA extracted from the cell can be purified more easily than DNA extracted from the cell. In particular, it is possible to use the fact that highly specialized organisms, such as vertebrates, have certain cells in certain places that are responsible for producing some of the protein in question. Alternatively, such a cell may be present at some stage in the development of the organism. Most of the mRNA isolated from the cells of such a cell contains the desired nucleotide sequence. Thus, the choice of cell to be isolated and method of isolation can take into account the advantages offered by the initial degree of purity of the mRNA to be isolated.

64640 12

In most tissues, glands, and organs, cells are connected by a fibrous connective tissue that is composed primarily of collagen, but may also contain other structural proteins, polysaccharides, and mineral stores, depending on the tissue. Isolation of cells from a particular tissue requires methods by which the cells can be detached from the connective tissue. Isolation and purification of a particular specialized cell type thus involves two main steps, which are to separate the cells from the connective tissue and to separate the desired cell type from other types of tissue cells. As an example, a method for isolating pancreatic islets of Langerhans suitable for isolating insulin mRNA is described.

Insulin-producing cells can also be derived from other sources, such as the pancreas of a fetal calf or cultured islet tumor cells. Isolation of pure islet cells is then much simpler, especially if pure cell cultures are used. The method of isolating islet cells described above would not be necessary in this case, but it is nevertheless a preferred method due to its general applicability.

It is often observed that the proportion of the desired mRNA can be increased if the cell's ability to respond to external stimuli is exploited. For example, hormone treatment may cause an increase in the production of the desired mRNA. Other methods include growing at a certain temperature and / or in a certain nutrient or other chemical substance. When isolating rat growth hormone mRNA, treatment of cultured rat pituitary cells with thyroid hormone and glucocorticoids synergistically significantly increased the amount of growth hormone mRNA.

2. Extraction of mRNA An important feature of the present invention is that RNase activity is virtually completely removed from the cell extract. The mRNA to be extracted is a single-stranded polynucleotide with no complementary strand. Thus, hydrolytic cleavage of any phosphodiester bond in the sequence would render the entire molecule 13,64640 incapable of transferring the intact genetic sequence to the microorganism. As mentioned above, the RN donkey is widespread and is very active and exceptionally stable.

It is on the skin, it withstands the usual methods of washing glassware and sometimes it contaminates organic chemicals. The difficulty in handling pancreatic cell extracts is very high, as the pancreas produces digestive enzymes and is thus highly RNase-rich. However, RNase aspiration applies to all tissues and the method of destroying RNase activity described herein can be applied to all cells. The exceptional efficiency of the method is demonstrated in the isolation of unchanged mRNA from pancreatic, islet cells.

The present invention uses a combination of a chaotropic anion, a chaotropic cation, and a disulfide bond cleavage reagent during cell disruption and all of the operations required to produce virtually protein-free RNα. The efficiency of the interaction of the above reagents has been demonstrated in practice by isolating virtually intact mRNA in good yield from islets of Langerhans isolated from rat pancreas.

The choice of suitable chaotropic ions depends on their water solubility and availability. Suitable chaotropic anions include e.g. guanidinium, carbamoylguanidinium, guanylguanidinium, lithium, etc. Suitable chaotropic anions include e.g. iodide, perchlorate, thiocyanate, diiodic salicylate, etc. The relative effectiveness of the salts formed by such anions and cations is determined by their solubility. For example, lithium diiodal salicylate is a more potent denaturant than guanidinium thiocyanate, but has a solubility of only about 0.1 M and is also relatively expensive. Guanidinium thiocyanate is a preferred cationic anion combination because it is readily available and has a solubility in aqueous solutions of about 5%. .

Thiol compounds, such as γ-mercaptoethanol, are known to disrupt the intramolecular disulfide bonds of proteins by the thiol disulfide exchange reaction. In addition to β-mercaptoethanol, several suitable thiol compounds are known, such as dithiothreitol, cysteine, propanol-dimercaptan, etc. Water solubility is a necessary requirement, as a large excess of thiol compound must be present compared to the intramolecular 14,64640 disulphides in order for the exchange reaction to take place. γ-mercaptoethanol must be given priority because it is readily available at a reasonable price.

The efficiency of a particular chaotropic salt is directly proportional to its concentration when it is desired to inhibit RNase in the extraction of RNA from cells or tissues. The preferred concentration is therefore the highest concentration that can be practiced. The success of the present invention in keeping the mRNA intact during extraction is thought to be due to the rate at which the RNase is denatured and the degree of denaturation. This apparently explains the superiority of guanidinium thiocyanate over hydrochloride, although the hydrochloride is only slightly weaker as a denaturant. The potency of a denaturant is therefore defined as the threshold concentration required for complete denaturation of the protein. On the other hand, the rate of denaturation of proteins often depends on the ratio of the denaturant concentration to the threshold value of 5 to 10 raised to the power, cf. Tanford, C.A. , Adv. Prot. Chein. 23, 121 (1968). Qualitatively, this ratio means that only a slightly more efficient denaturant than guanidinium hydrochloride can denature the protein many times faster at the same concentration.

The use of a disulfide-cleaving reagent in combination with a denaturant enables and enhances the latter effect because the RNase molecule becomes fully opened. Thiol compounds are thought to aid in the progress of the denaturation process because they prevent the rapid denaturation that can occur if the intramolecular disulfide bonds are left intact. In addition, the RNase impurity contained in the mRNA preparation remains virtually inactive even in the absence of denaturant and thiol. Disulfide-cleavage reagents having thiol groups are somewhat effective at any concentration, but it is preferable to use a large excess of thiol groups over the intramolecular disulfide bonds, as this will drive the exchange reaction toward the cleavage of the intramolecular disulfide bonds. On the other hand, since many thiol compounds are malodorous and it is uncomfortable to work with high concentrations, there is an upper concentration limit for practical reasons. When β-mercaptoethanol is used to separate intact RNA from rat pancreas, concentrations in the range of 0.05-1.0 M have been found to be effective, and the optimal concentration is considered to be 0.2 M.

64640 The pH of the medium can be anywhere from 5.0 to 8.0 when mRNA is extracted from cells.

After cell disruption, RNA is separated from cellular proteins and DNA. Several methods have been developed for this purpose, all of which are suitable and known to those skilled in the art. A common method previously used is ethanol precipitation, with which RNA is selectively precipitated. In the process of this invention, it is more preferable to omit the precipitation step and deposit the homogenate directly in a 5.7 M cesium chloride solution in a centrifuge tube and then perform centrifugation as described in Glisin, V., Crkvenjakov, R. and Byus, C., Biochemistry 13, 2633 (1974). This method is advantageous because the environment harmful to RNase can be preserved at all times and RNA free of DNA and protein is obtained in good yield.

By the method described above, the entire RNA of the cell homogenate is purified. However, only a fraction of this desired mRNA is present. Further purification takes advantage of the fact that in cells of higher organisms, mRNA is processed after transcription by the addition of polyadenyl acid. Such mRNA containing poly-A sequences can be selectively separated on a chromatography column packed with cellulose to which oligothymidylate has been added, cf. Avis, H. and Leder, P., supra. The method described above is suitable when virtually pure, undamaged and translatable mRNA from sources rich in RNase is required.

In certain cases, such as when using tissue culture cells as a source of rnRNA, the RNase impurity may be so low that the inhibition of RNase described above is not required. In this case, the previously known methods for removing RNase activity are sufficient.

3. Generation of cDNA Reference is made herein to Figure 1, which is a schematic representation of the remaining steps of the method. The first step now is to generate a DNA sequence complementary to the purified mRNA. Reverse transcriptase is selected as the enzyme for this reaction, although in principle any enzyme capable of forming a complementary strand of DNA could be used using mRNA as a template. The reaction can be performed under conditions known in the art by using mRMA as a template and a mixture of four deoxynucleoside triphosphates as a DNA strand. a precursor. It is preferred that one of the deoxynucleoside triphosphates is labeled at the oC position with a P atom, as it can be used to monitor the reaction, to serve as a marker in separation methods such as chromatography and electrophoresis, and to draw quantitative conclusions, cf. Efstratiadis, A., et al · 3 above.

As shown in the figure, the reverse transcriptase reaction result is a double-stranded hairpin structure in which the RNA strand and the DNA strand are joined together by a non-covalent bond.

The product of the reverse transcriptase reaction is removed from the reaction mixture by known methods. It has been found useful to use a combination of phenol extraction, chromatography ("Sephadex G-100", Pharmacia Inc., Uppsala, Sweden) and ethanol extraction.

Once cDIJA is enzymatically synthesized, the RNA template can be removed. Several methods are known for selectively degrading RNA in the presence of DNA. Base hydrolysis is a preferred method because it is highly selective and can be easily adjusted by pH.

After hydrolysis and neutralization, the P-labeled cDNA can be concentrated by ethanol precipitation if desired.

Such double-stranded hairpin cDNA is synthesized by a suitable enzyme such as DNA nolyme Ywas or reverse transcriptase. Reaction conditions are as previously described, / 32. . . . .

Including P-labeled nucleoside triphosphate. Reverse transcriptase is obtained from several sources. The avian myeloblastosis virus is a preferred source. The virus is available from Dr. D.J. Beard'ilt. Life Sciences Incorporated, St. Petersburg, Florida ', which manufactures the virus under an agreement with the National Institutes of Health.

Once the cDHA hairpin is formed, it may be advantageous to purify it from the reaction mixture. As previously mentioned, it has been found advantageous to use phenol extraction, chromatography ("Sephadex G-100") and ethanol precipitation when DNA is to be purified from protein impurities.

The 17,64640 hairpin structure can be converted to a standard double-stranded DNA structure by removing a single strand connecting the complementary strands. There are several enzymes capable of specifically hydrolyzing single-stranded portions of DNA. A well-suited enzyme for this purpose is S1 nuclease isolated from Aspergillus oryzae (manufactured by Miles Research Products, Elkhart, Indiana). Treatment of the DNA hairpin structure with S1 nuclease yields molecules with base pairs in good yield. Extraction, chromatography and ethanol precipitation are then performed as described above. Efstratiadis et al. have described in the above publication the synthesis of double-stranded cDNA transcripts of mRNA by reverse transcriptase and S. luclease.

The proportion of blunt-ended cDNAs can optionally be increased if treated with E. coli DNA polymerase I in the presence of four deoxynucleoside triphosphates. The interaction of the enzyme's xononuclease activity and polymorase activity works so that the protruding 3 'end is removed and the protruding 5' end is filled. This ensures that · as many cUHA molecules as possible are involved in the coupling reactions of the next step.

In the next step of the method of the invention, the ends of the cDNA product are processed so that a sequence of the restriction endonuclease recognition site is obtained at each end. Practical reasons determine the choice of DNA fragment to be inserted at the ends. The sequence to be added to the ends is selected based on the restriction endonuclease, which in turn is selected based on the DNA vector to which the cDNA is inserted. The plasmid of choice should have at least one site to which the restriction endonuclease cleavage effect may be directed. For example, plasmid pMB9 contains a single recognition site for the enzyme Kind III. Hind III is isolated from Haemophilus influenzae and purified by the following method: Smith, H.O. and Wilcox, K.'W. , J. Mol. Biol. 51, 379 (1970). The enzyme Hae III of Haemophilus aegyptious is purified by the following method: Middleton, J.H., Edgell, M.H. and Hutchison III, C.A., J. Virol. 10, 42 (1972). The enzyme of Haemophilus suis, Hsu I, catalyzes the same site-specific hydrolysis at the same recognition site as Hind III. Thus, these two enzymes can be considered functionally alternative.

18 64640

For insertion of double cDNAs at the ends, it is preferred to use a chemically synthesized double-stranded decanucleotide containing a Hind III recognition site. The sequence of the double-stranded decanucleotide is shown in Figure 1. Heyneker, H.L. , et al. and Scheller, R.H., et al. above. Several such recognition site sequences are available to those skilled in the art, and for this reason it is possible to select the ends of the double DNA to suit the restriction endonuclease required in each case.

The insertion of restriction site sequences into the ends of the cDNA can be accomplished by a method termed blunt end insertion and is catalyzed by a DNA ligase purified by the following method: Panet, A., et al., Biochemistry 12, 5045 (1973). The aforementioned Sgaramella, V., et al. have described the blunt-end coupling reaction. Coupling at the blunt end, where the reaction takes place with a blunt-ended cDNA and a large molar excess of a double-stranded decanucleotide containing a Hind III endonuclease recognition site, yields a cDNA with a Hind III restriction site sequence at each end. When the reaction product is treated with Hind III endonuclease, cleavage occurs at the restriction site and single-stranded self-complementing 5 'ends are formed, as shown in Figure 1.

4. Construction of a Recombinant DNA Transfer Vector Suitable transfer vectors currently accepted for use are rcuti. several bacteriophage lambda derivatives (see above. Blattner, F.R., Williams, B.G., Blechl, A.E., Denniston-Thompson, K., Faber, H.E., Furlong, L.A.,

Grunwald, D.J., Kiefer, D.O., Moore, D.D., Schuram, J.W.,

Sheldon, E.L. and Smithies, 0., Science 196, 161 (1977)) and col E1 plasmid derivatives (see, e.g., Rodriguez, RL, Bolivar, S., Goodman, HM, Boyer, HW, and Betlach, MN, ICN-UCLA Symposium on Molecular Mechanism, in the Control of Gene Expression, DP Nierlich, WJ Rutter, CE Γοχ, Eds. (Academic Press, NY, 1976) ss.

471-477). Plasmids derived from Col E1 are characterized by a relatively small size, having a molecular weight in the order of a few million, and a normal copy number of plasmid DNA per host cell of 20-40, but can be raised to a thousand or more, when host cells are treated with chloramphen-Koli. The ability of a host cell to increase the amount of gene it contains, under certain conditions, under the control of a researcher, makes it possible for the host cell to produce mainly the proteins encoded by the plasmid genes. Thus, such col E1 derivatives are preferred transfer vectors in the method of this invention. Suitable col E1 derivatives include e.g. plasmids pMB-9, the gene of which confers resistance to tetracycline, and pBR-313, pBR-315, pBR-316, p3R-317 and pBR-322, which contain the tetracycline resistance gene and the ampicillin resistance gene. The presence of the resistance-causing gene provides a suitable means of selecting cells that are infected with the plasmid, as such colonies grow in the presence of the drug, but in the absence of the plasmid, the cells do not grow. In the examples contained in this specification, a plasmid derived from col E1 containing e.m.

resistance gene and one Hind III recognition site.

Plasmid pBR-322 has been highly characterized.

Bolivar, F. et al. (Gene 2 (1977) 95) has described the synthesis and characterization of this plasmid. Plasmid e pBR322 has a molecular weight of 2.7 x 10 daltons (Bolivar F.,

Gene 4 (1978) 121) and contains ampicillin-resistant

R R

(Aβ) and tetracycline resistance (Te).

R

vat genes. There is one in the aforementioned Aβ gene

R

endonuclease Pst 1 recognition site. The Te gene has one recognition site for each of the following endonucleases:

Price III, Sal I and Bam HI. In addition, pBR322 contains one Eco R1 recognition site, two Hind II recognition sites, five Eco RII recognition sites, three Bgl I recognition sites, 12 Alu I recognition sites, 12 Hae II recognition sites, and 17 Search for the identification point of III. The recognition sites are labeled on the circular plasmid pBR322 on page 103

D

Bolivar et al. In. The Aβ gene is lost when the plasmid is cleaved from the Pst I recognition site and n foreign DNA is ligated to it. Similarly, Te is lost upon cleavage of pBR 322 by Hind III, Val I or Bam I endonucleases and insertion of foreign DNA into their recognition site.

20 64640

When DNA is ligated into the Pst I recognition site, recombinant molecules are formed that can be detected in strains that are both ampicillin-sensitive (Aβ) and tetracyclic.

R

ni resistant (Te) as well as by inserting DNA into the Sai I or Bam HI recognition site of Hind III, recombination molecules are formed that can be detected from strains that are both ampicillin resistant and tetracycline sensitive. A transfer vector containing foreign DNA linked to one of the four recognition sites mentioned above can be characterized by the drug resistance properties of the transfer vector, i.e., whether it is ampicillin-sensitive and tetracycline-resistant or ampicillin-resistant and tetracycline-sensitive. The transfer vector can be further characterized by removing the foreign DNA attached to it, determining the molecular weights of the two products formed, and comparing these to the molecular weights of the starting materials. In addition, the characterization can be supplemented by labeling the recognition sites of different endonucleases in the transfer vector. The sequence of the transferred DNA molecule can also be determined. The entire nucleotide sequence of said plasmid pBR322 is set forth in J. Sutcliffe's dissertation "Nucleotide Sequence of pBR322", Harvard University, Cambridge, Massachusetts, USA.

Similarly, plasmid pBR313 has been highly characterized. The synthesis and characterization of this plasmid has been described by Bolivar, F. et al. (Gene 2 (1977) 75).

g

Plasmid pBR313 has a molecular weight of 5.8 x 10 daltons and contains genes for ampicillin resistance (Aβ) and tetracycline resistance (Te). plasmid

P

The pBR313 Te gene has one recognition site for each of the following nucleases: Hind III, Sal I, and Bam HI. The recognition sites of plasmid pBR313 for the various endonucleases have been fully determined, and the resulting recognition site map is shown on page 8U of olivar et al. In. When ligating foreign DNA into Hind III.

At the recognition site of Sai I or Bam Hir, tetracycline resistance disappears. Thus, recombinant DNA molecules are found in 64640 strains that are both ampicillin-resistant and tetracycline-sensitive. The transfer vector can be characterized on the basis of drug resistance properties, the transferred DNA sequence, the restriction enzyme recognition site map, and the molecular weights mentioned above.

The third plasmid that has been highly characterized is pMB 9. This plasmid was prepared according to Rodriguez, R.L. et al. (above) and Bolivar, F. et al. (Gene 2 (1977) 75). Plasmid pMB 9 g has a molecular weight of 3.5 x 10 daltons and contains the gene that causes tetracycline resistance (Te). This plasmid contains one recognition site for each of the following endonuclei Eco R1, Hind III, Sal I and Bam HI. Of these, the recognition sites of the latter three are located in the Te gene. When foreign DNA was ligated into the Hind III, I or Bam HI recognition site, tetracycline resistance disappeared. A transfer vector containing foreign DNA at some of these sites can be characterized by this property. This transfer vector can be further characterized by sequencing the transferred DNA molecule, comparing molecular weights, and by recognition site analysis as described above.

Plasmid pSCIOI has been highly characterized. Cohen, S.H et al. (Proc. Nat. Acad. Sci. USA 70 (1973) 1293) have described the synthesis and preliminary characterization of this plasmid. The characterization has been supplemented by Cohen, S.N. et al., Proc. Nat.Acad. Sci. USA 70 (1973) 32 ^ Boyer, HW et al. (Recombinant Molecules), Beers, RF and Basset, EG ed., Raven Press, New York, p. 13 (1977). ) and Cohen, SN et al. (Recombinant Molecules, p. 91).

g

Plasmid pSCIOI has a molecular weight of 5.8 x 10 daltons,

R

and contains a gene that causes tetracycline resistance (Te). The Te gene contains one recognition site for each of the following endonucleases: Hind III, Sal I, and Bam HI. In addition, plasmid pSCIOI contains one Eco RI recognition site, one Hpa I recognition site, one Sma. I identification point and four Hinc II identification points. Tetracycline resistance is lost by cleavage of pSCIOI with Hind III, Sal I or Bam HI endo-nucleases and insertion of foreign DNA into their recognition site. By inserting 64640 22 DNA into Hind III, got I or Bam HI recognition site, recombination molecules are found in cultures that are tetracycline sensitive. A transfer vector containing foreign DNA ligated to one of the three sites mentioned above can be characterized based on the resistance properties of the transfer vector. The transfer vector can be further characterized by (a) deleting the transferred foreign DNA sequence, determining and comparing molecular weights, (b) constructing a recognition site map, and (c) determining the sequence of the transferred DNA molecule.

Several vectors can be used to transform Bacillus subtilis. These vectors are derived from the microorganism Staphylococcus aureus (see Fordanescu, S., J. Bakteriol. 124 (1974) 597 and Ehrlich, S. D., Pröc.

Nat. Acad. Sci. USA, 74 (1977) 1680). These plasmids have certain resistance and specific restriction endunuclease recognition sites. For example, plasmid pC194 confers resistance to chloramphenicol (Cm), contains a single Hind β site, and has a molecular weight of 23,64640 g at 1.8 x 10 daltons. The foreign DNA thus binds to the HindIII site of plasmid pC194. A transfer vector containing foreign DNA can be characterized by its resistance (Cm). The transfer vector can also be characterized by removing the ligated DNA by determining the molecular weight of the two products, relative to the molecular weight of the starting materials. The sequence of the ligated DNA can also be determined.

Recombinant plasmids are constructed by mixing restriction endonuclease-treated plasmid DNA and cDNA, the end groups of which have been treated accordingly. Plasmid DNA is used in large molar excess over cDNA to minimize combinations of cDNA segments with each other.

It is preferred to use a method that minimizes the number of colonies from which the recombinant plasmid is sought.

The method is performed by treating restriction endonuclease-digested plasmid DNA with alkaline phosphatase (manufactured by Worthington Biochemical Corporation, Freehold,

New Jersey). Alkaline phosphatase removes the phosphates at the 5 'end from the ends of the plasmid generated by the endonuclease and prevents self-ligation of the plasmid DNA. Thus, ring formation and concomitant transformation depends on the incorporation of a DNA fragment containing 5 'phosphorylated ends. The method described reduces the relative incidence of transformation without recombination to less than 1-10.

This invention is based on the fact that a DNA ligase-catalyzed reaction occurs between the 5 'phosphate end group of DNA and the 31' hydroxyl end group of DNA. In the absence of 5'-phosphate, no coupling reaction occurs. When double-stranded DNAs need to be combined, there are three situations, as shown in Table 1.

24 64640

table 1

Case Reactive compounds Ligase product 'i - I 3' 5 '3' 5 '- n; 1 H o ro- -opo-- -ΟΡΟ-, Η + 2 3HO-- -OPO - + 2H2 ° 5' 3 ' 5 '3' II 3 '5' 3 '5' -OH 1! 2C? -0 --- OPO-- -OH + OH --- OH HO - 2 ^ 5 '· 3' 5 '3' III 3 '5' _0Il _ no reaction -OH + HO- 5 '3'

In Table 1, double-stranded DNA is shown schematically as solid parallel lines and their respective 5 'and 3' end groups are labeled as hydroxyls (OH) or phosphates (OPO ^ H 2). In case I, both reacting molecules have 51-phosphate at their ends, and thus the two strands covalently join each other. In case II, only one of the ends to be joined has 5'-phosphate and thus only one of the strands to be joined is covalently attached to the other and a discontinuity point remains in the other strand. The covalently unjoined strand remains associated with the joined strand by hydrogen bridges present between the complementary base pairs of the opposite strands. In the case of ITI, there is no 5'-phosphate at either reactive end and no fusion reaction occurs.

Thus, the 5 'phosphate group must be removed from the end whose attachment to the other is to be prevented.

25 64640 Any method of removing the 51-phosphate group that does not otherwise damage the DNA structure is suitable for use.

Preferably, alkaline phosphatase catalyzed hydrolysis is used.

The method described above is also useful in the case where it is desired to cleave a linear DNA molecule into two subfragments, usually using a restriction endonuclease, and then reconstruct the original sequence. The subfragments can be purified separately and the desired sequence can be reconstructed by combining the subfragments. For this purpose, a DNA ligase can be used which catalyzes the annealing of the ends of the DNA fragments, cf. Sgaramella, V., Van de Sande, J.H. and Khorana, H.G., Proc. Natl. Acad. Sci.

USA 67, 1468 (1970). If the sequences to be joined are not blunt-ended, ligase from E. coli can be used, cf. Modrich, P. and Lehman, I.R., J. Biol. Chem 245, 3626 (1970).

Reconstruction of the original sequence from subfragments obtained by restriction endonuclease treatment is greatly enhanced if a method can be used which avoids the reconstruction of inappropriate sequences. An inappropriate result can be avoided if a homogeneous length cDNA fragment having the desired sequence is treated with a reagent capable of removing the 5 'terminal phosphate groups from the cDNA before the homogeneous cDNA is cleaved by restriction endonuclease. Here, alkaline phosphatase is the preferred enzyme. 51-terminal phosphates are a structural prerequisite for the binding activity of DNA ligase subfragments. Thus, ends that do not have a 5 'terminal phosphate cannot be covalently joined. DNA subfragments can only be ligated to ends containing a 5'-terminal phosphate formed as a result of the cleavage effect of the restriction endonuclease on the isolated DNA.

26 6 4 64 0

The procedure described above prevents the most inappropriate joining reaction, namely that the two fragments are joined in the reverse order, i.e. the back to the front and not the front to the back. Other possible side reactions, such as dimerization or ring formation, cannot be prevented because these occur according to reaction type II, cf. Table 1 above. However, such side reactions are less detrimental because they result in physically identifiable and separable products, whereas reverse recombination does not.

To illustrate the methods described above, the rat insulin cDNA code has been isolated and recombined with the plasmid. The bacterial strain transformed by the DNA molecules was E. coli X-1776. Transformed bacteria were isolated by culturing in tetracycline-containing medium. The DNA of a recombinant plasmid contained in the transformed cells was found to contain an associated DNA fragment of approximately 410 nucleotides in length. Other recombinants were also isolated and analyzed by the same method. The ligated fragments were excised from the plasmid by Hind III or Hsu I endonuclease treatment and their DNA sequence was analyzed by the following method: Maxam, A.M. and Gilbert, W., Proc. Natl. Acad. Sci.

USA 74, 560 (1977). The nucleotide sequence of the ligated DNA fragments was found to be too long and contained the code for rat whole proinsulin I and the prepeptide sequence for thirteen amino acids, out of a total of 23 amino acids. The following is the nature of this nucleotide sequence.

The method just described can be used to isolate and purify a gene of a higher organism, including a human gene, and transfer it to a microorganism in which it replicates. The description describes new recombinant plasmids containing all or part of the isolated gene.

64640

Example 1 This example describes the extraction and isolation of rat insulin mRNA, the synthesis and characterization of DNA complementary thereto. To prepare rat islet cells, the pancreas of anesthetized rat was infused with Hank's saline by infusing the posterior pancreatic duct. Hank's saline solution is a standard solution known to those skilled in the art, which is sold e.g. Grand Island Biological Supply Company,

Grand Island, New York. The pancreas was removed, ground in Hank's solution at 0 ° C, and the trypsin inhibitor of collagenase and soybean was allowed to act. All procedures were performed at 0-4 ° C unless otherwise indicated. The circumstances of the latter measure were very critical. Two ground rat pancreases in Hank's solution were placed in a 30 ml glass tube to a total volume of 8 ml. All glass tubes were pretreated with silicone ("Siliclad", Clay-Adams Division, Becton-Dickinson Inc., Parsippany, New Jersey). The incubation mixture contained 12 mg of collagenase, an enzyme prepared from Clostridium histolyticum (method of preparation: Mandi, I., Mackennan, JD and Howes, EL, J. Clin. Invest. 32, 1323 (1943)), type CLS IV, and which available from Worthington Biochemical Corporation, Freehold,

New Jersey, and 1 mg of soybean trypsin inhibitor available from Sigma Chemical Company, St. Louis, Missouri. The tube was shaken at 90 shakes per minute at 37 ° C for 25 minutes. The optimal progression of collagenase activity had to be monitored continuously.

If the incubation was too short, the detachment of the islet cells remained incomplete, and if the incubation time was too long, the islet cells began to dissolve. After incubation, the tube was centrifuged for 1 minute at 200 rpm. The mother liquor was decanted and the particles were washed with Hank's solution, and the centrifugation and washing were repeated a total of five times. After final centrifugation, the particles were suspended in 15 ml of "Ficoll" solution (Pharmacia Chemical Company, Uppsala, Sweden) at a density of 1.085. Then 8 ml of "Ficoll" solution with a density of 1,080, 5 ml of "Ficoll" solution with a density of 1,060, 2 * 6 4 6 4 0 were added and the tube was first centrifuged in a rocking cup rotor for 5 minutes at 500 rpm and then 5 minutes at 2000 rpm. As a result of this procedure, the vesicle cells remained at the bottom of the tube and the islet cells rose to the gradient and formed a front between the top two layers. The islet cell layer contained neurons, lymph nodes, and connective tissue as impurities. Large impurity particles were removed from the material in the bed. The rest of the material was placed in a dissecting microscope, and visible impurities were removed by hand with a micropipette. The cell preparation was then mixed with Hank's solution and centrifuged. The mother liquor was decanted and the cell material was stored in liquid nitrogen.

Islet cells harvested from 200 rats were homogenized at U ° C in 4M guanidinium thiocyanate ("Tridom",

Fluka AG Chemische Fabrik, Buchs, Switzerland) containing 1 MyV mercaptoethanol and buffered to pH 5.0. The homogenate was layered with 1.2 ml of 5.7 M CsCl containing 100 mM EDTA and centrifuged at 15 ° C for 18 hours at 37,000 rpm in a Beckman ultra-centrifuge rotor SW 50.1 (Beckman Instrument Company , Fullerton, California). RNA migrated to the bottom of the tube.

Polyadenylated RNA was isolated from the whole RNA preparation by chromatography on oligo (dT) cellulose by the method of Aviv, H. and Leder, P., cf. above.

All polyadenylated RNA from rat Langerhans islets was transcribed into cDNA by avian myeloblastosis virus reverse transcriptase obtained from Dr. D.J. From Beard (Life Science Inc., St. Petersburg, Florida). The reaction was performed in a mixture of 50 mM Tris-HCl, pH 8.3, 9 mM MgCl 2> 30 mM NaCl, 20 mM δ-mercaptoethanol, 1 mM each of three non-radioactive deoxyribonucleoside triphosphates, 250 μM

a fourth deoxynucleoside triphosphate labeled with Ot-P with a specific activity of 50-200 Ci / mol, 20 ug / ml oligo-dT - ^ 18 (Collaborative Research, Waltham, Massachusetts), 100 ug / ml ml of polyadenylated RNA and 200 units / ml of reverse transcriptase. The mixture was incubated at 45 ° C for 15 minutes. EDTA-Na 2 was then added to a concentration of 25 mM, the solution was extracted with a self-contained volume of phenol saturated with water, and then the aqueous phase was chromatographed on a Sephadex G-100 column (diameter 0.3 cm, height 10 cm). eluent with 10 mM Tris-HCl, pH 9.0, 100 mM NaCl and 2 mM EDTA. Ammonium acetate, pH 5.0, was added to the nucleic acid eluted with an interstitial volume to a concentration of 0.25 M, and precipitated with ethanol. The precipitate was collected by centrifugation, dissolved in 50 μl of freshly prepared 0.1 M NaOH and incubated for 20 minutes at 70 ° C so that the RNA was hydrolyzed.

The mixture was neutralized by the addition of 1M sodium acetate, pH 4.5, 3 2 and the P'-cDNA product was precipitated with ethanol and dissolved in water. A portion of the single-stranded cDNA was analyzed on a native polyacrylamide gel by the following method: Dingman, C.W. and Peacock, A.C., Biochemistry 7, 659, (1968).

32

The gels were dried and if-DNA was examined by autoradiography using "Kodak No-Screen NS-2T" film (Eastman Kodak Corporation, Rochester, New York). Judging by the electrophoresis pattern, the cDNA was heterodisperse. Judging by known standards, it contained at least one cDNA species of about 450 nucleotides.

Example la

Example 1 was repeated, however, using different concentrations of (i) mercaptoethanol in the homogenization step of the islet cells, namely 0.05 M, 0.2 M, 0.6 M and 0.8 M. All these concentrations gave the same result, i.e. mRNA degradation. There was no effect of RN: donkey.

Example Ib

Examples 1 and 1a were repeated, but using different pH in the islet cell homogenization step, namely pH 6.0, 7.0 and 8.0. The same result was obtained at all these pH values, i.e. no degradation of the mRNA by RNase occurred.

30 64640

Example 2 This example describes the synthesis and characterization of rat insulin double-stranded cDNA. To synthesize the complementary strand x, the single-stranded cDNA obtained in Example 1 was subjected to reverse transcription. The reaction mixture contained 50 μl of Tris-HCl, pH 8.3, 3 mM MgCl 2, 10 mH dithiothreitol, 50 mM each of three deoxyribonucleoside triphosphates, labeled 1 mM oi.-P-labeled nucleoside triphosphate with a specific activity of 1-10 Ci / mmol, 50 ug / ml cDNA and 220 units / ml reverse transcriptase. The reaction mixture was incubated for 120 minutes at 45 ° C. The reaction was quenched by the addition of EDTA-Na 2 to a concentration of 25 mM, extracted with phenol, chromatographed on Sephadex G-100 and precipitated with ethanol. A batch of the reaction product with an activity of 500-1000 pulses per minute was analyzed by gel electrophoresis as described in Example 1. Comparison with standard samples revealed a hetero-dispersed band of approximately 450 nucleotides in length.

An aliquot of the DNA reaction product of Example 1 and Example 2 was separately treated with restriction endonuclease Hae III and similar gel electrophoresis was performed. Both products were cleaved by endonuclease and two radioactive bands were detected by gel electrophoresis. The bands generated by cleavage of the double-stranded cDNA represented products of substantially the same length as those generated by cleavage of the single-stranded cDNA.

Example 3 This example describes how the Hind III decanucleotide recognition site is ligated to the double-stranded rat islet cDNA prepared in Example 2 by blunt end ligation. The double-stranded reaction product of Example 2 at a concentration of 2-5 ug / ml was first treated for 30 minutes at 22u and then for 15 minutes at 10 ° C with 30 units of S1 nuclease having an activity of 1200 units / ml (Miles Laboratories, Elkhart, Indiana), in a solution of 0.03 M sodium acetate, 4.5, 0.3 M sodium chloride and 4.5 mM ZnCl 2. The reaction was quenched by the addition of Tris base to give a final concentration of became 0.1 M, EDTA to a final concentration of 25 mM, and E. coli tRNA (prepared by the following method: von Ehrenstein, G., Methods in Enzymology, SP Colowick and NO Kaplan, Eds., Vol. 12A, pp. 588 (1967)) 40 μg / ml. The reaction mixture was extracted with phenol, chromatographed on Sephadex G-100 and the β-cDNA eluted at 32 volumes was ethanol precipitated. This treatment yielded, in good yield, cDNAs with the base pairs needed to attach the chemically synthesized decanucleotides to the blunt end.

Hind ΙΙΙ decamers were prepared by the following method: Scheller, R.H. Dickerson, R.E., Boyer, H.W.,

Riggs, A.D. and Itakura, K., Science 196, 177 (1977).

Incorporation of Hind ΙΙΙ decamers into cDNA was performed by incubation for 1 hour at 1 ° C in a solution of 66 mM Tris-HCl, pH 7.6, 6.6 mM MgCl 2, 1 mM ATP, 10 mM dithiothreitol, 3 mM Hind Decamer III with an activity of 105 beats per minute per picomole and about 500 units / ml T4 DNA ligase. The reaction mixture was then heated at 65 ° C for 5 minutes to inactivate the ligase. It was then treated for 2 hours at 37 ° C with 150 units / ml Hsu I or Hind® endonuclease and then potassium chloride was added to a final concentration of 50 mM, β-mercaptoethanol to a final concentration of 1 mM, and EDTA was added. that the final concentration became 0.1 mM New England Bio-Labs, Beverly, Massachusetts, sells Hind III and Hae III endonucleases. The reaction product was analyzed by gel electrophoresis as in Example 1 and a peak corresponding to a sequence of about 450 nucleotides was observed, as well as truncated fragments of Hind d decamers.

Example 3a

Eco-RI decanucleotide recognition sites were ligated to the cDNA of Example 2 as described in Example 3. (Eco-RI decamers were prepared according to the aforementioned article by Scheller et al. And had the sequence 5'-CCGAATTCGG-3 '). The product was then digested with Eco'RI using the same 32,64640 reaction conditions as above (see digestion with Hsu I and Hind III). Eco R1 was obtained from New England Prolabs. The reaction product was analyzed by gel electrophoresis (as described in Example 1), and a peak corresponding to a sequence of about 450 nucleotides was found, as well as fragments of Eco-RI decamers.

Example 4 In this example, a recombinant plasmid is generated and characterized after replication has occurred.

Plasmid pMB-9 DNA (method of preparation: Rodriguez, RL, Boliver, F., Goodman, HM, Boyer, HW and Betlach, M., ICN-UCLA Symposium on Molecular and Cellular Biology, DP Wierlich, WJ Rutter and CF Fox, Eds ., (Academic Press, New York 1976), pp. 471-477) was cleaved from the Hind III restriction site by Hsu I endonuclease and then treated with alkaline phosphatase BAPF (Worthington Biochemical Corporation, Freehold, New Jersey). The reaction mixture contained 0.1 units of enzyme per 1 ug of DNA and was incubated in 25 mM Tris-HCl, pH 8, at 65 ° C for 30 minutes, after which the phosphatase was removed by phenol extraction. After ethanol precipitation, phosphatase-treated plasmid DNA was added to cDNA with Hind III cohesive ends to a ratio of 3 moles of plasmid per mole of cDNA. The mixture was incubated for 1 hour at 14 ° C in a solution of 66 mM Tris, pH 7.6, 6.6 mM MgCl 2, 10 mM dithiothreitol, 1 mM ATP and 50 units / ml T4 DNA ligase.

The mixture was added directly to an E. coli X-1776 cell suspension prepared for transformation as follows. Cells were grown at 37 ° C to a cell density of about 2 x 10 6 cells / ml in 50 ml of nutrient solution containing 10 g / l tryptone, 5 g / l yeast extract, 10 g / l sodium chloride, 2 mM sodium hydroxide, 100 μl ug / ml diaminopimelic acid and 40 ug / ml thymine. Cells were harvested by centrifugation at 5,000 rpm for 5 minutes at 5 ° C, suspended in 20 ml of cold 10 mM sodium chloride solution, centrifuged as above, and resuspended in transformation buffer containing 75 mM CaCl 2, 140 mM NaCl, and 10 mM Tris, pH 7.5, and kept on ice for 5 minutes. The cells were then centrifuged at 64640 33 and resuspended in 0.5 of any transformation buffer. Transformation was performed by mixing 100 μl of cell suspension and 50 μl of recombinant DNA (1 μg / ml). The mixture was first incubated for 15 minutes at 0 ° C, then for H minutes at 25 ° C and finally for 30 minutes at 0 ° C. The cells were then transferred to agar plates for growth under selected conditions.

Screening of the recombinant plasmids was performed in the presence of 5 ug / ml tetracycline for Hind III site transformation. A specific recombinant, called PAU-Γ, was isolated. Impure plasmid preparations with 2-5 ug of DNA isolated from PAU-1 were treated with an excess of Hsu'I endonuclease. EDTA-Na 2 was added to a final concentration of 10 mM and sucrose to a final concentration of 10% (w / v) and the mixture was separated on a polyacrylamide gel (8%). DNA was found at a site corresponding to approximately the length of the HIO base pair.

Example Ha. (i) Example H was repeated except that the DNA of plasmid pBR-322 prepared by Bolivar et al. was used instead of the plasmid pMB-9 DNA. as described (Gene 2 (1977) 95). The conditions used were otherwise the same, except that the final selection of the recombinant clones was performed by culturing them in a medium containing 20 μg / ml tetracycline. The DNA sequence transferred to the recombinant clone was separated as previously described to give a DNA fragment of approximately HIO base pair in length, the nucleotide sequence of which is shown in Table 1.

(ii) Example H was repeated except that plasmid pMB-9 DNA was replaced with plasmid pBR313 DNA prepared by Bolivar et al. by the method described (Gene 2 (1977) 75). The conditions used were otherwise the same except that the final selection of recombinant clones was performed as described in (i) 34 64640 to obtain a DNA fragment containing approximately HIO base pairs, the nucleotide sequence of which is shown in Table 1.

(iii) Example 4 was repeated except that plasmid pMB-9 DNA was replaced with plasmid pSCIOI DNA prepared by Cohen et al. by the method described in (Proc. Nat. Acad. Sci.

USA 70 (1973) 1293). The conditions used (including the recombinant clone selection method) were as in Example 4. The DNA sequence transferred to the recombinant clone was separated to give a DNA fragment of about 410 base pairs, the nucleotide sequence of which is shown in Table 1.

Example 4b. Examples 4 and 4a were repeated except that E. coli RRI or E. coli H3101 was used instead of E. coli X-1776. The conditions used were the same as in Examples 4 and 4a, and the same results were obtained.

Example 5

The DNA of plasmid PAU-1 according to Example 4 was further purified by electrophoresis on a 6% polyacrylamide gel. The DNA was then eluted from the gel

. OO

and was labeled by incubation with P-ATP and polynucleotide kinase under the conditions described by Maxam and Gilbert (see Proc.Natl.Acad, Sci., USA, 74 (1977), pages 560-). The enzyme catalyzes the migration of the radioactive phosphate group from f1 'P-ATP to the 5' ends of the DNA. The enzyme was obtained from E. coli by the following method: Panet, A., et al., Biochemistry 12, 5045 (1973). The DNA thus labeled was digested with Hae III endonuclease as described in Example 2, and both fragments, one of about 265 and the other of about 135 base pairs, were separated on a polyacrylamide gel as described in Example 1. The isolated fragments were subjected to specific cleavage reactions and sequenced by the above-mentioned method of Maxam and Gilbert. Table 1 shows a combination of the results of this series of experiments and the results of similar series of experiments in which cDNA was examined using plasmid vectors derived from Col E1, such as pMB-9 and pBR-322. The 5 'end of the sequence has an undefined sequence of about 50-120 nucleotides, and the length of the poly-dA at the 3' end varies. The sequence is based on the best current information. The corresponding amino acid sequence of rat proinsulin I begins at the triplet site numbered 1 and ends at the triplet site numbered 86. The sequence marked with a dashed line is still slightly uncertain.

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Example 6 In this example, the nucleotide sequence of human insulin is isolated, purified, and transferred into a plasmid essentially as described in Examples 1-4 from human pancreatic tissue obtained, e.g., from a donated pancreas or a freshly dead body or pancreatic islet tumor. Substantially as described in Example 4, a microorganism containing the nucleotide sequence code of the A chain and the B chain of human insulin is prepared. The known amino acid sequence of the human insulin A chain is as follows: 1 10 Gly-Ile-Val-Glu-Gln-Cys-Cys-Thr-Ser-Ile-Cys-Ser-Leu-Tyr-.

20 ·

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

The known amino acid sequence of the human insulin B chain is as follows: 1 10 Phe-Val-Asn-Glu-His-Leu-Cys-Gly-Ser-His-Leu-Val-Glu-Ala-Leu-

Tyr-Leu-Val-Cys-Gly-Glu-Arg-Gly-Phe-Phe-Tyr-Thr-Pro-Lys-Thr

Amino acid sequences are numbered from the end with the free amino group, cf. Smith, L.F., Diabetes 21 (suppl. 2), 458 (1972).

Claims (1)

A method for producing a recombinant DNA molecule containing a nucleotide sequence encoding insulin and used in the manufacture of an insulin-producing microorganism, characterized in that a) mRNA is isolated from pancreatic tissue islet cells by homogenization in the presence of a ribonuclease inhibitor, e.g. a mixture of guanidinium thiocyanate and 0.05 to 1.0-m / J-mercaptoethanol at a pH of 5.0 to 8.0, so that no ribonuclease degradation of the mRNA occurs, b) prepared from the obtained mRNA in a manner known per se by means of a reverse transcriptase enzyme a cDNA having an insulin-encoding nucleotide sequence and said cDNA is double-stranded, c) ligation of restriction endonuclease recognition site sequences into the ends of the resulting double-stranded cDNA in a manner known per se, d) opening the bacterial plasmid vector with a restriction endonuclease, for example with Hind III, Hsu I or Eco R1, in a manner known per se, e.g. by incubation at pH 7.6 at 37 ° C for 2 hours. e) hydrolyzing the 51-phosphate end groups of the plasmid vector obtained in d) by treatment with, for example, alkaline phosphatase at pH 8.0 at 65 ° C for 30 minutes, wherein the cohesive ends mentioned in d) do not f) coupling the plasmid vector treated according to e) and the cDNA obtained in c) in a manner known per se by incubation in the presence of DNA ligase and ATP, e.g. at pH 7.6 at 14 ° C for one hour, using a molar excess a plasmid vector to obtain a fusion DNA molecule comprising a nucleotide sequence encoding insulin. 39 6 4 6 4 0 For use in the preparation of combinations of DNA molecules in which the nucleotide sequence of the insulin is isolated and which can be derived from the production of the insulin-producing microorganism, the kännetecknat därav, att a) mRNA isoleras ur öceller av buks the genome is homogenized when the ribon nucleus is inoculated into a single cell of t.ex. with a blending of 4-m guanidinium thiocyanate and 0.05 - 1.0-m / & - mercaptoethanol, at a pH of 5.0 - 8.0, the colors show a ribonuclease drop of the mRNA and then, b) ur denoted mRNA (a) a transcript of the reverse transcriptase sequence of the reverse transcriptase enzyme, the nucleic acid sequence of the insulin encoder, and a transcript of the transducer sequence; and for this purpose, the cDNA capacitance is selected from the group consisting of enzymes from the above. , t.ex. med Hind III, Hsu I eller Eco RI, pä och i för sig känt sätt t.ex. the genome is incubated at a pH of 7.6 and at a temperature of 37 ° C and 2 times, for which a plasmid vector is used in the cohesive region, e) at point d) a plasmid vector 5'-phosphate group hydrolyzes the genome t.ex. the alkaline phosphate is at a pH of 8.0 and a temperature of 65 ° C for 30 minutes, the color of point (d) being immediately adjacent to point (f) of point (e) of the plasmid vector and point (c) ), the cDNA is replicated at the same time as the nucleus of the DNA ligand and the ATP, exempted at pH 7,6 and the temperature.